chemical analysis of typical beverages and açaí …epubs.surrey.ac.uk/852962/1/thesis - fernanda...
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Chemical Analysis of Typical Beverages and Açaí Berry from South America
by
Fernanda Vanoni Matta
A thesis submitted to the Department of Chemistry in conformity with the
requirements for the Degree of Doctor of Philosophy
Faculty of Engineering and Physical Sciences
University of Surrey, Guildford, GU2 7XH
2019
i
Declaration of Originality
This thesis and the work to which it refers are the results of my own
efforts. Any ideas, data, images or text resulting from the work of others (whether
published or unpublished) are fully identified as such within the work and
attributed to their originator in the text, bibliography or in footnotes. This thesis
has not been submitted in whole or in part for any other academic degree or
professional qualification. I agree that the University has the right to submit my
work to the plagiarism detection service TurnitinUK for originality checks.
Whether or not drafts have been so-assessed, the University reserves the right to
require an electronic version of the final document (as submitted) for assessment
as above.
______________________________________
Fernanda Vanoni Matta
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Abstract
Brazil is a major producer of special natural foods and beverages that are commercialised and sold, locally and globally, as natural and processed products. Many are marketed as good sources of elements (minerals) and polyphenols, that play an important role in human health. At present, very few scientific studies have reported the chemical composition of these natural foods or beverages obtained in Brazil. The aim of this research was to determine the levels of elements and polyphenols in yerba mate, roasted coffee and açaí berries. The chemical composition was determined for the elemental content by inductively coupled plasma mass spectrometry and polyphenols by ultra-high performance liquid chromatography and ultraviolet–visible spectrophotometry. The elemental levels of non-commercial yerba mate leaves from the Barão de Cotegipe plantation (southern Brazil) had higher levels in the old leaves. New leaves grown on trees from an organic plantation had higher elemental levels, especially when compared with other plantations treated with NPK fertilisers. Moreover, higher elemental levels were found in plants grown in traditional organic plantations than in natural forests. The elemental levels of commercial yerba mate products from Brazil and Argentina were found to be similar. All levels were higher for commercial tea bag products than for green loose material. In Brazil, yerba mate is also sold as a roasted product (loose and tea bag) which had higher elemental levels than that for the green loose material. Infusions prepared using tea bag samples had higher elemental, polyphenol and xanthine levels than that for green loose regular infusions. Moreover, regular infusions made with green loose yerba mate had significantly higher levels of trace elements, polyphenols and xanthines in comparison with the roasted samples. All infusion methods (regular, Brazilian and bombilla) represented 0.1 to 5.0 % of the recommended daily allowance (RDA) of the trace elements measured. A regular infusion serving (1 cup of 200 mL) would provide 23.7 to 106.0 % for males and 30.3 to 135.5 % for females of the manganese RDA, depending on the type of yerba mate product. In terms of the total polyphenol intake, a regular infusion serving (200 mL) could contribute 4.0 to 14.5 % of the daily intake. The effect of roasting different coffee varieties (Obatã, Catuaí, Bourbon Amarelo and blend) collected from the Fazenda Palmares and Flor plantations (Amparo, São Paulo State) resulted in a slight increase of the elemental content of the beans during the roasting process. The total polyphenol content of coffee infusions, produced from beans collected at different times of the roasting process, showed a variation of 7.0 to 52.0 % higher levels in the dark roast (10 min) when compared to the green bean infusions (0 min). The chlorogenic acids and caffeine data showed a similar trend with an increase in the levels of the infusions prepared using the medium roast coffee. A cup of coffee (92 mL) can contribute up to 7.0 % of the estimated daily intake of polyphenols. Açaí berries obtained from the Amazon region are a major nutritional source for the local population and the processed pulp is becoming a major national and global ‘super-fruit’ product. The non-commercial purple mature pulp had a significantly higher concentration of
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total polyphenols and anthocyanins in comparison with the white samples (different variety). These samples were found to have high antioxidant activity due to the higher levels of total polyphenols and total anthocyanins when compared to the commercial purple and non-commercial white pulp samples. The strong antioxidant effect of açaí pulp was confirmed on mouse cells through the inhibition of producing radical oxygen species (ROS). A wound healing experiment performed using human fibroblast cells confirmed a migration effect on cells subjected to açaí pulp extracts. These results are very important, as such an experiment has never been reported, and implies that processed açaí pulp may have potential as a wound healing agent. There were no statistically significant differences in the elemental content between purple and white pulp samples. Processed açaí pulp, with less water added, had higher elemental levels (based on a fresh weight). Based on a regular consumption of purple açaí (500g), the dietary intake of total polyphenols would be more than 100% of the RDA. The consumption of açaí represents a good source of Mn (average of 1500% of the RDA), Cu (90%), Mg (30%), Ca (20%) and Zn (15%). In summary, this research provides a unique database of chemical values using analytically robust methods that can be used to evaluate the nutritional quality of Brazilian natural and commercial products and the impact of consumption on dietary intake and human health.
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“Eu sou a chuva que lança a areia do Saara
Sobre os automóveis de Roma,
Eu sou a sereia que dança, a destemida Iara,
Água e folha da Amazônia”
Caetano Veloso
v
List of Contents
Declaration of Originality .................................................................................. i
Abstract .............................................................................................................. ii
List of Figures ................................................................................................... x
List of Tables ................................................................................................... xv
Abbreviations ................................................................................................. xxi
Glossary ........................................................................................................ xxiv
Acknowledgements ..................................................................................... xxvi
Chapter 1. General Introduction ............................................................. 1
1.1. Overview of Brazil ................................................................................. 2
1.2. Trace Elements in the Human Diet and Health ................................. 5 1.2.1. Dose response curve and homeostasis of chemical elements .................... 7 1.2.2. Dietary intake – World Health Organisation (WHO) guidelines .................... 8 1.2.3. Deficiency and toxicity effects of chemical elements .................................... 9 1.2.4. Chemical elemental content of typical foodstuffs and beverages from Brazil
10 1.2.5. Manganese chemistry ................................................................................. 11
1.3. Polyphenols and Xanthines ............................................................... 14 1.3.1. Polyphenol chemistry .................................................................................. 15 1.3.2. Health effects of polyphenols ...................................................................... 17 1.3.3. Food total polyphenol range ....................................................................... 18
1.4. Polyphenol and elemental relationship............................................ 18
1.5. Analytical Methods and Challenges ................................................. 19
1.6. Aim and Objectives............................................................................. 24
Chapter 2. Methodology ........................................................................ 27
2.1. Introduction ......................................................................................... 28
2.2. Sample Collection ............................................................................... 29 Sample preparation ..................................................................................... 30
vi
2.3. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for Elemental Analysis ..................................................................................................... 31
Instrumentation – ICP-MS ........................................................................... 34 Internal standards – ICP-MS ....................................................................... 35 Limit of detection (LoD) and linear dynamic range (LDR) – ICP-MS .......... 36 Validation (accuracy and precision) – ICP-MS ........................................... 39
2.4. UV-Vis Spectroscopy for the Total Polyphenol Content Analysis 43
Instrumentation – UV-Vis ............................................................................ 44 Total polyphenol content by Folin-Ciocalteu analysis ................................. 45
2.5. High Performance Liquid Chromatography (HPLC) for Polyphenol Profile Analysis ........................................................................................................... 46
Instrumentation - HPLC............................................................................... 48
2.6. Statistical Analysis ............................................................................. 50 D'Agostino and Pearson normality test ....................................................... 51 Significance tests ........................................................................................ 51 Correlation coefficients................................................................................ 54
2.7. Summary .............................................................................................. 55
Chapter 3. Yerba Mate ........................................................................... 57
3.1. Introduction ......................................................................................... 58
3.2. General Introduction to Yerba Mate ................................................. 58 3.2.1. Natural occurrence ...................................................................................... 59 3.2.2. Production (plantation to processing plant) and products .......................... 60 3.2.3. Methods of consumption ............................................................................. 64
3.3. Health Effects of Yerba Mate Consumption .................................... 65 3.3.1. Chemical composition of yerba mate .......................................................... 66
3.4. Aim and Objectives............................................................................. 68
3.5. Non-Commercial Studies on Yerba Mate ......................................... 69 3.5.1. Description of the samples .......................................................................... 70 3.5.2. Materials and method.................................................................................. 71 3.5.3. Production by traditional plantations ........................................................... 71 3.5.4. Production by natural forest plantations ..................................................... 81
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3.5.5. Commercial processing plant ...................................................................... 84
3.6. Studies on Commercial Yerba Mate ................................................. 86 3.6.1. Description of the samples .......................................................................... 86 3.6.2. Materials and method.................................................................................. 87 3.6.3. Total elemental composition of commercial yerba mate ............................ 89 3.6.4. Elemental composition of yerba mate infusions ......................................... 94 3.6.5. Polyphenolic composition of yerba mate .................................................. 104
3.6.5.1 Total polyphenol of infusions ......................................................... 104 3.6.5.2 Chlorogenic acids, caffeine and theobromine levels in yerba mate
infusions 110 3.6.6. Link to dietary intake through consumption of yerba mate ....................... 114
3.6.6.1 Dietary intake of trace elements ..................................................... 114 3.6.6.2 Dietary intake of polyphenols ......................................................... 116
3.7. Conclusions....................................................................................... 119
Chapter 4. Chemical Composition of Roasting Brazilian Coffee ... 123
4.1. Introduction ....................................................................................... 124 4.1.1. Coffee production in Brazil ........................................................................ 124 4.1.2. Roasting of coffee beans .......................................................................... 125
4.2. Review of Roasting Coffee in Brazil (Elemental and Polyphenols) 126
4.3. Coffee Beans and the Roasting Process at Amparo, São Paulo State, Brazil 127
4.3.1. Sample collection and preparation of coffee beans .................................. 128 4.3.2. Roasting process ...................................................................................... 128 4.3.3. Grinding roasted coffee beans and particle size....................................... 129 4.3.4. Coffee infusions ........................................................................................ 129
4.4. Elemental Levels of Roasted Coffee .............................................. 130
4.5. Total Polyphenol and Chlorogenic Acid Levels in Roasted Coffee (infusions) 134
4.6. Effect of Pore Size of Ground Roasted Coffee .............................. 141
4.7. Chemical Composition of Roasted Coffee Infusions and Human Dietary Intake ............................................................................................................ 145
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4.8. Summary ............................................................................................ 146
Chapter 5. Brazilian Açaí ..................................................................... 149
5.1. Introduction ....................................................................................... 150
5.2. General Introduction to Açaí Berries from the Amazon Region, Brazil 150
Natural occurrence .................................................................................... 150 Brazilian açaí production and products ..................................................... 152 Health effects of açaí consumption ........................................................... 155
5.3. Chemical Composition of Açaí........................................................ 157
5.4. Aim and Objectives........................................................................... 161
5.5. Investigation of the Relationship between the Chemical Composition and Biological Activity of Açaí Samples ........................................ 162
Introduction................................................................................................ 162 Description of the samples ........................................................................ 162 Sample identification based on colour ...................................................... 163 Method development for the açaí extractions for organic analysis .......... 166 Total polyphenol and flavonoid content: Materials and method ............... 167 Total polyphenol and flavonoid content: Results and discussion ............. 168 Total anthocyanin content: Materials and method .................................... 174 Total anthocyanin content: Results and discussion .................................. 176 Total proanthocyanidin content: Materials and method ............................ 178
Total proanthocyanidin content: Results and discussion ........................ 178 Chemical antioxidant activity: Materials and method .............................. 180 Chemical antioxidant activity: Results and discussion ........................... 182 Elemental composition: Materials and method ....................................... 183 Elemental composition: Results and discussion ..................................... 183 Biological toxicity (cell viability assay): Materials and method................ 190 Biological toxicity (cell viability assay): Results and discussion ............. 190 Biological effect of açaí on radical inhibition assays: Materials and method
191 Biological effect of açaí on radical inhibition assays: Results and
discussion 193 Biological effect of wound healing in human cells: Materials and method
195
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Biological effect of wound healing in human cells: Results and discussion
197
5.6. Evaluation of the Amazon Geographical Variability and Industrial Processing on the Chemical Composition of Açaí ............................................... 200
Introduction................................................................................................ 200 Description of the samples ........................................................................ 200 Total polyphenol content: Materials and method ...................................... 205 Total polyphenol content: Results and discussion .................................... 205 Elemental composition: Materials and method ......................................... 208 Elemental composition: Results and discussion ....................................... 208
5.7. Link to Dietary Intake of Total Polyphenols and Minerals of Açaí 211
5.8. Conclusion......................................................................................... 217
Chapter 6. Conclusions and Future Work ........................................ 222
6.1. Overview ............................................................................................ 223
6.2. Yerba Mate ......................................................................................... 224
6.3. Chemical Composition of Roasting Brazilian Coffee ................... 227
6.4. Açaí ..................................................................................................... 228
6.5. Limitations ......................................................................................... 231
6.6. Future Work ....................................................................................... 232
Bibliography .................................................................................................. 233
x
List of Figures
Page
Figure 1.1 Map of South America with the highlighted production sites of yerba
mate, coffee and açaí (maps, 2019) 5
Figure 1.2 Biological dose response curve for elements (Underwood and Mertz,
1979) 8
Figure 1.3 Schematic of the Mn-forms as a function of Eh/pH in aqueous matrices
and aerobic conditions (Dorronsoro et al., 2006). 12
Figure 1.4 Schematic of the chemical formulae of xanthine, caffeine and
theobromine, important compounds present in yerba mate and coffee
(Merck, 2018).
15
Figure 1.5 Schematic of the chemical formulae of typical polyphenols found yerba
mate, coffee and açaí: (A) phenolic acids; (B) hydroxybenzoic acids; and
(C) flavonoids (Merck, 2018).
17
Figure 2.1 Analytical sequence adopted for the study of the chemical analysis of
typical beverages and açaí berries from South America. 29
Figure 2.2 Instrumentation of an inductively coupled plasma mass spectrometer
(ICP-MS) Agilent Series (Agilent, 2017). 32
Figure 2.3 Calibration curve for manganese (55Mn) using 115In as internal standard
for the Agilent 7800 ICP-MS in helium collision cell mode. 38
Figure 2.4 Gallic acid calibration curve obtained by the Folin-Ciocalteu assay on a
UV-Vis instrument (refer to section 2.4.2) 46
Figure 2.5 5-caffeoylquinic acid calibration curve obtained by the HPLC analysis
(refer to section 2.5.1). 50
Figure 3.1 Natural occurrence of yerba mate in South America. Adapted from
Maccari Junior (2005). 59
Figure 3.2 Main state producers of yerba mate in Brazil with their contribution (%) to
Brazilian production. Adapted from IBGE (2017). 61
Figure 3.3 Scheme of production of yerba mate. Adapted from Maccari Junior
(2005). 62
Figure 3.4 (A) Yerba mate tree; (B) Sapeco stage; (C) Yerba Mate cancheada for
the Brazilian and Argentine markets. Adapted from UFRS (2012). 63
Figure 3.5 Argentine (left) and Brazilian (right) green yerba mate commercial
samples. 64
Figure 3.6 Typical Brazilian Chimarrão (mate) consumption (Forma, 2016). 65
Figure 3.7 Schematic of the proposed bombilla method. Adapted from NGC (2015). 89
Figure 3.8 Manganese concentration (mg/200 mL) and rate of accumulation* (i.e.
potential intake) between 5 fractions (successive additions of hot water)
based on using the bombilla method (refer to section 3.6.2) for green
103
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loose yerba mate purchased in Brazil (n= 16) and Argentina (n=7). n is
the number of samples. The WHO set the upper limit for Mn in 11
mg/day (IOM, 2002). The analyses were determined using ICP-MS (refer
to section 2.3). *Calculated as the sum of the previous fractions.
Figure 4.1 Different maturation stages of the coffee cherry, showing green and the
mature red berries. 125
Figure 4.2 Roasting of Brazilian coffee beans from green (time = 0 minutes) and at
2 minutes intervals until the production of the dark roasted product (t =
10 minutes).
129
Figure 4.3 Typical Brazilian coffee infusion method. 130
Figure 4.4
Concentration of chlorogenic acids and caffeine (mg/L) of Brazilian
coffee infusions produced from beans sampled during the roasting
process (green t = 0 minutes, medium t = 6 min and dark roasted t = 10
min). The samples were the blend of the coffee varieties collected from
the Fazenda Palmares plantation (Amparo, São Paulo State) and
analysed by UHPLC (refer to section 2.5). n = 1.
135
Figure 4.5
The concentration of chlorogenic acids and caffeine (mg/L) of Brazilian
coffee infusions produced from beans sampled during the roasting
process (t = 0 to 10 min). The samples were of the Obatã coffee variety
collected from Fazenda Flor plantation (Amparo, São Paulo State) and
analysed by UHPLC (refer to section 2.5). n = 1.
137
Figure 4.6
The concentration of chlorogenic acids and caffeine (mg/L) of Brazilian
coffee infusions produced from beans sampled during the roasting
process (t = 0 to 10 min). The samples were of the Catuaí coffee variety
collected from the Fazenda Palmares plantation (Amparo, São Paulo
State) and analysed by UHPLC (refer to section 2.5). n = 1.
137
Figure 4.7
The concentration of chlorogenic acids and caffeine (mg/L) of Brazilian
coffee infusions produced from beans sampled during the roasting
process (t = 0 to 10 min). The samples were of the Bourbon Amarelo
coffee variety collected from the Fazenda Palmares plantation (Amparo,
São Paulo State) and analysed by UHPLC (refer to section 2.5). n = 1.
138
Figure 4.8
The concentration of chlorogenic acids and caffeine (mg/L) of Brazilian
coffee infusions as a function of the different bean particle sizes
according to the method of infusion: (1) coarse for French press; (2)
regular for siphon; (3) electric perk; (4) drip; (5) fine for Brazilian
infusions; and (6) espresso. The samples were collected at the medium
roast time of the process (t = 6 minutes), being a blend of the coffee
varieties sampled from the Fazenda Palmares plantation (Amparo- São
Paulo) and analysed by UHPLC (refer to section 2.5). n = 1.
140
Figure 4.9
The concentration of chlorogenic acids and caffeine (mg/L) of Brazilian
coffee infusions as a function of the different bean particle sizes
according to the method of infusion: (1) coarse for French press; (2)
regular for siphon; (3) electric perk; (4) drip; (5) fine for Brazilian
infusions; and (6) espresso. The samples were collected at the dark
roast time of the process (t = 10 minutes), being a blend of the coffee
141
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varieties from the Fazenda Palmares plantation (Amparo- São Paulo)
and analysed by UHPLC (refer to section 2.5). n = 1.
Figure 4.10 (A) to (F)
Scanning electron microscope images of Brazilian coffee beans sampled
during the roasting process (t = 0 to 10 min). The samples were the
blend of the coffee varieties collected from the Fazenda Palmares
plantation (Amparo, São Paulo State).
145
Figure 5.1 (A) Map of South America with Amazon forest in green (Fao, 2015); and
(B) Natural botanical distribution of two different species of açaí, namely,
Euterpe precatoria and Euterpe oleracea. Adapted from Yamaguchi et al.
(2015).
151
Figure 5.2 (A) The natural occurrence of açaí (E. oleracea) in the flooded forest
near Belém, Para State, Brazil; (B) the purple açaí fruit (pulp and seed
separated); and (C) the white açaí (‘greenish’) berries. Adapted from Potsch (2010).
152
Figure 5.3 (A) Harvesting of açaí by locals in the Amazon; and (B) a
‘despolpadeira’, machine used to mechanically extract the pulp of the
açaí berries. Adapted from Vida (2010).
155
Figure 5.4 Chemical structure of most predominant anthocyanin compounds found
in açaí berries being (A) cyanidin-3-glucoside and (B) cyanidin-3-
rutinoside (Yamaguchi et al., 2015).
158
Figure 5.5
Picture of the açaí berry samples where: (A) is the purple açaí whole
used as reference; (B) is the white açaí whole; (C) is the purple de-fatted
sample; (D) is the white de-fatted; (E) is the freeze-dried frozen pulp from
São Paulo; (F) is the commercial sample from São Paulo; and 7 is the
commercial sample bought in United Kingdom.
164
Figure 5.6 Illustration of the CIELAB colour space international parameters.
Adapted from Molino et al. (2013). 164
Figure 5.7 Molecular structures of the complexation between quercetin and
aluminium chloride used to determine the levels of total flavonoids.
Adapted from Frederice et al. (2010).
168
Figure 5.8 Box plots of the total polyphenol content (gallic acid equivalent mg/g) of
the açaí extracts determined using the Folin-Ciocalteu assay (refer to
section 5.5.5). The values relate to the type of sample (purple, n= 6;
white and commercial n= 4; n is the number of samples).
171
Figure 5.9 Standard curve of Cy-3-Glu concentration (mg/L) and the areas of the
peaks (mAU) used as the calibration curve for the determination of the
anthocyanin content of açaí extracts using a HPLC-DAD chromatogram
(at 520 nm).
175
Figure 5.10
The determination of the anthocyanin content of a purple non-
commercial açaí sample following methanolic extraction and using a
HPLC-DAD chromatograph (at 520 nm). The anthocyanin peaks are
cyandin-3-glucoside (retention time, tR = 17.458 minutes), cyandin-3-
rutinoside (tR = 21.069 min) and peonidin-3-rutinoside (tR = 26.237 min).
177
Figure 5.11 Total proanthocyanidin content (PAC) of açaí extractions presented as 179
xiii
B1 equivalents (B1E) via DMAC assay (refer to section 5.5.8) and
compared between the methanolic (70%MeOH) and aqueous (0.5%HAc)
extraction methods (n= 4, n, number of instrument replicates). Sample 1:
Purple açaí whole; 2: Purple açaí de-fatted; 3: white açaí whole; 4: white
açaí de-fatted; 5: oil extracted from white acai ; 6: oil extracted from
purple acai ; 7: pulp SP; 8: powder SP; 9: powder UK.
Figure 5.12 Antioxidant activity of açaí extracts determined by the ABTS assay, data
reported as Trolox equivalents (TE) (n=3; n, number of instrumental
replicates).
182
Figure 5.13
Box plots of the total elemental content of minor elements (mg/kg d.w.) of
açaí pulp samples using ICP-MS (refer to section 2.1) relating to the type
of sample (non-commercial: purple n= 6; and white n= 4; and
commercial: purple n= 4; n is the number of samples). The commercial
sample is a combination of pulp SP and powders SP and UK.
186
Figure 5.14
Box plots of the total elemental content of trace elements (mg/kg d.w.) of
açaí pulp samples using ICP-MS (refer to section 2.1) relating to the type
of sample (non-commercial : purple n= 6; and white n= 4; and
commercial: purple n= 4; n is the number of samples). The commercial
sample is a combination of pulp SP and powders SP and UK.
187
Figure 5.15 Formazan production levels in RAW 264.7 macrophage cells treated with
açaí extract solutions. Results expressed as mean ± st dev, n=3; n,
number of instrumental replicates).
191
Figure 5.16 Nitric oxide (NO) production in RAW 264.7 macrophage cells stimulated
with lipopolysaccharide (LPS). The cells were treated with 50 µg/mL açaí
extracts and dexamethasone (DEX). The results are expressed as the
mean ± st dev, n=3; n, number of instrumental replicates.
194
Figure 5.17
Radical oxygen species (ROS) production in RAW 264.7 macrophage
cells stimulated with lipopolysaccharide (LPS). The cells were treated
with 50 µg/mL açaí extracts and dexamethasone (DEX). Results are
expressed as the mean ± st dev, n=3; n, number of instrumental
replicates.
195
Figure 5.18 Cell migration determined using the OrisTM Cell 2-D migration of
adherent cells assay kit (Oris, 2017). 196
Figure 5.19
Florescence absorption of the radical oxygen species (ROS) production
in human dermal fibroblast cells (adult). The cells were treated with 50
µg/mL açaí extracts or 10% FBS (refer to section). Results are
expressed as the mean ± st dev, n=3; n, number of instrumental
replicates.
198
Figure 5.20 Fluorescence images of ‘wound healing’ of human dermal fibroblast cells (adult) between time 0 (A) and after 48 hours of incubation after
treatment with non-commercial white açaí whole sample (B).
199
Figure 5.21 (A) Picture of the open-air açaí market and (B) purple açaí fruits in Belém
(PA), Brazil. 201
Figure 5.22 Map of the sources where the açaí samples were harvested by the native 202
xiv
people. Adapted from Google Maps (2019).
Figure 5.23 Summary of the açaí processing steps and the differences between
companies, being Company I: Açaí Amazonas, Company II: Point do
açaí; and Company III: Açaí Santa Helena.
204
Figure 5.24
Box plots representing the total polyphenol (TP) content (gallic acid
equivalent mg/kg f.w.) of açaí extractions using the Folin-Ciocalteu assay
(refer to section 2.2.). The samples relate to the type of sample (non-
commercial, purple/whole: fruit, n= 7; seed, n=5; and processed freeze-
dried pulp, n= 8; where n is the number of samples).
206
Figure 5.25
Box plots representing the percentage intake (%) of minor elements
based on the consumption of a 500 g serving of açaí pulp (purple/whole)
(fresh weight). The data is compared with the World Health Organisation
(WHO) recommended daily allowance (RDA) for males (M) and females
(F). Note: WHO provide no gender data for Ca.
216
Figure 5.26 Percentage intake (%) of total polyphenol (TP) and trace elements of 500
g serving of açaí (fresh weight) in relationship to the recommended daily
allowance (RDA) for males (M) and females (F).
217
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List of Tables
Page Table 1.1
Symptoms of deficiency and toxicity of calcium, magnesium, iron, zinc
and copper (Strachan, 2010, Combs, 2013). 10
Table 1.2
Major sources and concentrations of calcium, magnesium, iron, zinc
and copper in the Brazilian diet (Unicamp, 2011). 11
Table 1.3
Manganese levels (mg/kg) in traditional beverages of Brazil analysed
by inductively coupled plasma optical emission spectrometry (ICP-
OES) (Unicamp, 2011).
14
Table 1.4 Analytical review of published studies on the elemental levels of
Brazilian yerba mate. 20
Table 1.5 Analytical review of published studies on the elemental levels of
Brazilian açaí 22
Table 1.6 Analytical steps involved in the analysis of polyphenols in plants and
food (Plaza et al., 2018). 24
Table 2.1 Typical operating conditions for the Agilent ICP-MS instruments. 35
Table 2.2 Investigated isotopes; limits of detection (LoD) in 1% HNO3 and
double-distilled deionised water (DDW) using 115In as internal standard
for the Agilent 7800 ICP-MS.
37
Table 2.3 Investigated isotopes and linear dynamic range (µg/L) used for each of
the type of samples (yerba mate, coffee and açaí) used in this study.
115In was used as internal standard for the Agilent 7800 ICP-MS
analysis.
39
Table 2.4 Evaluation of accuracy (comparison of measured and certified
elemental concentrations) and precision (relative standard deviation
(RSD %)) of for NIST SRM 1640a and CRM 3 (for Na, Mg, K and Ca).
41
Table 2.5 (a) Comparison of the certified reference values (CRM) with the calculated
concentration for the Tea Leaves INCT-TL-1 certified reference
materials.
42
Table 2.5 (b) Comparison of the certified reference values (CRM) with the calculated
concentration for the Peach Leaves SRM 1547 certified reference
materials (95% confidence level).
43
Table 2.6 Gradient programme for UHPLC analysis of yerba mate and coffee
infusions. 49
Table 3.1 Element content of yerba mate leaves for selected elements reported
in literature (weight basis not reported). 68
Table 3.2 Total elemental levels are reported as the mean and range (min –
max) of yerba mate leaves (based on age – new and old) for non-
commercial samples (mg/kg, dry weight) collected from traditional
plantations cultivated either using NPK fertilisers or non-chemical
73
xvi
(organic). The digested samples were analysed by ICP-MS (refer to
section 2.3).
Table 3.3
Statistical analysis using a two-tailed t-test (Miller et al., 2018) to
evaluate the relationship between the elemental levels of yerba mate
leaves (based on age – new and old) for non-commercial samples
collected from traditional plantations cultivated either using NPK
fertilisers or non-chemical (organic).
74
Table 3.4 Statistical analysis using a two-tailed t-test (Miller et al., 2018) to
evaluate the relationship between the elemental levels of yerba mate
leaves based on the use or non-use (organic) of NPK fertilsers during
traditional cultivation.
77
Table 3.5
Total elemental levels (mean ± standard deviation; mg/kg, dry weight)
of yerba mate leaves collected at different heights from the same tree
grown in a traditional plantation (cultivated as organic). The digested
samples analyses were analysed using ICP-MS (refer to section 2.3).
The number of samples, n = 2 replicates.
80
Table 3.6
Total elemental levels are reported as the mean and range (min –
max) of yerba mate leaves (based on age – new and old) for non-
commercial samples (mg/kg, dry weight) collected from traditional
cultivated organic plantations and grown between and beneath trees of
a native forest. The digested samples were analysed by ICP-MS (refer
to section 2.3)
82
Table 3.7 Statistical analysis using a two-tailed t-test (Miller et al., 2018) to
evaluate the relationship between the elemental levels of yerba mate
leaves (new and old) grown in traditional organic or native forest
plantations (refer to Table 3.6).
83
Table 3.8 Total elemental levels (mean ± standard deviation) of non-commercial
yerba mate samples (mg/kg, dry weight) during the commercial
processing of the harvested tree material. The analyses were
determined using ICP-MS (refer to section 2.3).
86
Table 3.9 Total elemental levels reported as mean and range (min – max) of
different types of commercial yerba mate samples (mg/kg, dry weight)
obtained from Brazil and Argentina. The analyses were determined
using ICP-MS (refer to section 2.3); n is the number of samples.
90
Table 3.10 Statistical analysis using a two-tailed Student t-test (Miller et al., 2018)
to evaluate the relationship between the origin (Brazil and Argentina);
packaging (loose and tea bags) and roasting (green and roasted) of
commercial yerba mate samples (refer to Table 3.9).
93
Table 3.11 Elemental levels (µg/200 mL), reported as mean and range (min –
max), of regular infusions of commercial yerba mate samples from
Brazil and Argentina. The analyses were determined using ICP-MS
(refer to section 2.3).
97
Table 3.12 Statistical analysis using a two-tailed Student t-test (Miller et al., 2018)
to evaluate the relationship between the origin (Brazil and Argentina); 98
xvii
packaging (loose and tea bags) and roasting process (green loose and
roasted) of regular infusions of commercial yerba mate (refer to Table
3.11).
Table 3.13 Percentage extraction (%) of regular infusions of commercial yerba
mate samples from Brazil and Argentina. Values reported as a mean
and range (min – max).
98
Table 3.14
Elemental levels (µg/200 mL), reported as the mean and range (min –
max), of Brazilian iced tea infusions prepared using commercial yerba
mate products from Brazil and Argentina. The analyses were
determined using ICP-MS (refer to section 2.3). n is the number of
infusion samples.
101
Table 3.15
Manganese levels (µg/200 mL) of bombilla infusions of commercial
green loose yerba mate samples from Brazil (n= 16) and Argentina (n=
7). n is the number of samples. The total manganese content refers to
a sum of the five fractions. The analyses were determined using ICP-
MS (refer to section 2.3).
102
Table 3.16
Total polyphenol content (mg/200 mL), reported as the mean and
range (min – max), of regular infusions of commercial yerba mate
samples from Brazil and Argentina. The samples were analysed by the
Folin-Ciocalteu method using a UV-Vis spectrometer (refer to section
2.4). n is the number of samples.
107
Table 3.17
Total polyphenol content (mg/200 mL), reported as the mean and
range (min – max), of Brazilian iced tea infusions of commercial yerba
mate samples from Brazil and Argentina. The analyses were
determined by the Folin-Ciocalteu method using a UV-Vis
spectrometer (refer to section 2.4). n is the number of samples.
108
Table 3.18
Total polyphenol content (mg/200 mL) of bombilla infusions of
commercial green loose yerba mate samples from Brazil (n= 16) and
Argentina (n= 7). The total content refers to the sum of the five
fractions. The samples were analysed by the Folin-Ciocalteu method
using a UV-Vis spectrometer (refer to section 2.4). n is the number of
samples.
109
Table 3.19
Chlorogenic acid, theobromine and caffeine content (mg/200 mL) of
regular infusions of commercial yerba mate samples from Brazil. The
samples were analysed by UHPLC (refer to section 2.5). For green
loose samples the results are presented as mean and range (min –
max). n is the number of samples.
111
Table 3.20
Chlorogenic acid content (mg/200 mL) of bombilla infusion fractions of
green loose commercial yerba mate products from Brazil. The samples
were analysed by UHPLC (refer to section 2.5). n = 4, n is the number
of samples. The percentage (%) refers to the contribution of the
fraction to the total (sum of the fractions).
113
Table 3.21 Theobromine and caffeine content of bombilla infusion fractions of
green loose yerba mate commercial products (mg/200 mL) from Brazil.
The samples were analysed by UHPLC (refer to section 2.5). n = 4, n
113
xviii
is the number of samples. The percentage (%) refers to the
contribution of the fraction to the total.
Table 3.22
Percentage intake (%) of manganese based on a serving (200 mL for
regular and Brazilian iced tea infusions; 1L for bombilla method) of
non-commercial yerba mate samples. The data is compared with the
World Health Organisation recommended daily allowance (RDA) of
manganese for males (M) and females (F).
116
Table 3.23
Percentage intake (%) of total polyphenol based on a serving (200 mL
for regular and Brazilian iced tea infusions; 1L for bombilla method) of
commercial yerba mate samples. The data is compared with the
values reported by (Fukushima et al., 2009) for the daily intake of
polyphenols.
118
Table 4.1 Element content of roasted coffee beans for selected elements
reported in the literature. Adapted from Pohl et al., (2013). 127
Table 4.2
Elemental levels (mg/kg, dry weight) of Brazilian coffee beans sampled
at different roasting times (minutes). The samples were of the Obatã
coffee variety collected from the Fazenda Flor plantation (Amparo, São
Paulo State) and analysed by ICP-MS (refer to section 2.3). Analysis in
duplicate.
131
Table 4.3
Elemental levels (mg/kg, dry weight) of Brazilian coffee beans sampled
at different roasting times (minutes). The samples were of the Catuaí coffee variety collected from the Fazenda Palmares plantation
(Amparo, São Paulo State) and analysed by ICP-MS (refer to section
2.3). Analysis in duplicate.
131
Table 4.4
Elemental levels (mg/kg, dry weight) of Brazilian coffee beans sampled
at different roasting times (minutes). The samples were of the Bourbon Amarelo coffee variety collected from the Fazenda Palmares plantation
(Amparo, São Paulo State) and analysed by ICP-MS (refer to section
2.3). Analysis in duplicate.
132
Table 4.5
Elemental concentration (mg/kg, dry weight) of roasted Brazilian coffee
beans. The different coffee varieties include those selected for their
quality (section 4.3.2) or as defected beans and were collected from
the Fazenda Palmares and Flor plantations (Amparo, São Paulo State)
and analysed by ICP-MS (refer to section 2.3). Analysis in duplicate.
133
Table 4.6 Total polyphenol content (mg/L) of Brazilian coffee infusions during
roasting time (minutes). The samples were from the different coffee
varieties from Fazenda Palmares and Flor (Amparo, São Paulo State)
and analysed by UV-Vis (refer to section 2.4). n = 1.
134
Table 4.7
The concentration of chlorogenic acids, caffeine and total polyphenol
(mg/L) of roasted Brazilian coffee infusions. The samples were
prepared from selected and defected beans (section 4.3.2) from
different coffee varieties collected from the Fazenda Palmares and Flor
plantations (Amparo- São Paulo) and analysed by UHPLC (refer to
section 2.5). n = 1.
139
xix
Table 4.8 Percentage intake (%) of total polyphenol based on the Brazilian daily
consumption (92 mL) of coffee infusion. The data is compared with the
values reported by Fukushima et al. (2009) for the daily intake of total
polyphenols.
146
Table 5.1 Literature review of the elemental content of açaí according to weight
basis, dry weight (d.w.) or fresh weight (f.w.) and sample type. 160
Table 5.2
Colour parameters of açaí samples where L* indicates lightness, a* the
red/green coordinate, b* the yellow/blue coordinate and ΔE the total colour difference (refer to Equation 5.1) determined by a reflectance
spectrophotometer (CR-400, Konica, Minolta, Japan). The data relates
to a pooled freeze-dried sample.
166
Table 5.3 Total polyphenol, flavonoid, anthocyanin (ANC) and proanthocyanidin
content (PAC); and chemical antioxidant activity (ABTS and DPPH) of
açaí pulp samples. The values are expressed as mean ± standard
deviation and dry weight. 169
Table 5.4 Literature values for the total polyphenol content (mg GAE/ 100g) and
antioxidant activities DPPH (g/g DPPH) and ABTS μmol Trolox/g of typical tropical berries from Brazil (dry weight). Table adapted from
Rufino et al. (2010).
172
Table 5.5 Literature review of the total polyphenol and total anthocyanin content
of other berries obtained by two different methods (HPLC and pH); and
the total proanthocyanidin (mg/100g) (fresh weight). Table adapted
from Rothwell et al. (2013).
174
Table 5.6 Gradient programme for the determination of the anthrocyanin content
of açaí extracts using an Agilent 1200 HPLC instrument. 175
Table 5.7 (a)
Total elemental levels (mean ± standard deviation) of essential trace
elements (mg/kg fresh weight) of açaí pulp samples determined using
ICP-MS (refer to section 2.1): data relates to the type of sample (non-
commercial : purple n= 6; and white n= 4; and commercial: purple n=
4; n is the number of samples).
184
Table 5.7 (b)
Total elemental levels (mean ± standard deviation) of non-
essential/toxic trace elements (mg/kg fresh weight) of açaí pulp
samples determined using ICP-MS (refer to section 2.1): data relates
to the type of sample (non-commercial : purple n= 6; and white n= 4;
and commercial: purple n= 4; n is the number of samples).
185
Table 5.8
Literature review of typical Brazilian fruits the total elemental content of
calcium, magnesium, manganese, iron, zinc and copper in (mg/kg)
(fresh weight). Data for açaí is reported as a commercial processed
material and all the others as raw natural typical Brazilian fruits. Table
adapted from Unicamp (2011).
189
Table 5.9 Total polyphenol (TP) content of non-commercial açaí (purple/whole)
samples, (mg GAE / g) determined by Folin-Ciocalteu analysis (refer to
section 5.6.3). Fruit and seeds refer to non-processed berries and the
pulp is processed material (fluid, medium and thick relates to the
207
xx
moisture content) (refer to section 5.6.2 for sample information).
Results are expressed as mean ± st dev in fresh weight, n is the
number of replicates, n = 3. Refer to Appendix 5.3 for code information.
Table 5.10
Total elemental concentration of açaí (non-commercial, purple/whole)
samples (mg/kg, fresh weight) analysed by ICP-MS (refer to section
5.6.4). Fruit and seeds refer to non-processed berries (section 5.6.2).
Results are expressed as a mean, n is the number of replicates, n = 3.
Refer to Appendix 5.3 for code information.
209
Table 5.11 Total elemental concentration in commercially processed açaí samples
(mg/kg, fresh weight) analysed by ICP-MS (refer to section 5.6.4).
Results expressed as a mean, n is the number of replicates, n = 3.
Refer to Appendix 5.3 for code information.
211
Table 5.12
Percentage intake (%) of total polyphenol and minor elements based
on the consumption of a 500 g serving (Heinrich et al., 2011) of the
commercial and non-commercial açaí pulp (reported on a fresh weight
basis). The data is compared with the World Health Organisation
recommended daily allowance (RDA) for males (M) and females (F).
213
Table 5.13
Percentage intake (%) of selected trace elements based on a 500 g
serving (Heinrich et al., 2011) of non-commercial or commercial açaí
pulp or powder (fresh weight). The data is compared with the World
Health Organisation recommended daily allowance (RDA) for males
(M) and females (F).
215
xxi
Abbreviations
% percentage m/z mass-to-charge ratio
℃ Celsius MAE microwave-assisted extraction
< less than mAU milli absorbance unit
Å angstroms max maximum
AA Açaí Amazonas MHz mega-hertz
ABIC Associação Brasileira da Industria
do Café min minutes
ABTS azino-bis(3-ethylbenzothiazoline-6-
sulphonic) acid min minimum
AC alternating current mm millimeters
ACN acetonitrile MS mass spectroscopy
AI adequate intake MTT 3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide
ANC anthocyanin mΩ milli-ohm
ANOVA analysis of variance n number
ARG Argentina n.r. not reported
BRA Brazil NCSU North Carolina State University
CAD charged aerosol detection NIR near-infrared
calc calculated NIST National Institute of Standards
and Technology,
CIELAB Commission internationale de
l'éclairage L*a*b* nm nanometer
CPS counts per second NO Nitric oxide
crit critic NPK nitrogen, phosphorous and
potassium
CRM certified reference materials org organic
CXLE carbon dioxide-expanded liquid
extraction; ORS Octopole Reaction System
d.w. dry weight PA Para
DAD diode array detection PAC proanthocyanidin
DC direct current PAM Produção Agricola Municipal
DCF 2′,7′-dichlorofluorescein PBS phosphate-buffered saline
DDW double-distilled deionised water PCA principal component analysis
DEX dexamethasone PEF pulse electric field
DEX dexamethasone pH potential of Hydrogen
DMEM Dulbecco’s modified Eagle’s medium
PIXE particle-induced X-ray emission
DMSO dimethyl sulfoxide PLE pressurized liquid extraction
xxii
DNA deoxyribonucleic acid ppb parts per billion
DOU Diário Oficial da União ppm parts per million
DPPH diphenyl-1-picrylhydrazyl psi pound force per square inch,
EC50 concentration required to obtain a
50% antioxidant effect RDA recommended daily allowance
eH Redox potential redox reduction-oxidation
EMBRAPA Empresa Brasileira de Pesquisa
Agropecuária RF radio-frequency
EVM Expert group on Vitamins and
Minerals, ROS reactive oxygen species
F female RP reverse phase
f.w. fresh weight rpm Revolutions per Minute
FAAS flame atomic absorption
spectroscopy rps revolution per second
FAES flame atomic emission spectroscopy RSD relative standard deviation
FAO Food and Agriculture Organization s second
FBS foetal bovine serum SCAA Specialty Coffee Association of
America
FD freeze-dried SFE supercritical fluid extraction
fert fertilsers SH Açaí Santa Helena
GAE gallic acid equivalents SLE solid-liquid extraction
GDP gross domestic product SP São Paulo
GF-AAS graphite furnace atomic absorption
spectroscopy SPE solid phase extraction
HDFa Human Dermal Fibroblast cells
(adult) SRM Standard Reference Material
HHPE high hydrostatic pressure extraction St dev standard deviation;
HPLC high performance liquid
chromatography SUS Sistema Único de Saúde
hr(s) hour(s) TACO Tabela Brasileira de
Composição de Alimentos
HSCC high-speed countercurrent
chromatography TE Trolox equivalents
IBGE Instituto Brasileiro de Geografia e
Estatística TP total polyphenol
ICP-MS inductively coupled plasma mass
spectrometry; tR retention time
ICP-OES inductively coupled plasma optical
emission spectrometry u atomic mass unit
INYM Instituto Nacional de la Yerba Mate UAE ultrasound assisted extraction
IOM Institute of Medicine UFPA Universidade Federal do Pará
IQ intelligence quotient UFRS Universidade Federal do Rio
xxiii
Grande do Sul
ISO International Organization for
Standardization UFRS
Universidade Federal do Rio
Grande do Sul
ISTD/IS internal standard UHPLC ultra-high performance liquid
chromatography
IU international unit UK United Kingdom
K kelvin UL upper level
Km kilometer UNICAMP Universidade de Campinas
LC liquid chromatography US(A) United States (of America)
LDL low-density lipoprotein UV ultra-violet
LDR linear dynamic range UV-Vis ultra-violet visible spectroscopy
LLE liquid-liquid extraction V volts
LoD limit of detection W watts
LoQ limit of quantification WHO World Health Organisation
LPS lipopolysaccharide α probability
LSGS Low Serum Growth Supplement ΔE total colour difference
M male 𝜇g/L ppb
xxiv
Glossary
accuracy Degree of closeness of measurements from
a true value (Ward, 2000).
analytical figures of merit Performance characteristics of an analytical
determination, such as limits of detection
and quantification (Skoog et al., 2017).
anti-proliferate Substance capable of avoiding the
accelerated grow of bacteria.
antioxidant Molecules that can prevent the oxidation of
biomolecules in biological systems (Bastos
et al., 2007).
bioavailability Fraction of the analyte which can be
absorbed and utilised for physiological
functions (Fairweather-Tait and Hurrell,
1996).
hydrophilicity Chemical molecules that are capable of
forming ionic or hydrogen bonds with water
molecules.
hydrophobicity Chemical molecules that repel water
molecules.
isomerisation Process where a molecule is transformed
into another maintaining the same number
of atoms, but in a different arrangement.
macronutrients Chemical substances that humans consume
in the large quantities, such as, fat, protein
and carbohydrate.
micronutrients Essential chemical nutrients required in
xxv
small quantities for the maintenance of
human health, such as, vitamins and
minerals.
minerals Chemical elements required as an essential
nutrient for human health.
nutraceuticals Products derived from food sources that can
provide health benefits.
precision Degree to which repeated measurements
present similar results (Miller et al., 2018).
repeatability Level of agreement between replicate
analysis of the same sample within the
same instrument conditions (Skoog et al.,
2017).
reproducibility Precision of replicate analysis under
different conditions (between-run) (Skoog et
al., 2017).
super-fruit Marketing term for fruits that promote health
benefits due to an outstanding nutritional
composition.
trace elements Elemental concentrations in biological or
environmental systems ranging from 0.01 –
100 mg/kg (Ward, 2000).
ultra-trace elements Elemental concentrations in biological or
environmental systems that are less than
0.01 mg/kg for ultra-trace elements (Ward,
2000).
xxvi
Acknowledgements
Initially, I would like to thank my sponsor, Science without Borders
(CAPES), for providing me the opportunity and necessary financial support for
my PhD. I would also gratefully acknowledge the funding received from the
University Global Partnership Network (UGPN) programme that allowed the
collaborative work for the Açaí project.
To my supervisors, Prof Neil I Ward and Dr Mónica Felipe-Sotelo, I would
like to show my appreciation for their enthusiasm, motivation and exceptionally
support throughout this PhD. Thank you for allowing me to grow as a scientist
and supporting me as a person.
To the members of the ICP-MS group and the University of Surrey, thank
you for your assistance and guidance with the practical activities. Special thanks
to Dr Catherine Donnelly, Dr Andrea Pedronda and Dr Jonathan Brown for their
helpful advice and support. My special appreciation to the Plants for Human
Health Institute group for their warmly welcome and support, in special Dr Mary
Ann Lila for all her valuable advice. I would also like to mention Dr Debora
Esposito for her incredibly helpful advice on both research and career wise. I
would like to acknowledge Dr Cassiana Nomura and Alexandrina Carvalho for
their appreciated collaboration. This research was only possible due to the
collaboration of all the local producers that I worked in Brazil (Barão de Cotegipe,
Cooperativa de cafeicultores de Amparo and açaí producers of Pará State) who
generously shared their wide knowledge and history with me.
Finally, my special appreciation and extreme thanks to my family, who
have always been my pillar and source of strength throughout this PhD. I would
like to dedicate this achievement to my parents and brother, whose through their
affection and tireless encouragement, supported me in these challenging years
so far away from home. Also, a huge thank to my all my special friends, who
were always there for me, thank you for all your patience, love and dedication to
our friendship.
1
Chapter 1. General Introduction
2
1.1. Overview of Brazil
Brazil is a federal republic, the largest country of Latin America, the fifth
largest in the World covering an area of 8511965 km2 and has a population of
212 million (IBGE, 2019). The gross domestic product (GDP) of the country is
US$ 2056 billion, which is linked to services (55%), industry (36%) and
agriculture (9%). This makes Brazil an advanced emerging economy (Reynolds
et al., 2019). Brazil is a rich supplier of a variety of natural resources, such as
minerals, water and natural foodstuffs. The availability of an adequate climate
and rainfall, besides the fertile nature of its soil, makes Brazil ideal for agriculture.
The major agricultural products in Brazil include sugar cane, corn, cassava,
soybean, oranges, coffee, cotton, tobacco, cocoa and fruit juices (FAO, 2018).
Brazil is the third largest agricultural exporter in the World, with trading activities
in products being worth more than US$ 100 billion (2018). The main trade
products are soybeans, green coffee, corn and fruit juices (IBGE, 2019).
The exportation and globalisation of the agricultural market is bringing
benefits to the economic expansion of the region by developing autonomous and
small businesses. However, the negative aspects of the globalisation process are
not yet fully understood, including for example, land use (deforestation) and the
impact on local health (Costa et al., 2019). In rural communities of Brazil, the
availability of natural resources extracted from the forests, small farming or
fishing have been able to guarantee a healthy source of calories for low-income
families (Mansur et al., 2016). The opening of the region to globalisation and
exportation of natural products has started a change in the eating habits of the
Brazilian population. This may be due to the increased cost of traditional
foodstuffs for the locals and an increasing trend (in emerging countries)
associated with the introduction of food products that are not part of the
traditional diet. This has resulted in the consumption of foreign imported and/or
highly processed foodstuffs (Brasil, 2006). A change in the consumption of food
associated with a traditional diet has already been observed in Brazil (and other
emerging countries), especially amongst the younger population (< 25 years)
3
(Costa et al., 2019). Moreover, an increased incidence of chronic diseases, such
as diabetes and hypertension, has also been reported, which may be linked to
the changes in diet (SUS, 2018).
A recent study about the intake of antioxidant nutrients (refer to glossary)
by the Brazilian population has reported an insufficient intake of antioxidant
nutrients, especially vitamins E, A and C. It was also found that the intake of
antioxidant nutrients varied based on nutritional status, gender and life stage
(Tureck et al., 2017). The Dietary Guidelines for the Brazilian Population (2017)
recommends that the population should return to the consumption of foodstuffs
linked with the traditional Brazilian diet. In summary, this means a diet based on
the preparation of cereals and legumes (rice and beans), fruits and vegetables,
as such foodstuffs provide the healthiest intake of nutrients, antioxidants and
minerals (refer to glossary) (Brasil, 2006).
Brazil is the World's third largest fruit producer, behind China and India.
Between 1990 and 2004 exports grew by 183% in value, 277% in quantity and
915% as a net value. The commercial production of Brazilian fruits has two
designations; unprocessed fruit, that represents 47% of the country's production,
and processed fruit, representing 53%. In terms of the total amount of
unprocessed fruit produced in Brazil, 31% is exported (EMBRAPA, 2017). The
European Union is the largest market for Brazilian fruits; almost 70% of the fruit
exported by Brazil is consumed by this economic bloc. Moreover, 34% of the
exports are destined to the Netherlands, however the country works not only as a
consumer, but also as a distributor to other countries of the European Union
(Cunha Filho and Carvalho, 2005).
Among all of the natural products that Brazil produces and exports, three
have gained special attention, namely, yerba mate, coffee and açaí, due to their
popularity, high quality and health claims (Heck and De Mejia, 2007, Abrahão et
al., 2010, Yamaguchi et al., 2015). Yerba mate (Ilex paraguariensis) is found in
South America and has been consumed by native peoples since pre-Columbian
times (Bracesco et al., 2011). It is consumed as a hot or cold infusion and is one
4
of the most popular beverages in South America, with an estimated 1 million
people consuming it due to its high levels of caffeine and antioxidants (de Morais
et al., 2009). The production of yerba mate is restricted to southern Brazil, as
shown in Figure 1.1, with an estimated exportation value of US$ 85 million in
2017 (IBGE, 2017). Coffee production in Brazil is responsible for about a third of
all global coffee, making Brazil the World's largest producer and exporter. The
estimated exportation value for 2017 was US$4 billion (IBGE, 2017). The crop
(Coffea arabica L.) first arrived in Brazil in the 18th Century and the country had
become a dominant producer by the 1840s, due to the local environmental and
climate being ideal growing conditions (Pendergrast, 2010). Coffee plantations
are mainly located in the south-eastern states of Minas Gerais, São Paulo and
Paraná, as shown in Figure 1.1. Coffee is one of the most commonly consumed
beverages in Brazil and one of the major sources of antioxidant intake in the daily
diet of Brazil (Tureck et al., 2017). Finally, the açaí berry (Euterpe oleracea) is a
native fruit from the Amazonian region of Brazil and has recently become very
popular due to its status as a ‘super-fruit’ (refer to glossary) related to the high
antioxidant activity of the fruit (Yamaguchi et al., 2015). The production of açaí is
still mainly an extractive activity from the Amazon forest, as shown in Figure 1.1
(Homma et al., 2006, Maciel et al., 2018, Tagore et al., 2018). Brazil is the main
producer and exporter of açaí, generating an estimated monetary source of
US$ 9 billion in 2017 (IBGE, 2017), exceeding the price per ton of common
exportation products, such as soybeans and Brazil nuts (Yamaguchi et al., 2015).
These important Brazilian products, namely, yerba mate, coffee and açaí, will be
discussed and investigated in chapters 3 (yerba mate); 4 (Brazilian coffee) and 5
(açaí), respectively.
5
Figure 1.1: Map of South America with the highlighted production sites of yerba
mate, coffee and açaí (Maps, 2019).
1.2. Trace Elements in the Human Diet and Health
Micronutrients (refer to glossary) play an important role in human
metabolism and health (Goldhaber, 2003, Strachan, 2010). There is a wide range
of elemental concentrations in biological systems, ranging from 100 mg/kg (or
parts per million) for minor elements, 0.01 – 100 mg/kg for trace elements, and <
0.01 mg/kg for ultra-trace elements (refer to glossary) (Versieck and Cornelis,
1980). The elements within biological systems can be further classified into
essential, non-essential and toxic elements (Strachan, 2010). Food and
beverages are the primary sources that provide the levels of the elements
(sometimes referred to as ‘metals’ or minerals) required to regulate various
chemical and biochemical reactions (Fraga, 2005). Frieden (1985) proposed a
biological classification of trace elements based on the amount in tissues. This
biological classification included: (i) essential trace elements (B, Co, Cu, I, Fe,
6
Mn, Mo and Zn); (ii) probably essential trace elements (Cr, F, Ni and V); and (iii)
physically promotive trace elements (Br, Li, Si, Sn and Ti) (Frieden, 1985). There
have been various definitions of what is an essential trace element, and many
include that they are ‘chemical micronutrients which are required in minute
quantities but play a vital role in maintaining the integrity of various physiological
and metabolic processes occurring within living tissues. The deficiency of any of
these trace elements may be apparent as a combination of various clinical
manifestations rather than a specific presentation as each trace element is
related to many enzyme systems’ (Bhattacharya et al., 2016). A recent report
stated that essential elements are defined in terms of their chemical state (as a
free ion, or as a variety of chemical compounds) that are included in all cells and
tissues of the human body (Skalnaya and Skalny, 2018). Physiological effects of
these elements depend on the dose. For each element there is an optimum
range of concentrations to perform vital functions (refer to section 1.2.1). At
deficiency or excessive accumulation levels of these elements, there is a
disturbance in the physiological activity associated with the element, which is
reversable with the addition of physiological amounts of the specific element – in
which chemical species is important (Mertz, 1981, Skalnaya and Skalny, 2018).
Essential ‘macro’ elements, whose concentration in the body exceeds 0.01%
(100 mg/L or ppm) include O, C, H, N, Ca, P, K, Na, S, Cl and Mg. Essential
‘trace’ elements, found at concentration ranges from 0.01% to 0.00001% (100 to
0.1 mg/L) includes: Fe, Zn, F, Mo, Cu, I and Mn. Finally, essential ‘ultra-trace’
elements are found at concentrations lower than 0.000001% (< 100 µg/L or ppb)
are Se, Co, V, Cr, As, Ni and Sn (Skalnaya and Skalny, 2018). There is some
debate about the essential roles of some trace elements, especially B and As
(Prashanth et al., 2015).
Essential trace elements have a number of roles in the maintenance of
human health. These elements have a key role within enzyme systems, as
metallo-enzyme complexes (Donnelly, 2015). Essential elements can also
participate in reduction-oxidation (redox) reactions by donating or accepting
electrons, or are involved in the transport of oxygen (iron) (Nielsen, 2007). Whilst
7
the diet is a major source of essential elements it can also be an exposure or
uptake route for non-essential and toxic elements. Non-essential or ‘possibly
essential’ elements are those for which no evidence has been published on an
essential role in the human body. This group includes: B, Al, Ba, Br, Bi, Li, Sr,
Rb, Sb, Ge, Be and Cs (Prashanth et al., 2015, Skalnaya and Skalny, 2018).
Toxic elements are those that have no nutritional value even at trace amounts
(Skalnaya and Skalny, 2018). They can be present in the regular human diet,
and include Cd, Pb and Hg, and are considered toxic even at low concentrations
(WHO, 1996).
Most plants obtain elements (minor, trace and ultra-trace) from the soil via
the soil solution into the root, but sometimes salts and minerals from fertilisers
can be taken up through the leaves (Kabata-Pendias, 2010). The formation of
soil chemistry is a result of the eroding of rock where its structure is broken down
into soluble compounds by physical processes (Garrett, 2000). These are
washed by rain and rivers where various reactions can occur between the
minerals (or elements/metals) and the organic matter from decomposing remains
of plants, animals and microorganisms. This increases the solubility of trace
elements (including the effect of pH and Eh – redox potential) and changes the
translocation to different parts of a plant (Garrett, 2000). The elemental content of
the soil can also change with the addition of fertiliser or compost, and from
human activities, such as, pollution, discharge of wastes or mining (Kabata-
Pendias, 2010).
1.2.1. Dose response curve and homeostasis of chemical elements
The main source of chemical elements for humans is through the diet. A
balanced diet will provide a normal person with an adequate supply of chemical
elements to maintain optimal health (Donnelly, 2015). Moreover, the human body
is capable of maintaining the content of these essential chemicals due to
homeostasis, which is a self-regulating mechanism that involves absorption,
8
storage and excretion processes (Nielsen and Hunt, 1989, Nielsen, 2007). Figure
1.2 represents the biological dose response curve (Underwood and Mertz, 1979).
If there is an inadequate supply of an element via the diet or the uptake is
compromised through disease, the physiological processes required by that
element are also reduced causing suboptimal health, with the manifestations of a
deficiency disease. Furthermore, if the essential elemental levels are too low
then death can occur (Nielsen and Hunt, 1989, Nielsen, 2007). Equally, a high
intake of an element that exceeds the homeostatic regulation leads to toxic
levels, and (if high enough) can also result in death (Nielsen and Hunt, 1989,
Nielsen, 2007). It is important to highlight that every element can produce signs
of toxicity at certain concentrations in the human body (Underwood and Mertz,
1979, Donnelly, 2015).
Figure 1.2: Biological dose response curve for elements (Underwood and Mertz,
1979)
1.2.2. Dietary intake – World Health Organisation (WHO) guidelines
In order to protect the health of the population, The World Health
Organisation (WHO) have undertaken several risk assessments of nutrient
intake, including for the chemical elements. The WHO have recommended levels
9
to prevent the population from inadequate and excessive intakes of the elements
(including major/ minor and trace – refer to section 1.2). In order to provide
guidance, a Recommended Dietary Allowance (RDA) has been set for different
population groups (Schumann, 2006), which is defined as the amount of a
nutrient sufficient to ensure the needs of nearly all of the population (97.5%)
(Schumann, 2006). If there is a lack of data to establish an RDA, an adequate
intake (AI) is proposed. Conversely, to protect against the toxic effects of over-
intake, a Tolerable Upper Level Intake (UL) has also been defined as the highest
average daily intake that is unlikely to provide a risk of toxic effects in almost all
of the general population (EFSA, 2006). It is important to highlight that the
bioavailability (refer to glossary) of these chemical elements is also critical for
considering the human dietary intake.
1.2.3. Deficiency and toxicity effects of chemical elements
In this study, calcium, magnesium, manganese, iron, zinc and copper are
highlighted because the concentration in the analysed products may provide a
significant contribution to the nutritional intake of these elements. Each one of
these essential elements follows a dose response curve (refer to Figure 1.2) and
has a nutritional recommended daily allowance (RDA) in order to prevent
deficiency (Goldhaber, 2003), or a tolerable upper level intake (UL) to prevent
toxicity effects (refer to section 1.2.2). Manganese, iron, zinc and copper are all
important micro-nutrients and are constituents of key proteins or enzymes in the
human body that provide a variety of functions (Combs, 2013). Moreover,
calcium and magnesium are essential macro-nutrients for structure and electro
regulation (Combs, 2013). Table 1.1 presents some of the symptoms of
deficiency or toxicity of the essential elements (calcium, magnesium, iron, zinc
and copper). Manganese will be further discussed in section 1.2.5.
10
Table 1.1: Symptoms of deficiency and toxicity of calcium, magnesium, iron, zinc
and copper (Strachan, 2010, Combs, 2013).
Element Deficiency symptoms Toxicity symptoms
Calcium Compromised bone structure and
nervous transduction
Bone and muscle weakness, kidney
stones, fatigue, cardiac arrhythmia
Magnesium
Compromised bone structure,
electrochemical regulation and
enzyme catalysis
Lethargy, nausea, muscle weakness,
urine retention, cardiac arrhythmia
Iron Anaemia, compromised immune
functions, fatigue
Hepatic cirrhosis, diabetes, heart
failure, arthritis
Zinc Poor growth, reduced testicular
development, osteoporosis risk Anaemia, copper depletion
Copper Anaemia, reproductive failure, bone
abnormalities, poor growth
Nausea, cirrhosis (hepatic
accumulation), gastroenteritis
1.2.4. Chemical elemental content of typical foodstuffs and beverages from Brazil
The TACO project (Tabela Brasileira de Composição de Alimentos,
Brazilian Table of Food Composition) was concluded in 2011 with the objective of
creating reliable analytical data for a national database on the composition of
typical Brazilian foods (Unicamp, 2011). TACO provided reliable information on
the food consumption of the Brazilian population that serves as a basis for
formulating policies and action plans for food and nutritional security (Galeazzi,
2001). Table 1.2 summarises the major sources of calcium, magnesium, iron,
zinc and copper in the Brazilian diet, using TACO as the reference. Manganese
will be discussed in section 1.2.5.
11
Table 1.2: Major sources and concentrations of calcium, magnesium, iron, zinc
and copper in the Brazilian diet (Unicamp, 2011).
Element Dietary source Concentration (mg/100 g)
Ca
Coriander (dried) 784
Fish (Lambari, raw) 1181
Milk (powder) 1363
Mg
Beans (Carioca, raw) 210
Soya (powder) 242
Brazilian nuts (raw) 365
Fe
Porridge (powder) 42.0
Beans (Rajado, raw) 18.6
Babaçu nut (raw) 18.3
Zn
Porridge (powder) 15.2
Beef (cooked) 8.1
Caruru leaves (raw) 6.0
Cu
Beef liver (raw) 9.01
Brazilian nuts (raw) 1.79
Papaya (Formosa, raw) 1.36
1.2.5. Manganese chemistry
The manganese levels in yerba mate and açaí berries have been reported
to be higher than any other common plant used for infusions (Bragança et al.,
2011, Wróbel et al., 2000) and other typical fruits (Unicamp, 2011). Therefore, in
this study, this particular element will be the focus of the research.
Manganese (Mn) has an atomic number of 25 and a molecular weight of
approximately 55 g/mol. It is a transition element located in group 7 and period 4
of the periodic table. It is not found in its elemental state in nature but is often a
component of a mineral, in combination with iron (ATSDR, 2000). Anthropogenic
(or man-made) contamination of Mn is usually associated with mining activities,
iron and steel production or via pollution associated with the combustion of fossil
fuels (Grygo-Szymanko et al., 2016). Manganese is an essential trace element
required for human health as it is a constituent of enzymes with many functions,
12
including immunity, regulation of blood sugar and cellular energy, blood clotting,
reproduction, digestion and bone growth (Roth et al., 2013). However, at high Mn
concentrations, this element is neurotoxic and chronic exposure may lead to a
condition known as manganism, a disorder that has symptoms similar to
Parkinson's disease (Michalke et al., 2007).
The most common oxidation states are from +2 to +7; with +2 being the
most stable and bioavailable; +3 is only present in complexes; +4 is insoluble
and found in particulates and colloids; + 5 and + 6 are instable in neutral
solutions; and + 7 is found in the permanganate ion (MnO4−). The behaviour of
manganese in water is affected by oxidation and reduction processes which have
a major influence on the species present (Dorronsoro et al., 2006). In aqueous
matrices and under aerobic conditions, Mn is more soluble in acidic (pH <6) and
insoluble in alkaline (pH >8) conditions, as shown in Figure 1.3.
Figure 1.3: Schematic of the Mn-forms as a function of Eh/pH in aqueous
matrices and aerobic conditions (Dorronsoro et al., 2006).
The guideline limit of manganese in drinking water is 400 µg/L according
to the World Health Organisation (WHO, 1996), although the limit in the UK is set
to 50 µg/L (EVM, 2003). This is mainly to avoid water colouration and deposition
in pipes rather than preventing negative effects on human health. Manganese is
absorbed in the human gut and excreted by bile, and because this system is not
13
fully developed in children, they are more susceptible to Mn toxicity (Neal and
Guilarte, 2012). There are several studies about toxic manganese exposure
through inhalation, which is common in mine workers, leading to problems in
intellectual and cognitive development (Rumsby et al., 2014). Although some
studies try to associate the same problems with Mn exposure in food and water,
there is a lack of conclusive correlations due to other cofactors in cognitive
development. A Canadian study has shown that exposure of Mn at levels in
drinking water, below that recommended by the WHO and above the UK limit,
can caused a decrease in the intelligence quotient (IQ) in children (Bouchard et
al., 2010). A study on rats found that exposure to Mn in drinking water results in
accumulation in the same tissues as that associated with inhalation and could
cause behavioural and locomotor effects (Reichel et al., 2006). Furthermore, Mn
is a key element in photosynthesis. The elemental content of a plant material
usually reflects the conditions where it was grown and could be influenced by
many factors, such as soil chemistry, environment and age (Saidelles et al.,
2010). In the case of manganese, it is rapidly taken up from the soil by plants and
distributed to the leaves and chloroplasts, where it plays an important role in
oxygen evolution and electron transport (Kabata-Pendias, 2010).
The recommended dietary intake levels for manganese are set at 2.3 and
1.8 mg/day for men and women respectively; and the tolerable upper intake level
is at 11 mg/day (IOM, 2002). Table 1.3 summarises the reported levels of
manganese in popular beverages from Brazil. It must be noted that fruit juices in
Brazil are made from frozen fruits (pulp) blended with water. The Brazilian
beverages that could contribute to the daily intake of manganese are açaí and
yerba mate.
14
Table 1.3: Manganese levels (mg/kg) in traditional beverages of Brazil analysed
by inductively coupled plasma optical emission spectrometry (ICP-
OES) (Unicamp, 2011).
Beverages Mn (mg/kg) Açaí, pulp, guaraná and glucose 32.86
Coffee, roasted, powder 25.79
Pineapple, pulp, frozen 10.21
Coconut water 2.53
Sugarcane, juice 2.06
Cappuccino, powder 1.71
Cupuaçu, pulp, frozen 1.70
Mango, pulp, frozen 1.19
Black infusion, 5% infusion 0.89
Cajá, pulp, frozen 0.70
Passion fruit, pulp, frozen 0.70
Graviola, pulp, frozen 0.56
Caju, pulp, frozen 0.54
Pitanga, pulp, frozen 0.53
Umbu, pulp, frozen 0.49
Coffee, 10% infusion 0.38
Acerola, pulp, frozen 0.34
Orange, juice 0.17
Lime, juice 0.10
Milk, whole < 0.05
Cachaça < 0.05
Beer 0.05
Soda < 0.05
1.3. Polyphenols and Xanthines
Polyphenols are plant metabolites characterised by the presence of at
least one aromatic ring with one or more hydroxyl groups (phenol structural units)
attached (Campos‐Vega and Oomah, 2013). In plants, both natural phenols and
15
the larger polyphenols play important roles in the ecology of most plants. Their
functions in plant tissues can include giving colour, acting as an insect or as a
mammal feeding deterrent. The end result is to help protect the plant against
stress from ultra-violet (UV) radiation damage, temperature, oxidative activity,
and tolerance to heavy metals through chelation of metal ions, or as a defense
against pathogens (Gould and Lister, 2005, Stevenson and Hurst, 2007,
Donnelly, 2015).
Xanthine or 3,7-dihydropurine-2,6-dione, is a purine base found in most
biological systems. A number of stimulants are derived from xanthine, including
caffeine and theobromine, as shown in Figure 1.4 (Voet et al., 2008). In plants,
caffeine is found in the seeds, nuts, or leaves of a number of plants and helps to
protect them against predator insects and to prevent germination of nearby
seeds (Saxena et al., 2013). Theobromine is a bitter alkaloid found in chocolate,
leaves of the tea plant and in coffee beans (Martínez-López et al., 2014).
Caffeine differs from theobromine in having an extra methyl group, as shown in
Figure 1.4.
Figure 1.4: Schematic of the chemical formulae of xanthine, caffeine and
theobromine, important compounds present in yerba mate and
coffee (Merck, 2018).
1.3.1. Polyphenol chemistry
Polyphenols can be divided into classes, namely, phenolic acids,
hydroxycinnamic acids and flavonoids (Crozier, 2003). Phenolic acids or
16
hydroxybenzoic acids have a C6-C1 structure, such as gallic acid and salicylic
acid, as shown in Figure 1.5. During the different stages of plant maturation and
growing conditions the concentration and type of phenolic acids change. The
functions of phenolic acids in plants range from nutrient uptake to protein
synthesis, enzyme activity, photosynthesis and structural components (Donnelly,
2015). Hydroxycinnamic acids have a general C6-C3 structure, as shown in
Figure 1.5. One of the most important derivative groups of hydroxycinnamic acids
is the chlorogenic acids or quinic acid conjugates, which are present in fruits and
yerba mate leaves (Manach et al., 2004, Bravo et al., 2007). Finally, flavonoids
have a C6-C3-C6 structure (Figure 1.5) and are the most common family of
polyphenols present in plants. Flavonoids can be further divided in 6 main sub-
classes, namely, flavones, isoflavones, flavonols, flavan-3-ols, flavanones and
anthocyanidins (Manach et al., 2004). The most widely distributed and diverse
group of flavonoids in nature is the flavonols, such as quercetin, kaempferol,
myricetin and isorhamnetin (Crozier, 2003). In fruits and vegetables quercetin
compounds are the most commonly occurring (Saltmarsh and Goldberg, 2003,
Kyle and Duthie, 2005, Donnelly, 2015). Flavan-3-ols are the most complex
group of flavonoids which occur as simple monomers or in complex polymeric
forms, such as proanthocyanidins. They are found in red wine, cocoa and berries
(Skates et al., 2018, Crozier et al., 2009). Anthocyanidins are present mostly in
fruits and flowers and are responsible for the red, blue and purple colouration.
The main source of anthocyanidins in the human diet is from fruits, especially
berries (Ovaskainen et al., 2008, Crozier et al., 2009). They play a major function
in plants mainly attracting pollinating insects and protecting plants from damaging
light (Gould and Lister, 2005).
17
Figure 1.5: Schematic of the chemical formulae of typical polyphenols found in
yerba mate, coffee and açaí: (A) phenolic acids; (B) hydroxybenzoic
acids; and (C) flavonoids (Merck, 2018).
1.3.2. Health effects of polyphenols
The polyphenols have an increasing recognition as an emerging field of
interest in nutrition in recent decades (Cory et al., 2018). The polyphenol
consumption may play a major role in the maintenance of human health, mainly
due to their antioxidant activity, by removing free radicals and reactive oxygen
species of the biological system (Donnelly, 2015). Moreover, polyphenols help
regulate the metabolism, weight, susceptibility to chronic disease and cell
proliferation in humans (Cory et al., 2018). Studies on biological systems have
shown that numerous polyphenols also have anti-inflammatory and antioxidant
activities, that could prevent and/or have therapeutic effects for
neurodegenerative disorders, cancer, cardiovascular disease and obesity (Pérez-
Jiménez et al., 2010, Singh et al., 2011). There is also evidence that the long-
term consumption of polyphenols helps protect humans against type-2 diabetes,
osteoporosis, pancreatitis, gastrointestinal problems and lung damage (Fraga et
al., 2010, Martín‐Peláez et al., 2013, Xiao and Hogger, 2015).
A number of studies have acknowledged cellular targets that can be
involved in the health claims of polyphenols. However, the mechanism of the
molecular interactions of polyphenols with cellular targets remains mostly
18
speculative (Fraga et al., 2010, Cory et al., 2018). Some of the mechanisms
proposed are free radical scavenging, metal sequestration and the interactions of
polyphenols with membranes, enzymes, transcription factors or receptors (Fraga
et al., 2010). The health benefits alleged to polyphenols are derived from the
polyphenol or the phenolic metabolites resulting from the transformation of the
compounds in the gut microbiota (Selma et al., 2009).
1.3.3. Food total polyphenol range
It is not easy to estimate the daily intake of total polyphenols, due to the
variation in the structural diversity of phenolic compounds for a particular
foodstuff (Scalbert and Williamson, 2000). Moreover, there is a variation in the
dietary intake of polyphenolic compounds between geographical regions and
consumption age groups. Several studies have agreed on a proposed range of 1
g of total polyphenols per day (Kühnau, 1976, Faller and Fialho, 2009, Landete,
2013, Fukushima et al., 2009). Beverages, such as coffee, wine, fruit juices and
tea, are the largest contributors to the dietary sources of polyphenols (Saura-
Calixto and Goñi, 2006). The total polyphenol content of foods is usually
determined by the Folin-Ciocalteu assay, described in section 2.4.2. In relation to
typical beverages, the total polyphenol content of green tea ranges from 8.7 to
25.8 g/100 g; black tea 8.0 to 26.3 g/100 g, and brewed coffee reported that the
total polyphenol content of coffee prepared in a commercial brewer was 0.96 to
2.27 g/L (Lakenbrink et al., 2000, Obuchowicz et al., 2011, Stodt and Engelhardt,
2013). The literature values for the total polyphenol content of typical tropical
berries from Brazil is presented in Chapter 5, Table 5.5.
1.4. Polyphenol and elemental relationship
The elements in solution can exist as free ions or complexes with naturally
occurring bioligands. Polyphenols are one of these natural ligands, which can
complex elements (or metals) through hydroxyl, carboxylate and phenolate
19
groups (Pohl and Prusisz, 2007, Khokhar and Owusu Apenten, 2003). There is
only a limited number of studies investigating the relationship between the
chemical interactions of elements and organic compounds in beverages. These
interactions could interfere on the bioavailability of not only the elemental
species, but also the polyphenols. Pohl and Prusisz (2007) reported that this
chelation between elements and flavonoids considerably reduced the
bioavailability of the metal for the body or completely impaired the absorption of
these chemicals. Recent studies have shown that iron is not bioavailable in
beverages and foodstuffs that are rich in polyphenols, tannins or fibers (Yuyama
et al., 2002, Toaiari et al., 2005; Perron and Brumaghim, 2009). However, there
are other compounds, such as vitamin C, that could enhance the bioavailability of
iron (Silva et al., 2004).
Therefore, the bioavailability ofelements and polyphenols may impact on
the human intake of these chemicals through the consumption of food products,
thereby increasing or decreasing the potential bioinorganic effect on the human
body (Fairweather-Tait and Hurrell, 1996).
1.5. Analytical Methods and Challenges
Having reviewed the chemistry of the Brazilian foodstuffs and beverages
under investigation, it is now important to evaluate the analytical methods that
are traditionally used in previously published studies looking at the levels of
elements and polyphenols in foodstuffs and infusions of products from Brazil.
Moreover, one of the important parts of analytical chemistry is the analytical
sequence which defines the methodology from establishing a project hypothesis
through to the critical analysis of the data and what actions should be taken to
address the hypothesis (refer to Figure 2.1) (Ward, 2000). The main stages of
the sequence involve the hypothesis, sample selection, sample preparation,
chemical analysis (including optimisation, calibration and validation of
instruments and methods) and the statistical analysis and presentation of data
(Ward, 2000).
20
Table 1.4: Analytical review of published studies on the elemental levels of
Brazilian yerba mate.
Author Year Elements Sample selection
Sample preparation
Chemical analysis Data analysis
Barbosa et
al. 2015
C, K, N, Mg, Ca,
P, Al, Na, Zn,
Mn, Fe, Ba, Cu,
Ni, Mo, Pb, Cr,
As, Co, Ag, V
and Cd.
non-
commercial
sample
acid digestion
and dry weight
element
analyser;
FAES; UV-Vis;
ICP-OES
mean and st
dev
Barbosa et
al. 2018
C, N, P, K, Ca,
Mg, Fe, Mn, Zn,
Cu, Ni, B, Mo,
Co, As, Cd, Pb,
Ba, Cr and V
non-
commercial
sample
acid digestion
and dry weight
element
analyser and
ICP-OES
mean and ratio
Bastos et
al. 2014
P, K, Ca, Mg,
Na, Fe, Mn, Cu
and Zn
non-
commercial
sample and
soluble extracts
dry ashing and
water
extraction
FAES
UV-Vis and
FAAS
mean and %
solubility
Giulian et
al. 2007
Mg, Al, Si, P, S,
Cl, K, Ca, Ti, Mn,
Fe, Cu, Zn and
Rb
commercial
and infusion
pellets;
bombilla method and
water
extraction
PIXE (X-ray)
metal
extraction
values
Heinrichs
et al. 2001
N,
P, K, Ca, Mg, S,
B, Cu, Fe, Mn,
Ni, Zn, Al, Cd,
Co, Cr, Na and
Pb
commercial
and infusion
acid digestion
and tea-based
method
ICP-OES
min, max,
mean, RSD
and %
solubility
Jacques et
al. 2007
K, Ca, Na, Mg,
Mn, Fe, Zn, and
Cu
non-
commercial dry ashing
FAAS and
FAES
mean, st dev
and ANOVA
Malik et al. 2008
Al, B, Cu, Fe,
Mn, P, Zn, Ca, K
and Mg commercial dry ashing
FAAS and
ICP-OES
mean and st
dev
Magri et
al. 2019
Mn, Al, Fe, Zn,
Cu, Ni, Cd and
Pb
non-
commercial microwave
ICP-OES and
GF-AAS
mean, RSD
and ANOVA
Milani et
al. 2019
Al, As, Ba, Cd,
Cr, Cu, Fe, Mn,
Ni, Pb, Se and
Zn
commercial
infusions
tea-bad
method ICP-MS
mean, min,
max and
ANOVA
Pozebon
et al. 2015
Al, Ba, Ca, Cu,
Fe, K, Mg, Mn,
P, Sr, Zn, Li, Be,
Ti, V, Cr, Ni, Co,
commercial acid digestion
ICP-MS, ICP-
OES
LOD, LOQ,
spike recovery
mean and st
dev
21
Author Year Elements Sample selection
Sample preparation
Chemical analysis Data analysis
As, Se, Rb, Mo,
Ag, Cd, Sb, La,
Ce, Pb, Bi and U
and CRM
Rossa et
al. 2015
C, N, K, Ca, Mg,
Na, Fe, Mn, Cu
and Zn
non-
commercial dry ashing
NIR and UV-
Vis
min, max, st
dev and
ANOVA
FAES: flame atomic emission spectroscopy; FAAS: flame atomic absorption spectroscopy; UV-Vis: ultraviolet–visible spectroscopy; ICP-OES: inductively coupled plasma optical emission spectrometry; ICP-MS: inductively coupled plasma mass spectrometry; GF-AAS: graphite furnace atomic absorption spectroscopy; PIXE: particle-induced X-ray emission; NIR: near-infrared spectroscopy; st dev: standard deviation; min: minimum; max: maximum; RSD: relative standard deviation; ANOVA: analysis of variance; LOD: limit of detection; LOQ: limit of quantification; CRM: certified reference material.
Table 1.4 reports a review of published studies on the elemental levels of
Brazilian yerba mate and açaí (Table 1.5), with a critical assessment of the
important stages of the analytical sequence (see above). In summary, there has
only been a limited number of published studies (n = 11 for yerba mate and n = 7
for açaí), with the focus being on major/minor elements and for non-commercial
and commercial products.
In relation to the yerba mate infusions the method is normally based on
water extraction, a simulation of the bombilla (traditional method) or the regular
tea-based methodology (refer to Table 1.4). There is a lack of details provided on
the mass or volume of commercial yerba mate products used to prepare
samples, whether the solution was filtered and the method of pre-analysis
storage.
22
Table 1.5: Analytical review of published studies on the elemental levels of
Brazilian açaí
Author Year Elements Sample selection
Sample preparation
Chemical analysis
Data analysis
Menezes
et al.
2008
a
Na, Mg, Al, Mn,
Co, Ni, Cu, Al,
As, Rb, Mo, O,
Ca, Se, Ag, Cd,
Ba, Hg, Pb, Th,
U, K, Sr, Sb and
Fe
commercial
processed pulp n.r. ICP-MS value
Unicamp 2011
Ca, Fe, Mg, Mn,
P, Na, K, Cu and
Zn
commercial
processed pulp dry ashing ICP-OES mean
Yuyama 2011 Na, Ca, K, Fe,
Zn, B, Co and Cr
non-
commercial
processed pulp
n.r. NAA and
validation
mean and st
dev
Llorent-
Martínez
et al.
2013
Ag, Al, As, Ba,
Be, Ca, Cd, Co,
Cr, Cu, Fe, K,
Mg, Mn, Mo, Na,
Ni, Pb, Sb, Se,
Tl, V, Hg and Zn
commercial
processed juice microwave
ICP-MS
Calibration,
validation,
spike recovery,
interferences
and LOD
min and max
Santos et
al.
2014
a
Mn, Ca, Cu, Fe,
Mg and Zn
non-
commercial
processed pulp
microwave
ICP-MS
Validation and
CRM
distribution
and ANOVA
Moreda-
Piñeiro et
al.
2018 Ca, Co, Cu, K,
Mg, Ni, P and Rb
commercial
supplement
enzymatic
hydrolysis -
microwave
ICP-MS
Recovery,
LOD
mean and st
dev
Santos et
al.
2014
b
Sm, Tb, Th, La,
Eu, Dy, Pr, Yb
and Tm
non-
commercial
processed pulp
microwave
ICP-MS
Validation and
CRM
PCA
ICP-OES: inductively coupled plasma optical emission spectrometry; ICP-MS: inductively coupled plasma mass spectrometry; NAA: neutron activation analysis; st dev: standard deviation; min: minimum; max: maximum; ANOVA: analysis of variance; PCA: principal component analysis; LOD: limit of detection; CRM: certified reference material; n.r.: not reported.
Moreover, in terms of the chemical analysis, there is a limited amount of
detail provided on the optimisation and calibration of instruments, with the
authors nominally referring to other referenced studies. A major criticism of most
published studies is the lack of validation data, with there being no evaluation of
the accuracy or precision (refer to glossary) of the measurements (refer to
glossary), analysis of certified reference materials or matrix-matched spiked
23
recovery tests (refer to chapter 2, section 2.3.4). There are two exceptions where
the publications provide details on the chemical analyses, namely, Pozebon et
al., 2015 for yerba mate and Llorent-Martínez et al., 2013 for açaí. The authors
present and discuss the limit of detection of the chosen methodology, spike
recovery of the analytes, and validation with the use of certified reference
materials. Also, very few studies report any critical statistical analysis of the
yerba mate data (refer to section 2.6). So, in conclusion the reported studies on
the elemental levels of yerba mate and açaí in Brazil, to date, may be questioned
in terms of the reliability of the data, which will be addressed in chapter 3 (yerba
mate) and chapter 5 (açaí).
In relation to the review of polyphenol analysis, researchers have
focussed on the appropriate extraction, separation, and identification of the
polyphenol compounds. The extraction methods should always be carefully
chosen, taking into consideration aspects, such as low use of organic solvents,
possible automation, effectiveness and selectivity (Plaza et al., 2018). Whist
physical extraction methods, such as filtration and grinding, can be simple and
effective for homogenising samples, chemical digestions can volatilise, change or
result in the loss of analytes. Moreover, the lack of commercially available
standards and the wide range of phenolic structures found in nature make the
identification of phenolic compounds a challenge (Plaza et al., 2018). Table 1.6
shows a scheme of the methodologies involved in the polyphenol analysis of
plants and foods, with a critical assessment of the important stages of the
analytical sequence (see above).
Furthermore, one of the limitations associated with sample selection is
that the research is usually performed on a single sample of plant or fruit
material. This represents a challenge to determine the representativity of the
levels of the polyphenol compounds in the plant species. The variability in the
different polyphenol levels can be due to agronomic and seasonal differences
(Timmers et al., 2017). Moreover, the variety of the different infusion methods,
also introduces a variability of the levels quantified. Finally, the selection of the
24
class of polyphenol analysed may result in an under-reporting and thereby an
under-estimating of the total polyphenol content (Donnelly, 2015).
Table 1.6: Analytical steps involved in the analysis of polyphenols in plants and
food (Plaza et al., 2018).
Sample pre-treatment Extraction Clean-up-
isolation Spectro-photometric
methods Advanced analytical
techniques
Grinding
Filtration
Centrifugation
Milling
Drying
Hydrolysis or
digestion
Conventional (SLE,
LLE, Soxhlet)
Advanced (SFE,
PLE, UAE, MAE,
CXLE, HHPE, PEF)
SPE
LLE
HSCCC
Total phenolics (Folin-
Ciocalteu assay)
Total flavonoids (AlCl3
assay)
Total
proanthocyanidins
(DMAC assay)
Total anthocyanins
(pH differential assay)
Total antioxidant
capacity (ABTS,
DPPH)
HPLC (UV/DAD,
FD, MS, CAD,
ECD)
Others (CE,
SFC, SFC, MS,
GC)
ABTS, 2,2′-azinobis-(3-ethylbenzothiazoline-6-sulphonate; CAD, charged aerosol detection; CE, capillary electrophoresis; CXLE, carbon dioxide-expanded liquid extraction; DAD, diode array detection; DMAC, dimethylaminocinnamaldehyde; DPPH, 2,2-diphenyl-picrylhydrazyl; ECD, electrochemical detection; FD, fluorescence detection; GC, gas chromatography; HHPE, high hydrostatic pressure extraction; HPLC, high performance liquid chromatography; HSCCC, high-speed countercurrent chromatography; LLE, liquid-liquid extraction; MAE, microwave-assisted extraction; MS, mass spectrometry; PEF, pulse electric field; PLE, pressurized liquid extraction; SFC, supercritical fluid chromatography; SFE, supercritical fluid extraction; SLE, solid-liquid extraction; SPE, solid phase extraction; UAE, ultrasound assisted extraction; UV, ultraviolet.
1.6. Aim and Objectives
The overall aim of this research was to generate and provide, especially to
the small producers, a knowledge of the chemical composition and associated
health claims of major natural foodstuffs and beverages of Brazil. Brazil is a
major agricultural producer of specialist foodstuffs for national and global
consumption. The Brazilian economy is US$ 14 billion for the combined
exportation of yerba mate, coffee and açaí. To this end, the aim was to
determine; (i) the levels of chemical elements (major, minor and trace) and
25
polyphenols of yerba mate (from the southern region), coffee (São Paulo state)
and açaí berries (Amazonian region); and (ii) to assess the impact of
consumption in terms of the daily dietary intake on providing an adequate
nutrient supply for humans. Moreover, a review of the literature confirmed that
there is a limited amount of reliable data. It is essential to establish an analytically
robust method(s) that enables an evaluation of: (i) the impact of sample selection
and treatment; (ii) the influence of diluting prepared sample solutions before
instrumental analysis; and (iii) the calculation, statistical analysis and reporting of
date in relation to what is traditionally consumed by individuals in Brazil. Finally, a
key aspect of this research was to establish a database for future yerba mate,
coffee and açaí production and marketing of natural products from Brazil.
The specific objectives of the study were to:
(i) undertake and evaluate an extensive literature review of the
reported elemental and polyphenol levels in typical foodstuffs and
beverages from Brazil;
(ii) source a wide range of non-commercial and commercial samples of
yerba mate, coffee and açaí in Brazil;
(iii) establish and validate a sample preparation method for the
determination of elements (major, minor and trace);
(iv) develop and validate a sample preparation method to analyse the
samples (for elemental and polyphenolic content) as consumed by
the typical consumer;
(v) validate the instrumental analysis by using certified reference
materials and analyte spike recoveries of the products;
(vi) determine the elemental levels of the materials and the infusions by
inductively coupled plasma mass spectrometry (ICP-MS);
26
(vii) determine the polyphenol content of typical infusions of yerba mate,
coffee and açaí extractions by ultra-high performance liquid
chromatography (UHPLC) and ultra-violet visible spectroscopy (UV-
Vis);
(viii) review the reported potential dietary intake of polyphenols from
typical beverages and fruits from Brazil;
(ix) examine the relationship between sample type, origin, processing
and chemical levels; and
(x) evaluate the potential contribution of elements and polyphenols in
infusions of yerba mate (green loose, roasted, and tea-bags)
coffee (roasted) and açaí pulp in terms of human dietary intake.
Chapter 2 provides the analytical sequence for this research (Figure 2.1)
and the various stages relating to the sampling strategies, sample selection and
preparation and instrumental techniques (calibration and validation) for the
chemical (elemental and polyphenolic) analysis of yerba mate, coffee and açaí.
Chapters 3, 4 and 5, respectively, cover the research studies on yerba mate from
Brazil and Argentina, coffee from the São Paulo State of Brazil and açaí berries
and pulp from the Amazonian region of Brazil. Finally, chapter 6 reviews the
findings and presents ideas about future areas of investigation based on the
conclusions of this research.
27
Chapter 2. Methodology
28
2.1. Introduction
This chapter provides the details of the analytical procedures and
techniques used to achieve the Aim and Objectives (as set-out in chapter 1,
section 1.5). The analytical plan presented in Figure 2.1, was designed to outline
the critical analytical steps involved in this research, starting from the sample
collection procedures for the various studies on Brazilian products, namely yerba
mate (chapter 3), coffee (chapter 4) and açaí (chapter 5). This chapter follows
the analytical organisation presented in Figure 2.1, namely, a description of the
sample collection steps (section 2.2), followed by the pre-analysis sample
preparation steps (section 2.2.1). The chemical analysis stages of this study and
used throughout the thesis included: (i) the elemental content of samples
determined by inductively coupled plasma mass spectrometry (ICP-MS), as
described in section 2.3; (ii) the total polyphenol content if samples by ultraviolet-
visible spectroscopy (UV-Vis), outlined in section 2.4; and (iii) the polyphenol
profile and caffeine levels determined by high performance liquid
chromatography (HPLC), reviewed in section 2.5. The instrumental results were
subjected to computational data handling and statistical evaluation before
presentation in this thesis. The instrumentation sections (refer to sections 2.3.1,
2.4.1 and 2.5.1) describes the theory, specific instrument information, and the
optimisation and validation for each instrument and associated data analysis.
Finally, a summary of the data treatment, including the statistical analysis used in
this thesis is presented in section 2.6.
29
Figure 2.1: Analytical sequence adopted for this study of the chemical analysis of
typical beverages and açaí berries from South America.
2.2. Sample Collection
Commercial samples of yerba mate (Ilex paraguariensis), Brazilian coffee
(Coffea arabica L.) and açaí (Euterpe oleracea) were purchased from retail
outlets and commercial suppliers of Brazil and the UK (and Argentina for the
yerba mate). The non-commercial samples were obtained directly from
30
producers in Brazil and brought to the UK. The unopened commercial and trace
element free polypropylene or paper sealed bagged samples were stored in a
fridge (< 4°C) or a closed storage unit (< 15°C) until analysis. A detailed list of the
samples is given in chapters 3 (yerba mate), 4 (coffee beans and products) and 5
(acai berries and products).
Sample preparation
Prior to analysis, the samples needed to be decomposed using suitable
methods of digestion for elemental (or mineral) analysis. The term ‘mineral’ is
included in this description as the study of nutrition uses this term for chemical
elements in relation to human health requirements. A decomposition step is
necessary to reduce the amount of residual carbon in a sample and also to
release the analytes into solution. This decomposition of the sample is a critical
step. Whilst dry ashing methods (i.e. using a muffle furnace) may lead to analyte
loss, wet or acid digestion (i.e. using a water bath or a microwave apparatus)
requires exposing the sample to concentrated acids (nitric, hydrofluoric or aqua
regia (3:1v/v HCl:HNO3) and monitoring the digestion for long periods of time
(typically hours for open vessel methods). For this reason, these steps were
carefully monitored and optimised by using certified reference materials, to
ensure that the processes of ashing or wet acid digestion did not result in an
analyte loss. The following sample digestion procedure was initially optimised for
yerba mate samples and the resultant procedure was also used in the other two
studies (coffee and açaí).
Samples of 0.2500 ± 0.0010 g homogenised sample were weighed on an
analytical balance, transferred to an acid pre-washed ceramic crucible (in
duplicate) and placed in a Carbolite AAF 1100 muffle furnace at 500°C for 12
hours. The resultant ash was homogenised with 1 mL nitric acid (PrimarPlus-
Trace Analysis Grade 68%, Fisher Scientific, Loughborough, UK) in a fume
cupboard. The digest was transferred to a 25 mL Sterilin™ polypropylene tube
and then diluted with double-distilled deionised water (DDW, 18 18 MΩ cm) until
31
25 g (weighed, ± 0.0001 g). Each final solution was filtered using a 0.45 μm
Millex-HA membrane filter (Merck Millipore, Germany) and the resultant solutions
were analysed.
A blank for each digestion was prepared in order to evaluate any possible
sources of elemental contamination or loss during the digestion process. A blank
solution was produced following the same steps as that outlined for the
preparation of the samples, but without any material. All of the standards and
dilutions in this study were corrected by mass using a 4 decimal point analytical
calibrated balance (± 0.0001 g) instead of the traditional procedures using
volumetric flasks, in order to provide a better level of accuracy. This is especially
important for low concentrations, where the error is magnified.
In addition to the determining the total chemical content of the digested
samples, this study also evaluated the total elemental or polyphenol content
available in a regular serving of the foodstuff or beverage. Therefore, a series of
laboratory simulations based on traditional consumption methods were
performed and are detailed in each chapter: (i) regular infusion; iced tea and
bombilla method for yerba mate (refer to chapter 3); (ii) Brazilian brewing for
coffee (chapter 4); and extractions for açaí (chapter 5).
2.3. Inductively Coupled Plasma Mass Spectrometry (ICP-MS) for Elemental Analysis
Inductively coupled plasma mass spectrometry (ICP-MS) was used in this
work to determine the total elemental composition of the samples of yerba mate
(chapter 3), coffee (chapter 4) and açaí (chapter 5). Furthermore, this technique
was also used to determine the elemental levels of the extracted fraction or
infusions of yerba mate and coffee samples.
ICP-MS is capable of measuring the elemental (metals or non-metals) at
very low concentrations, typically sub µg/L (Balcaen et al., 2015). The main
advantages of ICP-MS are: simultaneous multi-element analysis, a wide linear
32
dynamic range (which is the linear part of the calibration curve that is used to
calculate the concentration of an analyte in the solution), very low detection limits
(typically < 0.01 µg/L) and good levels of precision (typically a relative standard
deviation of < ± 10%). Therefore, ICP-MS has been widely used for a variety of
samples matrix, such as environmental, foods and beverages, waters, soils and
geological materials (Baroni et al., 2015, Rousseau et al., 2013). The main
drawback of ICP-MS is the high mantainance costs.
The ICP-MS instrument consists of five main stages: sample introduction,
sample ionisation by the plasma (ICP), interface, mass spectroscopy (MS)
analyser and detector (Agilent, 2017), as shown in Figure 2.2.
The sample (normally as a liquid) is taken up by the instrument through a
peristaltic pump to the nebuliser where it is converted to an aerosol (particles
smaller than 10 µm in the Ar carrier gas) and transported to the inductively
coupled plasma or ICP. Typically, only 1-2% of the sample volume is carried to
the plasma; the remaining larger particles are condensed by the spray chamber
and is drained to waste (Becker, 2007, Thomas, 2013).
Figure 2.2: Instrumentation of an inductively coupled plasma mass spectrometer
(ICP-MS) Agilent Series (Agilent, 2017).
The ICP-MS torch is assembled horizontally and is made of three
concentric quartz tubes where the argon gas flows. The plasma is produced
33
when the carrier gas is passed into the ICP torch and a spark is applied (from a
Tesla coil) which provides electrons that collide with argon atoms causing the
ionisation of the gas (electron collision reaction i.e. X + e- → X+ + 2e-). The
resultant species interact with an intense electromagnetic field generated by the
RF power (typically 750–1500 W) applied to the copper coil, resulting in the
oscillation of an alternating current (AC) within the coil at the frequency of the RF
generator (27 or 40 MHz). When the aerosol sample reaches the plasma, the
high temperature enables the desolvation, atomisation and ionisation of the
elements present in the solution (Dean, 2003, Linge and Jarvis, 2009,
Beauchemin, 2010).
Since the mass spectrometer or MS operates at low pressures and room
temperature; and the plasma is at atmospheric pressure and a high temperature
(6 to 8000 K), the interface between these two parts is critical (Becker, 2007). It
consists of a sampling cone with an orifice of 1.0 mm diameter and a skimmer
cone with a 0.75 mm diameter, and both are cooled by water. They are made of
nickel which gives a high thermal conductivity (Thomas, 2013). When the analyte
ions pass through the first orifice, the pressure is reduced to 1 torr and then
through the second sampler, where the pressure reaches a level that is similar to
that in the MS (Harris, 2010). The resultant ion beam is separated by a negative
potential between electrons and positive ions and focussed by electrostatic
lenses (ion optics), which also removes neutral species and photons (Harris,
2010).
The University of Surrey ICP-MS instruments also include a
collision/reaction cell which is a multipole (octapole, hexapole or quadrupole)
which operates in a radio-frequency (RF) mode only (Thomas, 2013). It is located
between the ion lenses and the mass analyser. The reason is that an added gas
will remove polyatomic spectral interferences before the ions enter into the MS
(Becker, 2007). In this study, an Octopole Reaction System (ORS) or collision
cell with helium (He) gas addition was used in order to eliminate interferences,
such as 35Cl40Ar+, which is replaced with 35Cl4He+ to prevent any overlap with
75As+ (the only isotope for arsenic – 100% natural abundance). Also, helium was
34
used because it is an inert gas and does not react with the sample analytes. The
principle of a collision cell is to reject the interferences by reducing their kinetic
energy when colliding with He gas. Only the analyte ions are transmitted to the
MS, because of the difference on the potential of the octopole and MS (Thomas,
2013, Yamada, 2015).
The next part is the mass analyser, which is a quadrupole mass filter that
separates the positive ions according to their mass-to-charge ratio (m/z). A
quadrupole has four cylindrical rods of the same size (length typically of 15 to 20
cm and diameter of 1 cm). The opposing rods are connected to a direct current
(DC) and the other 2 to an alternating current (AC). This difference in the voltage
(positive and negative) causes the passage of the analyte through the
quadrupole and the specific charges only allow the ions of specific m/z values to
reach the detector (Linge and Jarvis, 2009).
Finally, the detector converts the ions into electrical signals through a
channel electron multiplier. The analytes from the MS hit a surface coated with a
semiconductor material, has a negative potential at the first end and produce one
or more secondary electrons. These electrons move and hit another new surface
and emit more electrons until the generation of a pulse is detected (Thomas,
2013, Skoog et al., 2017).
Instrumentation – ICP-MS
The majority of the analysis performed on this study used an ICP-MS
Agilent 7800 Series (Agilent Technologies, UK) with an SPS 4 series
autosampler controlled through the use of Agilent software (MassHunter). This
Agilent ICP-MS instrument has an Octopole Reaction System (ORS). Sample
introduction is through the quartz, Peltier-cooled, Scott-type double-pass spray
chamber. Part of the studies (commercial samples of yerba mate and coffee;
refer to chapters 4 and 5) was also performed on a similar ICP-MS Agilent 7700x
Series (Agilent Technologies, UK) performed with the same features. The typical
operating conditions for both instruments are detailed in Table 2.1. The isotopes
35
were used to determine the concentration of the analyte elements. Instrument
optimisation was performed before each analysis using a 1 μg/L tuning solution
containing 7Li, 24Mg, 89Y, 140Ce, 204Tl and 59Co (Agilent Technologies, UK). The
sensitivity of the instrument for the selected elements under investigation was
enhanced before each analysis by the optimisation of the nebuliser flow rate and
RF power.
Table 2.1: Typical operating conditions for the Agilent ICP-MS instruments.
Part Parameter Agilent 7700x Agilent 7800
Sample
introduction
Nebuliser MicroMist MicroMist
Carrier gas flow 0.8 L/min 0.9 L/min
Nebuliser pump 0.3 rps 0.1 rps
Spray Chamber
Temperature 2 ºC 2 ºC
Plasma
condition
RF Power 1550 W 1550 W
RF Matching 1.95 V 1.80 V
Sampling Depth 8 mm 8 mm
Collision
cell
[1] ON He mode He mode
He gas flow 4.8 mL/min 4.3 mL/min
Isotopes
23Na, 24Mg, 39K, 40Ca, 51V,
52Cr, 55Mn, 56Fe, 59Co, 60Ni,
63Cu, 66Zn, 75As, 78Se, 95Mo
23Na, 24Mg, 39K, 40Ca, 51V,
52Cr, 55Mn, 56Fe, 59Co, 60Ni,
63Cu, 66Zn, 75As, 78Se, 95Mo
[2] OFF No gas mode No gas mode
Analyte elements 111Cd, 208Pb 111Cd, 208Pb
Detector
parameters
Type of detector Electron multiplier Electron multiplier
Pulse HV 980 V 925 V
Analog HV 1680 V 2163 V
Data
acquisition
Sample uptake time 50 s 40 s
Stabilisation time 30 s 25 s
Sample wash time 120 s 65 s
Internal standards – ICP-MS
Internal standards (ISTDs or IS) are used to correct for any drift in the
signal intensity resulting from an instrument issue, such as blockages, leaks and
36
any change in the instrument conditions during operation (Lord, 2014). The
correction of the data was performed by blank subtraction of the sample counts
per second (CPS), followed by this value being ratioed with the internal standard
CPS signal as shown in Equation 2.1:
𝑅𝑎𝑡𝑖𝑜 =𝐶𝑃𝑆 𝑎𝑛𝑎𝑙𝑦𝑡𝑒 𝑠𝑖𝑔𝑛𝑎𝑙 − 𝐶𝑃𝑆 𝑏𝑙𝑎𝑛𝑘 𝑠𝑖𝑔𝑛𝑎𝑙
𝐶𝑃𝑆 𝐼𝑆𝑇𝐷 𝑠𝑖𝑔𝑛𝑎𝑙
Equation 2.1
A 100 μg/L internal standard (IS) solution of indium (115In) was prepared
from a 1000 μg/mL stock solution (Aristar, UK) in 1% HNO3 (High Purity
Analytical Grade, Fisher Scientific, Loughborough, UK). In this study, indium was
used as the ISTD due to its mid-first ionisation potential and absence in the yerba
mate, coffee and açaí samples.
Limit of detection (LoD) and linear dynamic range (LDR) – ICP-MS
All instruments have a degree of noise associated with the measurements
that limits the precision of the background signal. Therefore, the instrumental limit
of detection (LoD) is the minimum concentration of the analyte that can be
determined to be different from the signal of the blank (Miller et al., 2018), and it
was calculated using Equation 2.2. In this study, the average of 10 replicate
signal values of 1% v/v HNO3 were used as a blank for the digested samples and
DDW for the yerba mate and coffee infusion samples. The instrumental LoDs for
the investigated elements are presented in Table 2.2:
𝐿𝑜𝐷 = 𝑦𝑏 + (3 𝑥 𝑆𝐷)
Equation 2.2
Where:
𝑦𝑏 is the mean blank signal; and
37
𝑆𝐷 is the standard deviation of the blank signal (Miller et al., 2018).
Table 2.2: Investigated isotopes; limits of detection (LoD) in 1% HNO3 and
double-distilled deionised water (DDW) using 115In as the internal
standard for the Agilent 7800 ICP-MS.
Isotope Natural Abundance (%) LoD in 1% HNO3 (µg/L) LoD in DDW* (µg/L) 23Na 100 0.09 0.06
24Mg 79.0 0.08 0.07
39K 93.3 0.07 0.05
40Ca 96.9 0.08 0.06
51V 99.7 0.02 0.05
52Cr 83.8 0.08 0.05
55Mn 26.1 0.03 0.03
56Fe 97.1 1.00 0.50
59Co 100 0.07 0.05
60Ni 26.1 0.06 0.04
63Cu 69.2 0.09 0.05
66Zn 27.9 0.20 0.10
75As 100 0.04 0.02
78Se 23.6 0.04 0.02
95Mo 15.9 0.09 0.09
111Cd* 12.8 0.03 0.03
208Pb* 52.4 0.03 0.03
*collision cell off.
The linear dynamic range (LDR) usually refers to the range of analyte
concentrations in which the detector produces a signal proportional to the
concentration of the analyte, as exemplified in Figure 2.3. A linear regression
equation (y = mx + c) can be calculated within the LDR using the signal (y-axis)
of a series of accurate standards with known concentrations (x-axis); this
equation will allow for the prediction of the concentration of unknown samples
(Miller et al., 2018). In order to prepare the calibration curves for the different
analytes, a range of 1 – 1500 µg/L multi-element solutions were prepared from a
38
1000 mg/L stock solution (TraceCERT®, Sigma-Aldrich, UK) for each element
and diluted in 1% v/v nitric acid.
Figure 2.3: Calibration curve for manganese (55Mn) using 115In as the internal
standard for the Agilent 7800 ICP-MS, in the helium collision cell
mode.
In this study, the range of elemental concentrations found in the samples
(digest solutions and infusions) differs significantly. Therefore, the use of the
appropriate LDR was evaluated to provide an accurate calculation procedure for
determining the analyte concentration of the unknown solution. This is a major
problem in many studies where a calculation curve over an LDR of say 1 to 1500
µg/L is used to calculate the concentration of an unknown solution that has a
digest solution value of 2 µg/L. The higher standards may have a significant
influence on the calculated level of accuracy, especially if the highest standards
are affecting the linearity of the curve. Therefore, the specific linear dynamic
ranges for each type of samples are presented on Table 2.3.
0 200 400 600 8000
500
1000
1500
Mn concentration (mg/L)
Rati
o C
PS
Mn
/ In
Y = 1.6619x + 6.4887R2 = 0.9995
Text
(µg/L)
39
Table 2.3: Investigated isotopes and linear dynamic range (µg/L) used for each of
the type of samples (yerba mate, coffee and açaí) used in this study.
115In was used as the internal standard for the Agilent 7800 ICP-MS
analysis.
Isotope Yerba mate (µg/L) Coffee (µg/L) Açaí (µg/L) 23Na 0 - 1500 0 - 1500 0 - 1500
24Mg 0 - 1500 0 - 1500 0 - 1500
39K 0 - 1500 0 - 1500 0 - 1500
40Ca 0 - 1500 0 - 1500 0 - 1500
51V 0 - 500 0 - 500 0 - 500
52Cr 0 - 750 0 - 500 0 - 500
55Mn 0 - 1500 0 - 1000 0 - 1500
56Fe 0 - 1500 0 - 1500 0 - 1500
59Co 0 - 500 0 - 500 0 - 250
60Ni 0 - 750 0 - 500 0 - 500
63Cu 0 - 1000 0 - 750 0 - 1500
66Zn 0 - 1000 0 - 750 0 - 1500
75As 0 - 250 0 - 250 0 - 250
78Se 0 - 250 0 - 250 0 - 250
95Mo 0 - 250 0 - 250 0 - 250
111Cd* 0 - 250 0 - 250 0 - 250
208Pb* 0 - 250 0 - 250 0 - 250
*collision cell off
Validation (accuracy and precision) – ICP-MS
Validation is an important step for the quality control evaluation of an
analytical technique, to assess if the method is fit-for-purpose. The precision or
repeatability (refer to glossary) describes the level of agreement between
replicate analysis of the same sample within the same instrument conditions. On
the other hand, reproducibility (refer to glossary) reflects the precision of replicate
analysis under different conditions (between-run), such as analysing the same
sample over different days (Miller et al., 2018). In this study, the instrumental
performance was validated by the comparative analysis of a water certified
40
reference material (CRMs), namely, NIST SRM 1640a (National Institute of
Standards and Technology, USA) for trace elements and CRM 3 Multielement
standard solution for ICP (Fluka Analytical, Sigma-Aldrich, Poole, UK) for Ca, Na,
K and Mg as shown on Table 2.4. The study was validated for the level of
accuracy, that is, how close the calculated mean value is from the true certified
value, by comparing the values in Table 2.4 and precision by the use of the
relative standard deviation (%). In general, there is a good agreement between
the certified and calculated values.
It is also important to validate the digestion methods against a matrix-matched
standard reference material. This material has to be analysed under the same
conditions as the samples and compared with the certified value. In this study,
two plant reference materials were used, Tea Leaves INCT-TL-1 (Instytut Chemi
i Techniki Jadrowej, Poland) and Peach Leaves SRM 1547 (National Institute of
Standards and Technology, USA). The accuracy of the elemental measurements
was determined comparing the measured concentration of each element and the
certified value of the CRM. The results are presented in Tables 2.5 (a) and (b). In
general, there is a good agreement between the certified and calculated values.
41
Table 2.4: Evaluation of accuracy (comparison of measured and certified
elemental concentrations) and precision (relative standard deviation
(RSD %)) of for NIST SRM 1640a and CRM 3 (for Na, Mg, K and
Ca).
Element Certified
value (µg/L)
Calculated value (µg/L) RSD (%)
n = 20a
n = 5b
n = 20a
n = 5b
Na 1000 ± 3 1034.58 ± 23.64 998.27 ± 17.84 2.3 1.8
Mg 400 ± 2 393.53 ± 12.52 399.56 ± 18.24 3.2 4.6
K 200 ± 3 188.28 ± 5.67 193.94 ± 4.99 3.0 2.6
Ca 2000 ± 5 1952.94 ± 23.63 1980.26 ± 21.74 1.2 1.1
V 14.93 ± 0.21 15.12 ± 0.43 15.07 ± 0.34 2.8 2.3
Cr 40.22 ± 0.28 38.58 ± 1.78 39.63 ± 0.94 4.6 2.4
Mn 40.07 ± 0.35 38.80 ± 1.30 39.78 ± 1.02 3.4 2.6
Fe 36.5 ± 1.7 40.10 ± 3.00 38.42 ± 1.52 7.5 4.0
Co 20.08 ± 0.24 19.57 ± 0.58 19.85 ± 0.84 3.0 4.2
Ni 25.12 ± 0.12 24.90 ± 1.23 25.01 ± 1.03 4.9 4.1
Cu 85.07 ± 0.48 84.42 ± 3.58 84.74 ± 2.51 4.2 3.0
Zn 55.2 ± 0.32 55.20 ± 0.62 55.01 ± 0.34 1.1 0.6
As 8.010 ± 0.067 7.88 ± 0.07 8.00 ± 0.09 0.9 1.1
Se 19.97 ± 0.16 19.31 ± 1.36 19.12 ± 1.07 7.0 5.6
Mo 45.24 ± 0.59 52.54 ± 2.64 43.92 ± 0.99 5.0 2.3
Cd 3.961 ± 0.072 3.92 ± 0.11 3.96 ± 0.19 2.8 4.8
Pb 12.005 ± 0.040 12.65 ± 0.65 11.93 ± 0.78 5.1 4.9
a : reproducibility, b: repeatability; n is the number of measurements; RSD is relative standard deviation (%).
42
Table 2.5 (a): Comparison of the certified reference values (CRM) with the
calculated concentrations for the Tea Leaves INCT-TL-1 certified
reference material.
Element Certified value (mg/kg)
Calculated value (mg/kg)
Na 24.7 ± 3.2 28.32 ± 5.16
Mg 2240 ± 170 2452.57 ± 183.28
K 17000 ± 1200 16449.19 ± 1374.10
Ca 5820 ± 520 5639.97 ± 630.38
V 1.97 ± 0.37 1.57 ± 0.85
Cr 1.91 ± 0.22 1.82 ± 0.95
Mn 1570 ± 110 1352.85 ± 129.19
Fe 432a 540.28 ± 19.20
Co 0.387 ± 0.042 0.50 ± 0.06
Ni 6.12 ± 0.52 5.82 ± 0.59
Cu 20.4 ± 1.5 21.73 ± 2.01
Zn 34.7 ± 2.7 30.57 ± 3.20
As 0.106 ± 0.021 0.09 ± 0.01
Se 0.076a 0.05 ± 0.02
Mo - -
Cd 0.030 ± 0.004 0.03 ± 0.01
Pb 1.78 ± 0.24 1.38 ± 0.43
ainformation value; all values reported on a dry weight basis.
43
Table 2.5 (b): Comparison of the certified reference values (CRM) with the
calculated concentration for the Peach Leaves SRM 1547
certified reference material.
Element Certified value (mg/kg)
Calculated value (mg/kg)
Na 23.8 ± 1.6 21.93 ± 2.43
Mg 4320 ± 150 4294.75 ± 146.23
K 24330 ± 380 25021.58 ± 353.92
Ca 15590 ± 160 14985.65 ± 156.28
V 0.367 ± 0.038 0.29 ± 0.06
Cr 1a 0.86 ± 0.08
Mn 97.8 ± 1.8 95.73 ± 2.54
Fe 219.8 ± 6.8 209.35 ± 8.30
Co 0.07a 0.03 ± 0.01
Ni 0.689 ± 0.095 0.79 ± 0.08
Cu 3.75 ± 0.37 3.45 ± 0.46
Zn 17.97 ± 0.53 16.36 ± 0.60
As 0.062 ± 0.014 0.05 ± 0.02
Se 0.120 ± 0.017 0.10 ± 0.03
Mo 0.0603 ± 0.0068 0.05 ± 0.02
Cd 0.0261 ± 0.0022 0.02 ± 0.00
Pb 0.869 ± 0.018 0.79 ± 0.04
ainformation value; all values reported on a dry weight basis.
2.4. UV-Vis Spectroscopy for the Total Polyphenol Content Analysis
Molecules can absorb part of the radiation, when exposed to light energy.
Consequently, electrons move from a lower energy state (ground) to a higher
one (excited). Ultraviolet–visible spectroscopy refers to the absorbed energy in
the ultraviolet and adjacent visible spectra and can be translated into an
absorbance spectrum (between the range of wavelengths of 200 to 700 nm)
(Pavia et al., 2014). Also, the extent of light absorbance is expressed by the
Beer-Lambert law, as presented in Equation 2.3:
44
𝐴 = log (𝐼𝑜
I ) = ε c l
Equation 2.3
Where:
𝐴 = absorbance;
𝐼𝑜 = intensity of incident light;
I = transmitted intensity;
ε = molar absorptivity of solute;
c = molar concentration of solute; and
l = path length of sample cell.
Since the path length of the sample cell is usually fixed and the molar
absorptivity is a specific constant, the absorbance of a sample is directly
proportional to the sample concentration. For most of the molecules, this
relationship is linear over a certain concentration range (Harvey, 2006).
Instrumentation – UV-Vis
The UV-Vis instrument has 3 main component parts – a light source, a
monochromator and a detector. As a light source, the UV-Vis spectrophotometer
has a deuterium lamp to emit light in the ultraviolet region and a tungsten
halogen lamp to emit light in the visible region. The light radiation is filtered
through an optical filter before passing through the slit to the monochromator in
order to purify the signal (Skoog et al., 2017).
The monochromator consists of a diffraction prism that disperses the light
radiation to produce a spectrum. If the prism is rotated, the desired wavelength
segment of the spectrum is selected. The radiation then passes through a beam
splitter which permits the radiation to pass through the reference cell or through
the sample cell (1 cm length transparent plastic cells). After the sample and
reference cells, the radiation passes through convex lens into the detector. When
the light radiation arrives at the detector, the light energy is converted into an
45
electrical current (Pavia et al., 2014). The extent of the electrical current is
proportional to the amount of light radiation arriving at the detector. A BioChrom
Libra UV-Vis spectrophotometer was used for the determination of the total
polyphenol content of samples in this study.
Total polyphenol content by Folin-Ciocalteu analysis
The Folin-Ciocalteu assay is used to quantify the total polyphenol content
of a sample throughout a reduction-oxidation (redox) reaction where the Folin-
Ciocalteu reagent reacts with the electrons from a reducing agent (e.g.
polyphenols) (Huang et al., 2005). The Folin-Ciocalteu reagent is a mixture of
phosphomolybdic and phosphotungstic acids. Although the chemistry of the
Folin-Ciocalteu is still unknown, the isopolyphosphotungstates are colourless
when fully oxidised, and the analogous molybdenum compounds are yellow
(Prior et al., 2005). The end-product reduction produces a blue coloured complex
with a maximum absorbance around 765 nm (Singleton et al., 1999b, Ainsworth
and Gillespie, 2007). Although the Folin-Ciocalteu assay is an excepted method
to determine the total polyphenol content, a limitation of the method is that the
reagent can also react with a series of interferences, such as ascorbic acid and
sugars, that could also be present in the samples. This may be especially true for
fruit juice samples (Prior et al., 2005). The samples analysed in this study did not
had a significant concentration of ascorbic acid or sugars.
The total polyphenol content of the yerba mate and coffee infusions and
açaí extracts was determined by the Folin-Ciocalteu assay, as detailed in ISO
14502-1 (ISO, 2005). A 1 mL aliquot of the diluted infusion or extract was placed
in a 15 mL polypropylene plastic test tube and 5 mL of 10% Folin-Ciocalteu
(Fisher Scientific, Loughborough, UK), freshly prepared in deionised water was
added and mixed by a vortex mixer. After 3 to 8 mins, 4 mL of 7.5 % w/v sodium
carbonate solution (Sigma-Aldrich, Poole, UK) was added and the tubes were
mixed using a vortex mixer. The tubes were left for 1 hour at room temperature
before the measurement at 765 nm. All the sample analyses were performed in
46
duplicate due to the limited amount of available sample. The results are
expressed in gallic acid equivalent (Sigma-Aldrich, Poole, UK), which was used
as a calibration over the range of 0 to 50 µg/mL, as shown in Figure 2.4. The
infusion and extract samples were diluted appropriately in deionised water to fit
the absorbance levels within the calibration range of the reference standards.
The limit of detection of the total polyphenol content was calculated for this assay
at 0.16 µg/mL gallic acid equivalent (based on the linear regression of the
calibration curve).
Figure 2.4: Gallic acid calibration curve obtained by the Folin-Ciocalteu assay
using a UV-Vis instrument (refer to section 2.4.2)
2.5. High Performance Liquid Chromatography (HPLC) for Polyphenol Profile Analysis
High performance liquid chromatography (HPLC) is an analytical
technique of separating, quantifying and identifying components in a mixture. In
HPLC, separation is based upon the differential distribution of analyte molecules
between two phases, a mobile and a stationary phase. Each component of the
mixture interacts differently with the two phases, and at different flow rates for
each component, leading to separation as they travel through the column
0 20 40 600.0
0.2
0.4
0.6
0.8
Gallic acid concentration (µg/mL)
Ab
so
rban
ce
y = 0.011x - 0.0008 R² = 0.9999
47
(Niessen, 2006). This technique relies on the pumping of a pressurised liquid
solvent containing the sample mixture (mobile phase) through a column filled
with a adsorbent material (stationary phase) (Snyder et al., 2011, García-
Álvarez-Coque et al., 2017). The resolution of the analytes in chromatography
depends on three parameters: efficiency, retention and selectivity as described in
Equation 2.4. Method development can maximise the resolution by optimising
each of these terms:
𝑅 = (√𝑁4 ) (
𝜅𝜅 + 1) (
𝛼 − 1𝛼 )
Equation 2.4
where:
𝑅 = resolution;
𝑁 = number of theoretical plates;
𝜅 = the retention or capacity factor; and
𝛼 = the separation factor (Dolan and Snyder, 2013).
An increase in the efficiency (refer to Equation 2.4), can be achieved by
increasing the length of the column or the porosity of the stationary phase or
reducing the thickness and particle size of the column (Ali et al., 2012, Cabooter
and Desmet, 2012, Skoog et al., 2017).
The retention factor (Equation 2.4), can be modified by variation of the
mobile phase, in order to have an effect on the hydrophobicity or hydrophilicity
(refer to glossary) interaction between the analytes and the mobile phase
(Fountain and Iraneta, 2012). Also, modification of the elution strength can be
achieved by changing the pH, thereby affecting the retention time (Harvey,
2006).
The selectivity (Equation 2.4) can be changed by modifying the type of
molecular interaction. For example, in the reversed phase (polar mobile phase
Efficiency Retention Selectivity
48
and non-polar stationary phase), the analytes are selected by the hydrophobic
interactions between the stationary and mobile phase. Additional polar functional
groups can change the selectivity of an analyte (Snyder et al., 2011, García-
Álvarez-Coque et al., 2017).
Instrumentation - HPLC
In this study, a reverse phase HPLC was preferred due to the polar nature
of the polyphenol compounds, providing a better separation of the analytes.
Therefore, the column contained a non-polar stationary phase and a polar
compound mobile phase.
The determination of the polyphenolic profile and caffeine levels of the
yerba mate and coffee infusions was performed following the optimised method
proposed by Donnelly (2015) for yerba mate infusions samples. This method was
developed in order to optimise the resolution (refer to Equation 2.4) by selecting
the best column (efficiency and selectivity factor) and mobile phase composition
and gradient (retention factor) (Donnelly, 2015). An ultra-high performance liquid
chromatography or UHPLC instrument was used with almost double the overall
operating pressure (to 15,000 psi) in order to obtain more rapid flow rates and
achieve better resolution separations in shorter time frames (de Souza et al.,
2010). A comparison of using HPLC and UHPLC instruments for chlorogenic acid
analysis of yerba mate was completed by Donnelly (2015), who showed that it
was possible to decrease the run time from 60 to 30 minutes. Therefore, a
Waters Acquity UPLC® (Waters, Milford, USA) instrument was used for the
yerba mate and coffee analysis. It was fitted with a binary solvent manager and
photodiode array detector (PDA) and controlled by Empower 3 chromatography
software (Waters, Milford, USA). The açaí polyphenolic analysis was performed
as described in the respective chapter (refer to section 5.5.7).
The method validation for the polyphenol analysis was performed by
Donnelly (2015). The matrix effects were determined using 3 independent
measurements of the reference materials at 5 different concentrations prepared
49
in water and methanol extracts of green tea, rooibos and hibiscus. The standard
solution curve was plotted against the standards prepared in the sample matrix.
The resultant slopes and intercept were calculated. The levels added were
equivalent to 65 – 87% of the matrix response of the extracts (Donnelly, 2015).
The recovery and precision of the method were also evaluated and ranged from
90 to 103 % and the coefficient of variation of 0.82 % (Donnelly, 2015).
The separation of the compounds was conducted by injecting a 5 μL
sample onto a Phenomenex (Macclesfield, Cheshire, UK) Kinetex© PFP column,
with dimensions 4.6 x 100 mm x 2.6 μm 100 A, held at a constant temperature of
25°C, a flow rate of 0.7 mL/min and controlled by a gradient programme, as
shown in Table 2.6. Data was collected at wavelengths of 280 and 320 nm.
Table 2.6: Gradient programme for UHPLC analysis of polyphenol and caffeine in
yerba mate and coffee infusions.
Time (min) 5% formic acid (%) 80% ACN*, 5 % formic acid (%) 0.1 95 5
8 92.5 7.5
22.3 60 40
24.6 0 100
27 0 100
28 95 5
29.5 95 5
* ACN = acetonitrile.
The polyphenol and caffeine levels were quantified using a 5-point
calibration curve for a blank and reference standards against the peak area (AU)
of a chromatogram. The reference materials of caffeine, 3-caffeoylquinic acid, 4-
caffeoylquinic acid and 5-caffeoylquinic acid were obtained from Sigma (Sigma-
Aldrich, Poole, UK) and prepared over the range of 0-150 mg/L for 5-
caffeoylquinic acid, 0-70 mg/L for 3-caffeoylquinic acid and 4-caffeoylquinic acid
and 0-40 mg/L for caffeine, as shown in Figure 2.5. The mixed standard solutions
were stored in a frozen state until used. The calculated limit of detection (based
on the linear regression of the calibration curve) calculated for caffeine was 0.047
50
mg/L, and the following for the polyphenols: 0.035 mg/L for 3-caffeoylquinic acid,
0.033 mg/L for 4-caffeoylquinic acid and 0.038 mg/L for 5-caffeoylquinic acid.
Figure 2.5: 5-caffeoylquinic acid calibration curve obtained by the UHPLC
analysis (refer to section 2.5.1).
2.6. Statistical Analysis
The statistical analysis applied in this study were carried out using the
statistical software packages GraphPad Prism 6 and IBM® SPSS® Statistics
version 20. After the analysis of the descriptive statistics, such as mean,
standard deviation and relative standard deviation; the D'agostino and Pearson
test was used to study the normality of the data (Miller et al., 2018). Where the
data followed a normal distribution, parametric tests were used, such as two-
tailed Student t-test, paired two-tailed t-test and analysis of variance; and non-
parametric tests for data not normally distributed, such as the Spearman‘s rank
(refer to chapter 5) (Miller et al., 2018).
The arithmetic mean of the measurements was calculated by the sum of
the values divided by the number of measurements (n). The standard deviation
describes the spread of the experimental values around the arithmetic mean and
is defined by Equation 2.5 (Miller et al., 2018):
0 50 100 1500
2
4
6
5-caffeoylquinic acid concentration (mg/L)
Peak A
rea x
10
6 (A
U)
Y = 0.040x + 0.023R2 = 0.9998
51
𝑠 = √∑(𝑥𝑖 − ��)2
𝑛 − 1
Equation 2.5
where:
𝑠 = standard deviation;
𝑥𝑖 = value;
�� = arithmetic mean; and
𝑛 = the number of samples.
The level of precision is then determined by calculating the relative
standard deviation or s/�� x 100 (%). The lower the % rsd value the better the
level of precision (Miller et al., 2018).
D'Agostino and Pearson normality test
In order to check if a set of data is normally distribuited or a quantification
of how far a set of data is from a Gaussian distribution, the D'Agostino and
Pearson test was used. In this test, the Skewness is first calculated relating to
the symmetry of the set of data and then the Kurtosis, which quantifies the peak
shape of the distribution. Finally, the D'Agostino and Pearson test combines both
results into a single value. Assuming that the data are not normally distributed; if
the probability or p value is small (< 0.05 for 95% confidence level) then the null
hypothesis will be rejected (Motulsky, 2014).
Significance tests
In order to compare the means of a certified population with the calculated
one a two-tailed Student t-test was performed, Equation 2.6 shows the
calculation of the parameter t (Miller et al., 2018). The null hypothesis in this test
52
is that the certified population is equal to the calculated one at a probability p =
0.05; if tcalc is lower than the tcrit value, then the null hypothesis is not rejected:
𝑡𝑐𝑎𝑙𝑐 =(�� − 𝜇) √𝑛
𝑠
Equation 2.6
where:
𝑡𝑐𝑎𝑙𝑐 = t calculated;
�� and 𝑠 = sample mean and standard deviation;
𝑛 = number of samples; and
𝜇 = mean of the certified population.
It is also important to compare the standard deviations between two
populations to evaluate the random error of two sets of data (Miller et al., 2018).
In order to test whether the difference between the variances is significant, a F-
test was performed by the ratio of the squares of the standard deviations. The
null hypothesis states the variance are not significant at a probability p = 0.05. If
the calculated value of F is lower than the critical value, then the null hypothesis
is not rejected (Miller et al., 2018).
In terms of evaluating the paired values from the same set of samples (i.e.
comparison of the results obtained for the same cohort of samples using two
different analytical methods), a paired two-tailed t-test was performed using
Equation 2.7 for the calculation of the parameter t (Miller et al., 2018). The paired
t-test was used to compare two extraction methods in the same set of samples in
Chapter 5. The null hypothesis in this test states that the means are not
significantly different at a probability p = 0.05; if tcalc is lower than the tcrit value
then the null hypothesis is not rejected:
53
𝑡𝑐𝑎𝑙𝑐 =�� √𝑛
𝑠𝑑
Equation 2.7
where:
𝑡𝑐𝑎𝑙𝑐 = t calculated;
�� and 𝑠𝑑 = mean and standard deviation of differences between the
paired values; and
𝑛 = number of samples.
In order to compare the means of sets of data with more than two
variables, a Kruskal-Wallis test was performed (refer to chapter 5) (Miller et al.,
2018). The values are arranged in an ascending order (1, 2,... to N; where N is
the number of samples) and given a rank value. The sum of the ranks was used
to calculate the chi-squared, as presented in Equation 2.8. The calculated value
is compared to the critical value. The null hypothesis in this test states that there
is no statistically significant difference between the variables; and the null
hypothesis is not rejected if the calculated value is less than the critical value
(Corder and Foreman, 2011):
𝑋2 = 12
𝑁 + 𝑁2 (𝑅𝑖
2
𝑁𝑖+ ⋯ +
𝑅𝑘2
𝑁𝑘) − 3 (𝑁 − 1)
Equation 2.8
where:
𝑋2= chi-squared;
𝑁 = number of samples; and
𝑅𝑖 = sum of the rank for a particular data set.
54
Correlation coefficients
In order to evaluate the direction and magnitude of a linear relationship
between sets of data (𝑥 and 𝑦); the Pearson product moment correlation
coefficient (𝑟𝑝, refer to Equation 2.9) was used if the data was normally
distributed and the Spearman rank correlation coefficient (refer to Equation 2.11)
if it was not normally distributed (Corder and Foreman, 2011, Miller et al., 2018).
The coefficient values range from -1 to +1, where -1 indicates a strong negative
correlation; 0 no correlation; and +1 a strong positive correlation. If the Pearson
product moment correlation coefficient is lower than 0.8, a two-tailed Student t-
test was used to determine the level of significance (refer to Equation 2.10). The
null hypothesis states that there is no correlation between the two sets of data. If
the calculated value exceeds the critical value, the null hypothesis is rejected and
the two data sets are significantly correlated:
Pearson product moment correlation
𝑟𝑝 =∑ {(𝑥𝑖 − ��)(𝑦𝑖 − ��)}𝑖
{[∑ (𝑥𝑖 − ��)2𝑖 ][∑ (𝑦𝑖 − ��)2
𝑖 ]}12
Equation 2.9
where:
𝑟𝑝 = Pearson product moment correlation coefficient;
𝑥𝑖 and 𝑦𝑖 = values of sets; and
�� and �� = average value of individual sets.
Two-tailed Student t-test
|𝑡| =|𝑟𝑝|√𝑛 − 2
√1 − 𝑟𝑝2
, 𝑤ℎ𝑒𝑟𝑒 𝑑𝑒𝑔𝑟𝑒𝑒𝑠 𝑜𝑓 𝑓𝑟𝑒𝑒𝑑𝑜𝑚 = 𝑛 − 2
Equation 2.10
55
where:
𝑡 = calculated t-test value;
𝑟𝑝 = Pearson product moment correlation coefficient; and
𝑛 = number of sample pairs.
Spearman rank correlation coefficient
𝑟𝑠 =6 ∑ 𝑑𝑖
2𝑖
𝑛(𝑛2 − 1)
Equation 2.11
where:
𝑟𝑠 = Spearman rank correlation coefficient;
𝑑𝑖 = difference between each ranking pair; and
𝑛 = number of sample pairs.
2.7. Summary
An overall analytical plan to address the aim and objectives of this study
was presented in section 2.1. The type and collection of the samples were
described in section 2.2 and the preparation for each analysis in section 2.2.1.
The total polyphenol content was determined by the Folin-Ciocalteu assay using
a BioChrom Libra UV-Vis spectrophotometer, as described in section 2.3.1. The
assay was described in section 2.3.2. The polyphenol analysis presented in this
study for the yerba mate (chapter 3) and coffee infusions (chapter 4) were
performed using a Waters Acquity UHPLC® instrument. The açaí extract
samples were analysed as outlined in chapter 5. The instrument and HPLC
theory were described in section 2.4. For all of the samples, elemental analysis
56
was performed by an Agilent 7800 inductively coupled plasma mass
spectrometer (ICP-MS). The theory and instrumentation for ICP-MS were
described in section 2.5.1. and the use of internal standards was explained in
section 2.5.2. Also, the analytical figures of merit (refer to glossary), such as, limit
of detection (LoD) and linear dynamic range (LDR) were described for all
elements in section 2.5.3. Furthermore, the validation of the instrument was
undertaken by the analysis of water certified reference materials (CRMs) and the
validation of the method through the analysis of plant certified reference
materials. These measurements were shown to be accurate for the purpose of
providing quality data (refer to section 2.5.4). Finally, the statistical analysis plan
used to evaluate the data presented in this study was summarised in section 2.6
and the normality test was outlined in section 2.6.1. Furthermore, the significance
tests applied in this study was presented in section 2.6.2, along with the
correlation analysis in section 2.6.3.
57
Chapter 3. Yerba Mate
58
3.1. Introduction
Yerba mate (Ilex paraguariensis), a native plant from the southern region
of Latin America, has been gaining attention due to its high levels of caffeine and
antioxidants. This chapter provides an overview of the literature available on
yerba mate (refer to section 3.2) and the relationship of its consumption with
human health (section 3.3). The aim and objectives of this chapter are outlined in
section 3.4. In this research, an evaluation of the non-commercial yerba mate
was carried–out with samples from Barão de Cotegipe, located in the Rio Grande
do Sul State, Brazil, according to the methodology described in sections 3.5.1
and 3.5.2. The elemental results are presented in sections 3.5.3, 3.5.4 and 3.5.5.
Furthermore, an investigation of the chemical composition of the commercial
yerba mate samples obtained from outlets in Brazil and Argentina is described in
section 3.6.1. All samples were analysed for the polyphenolic and elemental
content using the methodologies outlined in section 3.6.2 and the results are
presented in sections 3.6.3, 3.6.4 and 3.5.5. Also, a link to the dietary intake of
these chemicals was evaluated through the preparation of traditional infusions,
as outlined in section 3.6.6. Finally, a summary of the data is presented in
section 3.7.
3.2. General Introduction to Yerba Mate
Yerba mate (Ilex paraguariensis) is a native tree of South America that
has been consumed by indigenous peoples since pre-Colombian times and
adopted by the colonisers of South America (Bracesco et al., 2011). Nowadays, it
is consumed as a hot or cold infusion and is one of the most popular beverages
in South America, with an estimated 1 million people consuming around 1-2 L per
day of mate infusion (de Morais et al., 2009). Traditionally, yerba mate is
consumed using a gourd and a metal straw called a bombilla (Bracesco et al.,
2011). Although, it is gaining popularity in the USA, Europe, Germany and the
59
Middle East where the infusion is prepared using a regular tea bag (Heck and De
Mejia, 2007).
3.2.1. Natural occurrence
Yerba mate is native to the subtropical zone of three countries of South
America: Brazil, Argentina and Paraguay, as shown in Figure 3.1, and is naturally
grown within native forests. Even though the producers have successfully
cultivated yerba mate in plantations, there have been several unsuccessful
attempts at growing this plant in other parts of the World (Ilany et al., 2010).
Although the tree is usually harvest as a bush, it can reach up to 18 meters and
the leaves (evergreen and harvest for the yerba mate products) are up to 15 cm
long (Bracesco et al., 2011). The tree usually flowers from October to November
providing a small, greenish-white flower. Trees produce fruits from March to June
producing a dark red drupe from 4–6 millimeters (Heck and De Mejia, 2007).
Figure 3.1: Natural occurrence of yerba mate in South America. Adapted from
Maccari Junior (2005).
60
3.2.2. Production (plantation to processing plant) and products
The Worldwide production of yerba mate in 2017 was reported to be as
follows: Argentina – 689,196 tons (INYM, 2017); Brazil – 354,398 tons (IBGE,
2017); and Paraguay - 91,640 tons of green yerba mate (Aguinaga, 2017). Yerba
mate is produced in its native or natural state, where the tree grows between
forests, or in cultivated farms. One of the main differences between Argentinian
and Brazilian production is that in the former, all of the yerba is grown in the sun
(because it is cultivated), whilst in the latter, native trees are shaded within local
forests. These different types of cultivation may have an impact on the chemicals
present in yerba mate, especially polyphenols (Donnelly, 2015).
Although production has expanded in recent years, the Brazilian yerba
mate market is still very much restricted to the southern regions of the country.
The main production is undertaken by family units who cultivate the trees in local
forests (Balzon et al., 2004, Vasconcellos, 2012). According to data from PAM -
IBGE (Brazilian Institute of Geography and Statistics) the main region of yerba
mate plantation production is Rio Grande do Sul (IBGE, 2017). In contrast, forest
or native production occurs in Paraná, Santa Catarina and Mato Grosso do Sul
States on a smaller scale, as shown in Figure 3.2.
As the origin of the raw yerba mate material (cultivated or native)
influences the flavour of the final product, the production of yerba mate in Paraná
(mainly native) is more valuable than for other states of Brazil due to the low level
of bitterness - according to consumers (Maccari Junior, 2005). Yerba mate, an
important Brazilian export, is sent mainly to Uruguay, who do not have their own
yerba mate plantations. In 2017, revenue from the commercialisation of yerba
mate was estimated at 107 million US dollars (IBGE, 2017).
61
Figure 3.2: Main state producers of yerba mate in Brazil with their contribution
(%) to Brazilian production. Adapted from IBGE (2017).
There are several stages of yerba mate production, as shown in Figure
3.3. First, the leaves are harvested, which can take place at any time during the
year, however, to minimise damage to the plant, most producers harvest in Brazil
from May to September and in Argentina from April to September. During this
period about 65% of the total amount of yerba mate annual production is
harvested (Valduga et al., 2003). Since one of the most desirable characteristics
of yerba mate for Brazilian consumers is an intense green colour and as the
colour changes during storage, the Brazilian market requires constant production
throughout the year in order to maintain a constant supply of the fresh product
colour (Duarte, 2000).
After harvesting, when the leaves are picked by hand or mechanically, the
yerba mate is transported to the processing facility where it is classified and
stored until the sapeco stage, as shown in Figure 3.4. In this process, the leaves
are rapidly exposed to a flame that ruptures the leaf membranes and denatures
the enzymes thereby preventing further oxidation (Donnelly, 2015). Then, the
yerba is either triturated and dried in continuous rotary metal cylinders
(conventional method) or first dried at low temperatures using hot air and then
triturated (premium method). This process reduces the moisture content of the
leaves to 5 or 6% (Ward and Marcilla, 2003). The dried leaves, now called yerba
62
mate cancheada, are milled and packed for distribution to the market place in a
variety of loose or teabag packs. The teabag packaging found in the Brazilian
market contain the roasted yerba which is a further stage added to the yerba
mate cancheada stage, as shown in Figure 3.4. The differences in temperature,
time and the materials used during the production stages may all have an
influence on the quality and chemical compounds of the final product (Maccari
Junior, 2005).
Figure 3.3: Scheme of production of yerba mate. Adapted from Maccari Junior
(2005).
63
Figure 3.4: (A) Yerba mate tree; (B) Sapeco stage; (C) Yerba Mate cancheada
for the Brazilian and Argentine markets. Adapted from UFRS (2012).
There are various differences between Argentine and Brazilian production
including the different harvesting times. In the Argentinian product, the green
leaves are further aged for about 9 months in chambers, which leads to a change
in the colour of the final product. On the other hand, some of the Brazilian
commercial products are roasted. Moreover, the particle size of the final product
is also different, being around 5 mm for the Argentinian and 300 µm for the
Brazilian product, as show in Figure 3.5.
64
Figure 3.5: Argentinian (left) and Brazilian (right) green yerba mate commercial
samples.
3.2.3. Methods of consumption
Despite the great potential of using yerba mate in soft drinks, sweets,
cosmetics and medicines, mainly because of its antioxidant’s properties, the
dried leaves of the plant are mostly intended for a traditional type of infusion
consumed in South America. Infusions made from green yerba mate leaves are
widely consumed, primarily as chimarrão or mate (hot infusion) and tererê (cold
infusion) in Argentina, southern Brazil, Paraguay and Uruguay. Chimarrão is the
most popular form of yerba mate consumption in Brazil (as presented in Figure
3.6) followed by roasted leaf infusions sold in teabags, which contain only leaf
roasted material and are used to make hot and cold infusions (chá mate).
According to the Brazilian legislation, commercial packages of loose yerba mate
contain about 30% of twigs and 70% of leaves (Heinrichs and Malavolta, 2001).
65
Figure 3.6: Typical Brazilian Chimarrão (mate) consumption (Forma, 2016).
In chimarrão, the cup is made from a dried fruit from the calabash or bottle
gourd tree (Lagenaria siceraria). The “bomba” (Portuguese) or “bombilla”
(Spanish) is the metal straw, which has a filter at the lower end in order to
separate the infusion from the leaves.
The cup is usually filled with around 50 g of the loose green yerba mate
and 200 mL of water at 80 ºC; after consumption (seeping through the “bombilla”)
of the resulting infusion. The cup is then topped up with more hot water. This
cycle of infusion and consumption is repeated between 5 - 10 times, which
represents a unique and social form of consumption of the yerba mate. This is
very different from a regular infusion prepared with teabags, where approximately
3 g of green material (Argentina) and 1.8 g of roasted material (Brazil) are
brewed in a typical cup of 200 mL of water. This difference of consumption will be
evaluated in this study, namely, whether this influences the chemical intake
during the drinking of yerba mate, especially for trace elements and polyphenols
and what is the possible link to the daily dietary intake.
3.3. Health Effects of Yerba Mate Consumption
Yerba mate is regarded as offering various health benefits, it is well known
as a stimulant drink that eliminates fatigue and improves mental and physical
focus, mainly because of its high levels of caffeine (Bracesco et al., 2011). The
66
infusions also have significant levels of antioxidants, which are molecules that
can prevent the oxidation of biomolecules in biological systems (Bastos et al.,
2007).
Besides these important features, yerba mate is alleged to also help in
inflammatory and cardiovascular diseases (Schinella et al., 2005), promote
weight loss (Andersen and Fogh, 2001) and reduce sugar blood, cholesterol and
triglycerides levels (Filip and Ferraro, 2003).
However, negative effects on health have also been reported. Some
studies have indicated an alleged relation between the heavy consumption of
mate and cancer (Vassallo et al., 1985, Pintos et al., 1994, De Stefani et al.,
1996). The causal relationship between the consumption of yerba mate and
cancer has not been fully proven but it may be associated with the high
temperature of the water used for the infusion or other concurrent causes, such
as smoking and nutritional factors, instead of the yerba mate itself (Ramirez-
Mares et al., 2004).
3.3.1. Chemical composition of yerba mate
Some of the alleged health benefits of yerba mate could be explained on
the basis of its chemical composition; however, there is a lack of conclusive
studies. Among the organic compounds, xanthines (such as theobromine and
caffeine) have diuretic properties, can influence the relaxation of smooth muscle
and have been reported to cause myocardial stimulation (Filip et al., 2000,
Leborgne et al., 2002, Schinella et al., 2005).
One of the groups of antioxidants present in yerba mate is polyphenols
(refer to section 1.3), which are able to prevent oxidation of biomolecules (Colpo
et al., 2016). Antioxidant compounds present in yerba mate, such as
polyphenols, have been shown to inhibit lipid peroxidation, especially low-density
lipoprotein (LDL) oxidation (Gugliucci, 1996). In previous studies, the total
content of polyphenols in yerba mate was determined by the Folin-Ciocalteu
assay using gallic acid as a standard. Studies using this method have reported
67
levels of 51.3 - 72.9 mg gallic acid equivalents (GAE)/ 100 mL) in yerba mate
infusions which are similar to that found for green and black tea (Bravo et al.,
2007, Gorjanovic et al., 2012, Zielinski et al., 2014). On the other hand, the total
chlorogenic acid content, which is the main group of polyphenols present in
yerba mate, has been found to be higher than in tea (Camellia sinensis) and
similar to the content of filter coffee (Donnelly, 2015).
The principal polyphenol compounds present in yerba mate is the
chlorogenic acid group, including a range of mono-, di- and tri-acylated
compounds (Donnelly, 2015). The mono-chlorogenic acids found in yerba mate
include 1-, 3-, 4- and 5-caffeoylquinic acid as well 3-, 4- and 5-feruoylquinic acid
and p-coumaroylquinic acid (Jaiswal et al., 2010, Dugo et al., 2009, Bravo et al.,
2007). The predominant compounds present in yerba mate are 3-, 4- and 5-
caffeoylquinic acid and 3,5-, 4,5- and 3,4-caffeoylquinic acid with levels of 0.5 –
3% of the leaf material (Donnelly, 2015). Marques and Farah (2009) and Clifford
and Ramirez-Martinez (1990) also noted that the chlorogenic acid content of
roasted yerba mate (3%) decreases when compared to green yerba mate (9 –
10%).
Besides the polyphenol compounds, yerba mate also have xanthine
compounds, such as, caffeine and theobromine (Donnelly, 2015). A study using
the mass-to-ratio that simulates a traditional South American infusion method
found 1253 ± 72.5 μg/mL of caffeine and 53.1 ± 1.44 μg/mL of theobromine
(Murakami et al., 2013). Moreover, a regular infusion (European tea-based
method) presented 18.8 – 42.5 μg/mg of caffeine and 9.9 – 15.7 μg/mg of
theobromine (de Mejía et al., 2010).
Also found in yerba mate is high levels of aluminum, manganese, iron and
zinc in the leaves and infusions. A review of previous studies is presented in
Table 3.1. All of these studies were performed with different commercial samples
of yerba mate, consequently the elemental levels vary widely, which could be
influenced by different soil conditions, harvest periods, cultivation and processing
methods (Zeiner et al., 2015).
68
Table 3.1: Element content of yerba mate leaves for selected elements reported
in literature (weight basis not reported).
Author Year Concentration (mg/kg)
Mg Ca Mn Fe Cu Zn Barbosa
et al. 2015 2710 - 2730 5770 - 6800 129.0 - 232.6 47.8 – 54.1 7.2 – 8.2 24.9 – 56.6
Barbosa
et al. 2018 1600 - 1900 2900 - 3700 437 – 614 71 - 74 3.8 - 4.6 40 - 73
Bastos et
al. 2014 2700 - 4400 1800 - 2700 137.2 – 325.4 12.1 - 35.4 6.2 - 7.2 20.1 – 30.8
Donnelly
2015 - - 378.3 – 879.9 28.2 168.1 4.31 - 12.12 14.0 – 125.8
Giulian et
al. 2007 5025 ± 186 6785 ± 249 1315 ± 113 254 ± 27 14 ± 2 72 ± 5
Heinrichs
et al. 2001 4300 - 5200 6000 - 6600 665 – 1050 103 - 286 7.6 - 10.7 38 - 43
Jacques
et al. 2007 7447 -7977 3453 - 8520 1168 – 3542 54 - 120 6.20 -11.47 33 - 98
Malik et
al. 2008 3520 - 8115 9562 - 12720 309 – 1114 83.4 – 88.1 7.98 - 12.7 26.1 – 31.8
Magri et
al. 2019 - - 533 – 4865 58 - 173 8.0 - 20.0 13 - 181
Milani et
al. 2019 - - 1115 - 1811 103 - 437 9.5 - 12.2 21 - 26
Pozebon
et al. 2015 4587 - 5574 6947 - 7659 730 – 1368 154 - 226 31.9 - 38.3 44.2 – 79.4
Rossa et
al. 2015 80 - 9550 120 - 5450 236 - 1440 8.0 - 130.0 1.0 - 44.0 34.0 - 146.0
Marcelo et
al. 2014 4591 ± 842 6825 ± 842 1078 ± 377 205 ± 89.1 11.9 ± 2.06 63.6 ± 25.0
3.4. Aim and Objectives
The overall aim of this study was to investigate the chemical analysis of
different samples of yerba mate. Moreover, to analyse the elemental levels of
non-commercial samples in order to evaluate the effect of different plantation
methods. Finally, to perform a complete analysis of the commercial yerba mate
samples from the two main producers in the World, Brazil and Argentina. This
enabled an evaluation of the impact of different production methods, pre-
69
treatment of the commercial products and to assess the effect of the mode of
consumption on the uptake of polyphenols and elements.
The objectives were to:
(i) provide a literature review of reported elemental and phenolic levels of
yerba mate;
(ii) investigate the elemental profile of non-commercial and commercial
yerba mate samples;
(iii) assess the impact of different varieties; plantation processing
methods; and ages of tree and leaves on the total elemental analysis;
(iv) determine the total polyphenol and chlorogenic acids of commercial
samples of yerba mate from Brazil and Argentina;
(v) evaluate the chemical analysis of the yerba mate (loose and teabags)
material and infusion methods (regular and bombilla/traditional and
iced); and
(vi) investigate the impact of the consumption of yerba mate in terms of
the polyphenol and elemental dietary intake.
3.5. Non-Commercial Studies on Yerba Mate
This section provides an evaluation of the elemental content of non-
commercial yerba mate leaves collected in April 2017 from the Barão de
Cotegipe plantation. The description of the samples and methods used are
outlined in sections 3.5.1 and 3.5.2. An investigation of the production types
(traditional and natural); use of fertilisers (NPK and organic); age of leaves (new
and old) and height of the leaves in a tree (bottom, middle and top) is presented
in sections 3.5.3 and 3.5.4. Finally, an evaluation of the commercial processing
of the material is proposed in section 3.5.5.
70
3.5.1. Description of the samples
Samples were collected during a field-trip in April 2017 to Barão de
Cotegipe plantation, located in the Rio Grande do Sul State, Brazil. The company
is one of the biggest producers and exporters of yerba mate in Brazil. They
agreed to collaborate on this project to evaluate their different plantations and
processing methods in regard to the elemental composition of the samples. In
terms of the cultivation of the yerba mate, they have 3 different sites: (i)
traditional plantations: cultivated yerba mate planted between trees (Hovenia
dulcis and Araucaria angustifolia) and treated with NPK fertilisers; (ii) traditional
plantations: cultivated yerba mate planted under the sun and treated as an
organic form of farming; and (iii) natural forest: yerba mate trees grown between
and beneath natural forests, without any fertiliser treatments. A list of all samples
is presented in Appendix 3.1.
The yerba mate harvesting is usually performed by manually cutting the
branches of the tree to collect and process the material disregarding the age of
the leaves. An evaluation of the elemental composition of different yerba mate
trees in relation to the age of the leaves (new and old) was undertaken in this
study. Therefore, the small leaves at the end of each branch were collected as
new material and the mature larger leaves collected along the branch were
classified as old material. All of the leaves were collected at a medium height of
the tree (1.5 m).
The yerba mate trees from the Barão de Cotegipe plantation were pruned
to keep the same height (2.5 meters as average). Although, during the harvesting
period the leaves are collected from every height of the tree. An evaluation of the
elemental composition in relation to height where the leaves were collected was
proposed during the field-trip. Consequently, the new leaves from a tree grown
on the organic plantation were collected at the following heights: 0.5 – bottom;
1.5 - middle and 2.5 m- top.
Finally, an evaluation of the processing of the yerba mate leaves was
investigated. Samples were collected at each stage of the commercial
71
processing plant (refer to section 3.2.2): (i) harvested leaves: when the leaves
arrive at the processing plant; (ii) Sapeco: the leaves were collected after the
sapeco stage, where the material is exposed to an open fire for a short period of
time (refer to Figure 3.4); and (iii) dried: sample collected after the drying process
where the leaves were dried until the moisture content was reduced to 5-6%.
Due to the constant processing of yerba mate in the plant; the samples collected
do not refer to the same type of yerba mate production (different origin or
fertiliser treatment).
3.5.2. Materials and method
The determination of the total elemental composition of the yerba samples
was performed as described in section 2.3. The samples were fully digested at
500°C for 12 hrs using a muffle furnace and analysed by inductively coupled
plasma mass spectrometry or ICP-MS (refer to section 2.3).
3.5.3. Production by traditional plantations
This section will report a pilot investigation into the elemental levels of
yerba mate leaves cultivated in the traditional plantations of Barão de Cotegipe,
southern Brazil, with a view to evaluating the impact of different methods of
growing plants (with and without fertiliser; covered with native trees), harvest of
the leaves (height within a tree and if the leaves are new or old). The main
reason behind this investigation was two-fold, most reported studies focus only
on the elemental values of commercial products (refer to section 3.3.1), and to
advise the producers (Barão de Cotegipe) of the potential methods of cultivation
or harvest that may enhance the elemental and/or nutritional quality of the yerba
mate products. An evaluation of the difference between standard deviations
between the sets of data reported in this study was performed using a F-test
(refer to section 2.6.2) and is presented in Appendices 3.25 to 3.27. In general,
there is no statistically significant difference between the standard deviations at
p<0.05, that is the null hypothesis is retined as Fcal<Fcrit (Miller et al., 2018).
72
(i) Effect of cultivated yerba mate leaf age on elemental levels
The total elemental composition of the non-commercial yerba mate
samples cultivated in traditional plantations was evaluated following the method
proposed in section 2.3. The results of important trace elements (i.e. those
relating to human health) are presented in Table 3.2 grouped by the use of
fertilisers or not (organic) and by the age of the leaves (new and old), as
described in section 3.5.1. Magnesium, Ca, Mn, Fe, Cu and Zn were chosen
because the concentration in yerba mate products could have a significant
impact on the nutritional intake of these elements. Moreover, two types of
plantation were also investigated, namely one that uses NPK fertilisers as the
standard method of supporting the growth of yerba mate trees, and the other that
uses no commercial fertilsers (or organic production).
A preliminary inspection of the data (Table 2.3) shows that for both
traditional methods of yerba mate production, there is a wide variation in the
elemental concentrations of both the new and old leaves (refer to section 3.5.1
for the description on classifying the age of the material). In terms of the use of
fertiliser, Mg, Ca, Mn, Fe and Zn all have higher mean levels in the old leaves,
with the exception being for Cu (new > old). Moreover, the elemental ranges do
not overlap for Mg, Ca and Cu, whilst the other elements show some degree of
overlap in elemental levels for the two ages of leaves. As a contrast, organic
production has lower mean levels of Mn in the old leaves. Interestingly, all
elements now have an overlap in the elemental levels of the two leaf ages.
73
Table 3.2: Total elemental levels are reported as the mean and range (min –
max) of yerba mate leaves (based on age – new and old) for non-
commercial samples (mg/kg, dry weight) collected from traditional
plantations cultivated either using NPK fertilisers or non-chemical
(organic). The digested samples were analysed by ICP-MS (refer to
section 2.3).
Fertiliser Organic New leaves Old leaves New leaves Old leaves
n 4 4 9 8
Mg 3172
(2608 – 3724)
7015
(5710 – 8474)
4000
(2779 – 5914)
5180
(2356 – 8218)
Ca 2989
(2257 – 4160)
8631
(6511 – 10298)
4783
(2588 – 10534)
6671
(2536 – 10904)
Mn 623
(425 – 924)
949
(560 – 1394)
1195
(757 – 3395)
712
(349 – 1189)
Fe 47.09
(36.87 – 55.65)
66.13
(49.17 – 76.22)
34.28
(27.83 – 41.39)
41.16
(28.22 – 50.73)
Cu 28.67
(25.08 – 37.19)
14.67
(13.45 – 16.35)
15.10
(8.87 – 26.90)
9.90
(5.53 – 21.29)
Zn 79.50
(67.71 – 104.95)
166.14
(82.49 – 238.50)
90.15
(30.17 – 252.96)
107.47
(15.74 – 251.73)
n is the number of samples.
Statistical analysis of the data in Table 3.2 was undertaken using a two-
tailed Student t-test (Miller et al., 2018), based on the null hypothesis, that there
is no statistically significant difference in the elemental levels of the leaves (new
and old) collected from the same tree and grown in the two different yerba mate
plantations. Table 3.3 reports the statistical data.
74
Table 3.3: Statistical analysis using a two-tailed t-test (Miller et al., 2018) to
evaluate the relationship between the elemental levels of yerba mate
leaves (based on age – new and old) for non-commercial samples
collected from traditional plantations cultivated either using NPK
fertilisers or non-chemical (organic).
Fertiliser Organic n 8 12
tcrit 2.36 2.18
tcalc p Direction of
significance tcalc p
Direction of
significance
Mg 5.86 0.0011** new<old 1.76 0.1035 ns
Ca 6.25 0.0008*** new<old 2.49 0.0285* new<old
Mn 1.49 0.1863 ns 2.59 0.0236* new<old
Fe 2.56 0.0458* new<old 1.14 0.1858 ns
Cu 4.80 0.0030** new>old 1.91 0.0801 ns
Zn 2.18 0.0722 ns 0.36 0.7237 ns
n is the number of samples; tcalc is the calculated value (refer to Equation 2.6, section 2.6.2): tcrit is the critical value obtained for n-2 degrees of freedom, p is the level of probability; ns is that there is no statistically significant difference at p>0.05; * statistically significant at probability p<0.05; ** highly significant at probability p<0.01; and *** very highly significant p<0.001.
Yerba mate leaves grown in traditional plantations, cultivated with the use
of fertilisers, show a significant difference (p<0.05) in the levels of Mg, Ca and Fe
(new < old leaves) and Cu (new > old), confirming the initial inspection of the
data presented above. Moreover, the observed overlap in the elemental ranges
for Mn and Zn are associated with no significant difference (p>0.05). If the leaf
samples were collected from a traditional plantation, cultivated without any
treatment (organic), there was also a significant difference between the age of
the leaves from the same tree, namely, Ca and Mn only (new < old).
Although there is a limited number of leaf samples involved in this
investigation on the effect of leaf age (collected from the same tree), it shows that
for most of the elements (including those reported in Appendix 3.2 to 3.4 for V, Cr
and Se) the general trend is for the element to be at higher levels in the old
leaves.
75
The uptake, distribution and compartmentalisation and/or separation of
particular elements, especially in their soluble ionic form, are required by the
plant for optimal function (Leigh, 1997). Plant organs may have very different
concentrations of elements within tissues which may relate to the xylem vs.
phloem transport mechanisms, sites of complexation (for many heavy metals or
charge-dense ions), or tissue or cell-specific transport (Conn and Gilliham, 2010,
Marschner, 2011, Tester and Leigh, 2001). Therefore, in the yerba mate plant
(Illex paraguariensis), the results of this study confirm that for most of the
elements there is uptake and distribution to the leaves, with there being a
variation in the elemental concentrations for both new and old leaves (refer to
Table 3.2). This could be due to a matrix effect, but because the analysed
samples are from the same plant (different tissues), any possible matrix effects
would be minimal. In terms of explaining why many of these elements are found
at high levels in the older levels (with the exception being copper) various
authors have reported the accumulation of elements in older leaves of different
plants. Immobile or less mobile ions tend to accumulate in the older leaves
simply because the largest total amount of transpiration occurs through these
leaves (Tinker, 1981). There may be a tendency for elements to be deposited at
the leaf margins where transpiration is maximal (Tinker, 1981). De Maria and
Rivelli (2013) also commented on the largest accumulation of Cd, Zn and Cu in
plants being found in the leaves, mainly in the old ones, especially for mature
trees. Kabata-Pendias (2010) commented that zinc is likely to be concentrated in
mature leaves and if there is higher Zn concentrations in the soil, translocation
from the roots to the plant tops is enhanced. The reason why Cu is found at
higher levels in the new leaves (and also Mn for organic cultivation) is not clear,
although there are conflicting reports on the mobility of Cu in plants. Tinker
(1981) stated that Cu is fairly immobile in plants, tending to remain in the older
leaves. However, others have said that many heavy metals, including Cd, have
differential accumulation within the root system as opposed to the shoots (Puig
and Peñarrubia, 2009, Verbruggen et al., 2009). Therefore, the findings for Cu
76
and Mn at this time are not easy to justify. The same trend was found for the
reported levels of Na, K, Co, Ni, Cd and Pb (refer to Appendices 3.2 to 3.4).
(ii) Effect of yerba mate cultivation (with or without the use of
fertilsers) on the elemental levels of leaves (new or old)
The next question to be addressed is to know whether the use of
traditional cultivation methods, with or without (organic) the use of chemical
fertilisers, has a direct impact on the elemental levels of the leaves (for both new
and old). A statistical analysis was undertaken of the data in Table 3.2, using a
two-tailed t-test (Miller et al., 2018) with the null hypothesis being that there is no
significant difference in the elemental levels of new or old leaves grown in
plantations with/without the use of NPK fertiliser/organic, respectively (Table 3.4).
An initial evaluation of the data in Table 3.2 shows some interesting trends
in relation to the cultivation of yerba mate trees, with and without the use of
added fertiliser. At the time of sample collection, no information was available
from the producers about the type of NPK fertiliser or organic additives used on
the plantations. The statistical analysis of this data using a two-tailed Student t-
test is reported in Table 3.4. The new leaves grown on trees from the organic (no
chemical addition) plantation have higher levels of Mg, Ca, Mn (significant
p<0.05), and Zn (with same trend being observed for Co, Se and Cd, as reported
in Appendices 3.2 to 3.4). Only Fe (p<0.01) and Cu (p<0.05) have higher levels
(which are both statistically significant) in the new leaves from the trees grown
with the addition of fertilsers (with the same trend for Na, V, Ni, As and Pb, as
reported in Appendices 3.2 to 3.4). Conversely, for the older leaves from both
plantations, all of the elements reported in Table 3.2 are higher in the fertilser-
addition plantation, with Fe being statistically significant (fert > org; p<0.01). The
same trend was found for V, Cd and Pb; with the exception being for Na, K, Co,
Ni and Se, as reported in Appendices 3.2 to 3.4.
77
Table 3.4: Statistical analysis using a two-tailed t-test (Miller et al., 2018) to
evaluate the relationship between the elemental levels of yerba mate
leaves based on the use or non-use (organic) of NPK fertilsers during
traditional cultivation.
New leaves Old leaves n 11 11
tcrit 2.26 2.26
tcalc p Direction of
significance tcalc p
Direction of
significance
Mg 0.70 0.5030 ns 1.39 0.1992 ns
Ca 1.08 0.3076 ns 1.03 0.3313 ns
Mn 2.50 0.0374* fert<org 1.85 0.0975 ns
Fe 3.31 0.0092** fert>org 4.54 0.0014** fert>org
Cu 3.20 0.0109* fert>org 1.65 0.1330 ns
Zn 0.50 0.6291 ns 1.01 0.3405 ns
fert – fertilsers addition; org – organic or no chemical addition; n is the number of samples; tcalc is the calculated value (refer to Equation 2.6, section 2.6.2): tcrit is the critical value obtained for n-2 degrees of freedom, p is the level of probability; ns is that there is no statistically significant difference at p>0.05; * statistically significant at probability p<0.05; ** highly significant at probability p<0.01.
Most NPK fertilisers added to soils increase the levels of N, P and K so as
to stimulate plant growth and hopefully the uptake of these major elements by the
roots and upper plant tissues (Adekiya and Agbede, 2009). Many NPK fertilsers
can contain trace levels of other minerals and minor/trace elements (Ca, Mg, Na,
Fe, Mn, B, Cu and Zn) (Mursito et al., Mursito et al., 2017). The addition of these
other elements is to stimulate plant growth and enhance the bioavailability of
elements to the aerial tissues of the plant. This will also depend on soil chemistry
(pH, organic matter and water content, and the presence of other elements) and
any microbial activity in the soil or soil-root interface (including mycchoriza fungi
and nitrogen-fixation bacteria in the root nodules) (Kabata-Pendias, 2010).
Moreover, the addition of some elements in fertilisers (i.e. Fe) may have an effect
on the nutrition of other elements in plants, including Mo, Cu, Mn and P (Han et
al., 2016). Some studies have reported that the addition of NPK fertilisers
78
decreases soil pH and thereby also influences the exchangeable levels of Ca
and Mg in the plant. Kabata-Pendias (2010) also stated that this may solubilise
Fe leading to large amounts in the plant. This can result in elemental deficiencies
in the younger leaves (Liu et al., 2010). This pH change may be explained by the
leaching of basic cations, such as Ca and Mg (and maybe other trace elements),
from the soil. Moreover, the long-term use of NPK fertilisers has been shown to
result in the deficiency of many essential nutrients in the soil (Bailey et al., 2004)
(Liu et al., 2010) reported that the addition of NPK fertiliser to yellow poplar
(Liriodendron tulipifera Lin.) increased the levels of available nutrients in the soil,
thereby increasing the biomass of the plant, but observed no changes in the
plant nutrient concentrations of the upper plant tissues. In this research, it
appears that for yerba mate plants the addition of NPK fertiliser is having no
effect on the uptake of any essential trace elements by the newer growing
leaves, with the exception of Fe and Cu. This may be because the NPK is
stimulating root growth and the mobilisation of other trace elements to the stems
or shoots (Loneragan, 1981). Moreover, if the NPK fertiliser has high levels of
soluble Fe (which will be influenced by a lowering of the soil pH), this not only
increases the amount available to the plant, but it seems to lead to a significant
increase in the Fe levels of the upper tissues, including the newer leaves.
Interestingly, copper tends to be immobile in plants (Kabata-Pendias, 2010), but
if you increase the supply of copper, this leads to an increase in mobility within
the plant, primarily to the shoots, although for yerba mate, it seems there is also
an uptake by the newer leaves. Finally, as stated above, many authors have
reported that immobile or less mobile ions tend to accumulate in the older leaves
simply because the largest total amount of transpiration occurs through these
leaves (Kabata-Pendias, 2010, Tinker, 1981). Clearly, the addition of NPK and
other minerals or elements in the fertiliser has resulted in accumulation within the
older yerba mate leaves. Since there is no information about the organic farming
method being used (i.e. the addition of animal or plant manure), it is not possible
to state whether there are any nutrients or elements being added to the trees in
the organic plantation.
79
(iii) Effect of collecting yerba mate leaves from a tree at different
heights on the elemental levels of new leaves
The harvesting of yerba mate leaves from trees grown in traditional
plantations (organic) involves collecting any leaf material from the tree,
irrespective of height or age. Therefore, an important question is whether the
elemental levels of the collected leaves (only new) are different if collected at
different heights within the tree (namely, at 0.5 - bottom, 1.5 - middle and 2.5 m-
top). As a result of time constraints only one tree was investigated, therefore only
replicate samples were collected at each height and the results in Table 3.5 will
not be subjected to statistical analysis.
Interestingly, the levels of Mg, Ca and Zn are highest in the new leaves
collected from the bottom of the tree (0.5 m height). Iron and Mn show a degree
of translocation of the element to the upper parts (2.5 m), whilst Cu is slightly
elevated in the leaves from the middle of the tree (1.5 m). The other elements
reported in Appendices 3.2 to 3.4 demonstrated the following trend in terms of
tissue accumulation: (i) bottom for Na, Co, and Cd; (ii) middle for Ni and Se and
(iii) top for K, V, Cr and Pb.
80
Table 3.5: Total elemental levels (mean ± standard deviation; mg/kg, dry weight)
of yerba mate leaves collected at different heights from the same tree
grown in a traditional plantation (cultivated as organic). The digested
samples analyses were analysed using ICP-MS (refer to section 2.3).
The number of samples, n = 2 replicates.
Bottom (0.5 m)
Middle (1.5 m)
Top (2.5 m)
Mg 5829 ± 1 3940 ± 30 4054 ± 24
Ca 11383 ± 48 5469 ± 181 5857 ± 12
Mn 1070 ± 4 1009 ± 82 1229 ± 21
Fe 39.10 ± 5.45 32.64 ± 5.05 51.33 ± 1.92
Cu 5.41 ± 0.40 7.68 ± 0.28 5.21 ± 0.17
Zn 81.79 ± 0.59 64.08 ± 1.09 65.29 ± 1.28
An interpretation of the above findings confirms, as reported by many
authors, that the mobilisation and accumulation of specific trace elements in plant
tissues varies from plant species to species, soil type and chemistry, and the
concentration of the elements and other chemicals (including organic matter) in
soils and plant organs. Unfortunately, no soil samples were collected as part of
this study. This makes it difficult to know what effect the soil may have in the
organic plantation on the uptake by roots and subsequent mobilisation of trace
elements throughout the tissues of the yerba mate trees. Moreover, only new
growth leaves were studied, and the accumulation of specific trace elements in
such leaves is dependent on the morphology and surface absorption area of the
leaves (Bu-Olayan and Thomas, 2009, Baker and Brooks, 1989). Olowoyo et al.
(2012) evaluated the uptake of trace elements in two different plant species in
South Africa and found a wide variation in the elemental levels of the roots,
stems and leaves of the plants. Furthermore, elements may be adsorbed or
occluded by the presence of carbonates, organic matter or other minerals (Oliva
and Espinosa, 2007). Therefore, the raised levels of Fe, Mn, K, V, Cr and Pb (top
2.5 m) and Cu, Ni and Se (middle 1.5 m) found in the yerba mate trees may be
81
related to many factors which at this time are not known in terms of the soils (pH,
organic content, chemistry of elements and other species), or factors influencing
the mobility for these particular elements in trees grown under organic conditions.
3.5.4. Production by natural forest plantations
The elemental composition of the non-commercial yerba mate samples
cultivated between natural forests was evaluated following the method proposed
in section 3.5.2. Table 3.6 presents the results of selected trace elements
(relating to human health) grouped by the age of the leaves (new and old), as
described in section 3.5.1.
The data in Table 3.6 confirms the findings reported for the elemental
levels of new and old leaves taken from yerba mate trees grown in NPK fertilser-
added and organic plantations (Table 3.2). In general, older leaves have higher
concentrations of Mg, Ca, Mn, Fe and Zn, with the exception being for Cu (new >
old). Additionally, Na, Ni and Pb presented the same trend, where the elemental
concentrations were higher in the old leaves, as reported in Appendices 3.2 to
3.4. The remaining elements (V, Cr, Co, As, Se, Mo and Cd) had similar levels
for both new and old leaves. Also, the older leaves tend to have higher elemental
levels for the trees grown in the traditional cultivated (organic) plantation (with Zn,
Na, K, V, Cd and Pb showing the opposite trend, as shown in Appendices 3.2 to
3.4). Moreover, for the new leaves, there is a similar pattern with Mg, Ca, Fe, Cu,
K, Co and Ni (traditional > native forest) and Mn, Zn, Na, V and Se (traditional <
native forest), as reported in Table 3.6 and Appendices 3.2.
82
Table 3.6: Total elemental levels are reported as the mean and range (min –
max) of yerba mate leaves (based on age – new and old) for non-
commercial samples (mg/kg, dry weight) collected from traditional
cultivated organic plantations and grown between and beneath trees
of a native forest. The digested samples were analysed by ICP-MS
(refer to section 2.3).
Traditional Native forests New leaves Old leaves New leaves Old leaves
n 9 8 3 3
Mg 4546
(2873 – 7846)
4928
(3782 – 6046)
4000
(2779 – 5914)
5180
(2356 – 8218)
Ca 5921
(3277 – 11139)
7005
(3867 – 8635)
4783
(2588 – 10534)
6671
(2536 – 10904)
Mn 1076
(982 – 1159)
1064
(805 – 1365)
1195
(757 – 3395)
712
(349 – 1189)
Fe 92.27
(91.15 – 94.07)
104.24
(69.63 – 153.99)
34.28
(27.83 – 41.39)
41.16
(28.22 – 50.73)
Cu 18.10
(9.35 – 22.87)
14.56
(11.62 – 20.18)
15.10
(8.87 – 26.90)
9.90
(5.53 – 21.29)
Zn 58.49
(32.83 – 75.71)
53.61
(39.57 – 71.86)
90.15
(30.17 – 252.96)
107.47
(15.74 – 251.73)
n is the number of samples. Table 3.7 lists the two-tailed Student t-test (Miller et al., 2018) data for the
above values in Table 3.6 and confirms the higher elemental levels for plants
grown in the traditional organic plantations, especially for Ca and Fe levels of
new leaves, with p<0.05 and p<0.001, respectively. Furthermore, the Mn and Fe
levels of old leaves were also statistically higher for plants grown in the traditional
plantations (p<0.01). Appendices 3.2 to 3.4 provides the data for the other trace
element levels of new and old leaves for yerba mate trees grown in traditionally
cultivated organic and native forest plantations.
This pilot study was undertaken at the producers request to evaluate
whether growing yerba mate trees within a native forest (between and beneath
larger trees) would influence the elemental quality of the yerba products. The
83
hypothesis was that such trees would have different soil chemistry (influenced by
the roots and leaf litter from the native trees), tree competition for nutrients from
the soil and the impact of reduced sunlight (and thereby photosynthetic activity).
Copper, magnesium, potassium, iron, manganese, molybdenum and zinc are
necessary for plant health and are associated with enzyme systems and
chlorophyll production relating to photosynthesis (Tränkner et al., 2018, Raven et
al., 1999). The role of many of these elements, especially Mg and K, also are
part of light-dependent photosynthetic processes (Tränkner et al., 2018). Clearly,
the yerba mate trees grown under a canopy of native forest is influenced not only
by reduced sunlight, but by nutrient and water competition with the larger trees.
The elemental leaves, in general, are found to be higher in both the new and
older leaves of the traditionally cultivated organic trees.
Table 3.7: Statistical analysis using a two-tailed t-test (Miller et al., 2018) to
evaluate the relationship between the elemental levels of yerba mate
leaves (new and old) grown in traditional organic or native forest
plantations (refer to Table 3.6).
New leaves Old leaves n 10 10
tcrit 2.31 2.31
tcalc p Direction of
significance tcalc p
Direction of
significance
Mg 1.50 0.1712 ns 0.70 0.5023 ns
Ca 2.54 0.0346* trad>nat 0.77 0.4614 ns
Mn 2.13 0.0661 ns 4.43 0.0022** trad>nat
Fe 23.18 <0.0001*** trad>nat 4.16 0.0032** trad>nat
Cu 0.37 0.7205 ns 2.14 0.0648 ns
Zn 1.00 0.3453 ns 1.11 0.2978 ns
trad – traditional organic plantation; nat – native forest plantation; n is the number of samples; tcalc is the calculated value (refer to Equation 2.6, section 2.6.2): tcrit is the critical value obtained for n-2 degrees of freedom, p is the level of probability; ns is that there is no statistically significant difference at p>0.05; * statistically significant, p<0.05; ** highly significant, p<0.01, and *** very highly significant p<0.001.
84
3.5.5. Commercial processing plant
An evaluation of the elemental content of yerba mate leaves during the
commercial processing of the material was proposed, as described in section
3.5.1. The results are presented in Table 3.8. The samples were collected from
different stages of the processing plant on the day of the field-trip to the
company. The harvested samples are a combination of new and old leaves from
cultivated trees within a native forest (organic) from another producer (Santa
Catarina State). Therefore, this study is a simulation of what actually happens
during the harvesting of the yerba mate tree where all the leaves are collected
irrespective of the age of the samples and the mixture used to produce the
commercial product. As a result of constraints in the processing plant which has
different materials being processed at the same time, only one sample was taken
at each phase of the process for chemical investigation. Therefore, the results
will not be subjected to statistical analysis due to the limited number of samples.
A primary evaluation of the data presented in Table 3.8 shows a trend in
relation to the pre-process (or post-harvest) samples and after the sapeco
(subjected to an open fire flame - refer to section 3.2.2). In general, Mg, Ca, Mn,
Fe and Zn all had higher elemental levels after the sapeco stage of the process.
The only exception is copper that had very similar levels as the pre-processing
and post-drying stages. Furthermore, the data confirms the findings reported for
the elemental levels of traditional and native plantations, where leaves from the
traditional cultivated plantations had higher elemental levels than for those
collected from the yerba mate trees grown between native forests (Table 3.6).
Moreover, the post-drying samples (the last stage of the processing plant
involves the yerba mate material being dried until a moisture content of 5 to 6 %)
had lower levels of Mg, Ca, Fe and Zn when compared with the sapeco yerba
mate sample.
During the sapeco stage, the yerba mate leaves are thrown into an open
fire for a short period of time (seconds). This leads to the rupture of the leaf
membranes and the denaturation of the enzymes, preventing oxidation
85
(Donnelly, 2015). Several studies have also reported that post the sapeco stage
there was an increase in the levels of bioactive compounds (Isolabella et al.,
2010, Esmelindro et al., 2002, Schmalko and Alzamora, 2001). One study stated
that the extraction of caffeine and caffeoylquinic acids was more effective in dried
processed leaves due to the cell disruption and mechanical impact during the
processing stages (Bastos et al., 2006). Giulian et al. (2009) also demonstrated a
similar trend for the elemental analysis of mate samples (increasing levels as a
result of the sapeco stage, and then decreasing post the drying stage). The
mechanism of the sapeco is related to a quick loss of mass at high temperatures,
where it is expected that most of the water and ‘light’ (lower atomic weight)
elements are released from the leaves, which would lead to a reduction of the
leaf mass and consequently an increase in the concentration of the remaining
elements (Giulian et al., 2009). During the drying process (which involves the
material being transported along a moving belt) the time involved is ~ 10 times
longer than the sapeco stage, whilst the temperature is five times less. This
changes the dynamics of the elemental balance in the yerba mate material,
which could lead to different outcomes (Giulian et al., 2009). The other elements
reported in Appendices 3.2 to 3.4 (Na, K, V, Cr, Co, Ni and Cd) demonstrated a
similar trend where the sapeco stage results in higher elemental levels than for
the pre-processed samples. There are a couple of exceptions with the As, Se,
Mo and Pb values being similar for both stages of the yerba mate process. In
relation to the sapeco and drying stages the following trends were observed for
the elements reported in Appendices 3.2 to 3.4, namely: (i) higher (Na, Cr and
Cd) and lower (K, Se and Pb) levels in the material post the sapeco stage; and
(ii) similar levels for Co, Ni, As and Mo post the sapeco and drying stages.
86
Table 3.8: Total elemental levels (mean ± standard deviation) of non-commercial
yerba mate samples (mg/kg, dry weight) during the commercial
processing of the harvested tree material. The analyses were
determined using ICP-MS (refer to section 2.3).
Pre-processed Santa Catarina State*
Sapeco**
Drying process Native Traditional
n 4 2 2 2
Mg 6754 ± 577 7206 ± 21 4403 ± 40 5962 ± 54
Ca 5463 ± 616 7997 ± 424 6058 ± 244 6771 ± 63
Mn 368 ± 20 632 ± 22 445 ± 1 729 ± 53
Fe 54.2 ± 3.79 132.95 ± 10.85 68.52 ± 4.72 121.61 ± 6.02
Cu 11.34 ± 0.78 10.20 ± 0.22 11.08 ± 0.33 11.55 ± 0.52
Zn 29.79 ± 4.02 69.30 ± 2.72 34.78 ± 3.00 75.75 5.60
* samples from Santa Catarina State plantations, **refer to section 3.5.1.
3.6. Studies on Commercial Yerba Mate
This section provides an evaluation of the chemical content of commercial
yerba mate samples (loose and tea bags) from the two main producers in the
World, namely, Brazil and Argentina. The description of the samples and
methods used are outlined in sections 3.6.1 and 3.6.2. An investigation of the
total elemental content and the assessment of the effect of the mode of
consumption on the uptake of these elements is proposed in sections 3.6.3 and
3.6.4. Furthermore, the phenolic content of the commercial infusions is outlined
in section 3.6.5. Finally, an evaluation of the link to the dietary intake of the
elemental and phenolic content of yerba mate infusions is presented in section
3.6.6.
3.6.1. Description of the samples
All of the samples, with the exception for Barão de Cotegipe (southern
Brazil), where commercial yerba mate products from Brazil, Argentina and
87
Uruguay (which are from Brazilian production) were bought in local
supermarkets. Barão de Cotegipe, agreed to collaborate with this study and sent
a wide range of their products that are available in the South American market:
(i) Premium, selected healthy leaves of the natural plantation, dried
through a bed dryer using hot air at low temperatures (eliminating the
exposure of the material to smoke);
(ii) Nativa, same drying process as Premium, but using non-selected
leaves from the natural plantation;
(iii) Traditional, yerba mate from the traditional and natural plantations
dried together in a rotating metal cylinder where the leaves are in direct
contact with the smoke produced;
(iv) Moida Grossa, same processing method as outlined for Traditional but
ground at a larger particle sizes in the final product;
(v) Exportação, same processing of Moida Grossa but the yerba is aged
for at least 8 months in chambers, and also has a lower content of
twigs;
(vi) Terere, same traditional drying process but with the specific particle
size suitable for Terere (cold infusion);
(vii) Tostada, same processing of Moida Grossa but the final product is
then roasted, suitable for regular infusions and Brazilian iced tea
infusions;
(viii) Cambona, a genetic selected plant dried by Traditional method.
The samples were divided and compared by country of origin (Brazil or
Argentina) and type (loose material or teabags; green leaves or roasted). A list of
all samples is presented in Appendix 3.5.
3.6.2. Materials and method
The determination of the total elemental composition of the yerba mate
samples was performed as described in section 2.3. The commercial samples
88
were fully digested at 500°C for 12 hrs and analysed by inductively coupled
plasma mass spectrometry or ICP-MS.
An evaluation of the chemical content of different types of yerba mate
infusions was proposed and the infusions were analysed for the elemental (refer
to section 2.3); total polyphenol (section 2.4.2) and chlorogenic acid content
(section 2.5).
(i) Regular infusions
Regular infusions of yerba mate (commercial loose and teabag) were
prepared following a standardised industry protocol that reproduces the average
volume of water, weight of material and brew time given as the pack label
instructions (Donnelly, 2015). Double distilled deionised water (DDW) was
preferred instead of tap water for all infusions in order to evaluate the chemical
contribution from only the yerba mate material. A volume of 200 mL of freshly
boiled DDW was added to 1 teabag or 2.000 ± 0.0010 g of loose material, stirred
for 5 seconds and brewed for 4 minutes. In the last 5 seconds, the teabags were
squeezed against the side of the beaker and removed or the loose leaf infusions
were filtered through a teabag tissue (provided by Tata Global Beverages). Each
final solution was filtered using a 0.45 μm filter and the resultant solutions were
analysed.
(ii) Brazilian iced tea infusions
One of the most traditional ways of consuming yerba mate in Brazil is by
making a strong infusion to be cooled and drunk as an iced infusion. To prepare
this solution, one teabag or 1.30 ± 0.01 g of loose material was infused with 200
mL of freshly boiled DDW for 7 minutes. The loose leaf solutions were filtered
through a teabag tissue and all the infusions were filtered using a 0.45 μm filter
and analysed.
(iii) Simulation of traditional mate infusion – bombilla method
It is important to reproduce the traditional way of consuming yerba mate in
South America because this reflects the real chemical intake by the population.
89
A novel method was also proposed in this study in order to simulate the
bombilla method of consumption. It consisted of weighing 5.00 ± 0.01g of loose
yerba mate onto a filter paper (No. 1, Whatmann™, UK) and placing them onto a
Büchner funnel, as shown in Figure 3.7. Then, 10 ± 2 mL of DDW at 80 ± 2ºC
was added, followed after 30 seconds, by the addition of another 10 ± 2 mL of
hot DDW. The funnel was allowed to stand for 30 seconds before turning on the
vacuum, which was maintained until the yerba mate was dry. The filtrate was
then further filtered through a 0.45 µm membrane filter into a polyethylene tube
(first fraction) prior to analysis. The procedure was repeated using the same
yerba mate material to produce fractions 2, 3, 4 and 5.
Figure 3.7: Schematic of the proposed bombilla method. Adapted from NGC,
(2015).
3.6.3. Total elemental composition of commercial yerba mate
The total elemental composition of the commercial yerba mate samples
from Brazil and Argentina was evaluated following the method proposed in
section 3.6.2. The results for important trace elements (significant impact on the
contribution to the nutritional intake) are presented in Table 3.9; grouped by the
90
type of sample (green or roasted), processing package (loose or tea bags) and
origin (Brazil or Argentina), as described in section 3.6.1.
In order to test if any difference exists between the variances of the sets of
data reported in this study, a F-test (refer to section 2.6.2) was performed and is
presented in Appendices 3.28 to 3.29. In general, there is no statistically
significant difference between the standard deviations at p<0.05, that is the null
hypothesis is retined as Fcal<Fcrit. This states that the variability between the
data is related to random errors (Miller et al., 2018).
During the processing of yerba mate for the Argentina commercial
products, the dried samples are aged for up to 24 months in chambers which
helps to develop the flavour preferred by Argentinian consumers. Conversely, the
Brazilian population prefers a fresh and greener product, packed after the drying
process (Isolabella et al., 2010, Heck et al., 2008).
Table 3.9: Total elemental levels reported as mean and range (min – max) of
different types of commercial yerba mate samples (mg/kg, dry weight)
obtained from Brazil and Argentina. The analyses were determined
using ICP-MS (refer to section 2.3); n is the number of samples.
Green loose Green tea bag Roasted loose Roasted tea bag Origin Brazil Argentina Argentina Brazil Brazil
n 16 7 19 3 4
Mg 4577
(2787 – 5934)
4958
(3466 – 5495)
6197
(5577 – 4614)
6140
(5188 – 6934)
5563
(4557 – 7488)
Ca 7375
(6027 – 8639)
7222
(5972 – 8091)
7674
(6302 – 9073)
9782
(9255 – 10122)
8486
(8180 – 9012)
Mn 646
(486 – 834)
545
(383 – 671)
731
(483 – 1098)
746
(658 – 889)
536
(397 – 879)
Fe 37.31
(10.62 – 83.49)
31.20
(10.58 – 61.18)
80.23
(18.21 – 228.13)
31.65
(20.70 – 53.03)
80.07
(47.77 – 109.63)
Cu 9.23
(7.28 – 11.76)
8.39
(7.15 – 10.81)
9.56
(7.97 – 15.00)
9.33
(9.07 – 9.61)
11.68
(11.06 – 12.32)
Zn 58.89
(29.42 – 98.96)
67.09
(36.83 – 104.92)
117.05
(54.24 – 601.58)
110.76
(65.14 – 164.38)
84.78
(78.04 – 98.31)
91
A preliminary inspection of the data listed in Table 3.9 shows that in
general the elemental (Ca, Mn, Fe and Cu) levels for green loose yerba mate
products obtained from Brazil are slightly higher than that for the Argentinian
products, with the exception of Mg and Zn. Donnelly (2015) also reported that
Brazilian green loose samples had higher elemental levels when compared to
Argentinian products. A two-tailed Student t-test (Miller et al., 2018) confirmed a
significantly higher level of copper (tcal = 3.35 for 20 degrees of freedom;
p<0.0032) in the Brazilian samples when compared to the Argentinian products
(Table 3.10). Conversely, the zinc levels were significantly higher in the
Argentinian products (Table 3.10). The remaining elements, reported in
Appendices 3.6 to 3.8, have higher Na, V, Cr, Co, Ni and Se levels for the
Argentinian green loose products and higher K, As, Cd and Pb levels for the
Brazilian samples. In summary, the elemental levels are basically similar for the
two countries, which is to be expected as yerba mate primarily grows between
the Paraná and Paraguay river basins in South America (refer to Figure 3.1).
This yerba mate production area includes regions of Argentina, Paraguay and
Brazil (Cardozo Jr et al., 2007, Heck and De Mejia, 2007). Bastos et al. (2006)
reported that during the ageing of commercial yerba mate, the concentration of
some elements slightly decreases, indicating potential losses during this process.
Furthermore, the Argentinian samples are cultivated in direct sunshine whilst
Brazilian plantations include trees that are shaded as part of a native forest
environment. Previous studies, such as, Reissmann et al. (1999), Caron et al.
(2014) and Barbosa et al. (2015), have also reported a similar trend as the data
in this study, that is, lower elemental concentrations in the leaves of yerba mate
trees cultivated under the sun (Argentina). This condition may also affect the
physiology, transpiration and elemental uptake by yerba mate trees as modern
practices do not resemble the traditional (former) cultivation methods which used
to be similar to that of Brazil (i.e. native forests) (Barbosa et al., 2015).
The next study involved an evaluation of Argentinian commercial yerba
mate products based on the preparation and consumption of infusions using
green loose material or tea bags. In general, all the elements have higher levels
92
for the tea bag products when compared to the green loose material. Statistical
analysis (two-tailed Student t-test) confirmed that Mg, Fe and Cu had significantly
higher levels (tea bags > green loose; p<0.05) as reported in Table 3.10.
Moreover, the same trend was found for the elements (Na, K, V, Cr, Co, Ni, As,
Se, Mo, Cd and Pb) reported in Appendices 3.6 to 3.8. This is not surprising as
the tea bags contain only leaf material and have been processed to produce finer
particles (of around 1 mm). Interestingly, the Argentinian loose material has
some twigs in the commercial yerba mate samples which has a slight impact in
reducing the elemental level of the product (Donnelly, 2015). It was noted that
the green tea bags contain mostly leaf material with very few stems compared to
the loose yerba mate samples (refer to Figure 3.5). This suggests that the
investigated elements are present at higher levels in the leaf material compared
with the stems or may be wholly contained within the leaf (Raguž et al., 2013,
Kabata-Pendias, 2010).
The Brazilian market also sells roasted yerba mate products (both as
loose and tea bags), mostly used to prepare iced ‘tea’ infusions (Heck and De
Mejia, 2007). In order to prepare roasted yerba mate, the leaf material undergoes
a further roasting process, similar to coffee, typically at 160 °C for 12 mins (de
Godoy et al., 2013, Bastos et al., 2006). The total elemental levels between
commercial green and roasted yerba mate samples from Brazil was evaluated in
this study. The roasted (loose and tea bag) samples have higher elemental levels
when compared with the Brazilian green loose material (Table 3.9). This was
confirmed using a two-tailed Student t-test (Table 3.10), where Mg, Ca, and Zn
have significantly higher levels for the roasted samples (green < roast; 20
degrees of freedom, p<0.05). Furthermore, Na, K, V, Cr, Co, Ni, Se, Mo, Cd and
Pb (refer to Appendices 3.6 to 3.8) also had higher levels for the roasted samples
when compared to the green loose material (with the exception of As). The
roasting process changes and/or degrades a series of organic compounds
present in the material, including chlorogenic acids, flavonoids and polyphenols
(Clifford and Ramirez-Martinez, 1990, Farah et al., 2005, Bastos et al., 2006,
Marques and Farah, 2009, Bragança et al., 2011). Furthermore, during the
93
roasting process of the sample, there is a mass loss (volatiles and moisture),
resulting in an ‘enrichment’ of the elemental content of the roasted material
(Geiger et al., 2005, Franca et al., 2009, Schwartzberg, 2002).
Donnelly (2015) investigated the elemental content of commercial yerba
mate samples following a similar methodology to the one presented in this study.
In general, the data of this study, agrees with the reported values of Donnelly
(2015). Although, the analysed samples were different, the same trends were
found in relation to the origin (Brazil, Argentina) and packaging (loose, tea bags)
of the yerba mate products.
Table 3.10: Statistical analysis using a two-tailed Student t-test (Miller et al.,
2018) to evaluate the relationship between the origin (Brazil and
Argentina); packaging (loose and tea bags) and roasting (green and
roasted) of commercial yerba mate samples (refer to Table 3.9).
Origin
(Brazil and Argentina)
Packaging (Argentina)
Roasting (Brazil)
n 22 25 22
tcrit 2.09 2.07 2.09
tcalc p Direction of
significance tcalc p
Direction of
significance tcalc p
Direction of
significance
Mg 0.90 0.3750 ns 2.78 0.0105* loose<bag 2.62 0.0161* green<roast
Ca 0.92 0.3672 ns 0.28 0.7818 ns 4.55 0.0002*** green<roast
Mn 0.03 0.9755 ns 1.49 0.1489 ns 0.19 0.8535 ns
Fe 2.08 0.0507 ns 2.61 0.0156* loose<bag 1.74 0.0971 ns
Cu 3.35 0.0032** Bra>Arg 3.45 0.0022** loose<bag 1.95 0.0652 ns
Zn 3.54 0.0020** Bra<Arg 1.14 0.2666 ns 4.24 0.0004*** green<roast
Bra – Brazil; Arg – Argentina; n is the number of samples; tcalc is the calculated value (refer to Equation 2.6, section 2.6.2): tcrit is the critical value obtained for n-2 degrees of freedom, p is the level of probability; ns is that there is no statistically significant difference at p>0.05; * statistically significant, p<0.05; ** highly significant, p<0.01, and *** very highly significant p<0.001.
Furthermore, Table 3.1 lists the elemental values for yerba mate products
cited by other studies. Overall, there is good agreement with the reported values,
especially for Mg (Bastos et al., 2014, Giulian et al., 2007, Heinrichs and
Malavolta, 2001, Malik et al., 2008, Pozebon et al., 2015, Rossa et al., 2015,
94
Alexandre Marcelo et al., 2014); Ca (Jacques et al., 2007, Pozebon et al., 2015);
Mn (Donnelly, 2015, Heinrichs and Malavolta, 2001, Malik et al., 2008, Magri et
al., Rossa et al., 2015); Fe (Barbosa et al., 2015, Bastos et al., 2007, Donnelly,
2015, Jacques et al., 2007, Malik et al., 2008, Magri et al., Rossa et al., 2015);
Cu (Heinrichs and Malavolta, 2001, Jacques et al., 2007, Milani et al., 2016) and
Zn (Barbosa et al., 2018). Interestingly, very few studies have cited data for Cd
and Pb, which in terms of this research will be looked at in depth in the next
section on yerba mate infusions. Moreover, many of the above studies were
conducted only on Brazilian samples, and none reported the various sub-studies
of this research, namely, the effect of processing (roasting) or type of commercial
product (green loose/tea bag) using the same analytical procedures and
instrumental analysis. This study also provides data for various elements not
reported in the literature for yerba mate products (with the exception of Donnelly
(2015), who conducted a study at the University of Surrey, but using different
commercial samples from Argentina and Brazil (Mn, Fe, Cu, Zn, V, Cr, Co, Ni,
As, Se, Mo Cd and Pb).
3.6.4. Elemental composition of yerba mate infusions
The following studies involved an investigation into the elemental levels of
infusions, which are very important as this is the main form of consumption of
yerba mate. Moreover, this data will then be used to assess the human dietary
intake of specific trace elements through the different types of yerba mate
consumption (regular ‘tea-based’/normal and iced, or bombilla), which will be
reviewed in section 3.6.6.
(i) Regular ‘tea-based’ infusions
The total elemental composition of commercial yerba mate products (from
Brazil and Argentina) prepared as regular ‘tea-based’ infusions was evaluated
following the method proposed in section 3.6.2. It should be stressed that in this
research a standardised ‘tea-brewing’ method was followed, according to that
published by Donnelly (2015). The results of the selected trace elements are
95
shown in Table 3.11, grouped according to the type of sample (green or roasted);
processing package (loose or tea bags) and origin (Brazil or Argentina), as
described in section 3.6.1. The results are presented as µg/200 mL representing
the elemental intake in a typical serving (i.e. a cup of 200 mL of infusion). Only
the trace element composition (not including the major elements) was
investigated in this study involving yerba mate infusions. Statistical analysis of
the data in Table 3.11 was undertaken using a two-tailed Student t-test (Miller et
al., 2018), based on the null hypothesis, that there is no statistically significant
difference in the trace elemental levels of the products grown in different
countries (Brazil and Argentina), type of packaging (loose and tea bags) or
roasting process (green and roasted). Table 3.12 reports the statistical data.
The rate of extraction of the trace elements present in yerba mate material
was determined by calculating the concentration in the infusions per gram of leaf
used to prepare the infusion, and expressing this as a percentage (Donnelly,
2015). The mean value of the calculated rate of extraction for the types of yerba
mate products from Brazil and Argentina are reported in Table 3.13.
In relation to the country of origin of the yerba mate (Brazil and Argentina),
the regular infusions of yerba mate (green loose) from Brazil had slightly higher
elemental levels. This is in agreement with the reported results for the total
elemental content, presented in section 3.6.3. As was discussed, this is probably
due to the ageing process of the commercial Argentinian products (Bastos et al.,
2006). Interestingly, the difference in the copper levels of the commercial yerba
mate products from Brazil and Argentina (with Brazil being significantly higher,
p<0.01, as reported in Table 3.10) was not as apparent in the prepared regular
infusions (p<0.05, refer to Table 3.12). Furthermore, the reverse trend was found
for zinc. These differences may be due to the rate of extraction of the elements,
similar for copper and higher in the Brazilian samples for zinc (refer to Table
3.13). Another possible explanation may be due to the difference in the particle
size of the commercial yerba mate products, where the material from Brazil is
powdered during the processing stages, whereas the samples from Argentina
are milled, as shown in Figure 3.5. In terms of the other trace elements reported
96
in Appendices 3.9 and 3.10, Cr, Co and Ni were found at higher levels in the
Argentinian green loose regular infusions. The rest of the elements (V, As, Se,
Mo, Cd and Pb) had slightly higher levels in the Brazilian regular infusions.
The next study involved an evaluation of the infusion process involving
green loose and tea bag products from Argentina. When comparing the
elemental content of a regular infusion associated with a single serving (cup of
tea) using green loose and tea bags, there was a highly significant difference
between the samples. All the elements (Mn, Fe, Cu, Zn presented in Table 3.11
and for V, Cr, Co, Ni, As, Se, Mo Cd and Pb, reported in the Appendices 3.9 and
3.10) had higher levels for the tea bag samples when compared to the green
loose regular infusions. This finding suggests that the trace element extraction
efficiency (based on the calculated percentage extraction) of the material
packaged in the tea bags is higher when compared to the green loose yerba
mate products. The difference in the particle size of the material (loose: 3 – 5
mm; and tea bags - less than 1 mm) could be responsible for this variance.
Furthermore, the higher elemental content determined in the infusion of the
green tea bag samples may be a reflection of the ease of extraction from the
leafier plant parts found in the tea bags than the mixture of leaf and stems found
in the green loose material (Donnelly, 2015). The higher extraction rates of these
elements for yerba mate sold as green tea bags suggests, for example, that the
Mn, Cu and Zn are more easily extracted from the leafy parts of yerba mate than
the stems (refer to Table 3.13).
The elemental levels of green loose and roasted (from Brazil) regular
infusions from Brazil were evaluated, as presented in Table 3.11. Interestingly,
the opposite trend was found for the digested commercial samples when
compared with the regular infusions. In general, the regular infusions made with
green loose yerba mate had highly significant levels of Mn, Cu and Zn (two-tailed
Student t test, p<0.001) than the infusions prepared with roasted samples. The
same trend was found for Co, Ni, Mo, Cd and Pb, as reported in Appendices 3.9
and 3.10. Iron, V, Cr, As and Se had similar levels for both regular infusions
prepared with commercial green loose and roasted yerba mate. The elemental
97
levels in these infusions could depend on the levels of organic chelating species,
such as, chlorogenic acids, tannins, and other phenolic compounds present in
yerba mate, which may be modified or degraded during the roasting step
(Bragança et al., 2011).
Table 3.11: Elemental levels (µg/200 mL), reported as mean and range (min –
max), of regular infusions of commercial yerba mate samples from
Brazil and Argentina. The analyses were determined using ICP-MS
(refer to section 2.3).
Green loose Green tea bag Roasted loose Roasted tea bag Origin Brazil Argentina Argentina Brazil Brazil
n 16 7 19 3 4
Mn 1409
(473 – 3612)
1331
(519 – 4256)
2439
(1282 – 3937)
431
(373 – 496)
632
(491 – 861)
Fe 5.38
(2.53 – 9.66)
5.03
(1.91 – 8.21)
15.43
(8.74 – 20.48)
3.32
(2.22 – 4.38)
7.84
(5.85 – 10.65)
Cu 9.11
(4.69 – 13.12)
7.40
(4.15 – 11.53)
12.53
(3.62 – 20.37)
0.54
(0.30 – 0.86)
0.29
(0.08 – 0.67)
Zn 57.56
(33.67 – 82.01)
58.97
(44.85 – 93.13)
143.36
(100.84 – 191.92)
23.73
(9.47 – 35.76)
23.83
(18.97 – 30.05)
n is the number of samples
98
Table 3.12: Statistical analysis using a two-tailed Student t-test (Miller et al.,
2018) to evaluate the relationship between the origin (Brazil and
Argentina); packaging (loose and tea bags) and roasting process
(green loose and roasted) of regular infusions of commercial yerba
mate (refer to Table 3.11).
Origin
(Brazil and Argentina)
Packaging (Argentina)
Roasting (Brazil)
n 22 26 22
tcrit 2.09 2.07 2.09
tcalc p Direction of
significance tcalc p
Direction of
significance tcalc p
Direction of
significance
Mn 0.97 0.3441 ns 4.59 0.0001*** loose<bag 4.60 0.0002*** green>roast
Fe 1.46 0.1612 ns 6.42 <0.0001*** loose<bag 0.19 0.8512 ns
Cu 2.30 0.0325* Bra>Arg 3.13 0.0046** loose<bag 9.36 <0.0001*** green>roast
Zn 0.77 0.4497 ns 7.13 <0.001*** loose<bag 5.83 <0.0001*** green>roast
Bra – Brazil; Arg – Argentina; n is the number of samples; tcalc is the calculated value (refer to Equation 2.6, section 2.6.2): tcrit is the critical value obtained for n-2 degrees of freedom, p is the level of probability; ns is that there is no statistically significant difference at p>0.05; * statistically significant, p<0.05; ** highly significant, p<0.01, and *** very highly significant, p<0.001. Table 3.13: Percentage extraction (%) of regular infusions of commercial yerba
mate samples from Brazil and Argentina. Values reported as a mean
and range (min – max).
Green loose Green tea bag Roasted loose Roasted tea bag Origin Brazil Argentina Argentina Brazil Brazil
n 16 7 19 3 4
Mn 99
(44 – 160)
82
(44 – 116)
113
(73 – 184)
29
(28 – 31)
47
(31 – 93)
Fe 7
(4 – 25)
12
(6 – 25)
10
(3 – 33)
6
(4 – 8)
5
(4 – 9)
Cu 50
(29 – 80)
44
(29 – 80)
45
(15 – 74)
3
(2 – 4)
1
(0 – 3)
Zn 55
(28 – 80)
40
(28 – 51)
53
(9 – 80)
10
(7 – 12)
11
(11 – 17)
99
Comparative data in the literature is limited due to the lack of studies
reporting data on yerba mate infusions and the variability of the methods used for
infusions. However, Donnelly (2015) reported the elemental composition of
commercial yerba mate infusions, prepared using the same methodology as
proposed by this study. In general, the trace element concentrations in this study
are similar to the limited number of reported levels (Donnelly, 2015). The
exceptions include, the lower levels of copper in the Brazilian roasted and lower
levels of zinc in the green loose infusions, found in this study.
South American countries have set a limit for the concentration of toxic
metals in yerba mate products of 0.6 mg/kg for arsenic and lead and 0.4 mg/kg
for cadmium (DOU, 2013). All of the commercial yerba mate products analysed
in this study had As, Pb and Cd concentrations below these limits. Moreover, the
mean ‘toxic’ metal levels determined in this study for the regular infusions of
green loose: 0.025 µg/200 mL As; 0.04 µg/200 mL Pb and 0.07 µg/200 mL Cd;
and tea bags: 0.09 µg/200 mL As; 0.04 µg/200 mL Pb and 0.15 µg/200 mL Cd,
respectively. Therefore, the consumption of commercial yerba mate infusions
does not pose a risk of ‘dietary’ exposure to these specific toxic metals (Barbosa
et al., 2015, Pozebon et al., 2015). The lower levels of cadmium and lead in the
yerba mate infusions may be explained by the low levels in the leaf material
(refer to Appendix 3.10) and the ability of these elements to form complexes and
be retained in the plant material, even after the extraction with hot water (Michie
and Dixon, 1977).
(ii) Brazilian iced tea
In Brazil, one of the most popular methods of consuming yerba mate is by
iced tea infusions (refer to section 3.2.2). In order to evaluate the elemental
extraction for Brazilian yerba mate iced tea infusions, solutions were prepared
according to the method reported in section 3.6.2. The results are presented in
Table 3.14. In terms of comparative analysis, two methods were investigated,
namely, the regular infusion (refer to the section above) and the Brazilian iced
tea method. The two methods have different brewing times (regular infusions – 4
100
minutes; iced tea – 7 minutes) and proportions of yerba mate sample (mass of
solid to volume of water) (refer to section 3.6.2). In general, the Brazilian iced
infusion method resulted in higher elemental levels per serving associated with
the more concentrated infusions in comparison with the regular method (Tables
3.11 and 3.14).
As a result of constraints of time, selected samples (n = 3) of each group
were investigated by this infusion method. Therefore, the results will not be
subjected to statistical analysis due to the limited number of samples. A primary
evaluation of the data presented in Table 3.14 shows a trend where the Mn, Fe
and Cu levels were higher in the iced tea infusions (using Brazilian green loose
yerba mate) when compared to that using Argentinian material. An exception
was found for Zn where the Argentinian material resulted in higher levels in the
infusions; the same trend was found for the analysis of the commercial product
material (refer to section 3.6.3). A similar trend was found for Cr and Ni; but the
rest of the elements reported in Appendices 3.11 and 3.12 did not show a
significant difference between the countries.
Furthermore, the iced tea infusions produced using yerba mate material
packaged as tea bags had higher levels of Mn, Fe, Cu and Zn (Table 3.14), and
V, Cr, Co, Ni, As, Se, Mo Cd and Pb (Appendices 3.11 and 3.12) when
compared to the green loose material. Finally, the Brazilian green loose yerba
mate had higher levels of Mn, Cu and Zn when compared to the roasted
samples. The same trend was found for Co, Ni and Mo, as reported in
Appendices 3.11 and 3.12. Iron, V, Cr, As, Se, Cd and Pb (Appendices 3.11 and
3.12) had similar levels for iced tea infusions prepared with Brazilian green loose
and roasted yerba mate products.
101
Table 3.14: Elemental levels (µg/200 mL), reported as the mean and range (min
– max), of Brazilian iced tea infusions prepared using commercial
yerba mate products from Brazil and Argentina. The analyses were
determined using ICP-MS (refer to section 2.3). n is the number of
infusion samples.
Green loose Green tea bag Roasted loose
Roasted tea bag
Origin Brazil Argentina Argentina Brazil Brazil n 3 3 3 3 3
Mn 2276
(1721 – 2660)
1596
(1295 – 1964)
2716
(2061 – 3227)
820
(672 – 899)
650
(542 – 811)
Fe 10.66
(9.12 - 13.04)
6.28
(6.14 – 6.41)
28.35
(17.77 – 38.93)
6.93
(6.88 – 6.98)
17.51
(14.80 – 20.21)
Cu 20.85
(16.38 – 23.62)
12.12
(10.95 – 14.45)
21.62
(12.47 – 28.44) <LOD <LOD
Zn 79.76
(69.91 – 89.24)
93.04
(78.43 – 109.22)
236.65
(139.64 – 316.00)
38.39
(15.87 – 59.64)
30.41
(26.18 – 34.89)
(iii) Bombilla method
In order to simulate the traditional consumption of yerba mate in South
America, a bombilla method was performed following the procedure described in
section 3.6.2. The results for manganese are shown in Table 3.15, grouped by
country of origin and the concentration for the various fractions (that is the
successive additions of hot water, using DDW, to the same yerba mate material
in the cup). Manganese was selected because of the high levels found in yerba
mate products (refer to Table 3.9) and the importance of this trace element in
human health (refer to section 1.1). The full data for all of the elements are
reported in Appendices 3.13 to 3.19.
102
Table 3.15: Manganese levels (µg/200 mL) of bombilla infusions of commercial
green loose yerba mate samples from Brazil (n= 16) and Argentina
(n=7). n is the number of samples. The total manganese content
refers to a sum of the five fractions. The analyses were determined
using ICP-MS (refer to section 2.3).
Brazil Argentina
Fraction Mean Contribution (%) Range Mean Contribution
(%) Range
F1 13906 40.8 3120 – 30592 5312 22.6 2555 – 7165
F2 9801 28.7 3025 - 16964 5828 24.8 2564 – 9003
F3 5250 15.4 1449 – 7806 4596 19.5 1804- 6904
F4 3089 9.1 741 – 5869 5039 21.4 2537 – 13747
F5 2047 6.0 437 - 3756 2750 11.7 1102 - 4311
Total 34093 100% 23526 100%
Table 3.15 shows that there is a variation in the Mn levels of the fractions
(F1 to F5) based on the successive addition of hot water to the original mass of
yerba mate (green loose). This is not surprising as the elemental analysis of the
commercial material from Brazil and Argentina (Table 3.9) also showed a range
of Mn levels; i.e. for Brazilian green loose (486 to 834 mg/kg, Mn, d.w.) and
Argentinian (383 to 671mg/kg Mn d.w.). Moreover, the regular infusion data for
these commercial products had a similar variance in the Mn levels (Table 3.11).
The Brazilian green loose yerba mate initially provides more manganese in the
infusion fraction (F1) than that for the Argentinian product (based on the addition
of 100 mL of hot water). However, after subsequent additions of hot water
(fractions F2 to F5) the Mn levels are basically the same in the resultant
infusions, as shown in Figure 3.8. On the other hand, the accumulation (potential
intake level) of the element after the first fraction (F1) of yerba mate, purchased
from both countries, results in a similar pattern in terms of the Mn level (refer to
Figure 3.8). Both products result in a higher level of Mn intake through the
consumption of green loose yerba mate using this bombilla method, when
103
compared with the upper limit level for Mn intake of 11 mg/day (IOM, 2002). The
potential health impact of this will be discussed in section 3.6.6.
Figure 3.8: Manganese concentration (mg/200 mL) and rate of accumulation*
(i.e. potential intake) between 5 fractions (successive additions of hot
water) based on using the bombilla method (refer to section 3.6.2) for
green loose yerba mate purchased in Brazil (n= 16) and Argentina
(n=7). n is the number of samples. The WHO set the upper limit for
Mn in 11 mg/day (IOM, 2002). The analyses were determined using
ICP-MS (refer to section 2.3). *Calculated as the sum of the previous
fractions.
In terms of the other trace elements (refer to Appendices 3.13 to 3.19)
there is a similar pattern in terms of the fraction levels and the accumulation (F1
to F5 fractions). In general, all the elemental (Fe, Cu, Zn, Ni, Cr, V, Co, Cd and
Pd) concentrations steadily decrease from F1 to F5. The exception is for As, Se,
Mo where all of the fractions have similar concentrations (each fraction
contributes to approximately 20% of the sum of all fractions). Comparison with
the sum of the elemental intake of the 5 fractions for these trace elements leads
to the following order of intake: Zn > Cu > Ni > Fe > Cr > Co > Cd > Pb > Mo >
0
5
10
15
20
25
30
35
40
1 2 3 4 5 6
Con
cent
ratio
n of
Mn
(mg/
200m
l)
Fraction
ArgentinianFractionsBrazilianfractionsBrazilaccumulationArgentinaaccumulationWHO Upperlimit
104
As > Se > V. This refers to the specific solubility or ‘bioavailability’ of each
element through a hot water sequential extraction. Moreover, for As, Pb and Cd,
although there is an increase in the levels per fraction (which was previously
reported in section 3.6.3), this still did not present a significant impact on the
dietary intake levels). Finally, an assessment of the dietary intake of these
elements is presented in section 3.6.6.
3.6.5. Polyphenolic composition of yerba mate
Polyphenols are plant metabolites that have at least one aromatic ring with
one or more hydroxyl groups attached (Campos‐Vega and Oomah, 2013). These
molecules have been proven to be linked to a series of health benefits relating to
the antioxidant activity (Bastos et al., 2007, Colpo et al., 2016). Many studies
have demonstrated the strong antioxidant ability of these molecules in in vitro
analyses, by removing free radicals and reactive oxygen species that may have
harmful effects (Donnelly, 2015). Beverages, fruits and vegetables, may make a
significant contribution to the dietary intake of polyphenols (Fukushima et al.,
2009, Vanamala et al., 2006), as discussed in section 1.2. The measurement and
health beneficial effects of polyphenols in yerba mate infusions have been
reported, as reviewed in section 3.3.
3.6.5.1 Total polyphenol of infusions
In order to evaluate the potential intake of polyphenols in different infusion
methods of yerba mate (regular ‘tea-based’, Brazilian iced tea, or the traditional
bombilla), the total polyphenol content of the infusions was analysed by the Folin-
Ciocalteu assay involving a UV-Vis spectrometer, as described in section 2.4.
Moreover, this data will then be used to assess the intake of total polyphenol
content through the different types of yerba mate (green loose, tea bags and
roasted) in section 3.6.6. The full data for all of the samples are reported in
Appendices 3.20 to 3.22.
105
(i) Regular infusions
The total polyphenol content of commercial yerba mate products (from
Brazil and Argentina) prepared as regular ‘tea-based’ infusions was evaluated
following the method proposed in section 3.6.2. The regular infusion method was
that reported as standardised ‘tea-brewing’ (Donnelly, 2015). The results are
presented in Table 3.16, grouped according to the type of sample (green or
roasted); processing package (loose or tea bags) and origin (Brazil or Argentina).
The results are once more presented as µg/200 mL representing the total
phenolic intake in a typical serving (i.e. a cup of 200 mL of infusion).
A preliminary study of the data presented in Table 3.16 shows that the
commercial green loose yerba mate samples from Brazil have a slightly higher
mean total polyphenol content when compared to the Argentinian green loose
material. Although, the range of results are similar between the two countries, a
two-tailed Student t-test analysis confirmed that there is not a statistically
significant difference between the Brazilian and Argentinian green loose infusion
levels (n = 22, tcrit = 2.09 > tcalc = 1.54; p = 0.1391). This trend is similar to that
found for the elemental content of the yerba mate material (refer to section 3.6.3)
and infusions (refer to section 3.6.4). One of the main differences between
Argentinian and Brazilian production is that in the former country, the yerba is
grown in the sun (because it is cultivated), whilst in the latter, trees are shaded
within local forests. It has been reported that the different types of cultivation may
have an impact on the chemicals presents in yerba, especially polyphenols
(Donnelly, 2015). Furthermore, the Argentinian products are aged for up to 24
months in chambers, whilst the Brazilian commercial yerba mate is freshly
packed. The polyphenols are molecules that can easily degrade or change
during long storage periods or following exposure to high temperatures (Klimczak
et al., 2007). Therefore, it is interesting to note that whilst there are differences
between the two countries, in terms of yerba mate cultivation and processing, the
samples have similar total polyphenol levels.
106
The Argentinian commercial yerba mate packed as green loose or as tea
bags, have higher levels of total polyphenols in the tea bags, as was previously
reported for the elemental content (refer to section 3.6.4). Statistical analysis
using a two-tailed Student t-test confirmed a very highly significant difference,
that is, the total polyphenol content of Argentinian tea bags > green loose (n =
26, tcrit = 2.07 < tcalc = 5.78 and p < 0.0001). The particle size of the yerba mate
packed in the tea bags is smaller (1 mm) than the product packed as green loose
material (4 - 5 mm). This suggests that the polyphenols are more easily extracted
when there is an increase in the leaf contact area. Furthermore, the amount of
stems present in yerba mate sold as green loose also influences the amount of
polyphenols and methyl xanthine compounds present (Donnelly, 2015). Products
that contained 12.4 to 15% stems results in 20 to 40% lower levels of total
polyphenols than products without stems (Tamasi et al., 2007). In addition, the
caffeine content has also been found to be negatively correlated with the amount
of yerba mate stems present in the product (Mazzafera, 1997).
Finally, an evaluation of the impact of the roasting process on the total
polyphenolic content was performed by a comparison between the Brazilian
green and roasted commercial products. There was a very highly significant
difference between these products (a two-tailed Student t-test, where n = 22, tcrit
= 2.09 < tcalc = 8.01 and p < 0.0001). The Brazilian green loose material
presented a much higher total polyphenolic content when compared to the
roasted material. This confirms that the heating process leads to the loss of some
polyphenols, as has been previously reported (Clifford and Ramirez-Martinez,
1990, Marques and Farah, 2009, Donnelly, 2015). Therefore, the roasting
process has a major impact on the presence and levels of polyphenols in the
yerba mate products, but not on the elemental content, as was discussed in
section 3.6.4.
It is difficult to compare this data with that reported in the literature,
because there is a variability in the methods of preparing an infusion. Different
mass-to-water ratios (from 1:82 to 1:100) and infusion times (from 5 to 10
minutes) have been used by different research groups (Gorjanovic et al., 2012,
107
de Mejía et al., 2010, Bravo et al., 2007, Bastos et al., 2006). In comparison,
Donnelly (2015) used the same method of infusion as this study and the reported
range of total polyphenols was found to be very similar (79.9 – 303.1 mg gallic
acid equivalents (GAE)/200 mL for green samples and 79.9 – 303.1 mg
(GAE)/200 mL for roasted samples).
Table 3.16: Total polyphenol content (mg/200 mL), reported as the mean and
range (min – max), of regular infusions of commercial yerba mate
samples from Brazil and Argentina. The samples were analysed by
the Folin-Ciocalteu method using a UV-Vis spectrometer (refer to
section 2.4). n is the number of samples.
Type Origin n Total polyphenol content
Green loose Brazil 15
142.6
(79.7 - 169.1)
Argentina 7 125.0
(81.9 - 159.0)
Green tea bag Argentina 19 215.7
(134.8 - 261.0)
Roasted loose Brazil 3 45.2
(44.0 - 46.7)
Roasted tea bag Brazil 4 70.2
(56.3 - 93.6)
(ii) Brazilian iced tea
An evaluation of the potential intake of total polyphenols through
consuming yerba mate from Brazil (as an iced tea infusion) was assessed in this
study (refer to section 3.2.2). The total polyphenol content of the Brazilian yerba
mate iced tea infusions was measured, as described in section 2.4, using
solutions prepared according to the method outlined in section 3.6.2. The results
are presented in Table 3.17. In order to compare the intake levels linked to the
drinking of regular infusions, it should be stressed that the methods have
different: (i) brewing times (regular infusions – 4 minutes; iced tea infusions – 7
108
minutes); and (ii) proportions of yerba mate sample (mass of solid) to volume of
water (refer to section 3.6.2). Interestingly, the Brazilian regular and iced tea
infusion methods resulted in similar levels of total polyphenols. This suggests
that the polyphenols present in the yerba mate material are equally extracted by
the two methods.
Table 3.17: Total polyphenol content (mg/200 mL), reported as the mean and
range (min – max), of Brazilian iced tea infusions of commercial
yerba mate samples from Brazil and Argentina. The analyses were
determined by the Folin-Ciocalteu method using a UV-Vis
spectrometer (refer to section 2.4). n is the number of samples.
Type Origin n Total polyphenol Green loose Brazil 1 112.2
Green tea bag Argentina 3 275.5
(216.5 – 312.5)
Roasted loose Brazil 3 35.5
(29.2 – 41.7)
Roasted tea bag Brazil 3 71.5
(64.0 – 83.0)
(iii) Bombilla method
A bombilla method was performed in this study, in order to simulate the
traditional consumption of yerba mate in South America, as described in section
3.6.2. The results for the total polyphenol content are shown in Table 3.18, grouped by country of origin and the concentration for the various fractions
(successive additions of hot water to the same yerba mate material in the cup). In
comparison with the regular infusion method, the concentrations of the total
polyphenols in the first fractions are 10 times higher in the bombilla method. As
previously discussed, there was also a difference in the total polyphenol content
of the commercial material from Brazil and Argentina. Furthermore, the regular
infusion data for these commercial products had a similar variance in the levels
of total polyphenols (Table 3.16). In terms of the elemental data for the bombilla
109
infusions (presented in Table 3.15 and Figure 3.8), the percentage contribution to
the total concentration (the sum of the fractions, F1 to F5) is very similar to the
total polyphenol content (Table 3.18). Data reported for the elemental levels of
plants, and in prepared beverages, concluded that the elements can exist in the
infusions as free ions or complexes with naturally occurring bioligands. This is
particularly true for polyphenols, which can complex elements (or metals) through
hydroxyl, carboxylate and phenolate groups (Pohl and Prusisz, 2007, Khokhar
and Owusu Apenten, 2003). The findings of this study suggests that the
elements in these infusions could be chelated to the phenolic compounds
present in yerba mate (Bragança et al., 2011). Pohl and Prusisz (2007) proposed
that element-binding by flavonoids considerably reduces the bioavailability for the
body and/or impairs the absorption of these chemicals.
Table 3.18: Total polyphenol content (mg/200 mL) of bombilla infusions of
commercial green loose yerba mate samples from Brazil (n= 16)
and Argentina (n=7). The total content refers to the sum of the five
fractions. The samples were analysed by the Folin-Ciocalteu
method using a UV-Vis spectrometer (refer to section 2.4). n is the
number of samples.
Brazil Argentina
Fraction Mean Range Contribution (%) Mean Range Contribution
(%) F1 1559.7 475.4 - 2451.4 41.0 882.5 610.0 - 1011.9 29.4
F2 997.8 421.8 - 1215.6 26.2 828.2 565.5 - 999.7 27.6
F3 591.9 300.4 - 807.1 15.5 578.9 444.3 - 717.1 19.3
F4 383.8 194.4 - 552.8 10.1 412.8 302.2 - 473.3 13.7
F5 274.0 129.8 - 386.8 7.2 303.3 226.7 - 340.9 10.1
Total 3807.2 100% 3005.6 100%
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3.6.5.2 Chlorogenic acids, caffeine and theobromine levels in yerba mate infusions
The predominant type of polyphenol present in yerba mate is the
chlorogenic acid group, including a range of mono-, di- and tri-acylated
compounds. The main chlorogenic acids present in yerba mate are 3-, 4- and 5-
caffeoylquinic acids (Donnelly, 2015). Furthermore, there are important
xanthines, namely, caffeine and theobromine, that are also present in yerba
mate. They are related to a series of health claims linked with the consumption of
yerba mate infusions (refer to section 3.3).
The amount of these compounds in the regular infusions of commercial
yerba mate products from Brazil was determined by ultra-high performance liquid
chromatography (UHPLC) (refer to section 2.5). The regular ‘tea-based’ infusions
were prepared following the method proposed in section 3.6.2., according to that
published by Donnelly (2015). The results are shown in Table 3.19, grouped
according to the type of sample (green or roasted) and processing package
(loose or tea bags), as described in section 3.6.1. Only the commercial yerba
mate from Brazil was investigated in this study because the data for the
Argentinian material is already available (Donnelly, 2015). The full data for all of
the samples are reported in Appendices 3.23 and 3.24.
This study involved an evaluation of the yerba mate infusions involving
green loose and roasted (loose and tea bags) products from Brazil. It is clear
from the data presented in Table 3.19 that all of the organic compounds
(caffeoylquinic acids, caffeine and theobromine) are changing or suffering
degradation during the roasting process. This was to be expected since it was
proven that the roasting process (high temperatures) can change or degrade
organic compounds, such as, chlorogenic acids and xanthine (Clifford and
Ramirez-Martinez, 1990, Farah et al., 2005, Bastos et al., 2006, Marques and
Farah, 2009, Bragança et al., 2011).
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It was also noted from Table 3.19 that the roasted loose samples have
lower levels than the roasted tea bags of caffeoylquinic acids, caffeine and
theobromine. The yerba mate packed in the tea bags has more leaf material, as
previously discussed. This indicates that the chlorogenic acids and xanthines are
present at higher levels in the leaf compared with the stems (Donnelly, 2015,
Tamasi et al., 2007).
Table 3.19: Chlorogenic acid, theobromine and caffeine content (mg/200 mL) of
regular infusions of commercial yerba mate samples from Brazil. The
samples were analysed by UHPLC (refer to section 2.5). For green
loose samples the results are presented as mean and range (min –
max). n is the number of samples.
Type Green loose Roasted loose Roasted tea bag n 4 1 1
3-Caffeoylquinic acid 46.84
(41.44- 55.62) 2.86 3.98
Theobromine 4.02
(3.31- 5.03) 1.13 1.53
4-Caffeoylquinic acid 14.29
(12.99- 15.73) 2.03 3.16
5-Caffeoylquinic acid 24.01
(19.43- 29.09) 2.79 3.62
Caffeine 25.49
(18.57- 34.58) 5.14 7.58
Donnelly (2015) also reported the following concentrations (mg/200 mL)
for the commercial green loose yerba mate samples from Argentina: 33.37 of 3-
caffeoylquinic acid; 13.35 of 4- caffeoylquinic acid; 22.10 of 5- caffeoylquinic
acid; 1.67 of theobromine and 11.62 of caffeine. The green loose yerba mate
from Brazil has higher levels of caffeoylquinic acids, caffeine and theobromine
(refer to Table 3.19) than the Argentinian samples. The cultivation practices and
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processing of yerba mate leaf have an impact on the amount of polyphenol and
xanthine compounds (Donnelly, 2015). Sunlight exposed yerba mate plantations
normally lead to higher amounts of caffeoylquinic acids, caffeine and
theobromine, than for samples collected from shaded plantations (Dartora et al.,
2011). Streit et al. (2007) reported lower levels of chlorogenic acids and caffeine
in yerba mate infusions produced from Brazilian products collected from
reforested rather than native plantations.
(ii) Bombilla method
In order to evaluate the traditional consumption of yerba mate in South
America, a bombilla method was performed following the procedure described in
section 3.6.2. The results for chlorogenic acids are shown in Table 3.20 and for
xanthines (theobromine and caffeine) in Table 3.21; grouped by the
concentration for the various fractions, F1 to F5 (that is the successive addition of
hot water to the same yerba mate material). In comparison with the regular
infusion method (refer to Table 3.19), the bombilla method resulted in much
higher concentrations of the analysed compounds (10 times higher in the first
fractions). The percentage contribution of all three chlorogenic acids follows the
same pattern for F1 to F5 fractions (refer to Table 3.20), suggesting comparable
extraction behavior. The same trend was also found for the xanthines (refer to
Table 3.21). This study provides important data showing that any differences
relating to the various infusion methods (regular infusion and bombilla) must be
taken into consideration when using the data to predict the potential intake of
these chemicals through consuming yerba mate by individuals in South America.
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Table 3.20: Chlorogenic acid content (mg/200 mL) of bombilla infusion fractions
of green loose commercial yerba mate products from Brazil. The
samples were analysed by UHPLC (refer to section 2.5). n = 4, n is
the number of samples. The percentage (%) refers to the contribution
of the fraction to the total (sum of the fractions).
3-Caffeoylquinic acid 4-Caffeoylquinic acid 5-Caffeoylquinic acid
Mean (%) Range Mean (%) Range Mean (%) Range
F1 516.61 36.8 305.08 - 761.37 145.76 34.1 91.04 - 174.31 239.59 33.5 136.81- 284.63
F2 441.82 31.8 395.68 - 483.18 140.75 32.6 116.46- 160.69 232.56 32.5 184.65- 263.42
F3 242.32 17.6 224.83 - 269.4 79.78 18.5 62.11 - 87.45 134.64 18.7 98.18- 159.57
F4 121.22 8.8 82.47 - 198.35 40.23 9.3 29.25 - 64.17 66.97 9.5 46.75- 96.41
F5 72.12 5.2 47.12 - 133.68 24.28 5.6 17.04 - 42.76 40.69 5.8 27.16- 64.82
Total 1394.08 100% 430.8 100% 714.45 100%
Table 3.21: Theobromine and caffeine content of bombilla infusion fractions of
green loose yerba mate commercial products (mg/200 mL) from
Brazil. The samples were analysed by UHPLC (refer to section 2.5).
n = 4, n is the number of samples. The percentage (%) refers to the
contribution of the fraction to the total.
Theobromine Caffeine
Mean (%) Range Mean (%) Range
F1 41.5 36.5 23.8 - 52.43 258.76 35.8 144.13 - 425.62
F2 34.52 30.8 30.66 - 40.50 211.81 30.3 163.09 - 267.67
F3 19.89 17.7 17.31 - 25.46 124.3 17.9 92.96 - 144.85
F4 10.22 9.2 6.87 - 14.68 67.27 9.7 37.30 - 97.79
F5 6.35 5.8 4.25 - 10.51 43.68 6.3 23.88 - 70.90
Total 112.48 100% 705.82 100%
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3.6.6. Link to dietary intake through consumption of yerba mate
One of the aims of this study was to evaluate the effect of the mode of
consumption of yerba mate in terms of the nutritional intake from consuming
infusions of the products. This study will focus only on the potential intake of the
total levels of elements and polyphenols.
3.6.6.1 Dietary intake of trace elements
An evaluation of the contribution of different infusions prepared from
commercial samples of yerba mate from Brazil and Argentina, in terms of the
levels of trace elements and polyphenols, was undertaken using the data
presented in this chapter. It is important to highlight the differences in the serving
size of the different infusion methods. The serving size for regular tea-based and
Brazilian iced tea infusions is a cup (200 mL). The bombilla method is
traditionally performed with 50 g of yerba mate material and 1 litre of hot water
(added as successive fractions of 200 mL). Therefore, the daily amount of yerba
mate infusion consumed using a bombilla is close to 1 litre.
The recommended daily intake (RDA) of essential elements is defined by
the World Health Organisation or WHO (WHO, 1996) for males (M) and females
(F). The WHO RDA guidelines are compared with the calculated % intake of the
chemicals for the consumption of yerba mate infusions. All of the trace elements
determined in this study represent from 0.1 to 5% of the RDA, for all the infusion
methods (regular, Brazilian and bombilla) as reported in Appendices 3.9 to 3.19.
The exception is for manganese. Table 3.22 compares the different methods of
yerba mate infusion in relation to the recommended intake and upper limit of
manganese on a daily basis. A regular infusion serving (1 cup of 200 mL) can
provide 23.7 to 106.0 % for males and 30.3 to 135.5 % for females, of the daily
recommended manganese intake; depending on the type of yerba mate product.
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Even at the highest Mn concentrations in regular infusions the contribution to the
Mn uptake level is still below the upper limit value (WHO, 1996). The Brazilian
iced tea infusions provided a slightly higher contribution to the intake of
manganese. In terms of the bombilla method the daily amount of yerba mate
infusion consumed is close to a volume of 1 litre. This provides the
recommended daily intake of manganese and could provide concentrations
higher than the recommended upper consumption limit for this element per day
(refer to Table 3.22). It must be noted that this is a potential intake study
regarding the determination of the total concentration of manganese for these
different yerba mate infusion methods and further research needs to be
undertaken to evaluate the possible toxicity of manganese in relation to
consuming yerba mate using the bombilla method. In relation to the human
exposure of elements and polyphenols in food, it is important to also consider the
bioavailability of the compound or element. Bioavailability is defined as the
fraction of the analyte which can be absorbed and utilised for physiological
functions (Fairweather-Tait and Hurrell, 1996). Many factors can have an impact
on the absorption of elements, such as, the intake of polyphenols and dietary
fiber which can decrease the level of elemental absorption (Finley et al., 2011).
The manganese data reported in Table 3.11 and the total polyphenol values in
Table 3.16 for regular infusions of yerba mate presents a tendency of the
respective concentrations to increase together (Spearman’s correlation; R= 0.24,
p=0.23 n= 26 and = 0.05). This correlation indicates that the amount of total
polyphenol content could be potentially related to the manganese content of the
yerba mate infusions, influencing the bioavailability of both chemicals. In
particular, it is important to now develop and apply a method to determine the
bioavailability of manganese in these infusions and how such chemical forms
may be influenced by the presence of polyphenols in the infusions. The
concentration of manganese in yerba mate is the highest when compared to
other typical beverages, such as, coffee or green and black tea (Unicamp, 2011).
Moreover, based on the low levels of the toxic elements in the yerba mate
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infusions (regular, Brazilian and bombilla), the daily intake of As, Cd and Pb were
found to not be significant (refer to Appendices 3.10, 3.12 and 3.17 to 3.19).
Table 3.22: Percentage intake (%) of manganese based on a serving (200 mL for
regular and Brazilian iced tea infusions; 1L for bombilla method) of
non-commercial yerba mate samples. The data is compared with the
World Health Organisation recommended daily allowance (RDA) of
manganese for males (M) and females (F).
WHO RDA* Upper limit* M F
Type Origin 2.3 1.8 11
Regular infusions
Green loose Brazil 67.6 86.4 14.1
Argentina 44.3 56.6 9.3
Tea bags Argentina 106.0 135.5 22.2
Roasted Brazil 23.7 30.3 5.0
Brazilian iced tea
Green loose Brazil 82.3 105.1 17.2
Argentina 86.1 110.0 18.0
Tea bags Argentina 118.1 150.9 24.7
Roasted Brazil 32.0 40.8 6.7
Bombilla method
Green loose Brazil 1482.3 1894.0 309.9
Argentina 1022.9 1307.0 213.9
* World Health Organisation Recommended Daily Allowance and Upper limit (reported in mg/day) (WHO, 1996).
3.6.6.2 Dietary intake of polyphenols
An estimation of a recommended daily intake for total polyphenols is
difficult due to the variation in the levels of the phenolic compounds in a particular
foodstuff, structural diversity of the phenolic compounds, or lack of standardised
analytical methods (Scalbert and Williamson, 2000). Many studies have agreed
on a range of 1 g of total polyphenols per day (Kühnau, 1976, Faller and Fialho,
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2009, Landete, 2013). Most authors refer to Fukushima et al. (2009), to evaluate
the intake of polyphenols. The author calculated a daily consumption of 1492 mg
(fresh weight) of polyphenols, based on a balanced Japanese diet. Table 3.23
compares the different methods of yerba mate infusion in relation to the daily
intake of total polyphenols (Fukushima et al., 2009). A regular infusion serving (1
cup of 200 mL) will contribute 4 to 14.5 % of the daily intake of total polyphenols.
The Brazilian iced tea infusions provided a similar contribution to the intake of
total polyphenols. In the bombilla method the daily amount of yerba mate infusion
serving is 1 litre. This traditional method of consuming yerba mate in South
America can provide up to twice the amount of the adequate daily intake of total
polyphenols (refer to Table 3.23).
The range of total polyphenols in yerba mate regular infusions is similar to
that reported for green or black tea and fruit (grape, apple and orange) juices, but
is half of the total polyphenolic content of filter coffee (Donnelly, 2015). Although,
when the traditional method of consuming yerba mate in South America
(bombilla method) is evaluated, a single serving (1L) would make a significant
contribution to the total polyphenol intake for this population.
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Table 3.23: Percentage intake (%) of total polyphenol based on a serving (200
mL for regular and Brazilian iced tea infusions; 1L for bombilla
method) of commercial yerba mate samples. The data is compared
with the values reported by Fukushima et al. (2009) for the daily
intake of polyphenols.
Total polyphenol daily intake (mg/day)
Type Origin 1492
Regular infusion
Green loose Brazil 9.6
Argentina 8.4
Tea bags Argentina 14.5
Roasted Brazil 4.0
Brazilian iced tea Green loose Brazil 7.5
Tea bags Argentina 18.5
Roasted Brazil 3.6
Bombilla method
Green loose Brazil 255.2
Argentina 201.4
The total polyphenol data for yerba mate infusions was compared with the
European flavonoid database Phenol-Explorer (Pérez-Jiménez et al., 2010). In
this, filter coffee was ranked 37th (534 mg GAE/200 mL), black tea 58th (208 mg
GAE/200 mL) and green tea 67th (124 mg GAE/100 mL) in the top 100
polyphenol-containing foods (Pérez-Jimenez et al., 2010). The mean total
polyphenol content of yerba mate regular infusions of 135.7 mg GAE/200 mL,
Brazilian iced tea of 147.1 mg GAE/200 mL would place yerba mate infusions
above green tea in this ranking. Although, the bombilla method would provide
3406.4 mg GAE/1L and a serving of this method would place yerba mate above
all other beverages.
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3.7. Conclusions
This chapter presented the chemical analysis of different samples of yerba
mate. This research provided for the first time a comprehensive investigation of
the elemental composition of non-commercial, non-processed and processed
samples of yerba mate. Furthermore, this study provides an evaluation of the
elemental and polyphenolic levels of commercial samples and different infusions
methods of yerba mate from Brazil and Argentina. Finally, this study also
evaluated the potential contribution of the consumption of yerba mate to the
dietary intake of polyphenols and elemental nutrients. Manganese was
highlighted in this study due to its higher concentration in the yerba mate
samples and prepared infusions. As such, no study has assessed what the
potential intake of Mn would be through the consumption of different yerba mate
infusions in terms of the total dietary intake of this element by the South
American population.
The first investigation was to determine the element composition of non-
commercial yerba mate leaves collected from the Barão de Cotegipe plantation
in southern Brazil - April 2017 (refer to section 3.5). This pilot study was
undertaken at the producer’s request to evaluate whether growing yerba mate
trees within a native forest or in traditional plantations (including the use of
fertiliser or the impact of the age of leaves) would influence the elemental quality
of the yerba products. Overall, in terms of the age of the leaves (from trees
grown in both fertiliser and organic areas) collected from traditional plantations
(refer to section 3.5.3), the general trend was for the elements to be at higher
levels than in the old leaves (new < old; refer to Table 3.2). The next objective
was to evaluate the effect of yerba mate cultivation (with or without the use of
fertilsers) on the elemental levels of leaves (new or old). The new leaves grown
on trees from the organic plantation had higher levels of most of the elements,
especially Mn (a two-tailed Student t-test confirmed significantly higher levels at p
< 0.05) in comparison with the plantations treated with NPK fertilisers (new
leaves; fert < org). Conversely, for the older leaves from both plantations, all of
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the elements reported in Table 3.2 were higher in the fertilser-addition plantation
(old leaves; fert > org). An assessment of the elemental levels of leaves collected
at different heights of a yerba mate tree showed a variation between the bottom
(0.5 m), middle (1.5 m) and upper parts of the tree (2.5 m). Manganese values
confirmed a degree of translocation to the upper parts of the tree. The elemental
composition of non-commercial yerba mate samples cultivated between natural
forests was compared to samples from traditional plantations (refer to section
3.5.4). In general, higher elemental levels were found in plants grown in the
traditional organic plantations (refer to Table 3.6). In terms of the processing of
the yerba mate, the elements had higher elemental levels after the sapeco stage
(material is exposed to an open fire for a short period of time) of the process
(refer to section 3.5.5). Moreover, the post-drying samples (the last stage of the
processing plant involves the yerba mate material being dried until a moisture
content of 5 to 6 %) had lower levels of the elements when compared with the
sapeco yerba mate samples.
The trace elemental composition of commercial yerba mate products from
Brazil and Argentina was evaluated in terms of the type of sample (green or
roasted), processing package (loose or tea bags) and origin (Brazil or Argentina)
(refer to section 3.6.3). In general, the elemental levels for green loose yerba
mate products obtained from Brazil are slightly higher than that for the
Argentinian products. In summary, the elemental levels are basically similar for
the two countries, which is to be expected as yerba mate primarily grows
between the Parana and Paraguay river basins in South America (refer to Figure
3.1). The next study involved an evaluation of Argentinian commercial yerba
mate products based on the preparation and consumption of infusions using
green loose material or tea bags. All of the elements measured had higher levels
for the tea bag products when compared to the green loose material. This was
not surprising as the tea bags contain only leaf material and have been
processed to produce finer particles (of around 1 mm). It was also noted that the
green tea bags contain mostly leaf material with very few stems compared to the
loose yerba mate samples (refer to Figure 3.5). The total elemental levels of
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commercial green and roasted yerba mate samples from Brazil were also
evaluated in this study. The roasted (loose and tea bag) samples have higher
elemental levels when compared with the Brazilian green loose material (Table
3.9).
The elemental composition, total polyphenol content, chlorogenic acids,
caffeine and theobromine levels of commercial yerba mate products (from Brazil
and Argentina) prepared as infusions was evaluated in sections 3.6.4 and 3.6.5.
In relation to the country of origin of the yerba mate (Brazil and Argentina), the
regular infusions of yerba mate (green loose) from Brazil had slightly higher
chemical levels. This was in agreement with the reported results for the total
elemental content, presented in section 3.6.3. When comparing the chemical
content of a regular infusion associated with a single serving (cup of tea) using
green loose and tea bags from Argentina, there was a highly significant
difference (a two-tailed Student t-test) between the samples. All of the elements
(Table 3.11), polyphenols and xanthines (section 3.6.5) had higher levels for the
tea bag samples when compared to the green loose regular infusions. The
chemical analysis of regular infusions produced using green loose and roasted
samples from Brazil were evaluated, as presented in Table 3.11 and section
3.6.5. Interestingly, the regular infusions made with green loose yerba mate had
significantly higher levels (a two-tailed Student t-test) of trace elements,
polyphenols and xanthines.
Finally, an evaluation of the potential intake of trace elements and
polyphenols through the consumption of yerba mate products was proposed in
section 3.6.6. All the trace elements analysed in this study represent 0.1 to 5% of
the recommended daily allowance (RDA), for all the infusion methods (regular,
Brazilian and bombilla). An exception was found for manganese. A regular
infusion serving (1 cup of 200 mL) can provide 23.7 to 106.0 % for males and
30.3 to 135.5 % for females of the recommended daily intake of manganese,
depending on the type of yerba mate product (refer to Table 3.22). Although, in
terms of the bombilla method, the daily amount of yerba mate infusion consumed
is close to a volume of 1 litre. This results in Mn levels equating to all of the
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adequate recommended daily intake allowance and could provide concentrations
higher than the recommended upper consumption limit for this element per day
(refer to Table 3.22). In relation to the total polyphenol intake, a regular infusion
serving (1 cup of 200 mL) could contribute 4 to 14.5 % of the daily intake of total
polyphenols. Furthermore, the bombilla method (resulting in the drinking of about
1 litre of the infusion) can provide up to twice the amount of the adequate daily
intake of total polyphenols (refer to Table 3.23).
Finally, this study provides important new data about the chemical quality
of Brazilian and Argentinian yerba mate production and commercial products. It
is proposed that future research should evaluate the possible chemical
interactions between elements and organic compounds in the infusions, which
may influence the bioavailability of not only the elemental species, but also
provide another way of considering what may be the impact on human health
through consuming yerba mate (refer to chapter 6). Moreover, the data on
manganese levels in yerba mate infusions is worthy of further study especially in
relation to the drinking of other beverages, such as, coffee (refer to chapter 4) or
green and black tea.
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Chapter 4. Chemical Composition of Roasting Brazilian Coffee
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4.1. Introduction
Coffee is one of the most popular drinks in the World, with global trading
being worth more than 10 billion US dollars; the second most consumed
beverage after water and annual global consumption being approximately 500
billion cups (Clarke and Vitzthum, 2008, Butt and Sultan, 2011). Coffee is the
second largest commodity traded in the World, after petroleum (Butt and Sultan,
2011). The drink is prepared from the roasted seeds of the coffee plant; the
commercially important species being Coffea arabica L. (Arabica) and Coffea
canephora L. (variety Robusta) (Ludwig et al., 2014). It is often consumed, due to
its stimulatory effects, because of the high caffeine content present in the coffee
beans and related beverages. Furthermore, coffee is one of the largest
contributors, Worldwide, to the total dietary intake of polyphenols (Donnelly,
2015). This chapter presents an investigation of the chemical composition of the
roasting process of Brazilian coffee, using samples of small producers from
Amparo, São Paulo State, Brazil, according to the methodology described in
section 4.3.1. The results are presented in sections 4.4 and 4.5. An evaluation of
the physical changes of the beans during the roasting process is presented in
section 4.6. Finally, a study evaluating the link to dietary intake of elements and
total polyphenol derived from coffee infusions was performed and presented in
section 4.7, with the conclusions in section 4.8.
4.1.1. Coffee production in Brazil
The Coffea plant is native to tropical Africa, although more than 70
countries cultivate this plant, with Brazil, Colombia, Ethiopia and India being the
leading producers (Butt and Sultan, 2011). Brazil is the largest coffee producer,
responsible for a third of all coffee consumed Worldwide and the production is
mainly located in Minas Gerais, São Paulo, Espírito Santo and Bahia States
(Caldarelli et al., 2019). The main Coffea species in Brazil is Coffea arabica L.,
but there are several varieties (sub-species) cultivated through selective breeding
125
or natural selection of coffee plants. Such coffee varieties have different traits,
such as, bean size, yield, plant resistance against pathogens and maturation
stage (Clifford, 1985).
Brazilian coffee plantations are harvested during the dry season from June
to September, usually in one annual crop when the coffee berries are ripe, as
shown in Figure 4.1. In Brazil, due to the abundant sunshine, the fermentation of
the bean occurs when the berries are cleaned and dried under the sun for 8 to 10
days (depending on the weather conditions). The beans are further dried in rotary
dryer machines until a constant moisture content. The outer layer is then
removed, and the beans are selected and left to age for at least 6 months to
develop flavour. The green beans are then ready to be roasted and ground in
order to prepare the beverage. Brazil usually exports green coffee to be roasted
in the destination country.
Figure 4.1: Different maturation stages of the coffee cherry, showing green and
the mature red berries.
4.1.2. Roasting of coffee beans
The characteristic colour and aroma of coffee are produced during the
roasting process. Coffee oils, which accounts for 10% of the roasted beans, are
responsible for the aroma (Buffo and Cardelli‐Freire, 2004). The roasting process
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can be divided into three stages: (i) drying, where most of the moisture is
removed (endothermic); (ii) roasting, where numerous complex reactions take
place, changing the chemistry of the coffee beans, thereby also releasing a large
amount of carbon dioxide and producing hundreds of volatile compounds
related to the aroma and flavour of the coffee; and (iii) the cooling phase in order
to prevent the burning of the beans, using air as a cooling agent (Clifford, 1985,
Illy and Viani, 1995, Buffo and Cardelli‐Freire, 2004).
4.2. Review of Roasting Coffee in Brazil (Elemental and Polyphenols)
The chemistry of roasted coffee was investigated in the literature due to
the high popularity of the beverage all over the World. Furthermore, the
elemental content of roasted coffee in Brazil was investigated not only due to the
nutritional value, but also for food authenticity (Pohl et al., 2013). A review of
previous studies is presented in Table 4.1. All of these studies were performed
with different samples of roasted coffee. As a consequence, the elemental levels
vary, which could be influenced by different soil conditions, harvest periods,
cultivation and processing methods (Zeiner et al., 2015).
Among the organic compounds, xanthines (such as caffeine) and
antioxidant polyphenols may be responsible for the alleged health benefits
related to the consumption of coffee infusions. In previous studies, the total
content of polyphenols in coffee infusions was reported to be 0.96 – 2.27 g/L
(Lakenbrink et al., 2000). The major polyphenol compounds present in coffee are
the chlorogenic acid group, including caffeoylquinic acids, dicaffeoylquinic acids,
feruloylquinic acids and p-coumaroylquinic acids (Wei and Tanokura, 2015). The
roasting process has an impact on the amount of chlorogenic acids in the roasted
coffee beans. A reduction in the chlorogenic acid content has been reported as 8
to 10% for every 1% reduction in dry matter, resulting in losses of 60 to 70% for
medium roast and 90 to 95% for dark roast coffee. This results in the total
chlorogenic acid content of roasted coffee ranging from 1.8 to 80 mg/kg (Farah et
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al., 2005, Moon et al., 2009, Ferruzzi, 2010, Crozier et al., 2012, Ludwig et al.,
2014).
Table 4.1: Elemental content of roasted coffee beans for selected elements
reported in the literature. Adapted from Pohl et al. (2013).
Element Concentration (mg/kg) Reference
Mg 750 - 3100
(Martin et al., 1996, Martın et al., 1999, Suseela et al., 2001, Anderson and
Smith, 2002, Anthemidis and Pliatsika, 2005, Amorim Filho et al., 2007,
Grembecka et al., 2007, Santos et al., 2008, Ashu and Chandravanshi, 2011)
Ca 490 - 2200
(Martin et al., 1996, Martın et al., 1999, Anderson and Smith, 2002, Vega-
Carrillo et al., 2002, Anthemidis and Pliatsika, 2005, Amorim Filho et al., 2007,
Grembecka et al., 2007, Tagliaferro et al., 2007, Santos et al., 2008, Suseela
et al., 2001, Ashu and Chandravanshi, 2011)
Mn 6.6 – 320.0
(Martin et al., 1996, Martın et al., 1999, Suseela et al., 2001, Anderson and Smith, 2002, Anthemidis and Pliatsika, 2005, Zaidi et al., 2005, Amorim Filho
et al., 2007, Grembecka et al., 2007, Tagliaferro et al., 2007, Santos et al.,
2008, Ashu and Chandravanshi, 2011)
Fe 12.0 - 617.0
(Martin et al., 1996, Martın et al., 1999, Suseela et al., 2001, Anderson and Smith, 2002, Anthemidis and Pliatsika, 2005, Zaidi et al., 2005, Amorim Filho
et al., 2007, Grembecka et al., 2007, Tagliaferro et al., 2007, Santos et al.,
2008, Ashu and Chandravanshi, 2011)
Cu 0.4 - 30.1
(Martın et al., 1999, Suseela et al., 2001, Anderson and Smith, 2002, Anthemidis and Pliatsika, 2005, Amorim Filho et al., 2007, Grembecka et al.,
2007, Santos et al., 2008, Ashu and Chandravanshi, 2011)
Zn 1.2 – 803.0
(Martın et al., 1999, Suseela et al., 2001, Anderson and Smith, 2002, Vega-
Carrillo et al., 2002, Anthemidis and Pliatsika, 2005, Zaidi et al., 2005, Amorim
Filho et al., 2007, Grembecka et al., 2007, Tagliaferro et al., 2007, Santos et
al., 2008, Ashu and Chandravanshi, 2011)
4.3. Coffee Beans and the Roasting Process at Amparo, São Paulo State, Brazil
An evaluation of the chemical effects of the roasting process for Brazilian
coffee was selected as the main topic of research in this study. Therefore, it was
important to analyse samples from the same location in Brazil and using a
specific roasting method. The samples were collected during two field-trips in
April 2017 and 2018, as outlined in section 4.3.1. The coffee beans were roasted
and ground following the procedure described in sections 4.3.2 and 4.3.3. Finally,
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coffee infusions were prepared in order to simulate the typical Brazilian
consumption of a cup of coffee, as described in section 4.3.4.
4.3.1. Sample collection and preparation of coffee beans
Green coffee samples were collected from two different plantations at
Amparo, northern São Paulo State, namely, Fazenda Palmares and Fazenda
Flor. The samples collected at Fazenda Palmares were from the varieties and
harvesting dates: (i) Catuaí from the 2017 harvest; (ii) Bourbon Amarelo from the
2017 harvest; and (iii) a blend of the different varieties from the previous harvest
in 2016. Fazenda Flor plantation provided a green coffee sample from the Obatã
variety (2017 harvest). The green coffee beans were produced according to the
Brazilian standard method (refer to section 4.1.1) where the mature coffee
cherries were harvest and fermented under the sun, dried until constant moisture
content in rotary dryers and aged for 6 months. The green coffee samples were
then roasted.
4.3.2. Roasting process
The green coffee bean samples were roasted in a commercial IR-5
Diedrich® Roaster at Fazenda Palmares and the samples were collected every 2
minutes over a total 10 minute period, which is equivalent to a bean colour
spectrum from green to medium and finally dark roast, as shown in Figure 4.2.
The roaster was set at 200 °C and when the green beans are added there is a
slight decrease in temperature. Throughout the roasting process the temperature
quickly returns to the set temperature and the only changes that occur are
related to the air flow through the roaster. After 10 minutes, the coffee was
cooled and the beans were manually selected. This procedure was performed by
a trained plantation worker. The final roasted beans were chosen based on the
commercial value of the beans, that is, the quality of the product based on size,
colour and an absence of any cracked bean defects. Also, the blended bean
129
sample from the Fazenda Palmares plantation was roasted for only 6 minutes to
produce the medium roasted sample and at 10 minutes to produce the dark
roasted sample.
Figure 4.2: Roasting of Brazilian coffee beans from green (time = 0 minutes) and
at 2 minutes intervals until the production of the dark roasted product
(t = 10 minutes).
4.3.3. Grinding roasted coffee beans and particle size
The blended coffee beans from the Fazenda Palmares plantation that
were roasted at 6 and 10 minutes (medium and dark roast), were ground in a
commercial Bunn® Coffee Mill, that grinds the coffee according to the infusion
method, namely: (1) coarse for French press; (2) regular for siphon; (3) electric
perk; (4) drip; (5) fine for Brazilian infusions; and (6) espresso. The roasted bean
particle sizes decrease with the infusion method (1 to 6).
4.3.4. Coffee infusions
The coffee infusions were prepared following the Brazilian traditional
method of consumption which is a percolate method where the hot water (boiled)
is put on the coffee and filtered by a paper filter, as shown in Figure 4.3. The
coffee to water ratio was used as set by the Specialty Coffee Association of
America (SCAA), being a standard cup of coffee. Double distilled deionised water
(DDW) was preferred instead of tap water for all infusions in order to evaluate the
130
chemical contribution from only the coffee. A volume of 215 mL of freshly boiled
DDW was added to 7.00 ± 0.05 g of freshly ground coffee, on a commercial filter
paper supported by a plastic Melitta® percolator, as shown in Figure 4.3. The
samples were left to cool down to room temperature and each final solution was
filtered using a 0.45 μm filter. The resultant solutions were analysed for elemental
and polyphenol levels (refer to sections 4.4 and 4.5).
Figure 4.3: Typical Brazilian coffee infusion method.
4.4. Elemental Levels of Roasted Coffee
The elemental levels found in the roasted coffee beans, collected as
described in section 4.3 and analysed by inductively coupled plasma mass
spectrometry (section 2.3), are reported in Table 4.2 for the Obatã, Table 4.3 for
the Catuaí and Table 4.4 for the Bourbon Amarelo coffee varieties.
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Table 4.2: Elemental levels (mg/kg, dry weight) of Brazilian coffee beans
sampled at different roasting times (minutes). The samples were of
the Obatã coffee variety collected from the Fazenda Flor plantation
(Amparo, São Paulo State) and analysed by ICP-MS (refer to section
2.3). Analysis in duplicate.
Elemental levels of Obatã (mg/kg, d.w.) Roasting time (min) Ca Mg Mn Fe Cu Zn
0 1025.3 1827.0 30.71 27.88 14.21 10.65
2 1261.9 1925.3 38.77 38.08 13.82 7.04
4 788.5 1733.9 29.46 25.94 13.08 8.00
6 1011.4 1759.9 33.80 24.47 13.21 8.42
8 778.9 1832.2 30.73 30.19 16.36 8.56
10 1016.9 2012.2 30.99 30.41 14.43 6.21
Table 4.3: Elemental levels (mg/kg, dry weight) of Brazilian coffee beans
sampled at different roasting times (minutes). The samples were of
the Catuaí coffee variety collected from the Fazenda Palmares
plantation (Amparo, São Paulo State) and analysed by ICP-MS (refer
to section 2.3). Analysis in duplicate.
Elemental levels of Catuaí (mg/kg, d.w.) Roasting time (min) Ca Mg Mn Fe Cu Zn
0 1776.4 1937.3 27.44 23.04 12.65 6.21
2 1409.3 1971.5 25.00 22.06 13.01 6.88
4 1512.1 1940.5 26.71 22.02 12.17 7.29
6 1324.2 1847.2 24.76 22.11 11.67 11.77
8 1401.9 2009.3 32.34 26.81 13.27 8.83
10 1552.3 2256.9 25.52 27.71 13.61 7.56
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Table 4.4: Elemental levels (mg/kg, dry weight) of Brazilian coffee beans
sampled at different roasting times (minutes). The samples were of
the Bourbon Amarelo coffee variety collected from the Fazenda
Palmares plantation (Amparo, São Paulo State) and analysed by ICP-
MS (refer to section 2.3). Analysis in duplicate.
Elemental levels of Bourbon Amarelo (mg/kg, d.w.) Roasting time (min) Ca Mg Mn Fe Cu Zn
0 1652.7 1842.3 18.32 24.88 15.23 7.88
2 1428.7 1914.7 25.75 24.69 14.14 7.99
4 1521.8 1924.6 22.93 26.67 17.96 6.59
6 1255.1 1908.0 19.82 27.12 17.87 6.09
8 1517.4 1888.2 23.75 25.45 17.48 5.68
10 1619.9 2141.6 26.60 29.95 16.47 10.6
The elemental content of the different coffee bean varieties (Obatã, Catuaí
and Bourbon Amarelo) fluctuates throughout the roasting period but the final
roasted bean product has either the same or a slightly higher elemental content
(mg/kg, dry weight). This is as would be expected, since all of the green bean
samples (t = 0 minutes) were sun-dried at the plantation and throughout the
roasting process there may be a slight modification in the residual moisture
levels. The final roasted beans (subjected to 200 °C) seem to result in a ‘pre-
concentration’ of the elemental levels, thereby leading to a slightly higher
elemental level for the roasted coffee product. The same observation was found
for the other trace elements, as reported in Appendices 4.2 to 4.4. In summary,
this data clearly shows there is no reduction in the elemental content (in fact a
slight increase) of the coffee beans as a result of the roasting process.
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Table 4.5: Elemental concentration (mg/kg, dry weight) of roasted Brazilian
coffee beans. The different coffee varieties include those selected for
their quality (section 4.3.2) or as defected beans and were collected
from the Fazenda Palmares and Flor plantations (Amparo, São Paulo
State) and analysed by ICP-MS (refer to section 2.3). Analysis in
duplicate.
Bean variety and method of selection (mg/kg, d.w.) Ca Mg Mn Fe Cu Zn
Obatã selected 1016.9 2012.2 30.99 30.41 14.43 6.21
Obatã defected 1140.2 1698.4 31.14 34.05 14.14 5.95
Catuaí selected 1552.3 2256.9 25.52 27.71 13.61 7.56
Catuaí defected 1627.7 2281.7 23.12 29.00 14.74 7.83
Bourbon Amarelo selected 1619.9 2141.6 26.60 29.95 16.47 10.60
Bourbon Amarelo defected 1580.4 2182.6 24.72 40.26 21.66 9.48
Table 4.5 clearly shows that the selection of the final coffee beans (post
the roasting period of 10 minutes) does not influence the elemental levels of the
coffee beans (selected – based on quality factors as outlined in section 4.3.2).
There are also only small differences in specific elemental levels for the different
coffee bean varieties (which may be related to the fact that beans were collected
from different plantations).
134
4.5. Total Polyphenol and Chlorogenic Acid Levels in Roasted Coffee (infusions)
Table 4.6 reports the total polyphenol levels (mg/L) in infusions produced
from the different varieties of coffee beans (Obatã, Catuaí and Bourbon Amarelo)
collected from the Fazenda Palmares and Flor plantations (Amparo, São Paulo
State). The data are reported as a function of the different roasting times (0 to 10
minutes). The total polyphenol levels were determined using the Folin-Ciocalteu
assay (refer to section 2.4).
Table 4.6: Total polyphenol content (mg/L) of Brazilian coffee infusions during
roasting time (minutes). The samples were from the different coffee
varieties from Fazenda Palmares and Flor (Amparo, São Paulo State)
and analysed by UV-Vis (refer to section 2.4). n = 1.
Roasting time (min) Total polyphenol of the coffee variety (mg/L)
Obatã Catuaí Bourbon Amarelo 0 661 859 712
2 764 967 794
4 1134 947 1173
6 962 1084 973
8 1038 1119 912
10 1004 916 963
In general, the effect of roasting the green coffee beans increases the total
polyphenol content until about 4 to 6 minutes (roasting time). Then there is a
slight reduction (depending on the variety of coffee bean) to a final roasted bean
level that is ~ 7% (Catuaí) to 52 % (Obatã) higher than the levels in the green
beans (t = 0 minutes). A possible explanation for this was investigated using
scanning electron microscopy (SEM), as presented in section 4.6, below.
The chlorogenic acids and caffeine levels were also measured using ultra-
high performance liquid chromatography (UHPLC), in the coffee bean samples
135
collected throughout the roasting process (section 2.5). Figure 4.4 reports the
levels of 3-, 4-, or 5- caffeoylquinic acid and caffeine (mg/L) in infusions prepared
from coffee at the different stages of roasting, namely, green (t = 0 minutes),
medium (t = 6 min) and dark roasted (t = 10 min). The samples were a blend of
the coffee varieties collected from the Fazenda Palmares plantation (Amparo,
São Paulo State). The overall trend is similar to that found for the total
polyphenol content (Table 4.6) where there is an increase in the chlorogenic acid
and caffeine levels of the infusions prepared using the freshly ground medium
roast coffee. Furthermore, the dark roast product showed lower levels than the
green coffee for the chlorogenic acids and caffeine. A further evaluation of the
roasting process with selected coffee varieties and time intervals was also
undertaken.
Figure 4.4: Concentration of chlorogenic acids and caffeine (mg/L) of Brazilian
coffee infusions produced from beans sampled during the roasting
process (green t = 0 minutes, medium t = 6 min and dark roasted t =
10 min). The samples were the blend of the coffee varieties collected
from the Fazenda Palmares plantation (Amparo, São Paulo State)
and analysed by UHPLC (refer to section 2.5). n = 1 due to the
limited amount of available sample.
Figures 4.5, 4.6 and 4.7 report the levels of chlorogenic acids and caffeine
(mg/L) for the coffee bean samples collected at 2 minute intervals throughout the
Green coffee Light roast Dark roast0
100
200
300
400
500
Roasting colour
Co
ncen
trati
on
(m
g/L
)
3- caffeoylquinic acid
4- caffeoylquinic acid
5- caffeoylquinic acid
caffeine
136
roasting process. All samples were analysed using UHPLC (refer to section 2.5).
The samples were the Obatã (collected from Fazenda Flor and presented in
Figure 4.5), Catuaí and Bourbon Amarelo coffee varieties (collected from
Fazenda Palmares and presented in Figures 4.6 and 4.7, respectively). The
roasting process did not present a significant effect on the caffeine levels.
Interestingly, the chlorogenic acids follow the same trend as outlined for the total
polyphenol levels (Table 4.6 and Figure 4.4), with the levels being higher in the
samples taken during the middle of the roasting process. This is more
predominant for the levels of 5-caffeoylquinic acid, but 3- and 4- caffeoylquinic
acids also show a similar trend at 8 minutes of the roasting time (but of less
magnitude). This study contradicts what has been reported in the literature which
mainly describes the loss of chlorogenic acids in coffee during the roasting
process (Trugo et al., 1985, Bennat et al., 1994, Schrader et al., 1996). It has
been suggested that this may be due to the breakage of the carbon-carbon
bonds with the high roasting temperatures, causing isomerisation (refer to
glossary) or degradation of the compounds (Farah et al., 2005). The peaks
associated with the 3- and 4 - caffeoylquinic acids have already been reported for
the medium roast conditions and it has been suggested that this is due to a
partial hydrolysis of di- caffeoylquinic acids and the isomerisation of the 5-
caffeoylquinic acid, which implies a decrease in the amount of this compound
(Trugo et al., 1985, Farah et al., 2005). An increase in the levels of the total
caffeoylquinic acids after the beginning of the roasting process has also been
reported as a consequence of ‘pre-concentration’ during the roasting process
(loss of moisture content and volatile compounds) (Fujioka, 2006). The difference
between this study and the literature is that this research involved the analysis of
coffee infusions in order to simulate the chemical intake of a cup of coffee,
whereas other studies analysed the coffee bean extractions undertaken using
organic solvents (Donnelly, 2015). This study also considered the physical effect
and the pore size associated with the coffee roasting process, as discussed in
section 4.6.
137
Figure 4.5: The concentration of chlorogenic acids and caffeine (mg/L) of
Brazilian coffee infusions produced from beans sampled during the
roasting process (t = 0 to 10 min). The samples were of the Obatã
coffee variety collected from Fazenda Flor plantation (Amparo, São
Paulo State) and analysed by UHPLC (refer to section 2.5). n = 1.
Figure 4.6: The concentration of chlorogenic acids and caffeine (mg/L) of
Brazilian coffee infusions produced from beans sampled during the
roasting process (t = 0 to 10 min). The samples were of the Catuaí
coffee variety collected from the Fazenda Palmares plantation
(Amparo, São Paulo State) and analysed by UHPLC (refer to
section 2.5). n = 1.
0 2 4 6 8 100
500
1000
1500
Roasting time (min)
Co
ncen
trati
on
(m
g/L
)
3- caffeoylquinic acid
4- caffeoylquinic acid
5- caffeoylquinic acid
caffeine
0 2 4 6 8 100
500
1000
1500
Roasting time (min)
Co
ncen
trati
on
(m
g/L
)
3- caffeoylquinic acid
4- caffeoylquinic acid
5- caffeoylquinic acid
caffeine
138
Figure 4.7: The concentration of chlorogenic acids and caffeine (mg/L) of
Brazilian coffee infusions produced from beans sampled during the
roasting process (t = 0 to 10 min). The samples were of the
Bourbon Amarelo coffee variety collected from the Fazenda
Palmares plantation (Amparo, São Paulo State) and analysed by
UHPLC (refer to section 2.5). n = 1.
The effect of manually selecting the final coffee beans as an estimate of
product quality (roasting time of 10 minutes, refer to section 4.3.2) was also
evaluated for the polyphenol and caffeine content, as shown in Table 4.7.
Interestingly, the total polyphenol levels decreased in the infusions prepared with
the defected beans. In relation to the levels of chlorogenic acids and caffeine,
there is no particular effect associated with the selection of the beans (based on
quality) after the roasting process.
0 2 4 6 8 100
500
1000
1500
Roasting time (min)
Co
ncen
trati
on
(m
g/L
)
3- caffeoylquinic acid
4- caffeoylquinic acid
5- caffeoylquinic acid
caffeine
139
Table 4.7: The concentration of chlorogenic acids, caffeine and total polyphenol
(mg/L) of roasted Brazilian coffee infusions. The samples were
prepared from manually selected and defected beans (section 4.3.2)
from different coffee varieties collected from the Fazenda Palmares
and Flor plantations (Amparo- São Paulo) and analysed by UHPLC
(refer to section 2.5). n = 1.
Coffee varieties (mg/L) Total polyphenol
3-caffeolquinic acid
4-caffeolquinic acid
5-caffeolquinic acid Caffeine
Obatã selected 1004 189 226 424 331
Obatã defected 675 90 111 746 235
Catuaí selected 916 59 58 89 269
Catuaí defected 749 74 74 104 264
Bourbon Amarelo selected 963 142 165 296 265
Bourbon Amarelo defected 765 151 170 277 247
The effect of the roasted coffee particle size on the concentration of
chlorogenic acids and caffeine was investigated, as outlined in section 4.3.3.
Figure 4.8 reports the levels of those compounds measured in samples
associated with the medium roast period (t = 6 minutes) and Figure 4.9 for the
dark roast period (t = 10 minutes). It is clear that the roasted coffee particle size
has an influence on the extraction of the analysed chemical compounds. The
efficiency of the extraction is indirectly proportional to the particle size of the
roasted bean product used in the infusions. Interestingly, the behavior of the
efficiency trend changes with the roasted product, being an exponential curve for
the medium roast infusions. A similar, but more linear trend was found for the
dark roast infusions. This could be explained by the physical differences in the
coffee pores produced in the beans during the roasting process, as described in
section 4.6.
140
Figure 4.8: The concentration of chlorogenic acids and caffeine (mg/L) of
Brazilian coffee infusions as a function of the different bean particle
sizes according to the method of infusion: (1) coarse for French
press; (2) regular for siphon; (3) electric perk; (4) drip; (5) fine for
Brazilian infusions; and (6) espresso. The samples were collected
at the medium roast time of the process (t = 6 minutes), being a
blend of the coffee varieties sampled from the Fazenda Palmares
plantation (Amparo- São Paulo) and analysed by UHPLC (refer to
section 2.5). n = 1.
0 1 2 3 4 5 60
200
400
600
Particle size
Co
ncen
trati
on
(m
g/L
)
3- caffeoylquinic acid
4- caffeoylquinic acid
5- caffeoylquinic acid
Caffeine
141
Figure 4.9: The concentration of chlorogenic acids and caffeine (mg/L) of
Brazilian coffee infusions as a function of the different bean particle
sizes according to the method of infusion: (1) coarse for French
press; (2) regular for siphon; (3) electric perk; (4) drip; (5) fine for
Brazilian infusions; and (6) espresso. The samples were collected at
the dark roast time of the process (t = 10 minutes), being a blend of
the coffee varieties from the Fazenda Palmares plantation (Amparo-
São Paulo) and analysed by UHPLC (refer to section 2.5). n = 1.
4.6. Effect of Pore Size of Ground Roasted Coffee
It has been reported that during the roasting process of coffee beans there
is a loss of water. Moreover, there is a release of gases associated with a high
internal pressure within the bean that changes the volume and porosity of the cell
walls (Schenker et al., 2000). The structure of the coffee beans collected during
the roasting process was evaluated through a series of scanning electron
microscope (SEM) images. The SEM measurements were performed on a Jeol®
JSM-7100F. Figure 4.10 (A) to (F) shows the changes in the physical structure of
the beans, (a blend of the coffee varieties collected from the Fazenda Palmares
0 1 2 3 4 5 60
100
200
300
Particle size
Co
ncen
trati
on
(m
g/L
)
3- caffeoylquinic acid
4- caffeoylquinic acid
5- caffeoylquinic acid
Caffeine
142
plantation, Amparo, São Paulo State). The samples were collected at 2 minute
roasting intervals and range from: (A) 0 to (F) 10 minutes. It is clear from the
images that the structure of the coffee bean changes during the roasting process
and the pores become larger. During the roasting, there is an expansion of the
coffee bean and micropores, and therefore a decrease in the coffee density
(Jokanovic et al., 2012). The change in the porosity of the beans may have an
impact on the efficiency of the chemical extraction in the coffee infusions, as
reported in sections 4.4 and 4.5. It has been suggested that the presence of fine
micropores associated with roasting-induced changes of the coffee bean, can
allow the mobilised coffee oil to migrate to the bean surface (Schenker et al.,
2000). Moreover, the volume increase of the bean and development of pores
during roasting are known to be highly dependent on the roasting conditions
(Ortolá et al., 1998). What has not been reported until this research study is the
effect this has on the levels of chemicals in the coffee beans that are available in
the associated infusions.
(A) t = 0 min
143
(B) t = 2 min
(C) t = 4 min
144
(D) t = 6 min
(E) t = 8 min
145
(F) t = 10 min
Figure 4.10 (A) to (F): Scanning electron microscope images of Brazilian coffee
beans sampled during the roasting process (t = 0 to 10
min). The samples were the blend of the coffee varieties
collected from the Fazenda Palmares plantation (Amparo,
São Paulo State).
4.7. Chemical Composition of Roasted Coffee Infusions and Human Dietary Intake
This section evaluates the levels of total polyphenols in the prepared
roasted coffee infusions and the effect on human dietary intake. Since there are
no regulatory values for chlorogenic acids, these chemicals were not included in
the study. Moreover, no data is available for the elemental levels of the roasted
coffee infusions as the diluted solutions caused a significant reduction in the ICP-
MS instrument performance during sample analyses. This was associated with
the high dissolved solids content and ‘colour chemicals’ blocking the sample
injector (of the ICP) and the interface cones and skimmers (refer to section 2.3).
146
The average annual consumption of coffee by the Brazilian population
was 839 cups (each of 40 mL) per person in 2018 (ABIC, 2018). This suggests
that the daily Brazilian consumption is an average of 92 mL of coffee per day.
The calculated daily consumption of 1492 mg (fresh weight) of polyphenols,
based on a balanced Japanese diet was used to evaluate the potential intake
from coffee of these compounds to the Brazilian diet (Fukushima et al., 2009).
Table 4.8 compares the different roasting times of the coffee beans used to make
infusions in relation to the daily intake of total polyphenols. An average daily
consumption of coffee (92 mL) could contribute 4 to 7 % of the daily intake of
total polyphenols. This range is a higher contribution when compared to the
corresponding values for the consumption of yerba mate in Brazil (refer to
section 3.6.6.2), green or black tea, and fruit (grape, apple and orange) juices
(Donnelly, 2015).
Table 4.8: Percentage intake (%) of total polyphenol based on the Brazilian daily
consumption (92 mL) of coffee infusion. The data is compared with
the values reported by Fukushima et al. (2009) for the daily intake of
total polyphenols.
Roasting time (min) Daily percentage intake of total polyphenols (%)
Obatã Catuaí Bourbon Amarelo
0 4.1 5.3 4.4
2 4.7 6.0 4.9
4 7.0 5.8 7.2
6 5.9 6.7 6.0
8 6.4 6.9 5.6
10 6.2 5.6 5.9
4.8. Summary
Coffee is one of the most popular beverages that is consumed all over the
World. Brazil is the largest producer and exporter of green coffee beans. At
147
present the roasting of green coffee beans is only done on a small scale in local
regions of Brazil, and other countries import the beans and produce their own
commercial roasted coffee products. In Brazil, little is known about the impact of
the coffee roasting process on the chemical composition of the roasted beans
and related beverages. A series of studies on the roasting process and effect on
the elemental and polyphenol content was undertaken in this chapter. The effect
of roasting different coffee varieties (Obatã, Catuaí, Bourbon Amarelo and a
blend) collected from the Fazenda Palmares and Flor plantations (Amparo, São
Paulo State) resulted in a slightly increase in the elemental content during the
roasting process (Tables 4.2, 4.3 and 4.4). This data suggests that there is a
small decrease in the moisture content (‘pre-concentration’) of the beans and
highlights that no elemental losses were found during the roasting process. Any
measured elemental variation, especially for Ca, Mg, Cu and Zn, may be linked
to the soil chemistry or growing features of a specific plantation site from which
the green coffee beans were collected (section 4.4).
The total polyphenol content of the coffee infusions produced from beans
sampled during the roasting time, was 7 to 52 % higher for dark roast (10
minutes) when compared to the levels in infusions produced from green beans (t
= 0 minutes) (Table 4.6). The chlorogenic acid and caffeine analysis showed a
similar trend where there was an increase in the chemical levels of the infusions
prepared using the medium roast coffee (refer to section 4.5; Figures 4.4 to 4.7).
The effect of the roasted coffee particle sizes on the concentration of chlorogenic
acids and caffeine in the infusions was investigated (section 4.5). A strong
inversely proportional relationship existed between the roasted bean particle
sizes and the chemical concentrations of the resultant infusions (Figures 4.8 and
4.9). A manual selection of the good or defected beans, post the roasting
process of 10 minutes (refer to section 4.3.2), did not result in any major
differences in the elemental or chlorogenic acid levels of the roasted coffee
product (Tables 4.5 and 4.7). Although, the selection of higher quality beans
(refer to section 4.2) did result in higher total polyphenol levels for the resultant
coffee infusions. Any possible physical structure changes of the coffee beans
148
were investigated and the scanning electron microscopy (SEM) images showed
that during the roasting process there is an expansion of the coffee bean
resulting in the pores becoming larger (Figures 4.10 (A) to (F)). Finally, an
evaluation of the potential intake of total polyphenols from these coffee infusions
showed that based on the average daily consumption of roasted coffee, as an
infusion, in Brazil (92 mL), would lead to a 4 to 7 % estimated daily intake of total
polyphenols. This means that the daily consumption of coffee as an infusion in
Brazil would lead to an average of 92.96 mg/92 mL intake of total polyphenols
(medium roast, t = 6 min) that is higher than yerba mate, green and black tea.
The total polyphenol content of the Coffea canephora L. produced in Vietnam
was reported to be slightly higher than the Coffea arabica L. from Brazil and
roasted in Europe or USA (Hečimovic et al., 2011).
In recent years another Brazilian fruit has gained international popularity
as a ‘super-fruit’, namely, açaí (Euterpe oleracea), which will be evaluated in
terms of the chemical composition, so it can be evaluated against yerba mate
(chapter 3) and coffee (chapter 4) as a daily dietary sources of these chemicals
for the Brazilian population.
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Chapter 5. Brazilian Açaí
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5.1. Introduction
The açaí berry, a native fruit from the Amazonian region of Brazil, has
recently become very popular due to its status as a ‘super-fruit’ (refer to
glossary). In this research, an investigation of the relationship between the
chemical composition and biological activity of açaí samples was performed in
February 2018, using non-commercial (Amazon) and commercial açaí samples
obtained from outlets in Brazil (São Paulo) and the United Kingdom (UK). All
samples were analysed for the polyphenol and elemental content using the
methodologies outlined in section 5.5. The results are also presented in section
5.7. Furthermore, an evaluation of the impact that Amazonian geographical
variability and commercial processing has on the chemical composition of açaí
was carried–out in April 2018. Samples were collected along the Amazonas river
delta, located in the Pará State, Brazil, according to the methodology described
in section 5.6. The results are presented in section 5.8.
5.2. General Introduction to Açaí Berries from the Amazon Region, Brazil
Natural occurrence
The açaí palm (Euterpe genus) is a native tree from the Amazon region,
northern South America, which has 28 known species, though only two, namely
Euterpe oleracea and Euterpe precatoria. (Yamaguchi et al., 2015) are used for
commercial products. One of the differences between the species, besides the
size and format of the tree and leaves, is where they are grown (refer Figure 5.1).
In general, E. oleracea is a native palm from the Amazonas river estuary, usually
found in flooded forest areas alongside the river (Lee et al., 1998). This species
is the most commercially valuable due to the consumer response regarding
product taste or colour (Schauss et al., 2006a).
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Figure 5.1: (A) Map of South America with Amazon forest in green (Fao, 2015);
and (B) Natural botanical distribution of two different species of açaí,
namely, Euterpe precatoria and Euterpe oleracea. Adapted from
Yamaguchi et al. (2015).
Alternatively, E. precatoria is prevalent in the Amazonas river basin, which
is closer to the Equatorial line (Pacheco-Palencia et al., 2009). The palm tree of
both species can grow up to 25 meters in height (Lee and Balick, 2008) and its
fruit is a black-purple berry, measuring from 0.9 to 1.3 cm in diameter. The seed
accounts for 80 to 95% (in volume) of the fruit and the edible purple mesocarp is
only 1 to 2 mm in thickness (Pompeu et al., 2009), as shown in Figure 5.2. Even
though the dark purple berry is the most common açaí variety, there are also
other naturally occurring varieties (Oliveira et al., 2002), such as the white açaí
berry, where the ripe fruit is ‘greenish’ in colour after maturation (Rogez, 2000, da
Silveira et al., 2017).
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Figure 5.2: (A) The natural occurrence of açaí (E. oleracea) in the flooded forest
near Belém, Para State, Brazil; (B) the purple açaí fruit (pulp and
seed separated); and (C) the white açaí (‘greenish’) berries. Adapted
from Potsch (2010).
The açaí berry is a key cultural symbol of the Amazonian region and has
often been referred to as “black gold” since pre-colonisation ages (Andrade,
2014). For many years açaí has been an important dietary source of nutrients for
both urban and rural Amazonian communities, contributing up to 43% of their
dietary dry weight basis and 30% of their energy intake (Heinrich et al., 2011). It
is normally consumed as a side dish for fish, prawns and tapioca (manioc flour).
Interestingly, not only are the berries consumed but other parts of the palm tree
are used for medicinal purposes, namely, the roots, ‘heart of the palm’, leaves
and seeds (Yamaguchi et al., 2015).
Brazilian açaí production and products
The production of açaí, which is an extractive activity from the floodplain of
the Amazon forest, has been constantly growing in the past 20 years, due to its
increased popularity for human consumption, and the manufacture of cosmetic
products (Homma et al., 2006, Maciel et al., 2018, Tagore et al., 2018).
Nowadays, açaí is not only consumed by locals of the northern regions of Brazil,
but it has gained popularity in South America and internationally due to its
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classification as a ‘super-fruit’ (Schauss, 2013). Brazil is the main producer and
exporter of açaí, generating an estimated monetary source of 9 billion US dollars
per year (IBGE, 2017), exceeding the price per ton of soybeans and Brazil nuts
(Yamaguchi et al., 2015). Açaí is considered to be the most important export
product of the Amazon estuary. It is estimated that 200,000 tonnes of açaí are
extracted per year, and the industry is projected to grow by more than 12% over
the period from 2017 to 2025 (Research, 2018). The demand for açaí has been
increasing in southern Brazil and it is usually linked with the younger population
due to its appeal as an energetic, health and nutritional product (Rogez, 2000).
As a result of its increasing value in the market and the low processing yield (due
to the mass of the seed per berry), the processing of açaí berries can lead to
adultered products, especially for economic gain. The palm is also a source of
‘palm hearts’ (the inner core and growing bud of the tree) for the food industry,
where Brazil is the major exporter of this highly valued product. However, the
harvesting of this food source is also a threat to the production of the açaí berry,
since the extraction of the palm hearts can potentially damage the entire tree
(Jardim, 2002).
The present demand for açaí is mainly for the food industry, but it is
expected that other activities, such as the development of nutraceuticals (refer to
glossary), cosmetics and personal care products, will increase significantly in the
near future (Research, 2018). Recent studies reporting the anti-aging properties
of the berries and açaí extracts or oils have led to the increased use of these
materials in cosmetics, such as, anti-wrinkle and body hydrating creams or
products that prevent cutaneous disorders (Herculano, 2013). According to
published details Córdova-Fraga et al. (2004), açaí has been reported to have
potential as a contrast agent for the magnetic resonance examination of the
gastrointestinal tract (primarily linked to the non-toxicity properties of the berries).
The harvesting of açaí berries is still manually undertaken by local people
that go inside the forest, climb the trees and bring the berries to the city by boat
to sell (as shown in Figure 5.3). This harvesting activity, coupled with the natural
occurrence of açaí being in the flooded areas of the delta, restricts the production
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of the berry to the dry season of the Amazon region, namely, from June to
November (Rogez, 2000). After harvesting, the açaí berries have to be
processed within 24 hours in order to prevent oxidation of the berries. The fruits
are transferred to a processing plant where they are selected according to the
maturation state, size and then washed twice with water in order to remove any
other material (twigs and leaves). Then, the fruits are usually left in a warm water
tank to soften the pulp. The berries are transferred to a specific machine
(despolpadeira PT) as shown in Figure 5.3. This acts like a mild blender that
removes the softened pulp from the seeds with the assistance of a certain
amount of added water. The final product is then packed, ready to be frozen and
consumed mainly by the beverage industry as frozen pulp, or as a smoothie and
as juice. The seeds are treated as a by-product of the industry, or used as
biomass or as a natural fertiliser in the plantation (Schauss et al., 2006b,
Pacheco-Palencia et al., 2008, Pacheco-Palencia et al., 2009, Schauss, 2013).
Açaí pulp can also be dehydrated using the process of lyophilisation of the
product (Carneiro et al., 2015). The resultant açaí powder is used for many
applications, such as, food supplements or additives, and as a natural colour
agent (Bobbio et al., 2000).
The processing of the açaí berries may also include a decontamination
step, which involves an additional washing step with an ozonated or hypochlorite
solution, or pasteurisation of the final product. The latter is the preferred method
for the international market. This is undertaken by changing the temperature
which leads to the oxidation of the pulp, thereby changing the colour and taste of
the final product. This step became important after some reported cases in the
Amazon region relating to the consumption of açaí berries with Chagas disease,
a tropical parasitic condition. This disease is transmitted by a protozoa parasite
(Trypanosoma cruzi), present in the faeces of the insect barbeiro (Tiatoma
brasiliensis) (Hamilton et al., 2012).
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Figure 5.3: (A) Harvesting of açaí by locals in the Amazon; and (B) a
‘despolpadeira’, machine used to mechanically extract the pulp of
the açaí berries. Adapted from Vida (2010).
The amount of added water is determined by the market requirements and
is classified according to a Brazilian regulation, based on the solid content of the
final product: (i) thin or popular açaí from 8 to 11% of total solids; (ii) regular or
medium açaí from 11 to 14% of total solids; and (iii) special or thick açaí with
more than 14% of total solids (Rogez, 2000, Homma et al., 2006). Furthermore,
due to the limited regulation control of the açaí processing, each açaí company
has their own method of processing the berries. This may be based on a
separate cleaning stage, where the temperature of the water is changed, or the
acidity of the final product is modified by adding citric acid, or further
pasteurisation is undertaken, as presented in Figure 5.11. Nowadays, with the
increasing demand of açaí, there is a need for quality standards to be used in the
production of the fruit, especially for pulp that is targeted for the international
markets (Pagliarussi, 2010).
Health effects of açaí consumption
Blackcurrants, blueberries and açaí berries are frequently classified as
‘super-fruits’ that can help to improve or maintain health (Esposito et al., 2014,
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Timmers et al., 2017, Skates et al., 2018). The increasing use of the açaí berry
as an energetic and antioxidant drink has led to an expansion in the amount of
scientific research. Furthermore, the consumption of açaí pulp has been linked to
offering various health benefits, such as anti-inflammatory (Schauss et al.,
2006a, Jensen et al., 2008), cardio protective (Rocha et al., 2007, de Souza et
al., 2010), anti-tumor (Hogan et al., 2010), a weight loss agent (Marcason, 2009),
antioxidant (Mertens-Talcott et al., 2008) and also has anti-proliferate (refer to
glossary) properties (in relation to bacteria) (Ribeiro et al., 2010). As stated
above, other parts of the palm tree are also used as traditional medicines in Latin
America. This has been linked with relieving pains, acting as an anti-diarrheal
(Galotta and Boaventura, 2005) or anti-malarial agent (Ruiz et al., 2011).
Various studies have been reported on the chemical composition of the
bioactive molecules of açaí, and related biological activities (Heinrich et al., 2011,
Yamaguchi et al., 2015). The antioxidant activity of açaí has been studied in
cellular models and in-vivo studies through the use of different assays, such as,
scavenging free radicals (Hassimotto et al., 2005, Lichtenthäler et al., 2005b,
Schauss et al., 2006a, Rufino et al., 2011, Kang et al., 2012) and the inhibition of
oxidation in cell cultures (Matheus et al., 2003, Matheus et al., 2006). Although,
the pulp has been shown to have a wide antioxidant activity, the phenolic
compounds were found to not be correlated with this property. This implies that
other compounds, still to be identified, may also contribute to the antioxidant
properties of the açaí berries (Yamaguchi et al., 2015). Also, there have been
some biological assays that were used to investigate the anti-inflammatory
effects of the berries, even though the mechanisms are still unknown (Poulose et
al., 2012, Xie et al., 2012). When compared to other Amazonian fruits, the açaí
berry has been shown to exhibit the best retention capacity for free radicals
(Canuto et al., 2010). Furthermore, one study on açaí has led to a proposal that
açaí extracts have a positive effect on different cellular models, such as, on the
brain cells of rats. This may be due to chemicals that can lead to protection
against neurodegenerative diseases (Spada et al., 2009, Poulose et al., 2012).
Another study, using the vascular cells of rats, found a reduction in the risk of
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cardiovascular disease (Rocha et al., 2007). It has also been reported that açaí
juice may have an athero-protective effect, that is, it aids in protecting an
organism against atherosclerosis and regulates inflammation (Xie et al., 2011).
Moreover, a research study found that feeding rats with açaí juice resulted in a
balance of levels of high and non–high density cholesterol (de Souza et al.,
2010). Açaí has also been found to be an anti-tumoral, anti-genotoxical and anti-
proliferate agent due to the protection against deoxyribonucleic acid (DNA)
damage and the prevention of the formation of reactive species (Hogan et al.,
2010, Ribeiro et al., 2010, Fragoso et al., 2012). Human consumption trials have
also confirmed the positive effects of an açaí diet through an increase of the
antioxidant effects on human plasma (Jensen et al., 2008, Mertens-Talcott et al.,
2008).
5.3. Chemical Composition of Açaí
Açaí berries are considered to be a functional food due to being an
important source of fibers, anthocyanins, minerals, energy, fatty acids and
vitamin E (Pacheco-Palencia et al., 2008, Vera de Rosso et al., 2008, Darnet et
al., 2011, Yuyama et al., 2011). The main composition of açaí is 50% lipids, 25%
fibers and 10 % of proteins (on a dry mass basis). This represents an important
source of the nutritional components for the human diet (Unicamp, 2011). The
health benefits and the high biological activity associated with açaí are mainly
due to the chemical composition of the açaí berries and the presence of bioactive
compounds, such as, polyphenols and minerals (including elements)
(Schreckinger et al., 2010). The presence of these biomolecules has already
been correlated to the high biological activity of the açaí berries (Kuskoski et al.,
2006, Menezes et al., 2008b).
Anthocyanins, which is a class of flavonoids, are responsible for the
purple, red and blue pigments of the açaí berries, red wine or blueberries (Grace
et al., 2014). The levels of anthocyanin vary depending on the growing
conditions, origin, temperature exposure, seasonality and maturation (Timmers et
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al., 2017, Rogez et al., 2011). A study on the kinetics associated with the
accumulation of anthocyanin during the maturation of the açaí berry was
performed and demonstrated a relationship between the anthocyanin content of
the berries and the time of harvest (Rogez et al., 2011). Although the amount of
the anthocyanin may change between samples, the chromatographic profiles
found for açaí berries are similar when determined by high performance liquid
chromatography (HPLC). The most predominant anthocyanin compounds found
in açaí berries are cyanidin-3-glucoside and cyanidin-3-rutinoside and their
chemical structures, are presented in Figure 5.4. Other analytical methods have
been used to identify and quantify the other anthocyanin compounds in açaí
berries, such as, ultra-high performance liquid chromatography – photodiode
array (UHPLC-PDA), which has enabled the separation of peonidin-3-glucoside,
pelargonidin-3-glucoside and peonidin-3-rutinoside, and the subsequent
detection of cyanidin-di-O-gly-cosides (Dias et al., 2012).
Figure 5.4: Chemical structure of most predominant anthocyanin compounds
found in açaí berries being (A) cyanidin-3-glucoside and (B)
cyanidin-3-rutinoside (Yamaguchi et al., 2015).
Several studies have evaluated the polyphenol profile of açaí berries by
HPLC and mass spectrometry, and agreed on the presence of ferulic acid, p-
hydroxybenzoic, gallic, protocatechuic, ellagic, vanillic, p-coumaric acids and
ellagic acid glycoside (Del Pozo-Insfran et al., 2004, Gallori et al., 2004,
Lichtenthäler et al., 2005a, Ribeiro et al., 2010, Rojano et al., 2011, Gordon et
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al., 2012). The lipid content of açaí pulp accounts for 70 to 90% of the total
calories (fresh weight), where the fatty acids, namely, linoleic, oleic and palmitic
acid, were found to be the major poly- and mono-unsaturated fatty acids present
in the samples (Schauss et al., 2006a, Schauss et al., 2006b). The high
concentration of these fatty acids reinforces the impact of açaí in reducing
cholesterol, preventing cardiovascular diseases and suggests that the fruit is a
rich source of essential fatty acids for the Amazonian diet (de Lima et al., 2000,
Yuyama et al., 2011).
Açaí is also a source of minerals, such as manganese, iron, zinc,
phosphorous, sodium, copper, calcium, magnesium, potassium, nickel, boron
and chromium (Rogez, 2000, de Souza et al., 2010, Maria do Socorro et al.,
2010, Rufino et al., 2010, Costa et al., 2013). The term minerals is used in the
field of nutrition and includes elements (major, minor and trace). Unfortunately,
there have only been a few studies that have reported the elemental composition
of açaí, often focussing on the ‘chemical fingerprinting’ of the açaí samples,
namely, preventing adulteration and tracing the possible origins of the berries
(Santos et al., 2014b). However, there is still a gap in the knowledge on how the
consumption of açaí can affect the human nutritional intake of minerals. A
summary of the reported elemental levels in açaí berries is presented in Table
5.1.
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Table 5.1: Literature review of the elemental content of açaí according to weight
basis, dry weight (d.w.) or fresh weight (f.w.) and sample type.
Reference
Rogez, 2000
Menezes et al., 2008a
Unicamp, 2011
Yuyama et al., 2011
Llorent-Martínez et
al., 2013
Santos et al., 2014a
Moreda-Piñeiro et al., 2018
Weight basis d.w. d.w. - f.w. f.w. d.w. d.w.
Sample type juice pulp frozen pulp juice juice - supplement
Element Concentration (mg/kg)
Ca 3090 3300 350 159.9 –
578.5 230 4800 80.3
P 1470 545 160 - 180 1400 70.5
Mg 1780 1244 170 - 80 1400 62.5
K 9900 9000 1240 737.8 –
3766.9 1080 7400 460
Na 760 2850 50 2.7 –
139.2 800 - -
Zn 17.3 28.2 3 1634.3 –
5853.7 0.2 10.1 -
B 15.84 - - - - - -
Fe 20.59 45 4 4.6 –
11.16 8 - -
Se 13.21 <0.02 - - - - -
Mn 323 107.1 61.6 - 4 34.3 -
Cu 13.76 21.5 1.8 - 0.1 20.4 <0.58
Ni 2.03 2.8 - - 0.05 - 0.37
Cr 5.31 - - 229.0 –
1485.3 - - -
Cd 0.46 <0.02 - - - - -
Pb 0.408 0.14 - - - - -
Sr 44.66 7.9 - - - - -
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It has been reported that the açaí berry (freeze-dried pulp) is a good
source of K, Ca, Mg, Fe and Mn (Menezes et al., 2008a) and açaí-based health
supplements (Moreda-Piñeiro et al., 2018). Other studies have also reported the
levels of potential toxic elements (As, Cd, Hg, Pb, Sn and Tl) due to food safety
concerns (Llorent-Martínez et al., 2013). There are many inconsistences in the
reported results possibly due to the genetic variability of the samples analysed
(Menezes et al., 2008a), seasonal variation (Timmers et al., 2017) or the
conditions of industrial processing (Correia et al., 2017). Also, the reported
sampling of açaí berries in these studies did not show any systematic approach
in relation to the source of berries, the species, harvesting time or processing
methods (refer to Table 5.1).
5.4. Aim and Objectives
The overall aim of this work was to investigate the chemical
characterisation, antioxidant and biological activities of different samples of açaí.
Due to the Worldwide increase in consumption of açaí, this study was designed
to investigate the potential contribution of açaí pulp to the human dietary intake of
polyphenols and elemental nutrients. This information is especially important for
the Amazonian population that heavily relies on açaí as part of their diet, culture
and economy.
The objectives were to:
(I) provide an extensive literature review of the reported chemical values of açaí;
(II) source a wide range of açaí samples from different geographical locations including the Amazon region of Brazil; varieties; processing methods; and commercial locations;
(III) investigate the anthocyanin profile, quantification of the total anthocyanin and proanthocyanidin content and antioxidant activities in non-commercial and commercial açaí samples;
162
(IV) determine the total polyphenol and elemental content of açaí (non-commercial and commercial) samples;
(V) evaluate the difference in the chemical composition of non-commercial and commercial açaí samples;
(VI) characterise the chemical composition of different varieties of açaí and açaí products;
(VII) determine the cell viability and antioxidant capacities of açaí extractions using macrophage cells;
(VIII) investigate the wound healing capacity of açaí extractions on human fibroblast cells; and to
(IX) assess the impact of the consumption of açaí in terms of the dietary intake of polyphenols and elements.
5.5. Investigation of the Relationship between the Chemical Composition and Biological Activity of Açaí Samples
Introduction
This section provides a description of the methods used in relation to the
chemical analysis of the açaí berries collected for analysis in February 2018. The
preparation of açaí extracts is described in section 5.5.4, the chemical analysis
(sections 5.5.5 to 5.5.14) and the biological assays (sections 5.5.15 to 5.5.20) of
non-commercial and commercial açaí berries obtained from Brazil and the United
Kingdom.
Description of the samples
The initial analyses were performed with the following set of samples:
commercialised frozen pulp or pure açaí which was bought in São Paulo (Pulp
SP) and freeze-dried. In addition, the non-commercial açaí samples, both whole
(purple and white whole açaí) and de-fatted (purple and white de-fatted açaí)
berries were obtained directly from the Amazon region. The samples were
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freeze-dried and the oil was extracted using the supercritical carbon dioxide
method (Pessoa and Teixeira, 2012). Oil extraction was performed at the
Universidade Federal do Pará (UFPA). Commercial samples were bought as
pure açaí powder at local supermarkets in the United Kingdom (commercial UK)
and Brazil (commercial SP).
Sample identification based on colour
The non-commercial and commercial açaí samples showed a visible
difference in the colour of the material, as shown in Figure 5.5. It is known that
the purple colour present in natural materials, such as red wine and blueberries,
is related to the content of anthocyanin (Del Pozo-Insfran et al., 2004). Therefore,
the berries were identified according to their colour following the CIELAB colour
space international parameters (León et al., 2006).
The colour parameters of the berries were measured using a reflectance
spectrophotometer (CR-400, Konica, Minolta, Japan) calibrated with a regular
white tile according to the CIELAB colour space international parameter, as
shown in Figure 5.6 and calculated using Equation 5.1. This analysis was
performed at the Plants for Human Health Institute, North Carolina State
University (NCSU, USA).
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Figure 5.5: Picture of the açaí berry samples where: (A) is the purple açaí whole
used as reference; (B) is the white açaí whole; (C) is the purple de-
fatted sample; (D) is the white de-fatted; (E) is the freeze-dried frozen
pulp from São Paulo; (F) is the commercial sample from São Paulo;
and 7 is the commercial sample bought in United Kingdom.
Figure 5.6: Illustration of the CIELAB colour space international parameters.
Adapted from Molino et al. (2013).
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ΔE* = [ΔL^2 + Δa^2 + Δb^2]^1/2
Equation 5.1
where:
ΔE = total colour difference;
L = lightness (L);
a = greenness (−a) or redness (+a); and
b = blueness (−b) or yellowness (+b).
The non-commercial purple whole berries obtained directly from the
Universidade Federal do Pará (UFPA) were used as a reference sample and the
colour differences were calculated using Equation 5.1 so as to compare with a
particular açaí sample. The differences in the colour between the whole purple
berries and others are reported in Table 5.2. Commercial samples collected from
Brazil (SP) had a similar colour compared to the standard (lower total colour
difference), whilst the commercial samples bought in the UK were found to be
lighter (higher L), redder (higher a) and yellower (higher b). The oil extraction (de-
fatted samples) did not have a significant effect on the colour difference of the
purple samples. However, within the white samples, the de-fatted material was
slightly lighter (higher L) and yellower (higher b). This simple and low-cost
experiment can be used in the future to identify possible cases of fraud in the
açaí industry and might be useful to perform a qualitative analysis on the
anthocyanin content of açaí products.
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Table 5.2: Colour parameters of açaí samples where L* indicates lightness, a*
the red/green coordinate, b* the yellow/blue coordinate and ΔE the
total colour difference (refer to Equation 5.1) determined by a
reflectance spectrophotometer (CR-400, Konica, Minolta, Japan). The
data relates to a pooled freeze-dried sample.
L* a* b* ΔE Purple açaí whole (reference) 35.44 1.34 - 0.31 -
Purple açaí de-fatted1 35.94 5.17 - 0.52 7.48
White açaí whole 44.96 1.49 13.82 145.15
White açaí de-fatted 55.85 - 0.01 20.06 416.66
Pulp SP 33.33 2.65 0.23 3.23
Commercial SP 36.82 6.17 0.22 12.76
Commercial UK 51.46 10.88 6.66 198.12
1de-fatted (removal of the oil fraction); SP – Sao Paulo, Brazil; UK – United Kingdom.
Method development for the açaí extractions for organic analysis
In accordance with the literature about the extraction of regular berries for
polyphenolic analysis, the most common and efficient method is methanolic
extraction (Kapasakalidis et al., 2006, Castaneda-Ovando et al., 2009). In order
to evaluate what would be available for human intake of the chemicals present,
an extraction with water and mild acid is also proposed in this study. A
comparison of using both extraction solvents (methanolic and aqueous) was
undertaken. Furthermore, the anthocyanin compounds reported to be present in
açaí samples only exist as a stable ring structure under mild acidic solution
conditions (Albarici et al., 2006). Therefore, in order to protect the anthocyanin
structure, and to not further change the chemical structure, 0.5% of a weak acid
solution was added to the solvents. Samples of 0.20 ± 0.01g of ground freeze-
dried açaí were extracted (in duplicate) with 5 mL of solvent: acidified 70% v/v
High Pressure Liquid Chromatography (HPLC) grade methanol (Fisher Scientific,
Pittsburgh, USA) in 0.5% v/v HPLC grade acetic acid (HAc) (Fisher Scientific,
Pittsburgh, USA), or acidified water (0.5% v/v HAc). The mixtures were sonicated
167
for 10 minutes and centrifuged (Sorvall RC-6 plus, Asheville, NC, USA) for 10
minutes at 5000 rpm. The supernatant was transferred to a 25 mL volumetric
flask. The resultant pellet of açaí was further extracted (twice) and the extracts
were combined and diluted with double distilled water to a final volume of 25 mL.
Each final solution was filtered using a 0.2 μm PTFE syringe filter (Fisher
Scientific, Pittsburgh, USA) before analysis.
Total polyphenol and flavonoid content: Materials and method
The total polyphenol content of the açaí extractions (methanolic and
aqueous) was determined using the Folin-Ciocalteu assay, as previously
described in section 2.2. The assay was adapted for a micro-plate following the
Singleton method (Singleton et al., 1999a) and performed at NCSU. The
advantage of using a microplate reader, instead of cuvettes, is to reduce the
amount of reagents used (by the factor of 40) with consequent reduction of time
and cost. This results in an increase in the level of precision, because all of the
samples were prepared and read at the same time as the Folin-Ciocalteu
reaction is time-dependent. In a 96 well-plate, 75 µL of distilled water was added
to each well (in triplicate) along with 25 µL of the diluted sample or standard, and
25 µL of diluted (1:1 v/v volume) Folin-Ciocalteu reagent. After 6 minutes, 100 µL
of 7.5% v/v sodium carbonate solution was added. The plate was then left in the
dark for 90 minutes and read using a UV-Vis plate reader (SpectraMax® M3,
Sunnyvale, USA) at 765 nm. The results are expressed in gallic acid equivalent,
which was used as a standard reference (Singleton et al., 1999a).
The total flavonoid content can be determined using a colourimetric
method based on the complexation of the flavonoids with aluminium chloride
(AlCl3) (da Silva et al., 2015). An example of this coordination for the quercetin
flavonoid is shown in Figure 5.7. The resulting complexes present a different
coloration from the initial solution and can be determined spectrophometrically
(yellow to blue).
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Figure 5.7: Molecular structures of the complexation between quercetin and
aluminium chloride used to determine the levels of total flavonoids.
Adapted from Frederice et al. (2010).
The assay, previously described by Zhishen et al. (1999), was adapted for
a micro-plate assay. In a 96-well plate, 100 μL of açaí extracts or standard were
added to each well (in triplicate) in addition to 100 μL of 2% AlCl3 solution
(Sigma-Aldrich, St. Louis, USA), freshly prepared in methanol (HPLC grade,
Sigma-Aldrich, St. Louis, USA). The plate was mixed and left at 20 °C for 1h. The
absorbance was read using a UV-Vis plate reader at 415 nm. Results are
expressed as quercetin equivalent, which was used as the standard reference for
the calibration curve ranging from 0 to 100 mg/L quercetin equivalent. The
extracts were also diluted with the extraction solution as required to fit the
calibration curve.
Total polyphenol and flavonoid content: Results and discussion
The açaí extracts, collected as part of this study, were analysed in order to
evaluate the total polyphenol content using the Folin-Ciocalteu assay (refer to
section 5.5.5). The results are presented in Table 5.3, along with the data
obtained from the other assays performed on samples. The data is presented on
a dry weight basis (d.w.). In order to compare these values with reported
literature values, the data were converted to a fresh weight basis (f.w.). This
information does not exist for these samples, so an assumption of a 90%
169
moisture content was adopted, based on a procedure described by Pavan et al.
(2012).
Açaí extraction methods (aqueous and methanolic), as reported in section
5.5.4, were also evaluated using a paired two-tailed t-test (Miller et al., 2018).
The data confirmed that the null hypothesis was retained, and as such, confirmed
that there is no statistically significant difference in the total polyphenol content
(tcalc= 1.11 < tcrit = 2.30, p=0.3146, n=9, α =0.05). Therefore, an aqueous solution
was used for further analysis of the total polyphenol content. From the data
presented, it is clear that the samples that were de-fatted (i.e. removal of the oil
fraction) showed a significantly higher total polyphenol content for the non-
commercial samples (paired two-tailed t-test, tcalc= 5.60 > tcrit = 2.15, p<0.0001,
n=16, α =0.05).
Table 5.3: Total polyphenol, flavonoid, anthocyanin (ANC) and proanthocyanidin
content (PAC); and chemical antioxidant activity (ABTS and DPPH) of
açaí pulp samples. The values are expressed as mean ± standard
deviation and dry weight.
Samples Total
polyphenol (mg/g)*
Total flavonoid (mg/g)*
Total ANC
(mg/g) - 70%
MeOH extraction
DMAC Total PAC
(mg/g) - 0.5% HAc extraction
ABTS (mg/g)*
DPPH (mg/g)*
Number of replicates 6 6 2 3 6 6
Non-commercial
Purple Açaí whole 32.00 ±
1.03
6.39 ±
1.23
10.20 ±
0.24
6.10 ±
2.09
438.0
± 17.5
336.0
± 72.0
Purple Açaí de-fatted 39.40 ±
1.67
8.05 ±
0.81
14.33 ±
0.58
5.06 ±
0.68
529.0
± 57.0
419.0
± 69.5
170
Samples Total
polyphenol (mg/g)*
Total flavonoid (mg/g)*
Total ANC
(mg/g) - 70%
MeOH extraction
DMAC Total PAC
(mg/g) - 0.5% HAc extraction
ABTS (mg/g)*
DPPH (mg/g)*
White Açaí whole 9.40 ± 0.70 2.12 ±
0.27 <0.01
3.96 ±
2.39
83.0 ±
9.7
53.2 ±
15.1
White Açaí de-fatted 11.70 ±
0.24
2.38 ±
0.35 <0.01
2.60 ±
0.49
101.0
± 16.2
67.4 ±
10.7
Oil White 2.74 ± 1.13 1.42 ±
0.96 <0.01
1.54 ±
0.22 <15.3 <7.4
Oil purple 1.68 ± 0.52 0.87 ±
0.38 <0.01
3.40 ±
1.42 <15.3 <7.4
Commercial
Pulp SP 28.30 ±
0.64
5.00 ±
0.68
3.59 ±
0.12
4.75 ±
1.58
310.0
± 41.4
222.0
± 38.6
Powder SP 42.40 ±
1.53
6.07 ±
1.45
4.70 ±
0.01
4.47 ±
0.54
316.0
± 39.1
304.0
± 43.2
Powder UK 5.09 ± 0.42 1.88 ±
0.73 <0.01
5.48 ±
2.20
55.2 ±
31.4 <7.4
*Results presented as a combination of both aqueous and methanolic extracts. GAE: gallic acid equivalent; ANC: anthocyanin content; PAC: proanthocyanidin content; DMAC: Dimethylacetamide; ABTS: 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid); DPPH: 2,2-diphenyl-1-picrylhydrazyl; SP: São Paulo; UK: United Kingdom.
Figure 5.8 presents a comparison of the total polyphenol content of non-
commercial and commercial purple and white açaí freeze-dried berries. The
purple non-commercial samples have a significantly higher amount of total
polyphenol content in comparison with the white samples (two-tailed Student t-
test, tcalc= 5.95 > tcrit = 2.30, p=0.0003, n=9, =0.05). Furthermore, the
commercial purple berries have a higher level of variability (standard error of the
mean of 9.26 GAE mg/g) when compared with the purple pure açaí berries (2.90
GAE mg/g).
171
Figure 5.8: Box plots of the total polyphenol content (gallic acid equivalent mg/g)
of the açaí extracts determined using the Folin-Ciocalteu assay (refer
to section 5.5.5). The values relate to the type of sample (purple, n=
6; white and commercial n= 4; n is the number of samples).
The results for this study show a similar pattern in terms of the de-fatting
process. In general, the removal of the oil fraction results in the pre-concentration
of the chemicals in the de-fatted samples. Moreover, this indicates that the oil
does not have a significant level of the polar chemicals analysed. A previous
study on a range of fatty acids found in açaí oil confirm a similar pattern to the
data obtained here for the levels of total polyphenol, flavonoid and anthocyanin in
de-fatted açaí pulp (Pacheco-Palencia et al., 2008). This pre-concentration of the
chemicals will also have an effect on the antioxidant activity and therefore the
radical inhibition of the berries.
Purple
White
Comm
erci
al p
urple
0
10
20
30
40
50
To
tal p
oly
ph
en
ol co
ncen
trati
on
(G
AE
) m
g/g
172
Table 5.4: Literature values for the total polyphenol content (mg GAE/ 100g) and
antioxidant activities DPPH (g/g DPPH) and ABTS μmol Trolox/g of
typical tropical berries from Brazil (dry weight). Table adapted from
Rufino et al. (2010).
*concentration required to obtain a 50% antioxidant effect
Table 5.4 presents a literature review of the total polyphenol content and
antioxidant activities of typical tropical berries from Brazil. The data is reported on
a dry weight basis and presents a significant correlation between total polyphenol
and antioxidant activities (Spearman’s correlation; R= -0.81, p<0.0001 for DPPH
and R= 0.77, p=0.0002 for ABTS; n=18 and =0.05). This correlation indicates
Fruits Extractable polyphenols DPPH ABTS+ mg GAE/100 g EC50* (g/g DPPH) μmol Trolox/g
Açaí 3268 ± 527 598 ± 164 64.5 ± 19.2
Acerola 10280 ± 77.7 49.2 ± 2.5 953 ± 34.1
Bacuri 1365 ± 43.3 6980 ± 854 18.1 ± 3.7
Cajá, yellow mombim 579 ± 12.9 1064 ± 162 40.7 ± 2.2
Caju, cashew apple 830 ± 26.5 906 ± 78.2 79.4 ± 15.7
Camu-camu 11615 ± 384 42.6 ± 1.4 1237 ± 33.8
Carnaúba 830 ± 28.3 4877 ± 24.3 16.4 ± 0.2
Gurguri 1364 ± 24.8 360 ± 32.7 136 ± 20.1
Jaboticaba 3584 ± 90.9 138 ± 3.1 317 ± 2.7
Jambolão, java plum 1117 ± 67.1 938 ± 46.9 125 ± 10.8
Juçara 5672 ± 55.9 70.1 ± 4.8 606 ± 142
Mangaba 935 ± 37 890 ± 69.1 65.6 ± 7.4
Murici, nance 2380 ± 104 238 ± 17.7 412 ± 13
Murta 2055 ± 75.7 363 ± 27.4 166 ± 4
Puçá-coroa-de-frade 1047 ± 77 316 ± 2 161 ± 3
Puçá-preto 2638 ± 48.9 65.6 ± 2.4 346 ± 21.7
Umbu 742 ± 19 933 ± 109 77 ± 15.4
Uvaia 1930 ± 129 276 ± 22.2 182 ± 14.2
173
that the amount of total polyphenol content of the açaí berries could be
potentially related to the chemical antioxidant activity. The total polyphenol
content of the açaí pulp (Figure 5.8) is in accordance with the range reported in
the literature for açaí purple berries (Gordon et al., 2012, Augusti et al., 2016),
white berries (Lichtenthäler et al., 2005b) and oil (Pacheco-Palencia et al., 2008).
It should be pointed out that a direct comparison between the results presented
in this study and the literature is often difficult due to a lack of information relating
to the weight basis or origin and treatment of the samples (published data). The
non-commercial purple and commercial samples from São Paulo represent an
average level of the total polyphenol content, in comparison with other Brazilian
fruits, based on the classification reported by Rufino et al. (2010). The main
advantage of consuming açaí rather than the other fruits presented in Table 5.4
is associated with the easy access to açaí in all regions of the country (as a
frozen pulp). As such, the consumption of the processed frozen pulp is not
dependent on a seasonality factor (Tonon et al., 2009), in contrast to other
Brazilian fruits and seasonal berries. In comparison with other traditional berries,
presented on Table 5.5, the non-commercial purple açaí and the commercial
powder SP have a similar total polyphenol content to that of cranberries, higher
than raspberries and grapes and lower than blackberries, bilberries and
blackcurrants (Rothwell et al., 2013). The non-commercial white and the other
commercial açaí samples have lower values than the reported values for other
berries (refer to Tables 5.4 and 5.5).
The açaí extracts were also analysed by the AlCl3 assay (refer to section
5.5.6) in order to determine the total flavonoid content. The results for the total
flavonoid concentration are reported in Table 5.3 and have a similar pattern to
the data in Figure 5.8. Furthermore, a two-tailed paired t-test (Miller et al., 2018)
showed that the açaí extraction methods (aqueous and methanolic) do not
present a significant difference in terms of the flavonoid content (tcalc= 0.56 < tcrit
= 2.30, p=0.5907, n=9, =0.05).
174
Table 5.5: Literature review of the total polyphenol and total anthocyanin content
of other berries obtained by two different methods (HPLC and pH);
and the total proanthocyanidin (mg/100 g) (fresh weight). Adapted
from Rothwell et al. (2013).
Total polyphenol
Total anthocyanin
(HPLC)
Total anthocyanin (pH method)
Total proanthcyanidin
(HPLC) cranberry 315 49.89 32 -
blackberry 569.43 172.59 146.8 17.95
bilberry 525 - 299 -
raspberry 154.65 72.47 43.57
blackcurrant 820.64 592.22 225.04 138.21
grape (black) 184.97 62.1 - 61.2
low bush blueberry 471.55 187.24 149.17 333.1
strawberry 289.2 73.01 - 145
The total flavonoid content of açaí pulp extractions have a similar pattern
to that for the total polyphenol content, as would be expected because the
flavonoids are a subclass of polyphenols (Tsao, 2010). The data is also in
agreement with the available literature for commercial açaí samples (Rufino et
al., 2010, Horszwald and Andlauer, 2011).
Total anthocyanin content: Materials and method
The determination of the total anthocyanin content of the açaí extracts
was performed following the Grace et al. (2013) method using a high
performance liquid chromatography or HPLC instrument (Agilent 1200 HPLC)
with photodiode array detector (DAD). The separation was conducted using a RP
Supelcosil-LC-18 column, with dimensions 250 mm × 4.6 mm × 5 μm (Supelco,
Bellefonte, USA) held at a constant temperature of 30°C with a flow rate of 1
mL/min and the gradient programme, as shown in Table 5.6.
175
Table 5.6: Gradient programme for the determination of the anthrocyanin content
of açaí extracts using an Agilent 1200 HPLC instrument.
Time (minutes) 5% v/v formic acid (%) 100% methanol (%) 0 90 10
5 85 15
15 80 20
20 75 25
25 70 30
45 40 60
47 90 10
60 90 10
The quantification of the total anthocyanin content was based on the sum
of the integrated anthocyanin peaks from 0 to 60 minutes, calculated against the
standard curve of Cy-3-Glu (as shown in Figure 5.9).
Figure 5.9: Standard curve of Cy-3-Glu concentration (mg/L) and the areas of the
peaks (mAU) used as the calibration curve for the determination of
the anthocyanin content of açaí extracts using a HPLC-DAD
chromatogram (at 520 nm).
0 1 2 30
2
4
6
Cy-3-Glu concentration (mg/L)
Are
as o
f th
e p
eaks (
mA
U) Y = 1.908x + 0.07211
R2 = 0.9982
176
Total anthocyanin content: Results and discussion
Figure 5.10 reports the HPLC-DAD chromatogram at 520 nm used to
integrate the anthocyanin peaks and determine the total anthocyanin content of
the açaí samples, as described in section 5.5.7. The anthocyanin peaks, found in
the extracts for purple açaí (non-commercial and commercial) samples, are
cyandin-3-glucoside (retention time, tR = 17.458 minutes), cyandin-3-rutinoside
(tR = 21.069 min) and peonidin-3-rutinoside (tR = 26.237 min), respectively. The
identification of the anthocyanin peaks was performed using an cyanidin 3-
glucoside reference standard and the peak identification and elution order by the
method previously proposed Vera de Rosso et al. (2008). Only the purple
commercial SP, pulp SP and non-commercial purple açaí samples had
detectable anthocyanin peaks.
The quantification of the total anthocyanin content was performed, as
described in section 5.5.8 and is presented in Table 5.3. Statistical analysis
confirmed that there is no significance difference between the two extraction
methods (paired two-tailed t-test, tcalc= 2.79 < tcrit = 3.18, p=0.0684, n= 4,
=0.05).
177
Figure 5.10: The determination of the anthocyanin content of a purple non-
commercial açaí sample following methanolic extraction and using a
HPLC-DAD chromatograph (at 520 nm). The anthocyanin peaks are
cyandin-3-glucoside (retention time, tR = 17.458 minutes), cyandin-3-
rutinoside (tR = 21.069 min) and peonidin-3-rutinoside (tR = 26.237
min).
The anthocyanin profile and content of the purple açaí berries are in
agreement with previous studies (Del Pozo-Insfran et al., 2004, Lichtenthäler et
al., 2005b, Pacheco-Palencia et al., 2008, Gordon et al., 2012, Gouvêa et al.,
2012). The purple açaí samples were the only extracts that showed an
anthocyanin profile as it could be expected from the purple colour of the material,
which is due to the anthocyanin molecules (Del Pozo-Insfran et al., 2004). As
such, the white açaí berries were expected to not show significant levels of
anthocyanins, as confirmed by the results. Secondly, the pure açaí powder,
commercially purchased in the UK, did not show any significant anthocyanin
peaks, below the limit of detection. This suggests that the UK sample might have
some other material added to it or that the chemicals might have been lost during
commercialisation. The total anthocyanin content results are in accordance with
the qualitative analysis of the colour of the material, as presented in section
178
5.5.3. In comparison with other typical berries, as presented in Table 5.5, the
total anthocyanin content for the non-commercial purple açaí samples ranges
from 102.0 to 143.3 mg/100 g (f.w.) and for the commercial purple açaí samples
35.9 to 47.0 mg/100 g (f.w.) Therefore, açaí would occupy the fourth position in
ranking of the berries (Table 5.5) in terms of the total anthocyanin content of the
fruit.
Total proanthocyanidin content: Materials and method
The total proanthocyanidin (PAC) content was determined using the 4-
dimethylaminocinnamaldehyde (DMAC) method adapted for a micro-plate assay,
as previously described by Prior et al. (2010), where the DMAC reacts with the
terminal units of the PAC oligomers. In a 96 well-plate, 63 µL of the diluted
sample, standard or blank was added, in triplicate, to each well along with 189 µL
of the DMAC reagent (Sigma-Aldrich, St. Louis, USA). The plate was set on the
plate reader at 640 nm to read the absorbance value of the wells, at time
intervals of a minute for a total period of 30 minutes. The results are expressed in
procyanidin B1 dimer (Sigma-Aldrich, St. Louis, USA) equivalent, that was also
used as a standard reference. The extracts were also diluted with the extraction
solution to fit a calibration curve ranging from 0 - 100 mg/L B1 equivalent.
Total proanthocyanidin content: Results and discussion
The total proanthocyanidin (PAC) content of the açaí samples was
determined using the DMAC assay (refer to section 5.7.4). Interestingly, this was
the only assay that produced a statistically significant difference between the two
sets of extraction samples (i.e. aqueous vs methanolic) (paired two-tailed t-test,
tcalc= 4.16 > tcrit = 2.30; p=0.0031, n=9, =0.05), as shown in Figure 5.11. The
aqueous extraction resulted in significantly higher PAC concentrations, due to the
nature of the PAC compounds. Proanthocyanidin are oligomeric flavonoids (He
179
et al., 2008) and previous studies have shown that the polymers (more than 10
units) are the major PAC in freeze-dried açaí (Schauss et al., 2006b). Therefore,
it was expected that the polymers are more soluble in the aqueous rather than
the methanolic extracts (He et al., 2008). The difference in the PAC levels
between the purple and white samples were lower than that for the other assays
presented in this study. This supports the claim that PAC could be the class of
polyphenols responsible for the antioxidant activity of the white açaí berries.
Figure 5.11: Total proanthocyanidin content (PAC) of açaí extractions presented
as B1 equivalents (B1E) via DMAC assay (refer to section 5.5.8) and
compared between the methanolic (70% MeOH) and aqueous (0.5%
HAc) extraction methods (n= 4, n, number of instrument replicates).
Sample 1: Purple açaí whole; 2: Purple açaí de-fatted; 3: white açaí
whole; 4: white açaí de-fatted; 5: oil extracted from white açaí; 6: oil
extracted from purple açaí; 7: pulp SP; 8: powder SP; 9: powder UK.
The analysis of the oil extracts for both white and purple açaí berries
showed a significant level of PAC (typically 2 – 4 mg/g), confirmed by the fact
that the whole samples have a higher PAC content (6 mg/g for purple and 4 mg/g
for white) than that for the de-fatted samples (5 mg/g for purple and 3 mg/g for
white). The commercial samples also have PAC levels (typically 5 mg/g) similar
to that for the non-commercial purple whole sample (6 mg/g).
1 2 3 4 5 6 7 8 90
2
4
6
8
10
Samples
To
tal P
AC
(B
1E
) m
g/g
70% MeOH
0.5% HAc
180
In comparison with other ‘super-fruits’, the reported values of PAC levels
for açaí obtained in other studies is in agreement with the data in this study for
purple açaí samples. Furthermore, açaí values are similar to the values for
blueberries and cranberries; but higher than that for pomegranate and lower than
cocoa seeds (Crozier et al., 2011). The reported values of PAC for typical berries
are reported in Table 5.5 (based on a HPLC method). These literature values are
compared with the data of this study which were obtained using the DMAC
method, as described in section 5.5.8. Although any critical analysis of the values
would be questionable (based on the variation of techniques), it is interesting to
note that the non-commercial purple açaí samples of this study have similar PAC
levels to those reported for black grapes (Rothwell et al., 2013).
Chemical antioxidant activity: Materials and method
Free radicals and other oxidising molecules have been recently
considered as one of the main reasons linked to the onset of cancer,
Alzheimer’s, Parkinson and cardiovascular diseases (de Souza et al., 2010). The
excess of these free radicals can be balanced by antioxidants produced by the
body or acquired via the diet.
The antioxidant activity of a compound or natural product can be
measured by different mechanisms, such as electron transfer, reducing power,
hydrogen atom transfer or radical scavenging (Shahidi and Zhong, 2015). In this
study, the antioxidant activity of açaí was determined using two different
methods, namely, radical scavenging by the 2,2'-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid (ABTS) and 2,2-diphenyl-1-picrylhydrazyl
(DPPH) molecules.
Antioxidants can scavenge reactive oxygen species (ROS) or other free
radicals by hydrogen atom transfer or electron transfer. This activity is usually
expressed as the Trolox equivalent, which is a well-known antioxidant analog of
vitamin E (Arts et al., 2004).
181
The ABTS assay involves the conversion of 2,2'-azino-bis(3-
ethylbenzothiazoline-6-sulphonic acid) or the ABTS reagent to a radical through
reacting it with persulfate. The resultant solution is then colourimetrically reacted
with the antioxidant compounds present in the extracts, especially the
polyphenols. The antioxidant activity was measured by ABTS following the Re et
al. (1999) method. The 2.45 mM ABTS+• solution was prepared by reacting the
ABTS solution (Sigma-Aldrich, St. Louis, USA) with potassium persulfate (Sigma-
Aldrich, St. Louis, USA) and left to stand in the dark for 12 to 16 hours. The final
solution was diluted until the absorbance at 734 nm reached 0.70 0.02
absorbance units using a UV-Vis plate. In a 96 well-plate, 9 µL of açaí extract
was added to 271 µL of ABTS+• solution (in triplicate). The absorbance of the
plate was read after 10 minutes at 30C along with 9 µL of distilled water and 271
µL of ABTS+• solution, read as the blank. The results are expressed in Trolox
equivalent (Sigma-Aldrich, St. Louis, USA), which was used as a calibration over
the range of 100 to 500 µM.
The other radical scavenging assay involves the free radical 2,2-diphenyl-
1-picrylhydrazyl or DPPH• which reacts with an antioxidant molecule resulting in
a discoloration of the solution, measured at 515 nm using a micro-plate UV-Vis
reader (Truong et al., 2007). The radical 150 µM DPPH• solution was prepared
by mixing the DPPH reagent (Sigma-Aldrich, St. Louis, USA) in 80% v/v
methanol (HPLC grade, Sigma-Aldrich, St. Louis, USA). In a 96 well-plate, 180
µL of the radical solution was added to 20 µL of the açaí extract, standards or
70% methanol v/v, which was used as a blank. The plate was left in the dark for
40 minutes and read at 515 nm. The results were also quantified using a
calibration curve of Trolox (Sigma-Aldrich, St. Louis, USA) over the range of 100
to 500 µM.
182
Chemical antioxidant activity: Results and discussion
The antioxidant activity results are presented in Figure 5.12 and Table 5.3.
The data shows a significantly higher level of antioxidants for most of the
samples, with the exception being for the oil extraction samples. The extraction
methods did not show a significance difference, based on using a paired two-
tailed t-test (tcalc= 1.56 < tcrit = 2.45, p=0.1705, n=7, =0.05). The white and the
commercial açaí samples bought in UK, have significantly lower levels of
antioxidant activity when compared to the non-commercial purple açaí berries
(One-way ANOVA, r = 0.96, p<0.0001, n=40, =0.05). In addition, the
commercially bought açaí in the UK have a significantly lower activity when
compared to the other commercial purple samples (One-way ANOVA, r = 0.92,
p<0.0001, n=23, =0.05).
Figure 5.12: Antioxidant activity of açaí extracts determined by the ABTS assay,
data reported as Trolox equivalents (TE) (n=3; n, number of
instrumental replicates).
The DPPH assay was performed as described in section 5.5.6 and the
results are presented in Table 5.3. The freeze-dried pulp sample from São Paulo
had a slightly lower activity in this assay when compared to the ABTS assay. On
Purple
White
Comm
ercia
l purp
le0
200
400
600
AB
TS µ
mol
TE
/g
183
the other hand, the commercial sample bought in the UK did not show any
significant antioxidant activity.
The same trend was found for the de-fatted samples analysed by this
assay, with the whole açaí extracts showing a slightly lower antioxidant activity
due to the concentration of the material when removing the oils. These findings
are similar to the ABTS assay confirming the high antioxidant activity of the purple
non-commercial açaí material. The data relating to the antioxidant activity of the
açaí extracts are in agreement with the range of results reported in the literature
for açaí samples (Gordon et al., 2012, Augusti et al., 2016, Garzón et al., 2017).
Furthermore, in comparison with typical Brazilian fruits (refer to Table 5.4), açaí
has an average level of antioxidant activity.
Elemental composition: Materials and method
The determination of the total elemental composition of the açaí samples
was performed as described in section 2.3. The samples were fully digested and
analysed by inductively coupled plasma mass spectrometry or ICP-MS.
Elemental composition: Results and discussion
The total elemental composition of the açaí samples was evaluated
following the method proposed in section 5.5.10. The calcium, magnesium,
manganese, iron, zinc and copper; essential minor and trace elements found in
significant levels in açaí samples are presented in Table 5.7 (a) calculated as
fresh weight and shown on a dry weight basis in Figures 5.13 and 5.14.
Moreover, these elements were chosen because their levels in açaí products
may play a significant contribution to the nutritional intake of these minerals. It
should be stated that the powdered samples were obtained by freeze-drying, so
the same ‘conversion’ factor used to convert dry to fresh weight (based on 90%
of the material being water) was also applied to the data in Table 5.7. A full set of
data for all elements is presented in Appendices 5.1 and 5.2.
184
Table 5.7 (a): Total elemental levels (mean ± standard deviation) of essential
trace elements (mg/kg fresh weight) of açaí pulp samples
determined using ICP-MS (refer to section 2.1): data relates to the
type of sample (non-commercial : purple n= 6; and white n= 4; and
commercial: purple n= 4; n is the number of samples).
Samples Non-commercial Commercial
Purple Açaí
whole
Purple Açaí de-
fatted
White Açaí
whole
White Açaí de-
fatted Pulp SP Powder
SP Powder
UK
Ca 468.82 ±
14.15
527.34 ±
58.07
416.23 ±
2.20
525.11 ±
13.28
165.04 ±
16.12
202.93 ±
17.25
66.17 ±
15.65
Mg 233.23 ±
2.16
272.36 ±
4.15
246.92 ±
12.13
301.77 ±
9.00
202.21 ±
2.75
203.69 ±
2.14
85.92 ±
1.13
Mn 64.06 ±
0.93
80.92 ±
0.77
61.14 ±
0.60
80.89 ±
1.57
26.76 ±
1.51
54.70 ±
1.78
1.62 ±
0.01
Fe 3.01 ±
0.00
4.17 ±
0.12
3.65 ±
0.75
4.30 ±
0.10
3.00 ±
0.04
2.19 ±
0.03
0.23 ±
0.01
Zn 2.49 ±
0.07
3.24 ±
0.17
2.65 ±
0.03
3.62 ±
0.17
2.70 ±
0.65
2.32 ±
0.31
1.27 ±
0.12
Cu 1.81 ±
0.03
2.22 ±
0.01
1.72 ±
0.06
2.50 ±
0.10
1.52 ±
0.05
1.50 ±
0.09
0.35 ±
0.02
The difference between the elemental concentrations of the non-
commercial whole and de-fatted samples (section 5.5.2) follow the same trend as
the previous data for organics and biological analysis (section 5.7). Overall, for
both white and purple açaí pulp, all of the elemental values for the de-fatted
samples are slightly more concentrated than the whole material due to the
removal of the oil fraction. The enhancement of the elemental levels in the
defatted samples is on average 28% for purple and 38% for white samples.
However, there is no statistically significant difference for all the 15 analysed
elements for both purple (paired two-tailed t-test, tcalc= 1.76 < tcrit = 2.15,
p=0.1004, n=15, =0.05) and white samples (paired two-tailed t-test, tcalc= 1.69 <
tcrit = 2.15, p=0.1131, n=15, =0.05). Interestingly, the commercial pulp and
185
powder products purchased in São Paulo have similar elemental levels.
However, the powered sample purchased in the UK has either been subjected to
a different process or has been ‘adulterated’ as the elemental levels are typically
30% of the Brazilian powdered samples.
The commercial samples, especially the açaí pulp (purchased in São
Paulo) has lower (and a larger spread) elemental levels than the non-commercial
samples. For example, the manganese levels of the non-commercial pulp were
64.06 ± 0.93 mg/kg Mn (f.w.) for purple and 61.14 ± 0.60 mg/kg Mn (f.w.) for
white; whilst the commercial pulp (purple) was 26.76 ± 1.51 mg/kg Mn (f.w.).
Clearly, the commercial processing has reduced the Ca and Mn levels by about
50% and the commercial pulp has a significant variance (as shown by the
standard deviation values). This may be due to the origin of the berries used in
the commercial sample or the processing method used (which are both
unknown).
Table 5.7 (b): Total elemental levels (mean ± standard deviation) of non-
essential/toxic trace elements (mg/kg fresh weight) of açaí pulp
samples determined using ICP-MS (refer to section 2.1): data
relates to the type of sample (non-commercial : purple n= 6; and
white n= 4; and commercial: purple n= 4; n is the number of
samples).
Samples Non-commercial Commercial
Purple açaí
whole
Purple açaí de-fatted
White açaí whole
White açaí de-fatted Pulp SP Powder
SP Powder
UK
As <0.001 <0.001 <0.001 <0.001 <0.001 <0.001 <0.001
Cd 0.007 ±
0.0000
0.010 ±
0.001
0.009 ±
0.000
0.018 ±
0.001
0.004 ±
0.001
0.006 ±
0.000
0.001 ±
0.000
Pb 0.050 ±
0.000
0.070 ±
0.000
0.030 ±
0.000
0.050 ±
0.000
0.010 ±
0.000
0.003 ±
0.000
0.002 ±
0.000
186
The non-essential or toxic elements, arsenic, cadmium and lead (reported
in Table 5.7 (b)) are found at very low levels in both the commercial and non-
commercial açaí products (typically <0.01 mg/kg f.w.). The low concentration for
arsenic and lead are in agreement with what is normally found in typical tropical
fruits (<0.003 mg/kg for arsenic and <0.006 mg/kg for lead) (Avegliano, 2009,
Vannoort and Thomson, 2003). The cadmium levels found in this study are
higher than that for tropical fruits as described in the literature (<0.002 mg/kg).
However, the levels do not make a significant contribution to the daily dietary
intake of these elements (1.31 ± 0.16 μg/day for cadmium) (Colli, 2005,
Avegliano et al., 2011). This is encouraging in terms of the açaí products being
used for human consumption.
Figure 5.13: Box plots of the total elemental content of minor elements (mg/kg
d.w.) of açaí pulp samples using ICP-MS (refer to section 2.1)
relating to the type of sample (non-commercial: purple n= 6; and
white n= 4; and commercial: purple n= 4; n is the number of
samples). The commercial sample is a combination of pulp SP and
powders SP and UK.
Ca Mg
Purple White Commercial 0
2
4
6
8
Co
ncen
trati
on
of
Ca (
g/k
g)
Purple White Commercial 0
1
2
3
4
Co
ncen
trati
on
of
Mg
(g
/kg
)
187
Figure 5.14: Box plots of the total elemental content of trace elements (mg/kg
d.w.) of açaí pulp samples using ICP-MS (refer to section 2.1)
relating to the type of sample (non-commercial : purple n= 6; and
white n= 4; and commercial: purple n= 4; n is the number of
samples). The commercial sample is a combination of pulp SP and
powders SP and UK.
Figures 5.13 and 5.14 graphically report the data (as box plots) for the
non-commercial purple or white and commercial purple açaí samples (based on
a dry weight status). In the box plots, the pulp SP, powder SP and powder UK
samples were combined as commercial samples, causing a spread of the target
elemental concentrations. The data (now reported as on a dry weight basis)
relates to the literature presented in Table 5.1. For calcium, the reported values
are lower than that presented in this study for non-commercial purple samples,
with the exception of values published by da Silva Santos et al. (2014), who also
analysed a non-commercial purple açaí sample. The levels of manganese are at
Purple White Commercial
0
200
400
600
800
1000
Purple White Commercial 0
10
20
30
40
Purple White Commercial
0
10
20
30
40
50
Purple White Commercial 0
10
20
30
Mn Fe
Zn Cu
Co
ncen
trati
on
of
ele
men
t (m
g/k
g)
188
least 3 times higher than that reported in other study (Table 5.1). Iron, copper
and zinc have similar levels to that reported by Menezes et al. (2008a), who
analysed a commercial sample of açaí pulp purchased in Belém (Amazon -
Pará). When compared to the other 47 typical fruits, açaí has the highest content
of manganese, the 3rd highest of calcium, 4th of copper, 6th of magnesium, 11th of
zinc and 13th of iron content (Unicamp, 2011).
The elemental values for açaí and other typical Brazilian fruits are
summarised in Table 5.8 (Unicamp, 2011). The açaí sample used was a
commercial açaí frozen pulp obtained from São Paulo. Therefore, in order to
compare the values with the ones described in Table 5.7 (a), the commercial
pulp SP was selected. There was no significant difference between the published
values and the reported values of this study (two-tailed Student t-test, tcalc= 1.83
< tcrit = 2.57, p=0.3667, n=6, =0.05). In comparison to other fruits, açaí
represents good levels of manganese and high levels for the other elements (Ca,
Mg, Fe, Zn and Cu), with the exception of graviola and pequi (refer to Table 5.8).
The typical daily fruits of a Brazilian diet include mango and papaya, which when
compared with the results for açaí, have lower levels of the essential elements
(Unicamp, 2011).
Potassium, sodium, chromium, nickel, cobalt, vanadium, molybdenum and
selenium were also analysed in this study and are presented in Appendices 5.1
and 5.2. The typical level of potassium in tropical fruits is 10399 mg/kg (d.w.)
which is similar to the non-commercial açaí pulp samples. However, the açaí
pulp samples have higher levels of sodium (Colli, 2005, Avegliano et al., 2011).
In comparison with the typical value of trace elements in fruits presented by
Kabata-Pendias (2010), the açaí pulp showed higher levels of chromium
(average of 0.48 mg/kg f.w. against 0.08 mg/kg presented on the literature),
nickel (0.76 mg/kg f.w. against 0.06 mg/kg), and cobalt (0.008 mg/kg f.w. against
0.0016 mg/kg), and lower levels of vanadium (0.003 mg/kg f.w. against 0.33
mg/kg), molybdenum (0.01 mg/kg f.w. against 0.07 mg/kg), and selenium (below
the limit of detection against 0.04 mg/kg).
189
Table 5.8: Literature review of typical Brazilian fruits the total elemental content
of calcium, magnesium, manganese, iron, zinc and copper in mg/kg
(f.w.). Data for açaí is reported as a commercial processed material
and all the others as raw natural typical Brazilian fruits. Adapted from
Unicamp (2011).
Fruits Ca Mg Mn Fe Zn Cu Açaí (Euterpe oleracea) 351.8 170.4 61.6 4.3 2.7 1.8
Acerola (Crataegus azarolus) 125.5 131.3 0.7 2.2 1.5 0.7
Cocoa (Theobroma cacao) 121.0 246.2 0.4 2.6 5.9 1.5
Cajá (Spondias mombin) 127.4 112.8 0.5 1.5 1.8 0.2
Cashew fruit (Anacardium occidentale) 14.2 101.1 1.2 1.5 0.9 0.7
Carambola (Averrhoa carambola) 47.9 73.6 1.3 2.0 2.4 0.8
Ciriguela (Spondias purpurea) 274.1 179.6 0.6 3.6 5.3 1.2
Cupuaçu (Theobroma grandiflorum) 131.2 181.7 0.7 4.9 3.4 0.7
Guava (Psidium guajava) 44.5 68.9 0.9 1.7 1.3 0.4
Grape (black) (Vitis vinifera) 76.2 58.3 0.7 1.7 Trace 0.5
Graviola (Annona muricata) 401.2 235.0 0.8 1.7 1.3 0.4
Jaboticaba (Plinia cauliflora) 83.5 177.8 3.0 0.9 2.8 0.7
Jamelão (Syzygium cumini) 30.9 21.6 Trace 0.5 0.5 0.3
Mamão Papaya (Carica papaya) 224.2 221.8 0.1 1.9 0.7 0.2
Mango (Mangifera indica) 116.6 78.2 1.7 1.0 0.7 1.0
Passion Fruit (Passiflora edulis) 53.9 279.7 1.2 5.6 3.9 1.9
Pequi (Caryocar brasiliense) 324.4 297.7 6.4 2.7 9.6 2.1
Pitanga (Eugenia uniflora) 178.8 122.3 3.6 4.0 3.5 0.8
Pomegranate (Punica granatum) 47.5 127.0 1.3 2.6 6.7 1.9
Strawberry (Fragaria vesca) 109.0 96.7 3.3 3.2 1.8 0.6
Umbu (Spondias tuberosa) 115.6 113.5 0.3 0.9 4.2 0.4
The purple and white açaí have similar elemental levels. There was no
significant difference between the elemental levels of non-commercial purple and
white açaí (purple and white whole: paired two-tailed t-test, tcalc= 0.18 < tcrit =
2.15, p=0.8013, n=15, =0.05; purple and white de-fatted: paired two-tailed t-
test, tcalc= 1.48 < tcrit = 2.15; p=0.1622, n=15, =0.05). This suggest that the
190
samples might come from the same region, or from a similar soil composition
(Gonzálvez et al., 2009).
Biological toxicity (cell viability assay): Materials and method
The cell line mouse macrophage RAW 264.7 (ATCC TIB-71, American
Type Culture Collection; Livingstone, USA) used in the biological studies was
maintained in Dulbecco’s modified Eagle’s medium (DMEM, Life Technologies,
New York, USA), supplemented with 100 IU/mL penicillin/100 μg/mL
streptomycin (Fisher Scientific, Pittsburg, USA) and 10% foetal bovine serum
(Life Technologies, New York, USA) at a density not exceeding 5 × 105 cells/mL.
This was maintained at 37 °C in a humidified incubator with 5% of carbon dioxide
prior to the analysis.
The RAW 264.7 cells were seeded in a 96 well plate for the viability assay.
The cell viability was measured by the MTT (3-(4,5-dimethylthiazol-2-yl)-2,5-
diphenyl-tetrazolium bromide) assay, as previously described by Esposito et al.
(2014). In summary, the cells were seeded in a 96-well plate and treated with two
different doses of the açaí extracts, 50 and 250 µg/mL. The solvent vehicle or
dimethyl sulfoxide or DMSO (Sigma-Aldrich, St. Louis, USA) was used as a
positive control. After incubation, the media was discarded and 100 µL of DMSO
was added to dissolve the purple crystals. The resultant solution was quantified
spectrophotometrically at 550 nm using a microplate reader SynergyH1 (BioTek,
Winooski, USA).
Biological toxicity (cell viability assay): Results and discussion
The potential toxicity of the açaí pulp extracts (both methanolic and
aqueous) was determined using the cell viability assay, as described in section
5.5.10. The results, shown in Figure 5.15, were normalised for the concentration
of the formazan levels (which is the reduced purple product of 3-(4,5-
191
dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide or MTT measured by UV-
Vis spectrophotometer) of the blank (non-treated) cells. The normalised value of
the blank cells is related to the number of viable cells. The vehicle or solvent
used in the extractions was also used to treat the cells so as to confirm the non-
toxicity of the solvent. Dimethyl sulfoxide (DMSO) was used as a positive control,
due to its known toxicity to the cells at higher concentrations, as shown in Figure
5.15; where the viability of the cells decreases by half when treated with 5 µL of
DMSO. Neither of the açaí samples showed a significant toxicity response, even
at the higher concentration of 250 µg/mL (One-way ANOVA, r = 0.43, p=0.0757,
n=31, =0.05).
Figure 5.15: Formazan production levels in RAW 264.7 macrophage cells treated
with açaí extract solutions. Results expressed as mean ± st dev, n=3;
n, number of instrumental replicates).
Biological effect of açaí on radical inhibition assays: Materials and method
In order to evaluate the cellular antioxidant activities of the açaí extracts,
the amount of nitric oxide produced and released to the media of the cells was
measured. In infections and other inflammatory conditions, the macrophages are
Blank
Vehic
le
DMSO
(5ul)
Purple
White
Comm
erci
al
Purple
White
Comm
erci
al0.0
0.5
1.0
Fo
rmaza
n levels
F
old
in
cre
ase o
ver
bla
nk
50 mg/mL 250 mg/mL
192
activated in order to produce NO, an important mediator of the immunity system,
due to its regulatory and cytotoxic effects (Fang and Vazquez-Torres, 2002).
The cell line mouse macrophage RAW 264.7 (ATCC TIB-71, American
Type Culture Collection; Livingstone, USA) was also used in this study. The
nitrite production, a stable end product of NO production in activated
macrophages, was accessed colourimetrically at 540 nm and read on the
microplate reader. The cells were induced by lipopolysaccharides of bacteria
(LPS) to respond to an inflammatory process (Bannerman and Goldblum, 2003).
As a positive control, the cells were treated with dexamethasone (DEX), a well-
known compound that has an anti-inflammatory activity and inhibits the
production of NO. The ability of açaí pulp extracts to inhibit the nitric oxide radical
formation was determined according to Oliveira et al. (2010). A 100 μL portion of
the cell culture medium was added to 100 μL of the Griess reagent (1%
sulfanilamide and 0.1% naphthylethylenediamine in 5% phosphoric acid,
Promega, Fitchburg, USA), and the mixture was incubated at room temperature
for 10 min. The cells were treated with 50 µg/mL of the açaí extracts. The
absorbance was compared against a set of sodium nitrite standards (Promega,
Fitchburg, USA).
The in vitro radical oxygen species or ROS assay was used to evaluate
the capacity of the açaí extracts to decrease the production of ROS in the
stressed cells (Choi et al., 2007a). To this end, the cells were tagged with a
fluorescence dye and then induced with lipopolysaccharide (LPS) and treated
with the açaí extracts. The cell line mouse macrophage RAW 264.7 (ATCC TIB-
71, American Type Culture Collection; Livingstone, USA) was used in this study.
A known antioxidant compound, namely dexamethasone (DEX), was also used
as a positive control. In order to determine the in vitro reactive oxygen species
(ROS) generation, a fluorescent dye protocol was adapted (Choi et al., 2007b).
The RAW 264.7 macrophage cells were maintained as previously described and
seeded in a 24-well plate and incubated overnight at 37 °C. Cells were then
treated with 1 μL of 50 μM solution of dichlorodihydrofluorescein diacetate
acetylester (H2DCFDA, Molecular Probes, Eugene, USA), freshly prepared in
193
sterile phosphate-buffered saline (PBS, Sigma-Aldrich, St. Louis, USA) for 30
min. The fluorescent medium was aspirated, and the cells were exposed to 1 μL
of extract and 1 μL of lipopolysaccharide (LPS, from Escherichia coli 026:B6,
Sigma-Aldrich, St. Louis, USA), incubated for 24 h and the fluorescence of 2′,7′-
dichlorofluorescein (DCF) was measured at 485 nm (excitation) and 515 nm
(emission) on the microplate reader. A 10 μM aliquot of the antioxidant,
dexamethasone (DEX), was used as a positive control.
Biological effect of açaí on radical inhibition assays: Results and discussion
The radical inhibition levels of the açaí extracts were determined by NO
and ROS assays (refer to section 5.5.17). A paired two-tailed t-test (Miller et al.,
2018) confirmed that the null hypothesis was retained, that is, the different
extraction methods did not show a statistically significant difference in the radical
inhibition levels (tcalc= 0.14 < tcrit = 2.12; p=0.8939, n=17, =0.05). Therefore, the
results shown in Figure 5.16 represent the combination of the different extraction
methods. Comparison with the LPS induction study, confirmed that neither of the
açaí pulp samples (purple or white, and non-commercialised or commercialised)
showed any inhibition of the NO production by the extracts (One-way ANOVA; r =
0.33, p=0.2705, n=3, =0.05). Even though the samples did not show any
inhibition of the NO production it was only possible to evaluate that the presence
of the açaí extracts does not have an effect on the inducible nitric oxide synthase
(iNOS). This result contradicts previous studies (Matheus et al., 2003, Matheus
et al., 2006), although these researchers used a herbarium açaí pulp sample
cultivated in a different region than the natural occurrence of açaí.
194
Figure 5.16: Nitric oxide (NO) production in RAW 264.7 macrophage cells
stimulated with lipopolysaccharide (LPS). The cells were treated
with 50 µg/mL açaí extracts and dexamethasone (DEX). The
results are expressed as the mean ± st dev, n=3; n, number of
instrumental replicates.
The in-vitro reactive oxygen species (ROS) generation was determined
following the method described in section 5.5.11. The results shown in Figure
5.17 were normalised by the fluorescence levels of the cells induced by LPS only
and compared with the treated cells. It is clear that all of the açaí samples have a
positive effect on the inhibition of ROS generation, confirming the antioxidant
activity shown in the chemical assays. The data for the purple and white açaí
pulp samples, shown in Figure 5.17, are a combination of the non-commercial
whole and de-fatted samples, because they did not presented any significant
difference using a paired two-tailed t-test (for purple, tcalc= 0.28 < tcrit = 3.18;
p=0.9903, n=4, =0.05 and white, tcalc= 0.14 < tcrit = 3.18, p=0.4669, n=4,
=0.05). When compared with the LPS induced cells, all of the açaí pulp
samples confirmed a statistically significant difference (One way ANOVA, r =
0.55, p=0.0012, n=30, =0.05).
Control
LPSDEX
Purple
White
Comm
erci
al0.0
0.5
1.0
NO
pro
du
cti
on
F
old
in
cre
ase o
ver
LP
S
195
Figure 5.17: Radical oxygen species (ROS) production in RAW 264.7
macrophage cells stimulated with lipopolysaccharide (LPS). The
cells were treated with 50 µg/mL açaí pulp extracts and
dexamethasone (DEX). Results are expressed as the mean ± st
dev, n=3; n, number of instrumental replicates.
Biological effect of wound healing in human cells: Materials and method
The process of human wound healing is very complex, but it can be
divided into three main phases: inflammatory, proliferative and maturation (Wild
et al., 2010). The first occurs after body injury when many different inflammation
processes can occur, such as pain and swelling. The proliferative phase occurs
when the fibroblasts migrate from the tissue to the wound, so as to close the
injury. Then, finally at the last stage, collagen is deposited into the tissue (Wild et
al., 2010). There is no time frame for the duration of each one of these phases
and the body can go back and forward in this healing process, based on different
factors. Positive factors that help to improve or accelerate the wound healing
process are: vitamins A, C and E, iron, zinc and fats (Sanchez and Watson,
2016).
Control
LPSDEX
Purple
White
Pulp S
P
Comm
erci
al S
P
Comm
erci
al U
K0.0
0.5
1.0
RO
S p
rod
ucti
on
F
old
in
cre
ase o
ver
LP
S
196
In this study, the wound healing process was evaluated by investigating
the effect of the açaí pulp extracts on enhancing the proliferation phase of the
wound. The cell line Human Dermal Fibroblast cells (adult) – HDFa (Invitrogen C-
013-5C, Thermo Fisher Scientific Massachusetts, USA) was maintained in
Medium 106 (Invitrogen M-106-500, Thermo Fisher Scientific Massachusetts,
USA) supplemented with Low Serum Growth Supplement – LSGS (Invitrogen S-
003-10, Thermo Fisher Scientific Massachusetts, USA) and 1% of the antibiotic
penicillin /streptomycin solution; 10,000 IU/10,000 μg/mL (Fisher MT-30-002-CI,
Fisher Scientific, Pittsburgh, USA) at a density minimum of 2.5 x 104 viable
cells/mL and was maintained at 37 °C in a humidified incubator with 5% of
carbon dioxide prior to the analysis.
The cell migration assay was performed using the OrisTM Cell 2-D
migration of adherent cells assay kit (AMSBio, Cambridge, USA), where stoppers
were used to simulate the wound, as shown in Figure 5.18. Furthermore, the
cells were dyed with NucBlue® Live Cell Stain (Thermo Fisher Scientific
Massachusetts, USA), which is a reagent that bounds to DNA and can be excited
by UV light at 360 nm, with an emission maximum at 460 nm.
Figure 5.18: Cell migration determined using the OrisTM Cell 2-D migration of
adherent cells assay kit (Oris, 2017).
The stoppers were applied to a 96-well plate, where 50 μL of suspended
dyed cells and 1 μL of the açaí extracts (10 mg/mL) were added to each well,
with the exception for the controls that were ran at the same time. For the blank
197
readings only, media was added to the wells; 10% of foetal bovine serum (FBS,
Thermo Fisher Scientific Massachusetts, USA) was used as a positive control;
and for the full cell readings, the stoppers were never added to the wells,
allowing the cells to migrate. The plate was incubated for 2 hours and the
fluorescence was read, and blank corrected, on the micro-plate reader
SynergyH1 (BioTek, Winooski, USA) at time zero. Moreover, the images of the
cells were evaluated using the microscope EVOS FL Cell Imaging System
(Thermo Fisher Scientific Massachusetts, USA). The cells were further incubated
for another 48 hours and the migration of the cells were evaluated both via
florescence and imaging.
Biological effect of wound healing in human cells: Results and discussion
The wound healing experiment was performed as described in section
5.5.19 and the results are shown in Figure 5.19 as the difference between the
fluorescence values at t = 48 hours minus t= 0 hour of incubation. The results are
normalised to the values of the positive control, 10% FBS, and combine the two
extraction methods (refer to section 5.5.4), because they did not show a
significant difference, based on a paired two-tailed t-test (tcalc= 1.56 < tcrit = 2.30;
p=0.1565, n=9, =0.05). Furthermore, the purple açaí results (a combination of
non-commercial purple whole and de-fatted samples) showed no significant
difference based on using a paired two-tailed t-test, tcalc= 0.20 < tcrit = 2.36,
p=0.8449, n=8, =0.05; and the commercial the pulp SP, commercial SP and
commercial UK samples also did not show a significant difference using a one-
way ANOVA test (r = 0.19, p=0.1148, n=21, =0.05).
198
Figure 5.19: Florescence absorption of the radical oxygen species (ROS)
production in human dermal fibroblast cells (adult). The cells were
treated with 50 µg/mL açaí pulp extracts or 10% FBS (refer to
section 5.5.19). Results are expressed as the mean ± st dev, n=3;
n, number of instrumental replicates.
In order to investigate the migration of the cells, a picture of each sample
was taken, as described of section 5.5.19. Figure 5.20 shows a visual difference
of the migration of the cells between time 0 and after 48 hours of incubation ,
following the treatment with the non-commercial white açaí whole sample. This
was the sample that showed the highest potential of wound healing, as shown in
Figure 5.19 (0.5 fold increase over 10% FBS).
Blank
10%
FBS
Full ce
lls
Purple
White
whole
White
de-
fatte
d
Oil
White
Oil
purple
Comm
erci
al0.0
0.5
1.0
1.5F
lore
cen
ce A
bso
rpti
on
F
old
in
cre
ase o
ver
10%
FB
S
199
Figure 5.20: Fluorescence images of ‘wound healing’ of human dermal fibroblast
cells (adult) between time 0 (A) and after 48 hours of incubation
after treatment with non-commercial white açaí whole sample (B).
The results for this assay demonstrated the potential of the açaí pulp
extracts as a wound healing agent. In contrast to the other experiments reported
above in this study, the samples where the oil was present presented a positive
effect on the wound healing. This indicates that the oil plays an important role in
the wound healing process. Previous studies that have analysed the fatty acid
composition of the açaí oil, reported levels of 60% oleic acid, 22% of palmitic
acid, 12% of linoleic acid, 6% of palmitoleic acid and traces of other fatty acids
(Pacheco-Palencia et al., 2008). It has already been established that fatty acids
play an important function in the migration of cells (Sanchez and Watson, 2016).
A further study has also reported the influence of fatty acids on the acceleration
of the wound healing process (Arnold and Barbul, 2006).
200
5.6. Evaluation of the Amazon Geographical Variability and Industrial Processing on the Chemical Composition of Açaí
Introduction
It has been reported that the elemental content of a material is related to
the origin of the samples and that it is possible to track the origin or source of the
samples via ‘a fingerprint’ of the elemental content (Santos et al., 2014b). Also,
the antioxidant molecules are unstable and their content can possibly change
after the harvesting and processing stages of a sample (Timmers et al., 2017).
Therefore, it was important to analyse samples with a known record of the
harvesting and processing dates and the origin or location of these activities. The
samples listed in section 5.6.2 were analysed for the total polyphenol content, as
outlined in section 2.2 and elemental content (section 2.1). The results (fresh
weight basis) are reported in sections 5.8.1 and 5.8.2. The full set of data for both
dry and fresh weight and their moisture content are presented in Appendices 5.4
to 5.7.
Description of the samples
Samples were collected from a local market and companies along the
Amazonas river delta (Pará State). The fresh açaí berries were collected by
native residents from different areas of the Amazon forest, and at specific time
periods. The local market, as shown in Figure 5.21, is an open-air area in Belém,
that operates daily at dawn, where the local Amazonian people (from the
Amazon forest areas or islands of Belém or along the Amazonian rivers) unload
the berries from their boats for sale in the market.
201
Figure 5.21: (A) Picture of the open-air açaí market and (B) purple açaí fruits in
Belém (PA), Brazil.
The market supplies the city with the açaí berry in natura, which is
immediately processed during the morning. The resultant pulp is either ready for
consumption or frozen and shipped to the rest of the country or abroad.
Information was available for all of the açaí samples included in this study,
including source (Figure 5.22) and processing methods, as shown in Figure 5.23.
The full sample list is reported in Appendix 5.3.
The processed pulp samples were collected from 3 companies that have
different processing methods, as described in Figure 5.23.
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Figure 5.22: Map of the sources where the açaí samples were harvested by the
native people. Adapted from Google Maps (2019).
The Açaí Amazonas (Company I) has the only açaí plantation of the
country. They have two different sites, one in Mangau, which is an organic
plantation and the other one in Macupixi, where the crop is treated with fertilisers.
Products from this company are usually a blend of the two plantations. As such,
the açaí palm has aerial roots and it normally grows in very humid and muddy
soils close to rivers. In order to keep the roots protected, Açaí Amazonas uses
Mombaça, a type of grass, to cover the roots and they also use an artificial
dripping irrigation system to keep the soil humid. Açaí Amazonas also cultivate
açaí BRS, a variety genetically modified by the Brazilian Agricultural Research
Corporation - Embrapa (Homma et al., 2006). In general, harvesting is still
manual, although the palms are usually pruned to maintain a medium height to
aid harvesting operations. The fruits from these plantations are transferred to the
Açaí Amazonas processing plant where the berries are selected according to the
maturation state and size. Post-selection, the material is washed with ozoned
water in order to make the fruits safe for human consumption. Then, after the
softening of the pulp (or outer layer of the fruit), the berries are transferred to a
specific machine (despolpadeira) where the pulp is taken off from the seeds with
a certain amount of added water. The amount is determined by the market and
Brazilian legislation. The separated thick pulp juice is finally packed and frozen,
203
and the remaining seeds are used as biomass or thrown back into the plantation
as a natural fertiliser.
The ‘Point do Açaí’ (Company II) is well known in Belém city and their
production focusses on the local market of processed açaí for the citizens of
Belém. Every day, they receive freshly harvested native açaí grown in the small
islands close to city. When the fruits arrive at the processing plant, they are
selected and washed in a tank with water, then with a hypochlorite solution in
order to make it safe for human consumption. The final step is to repeat the
procedure using tap water to remove the remaining hypochlorite. After the
washing stage, the fruits are softened by thermal shock and the pulp is extracted
with the addition of a pre-determined quantity of water. The thick pulp is
extracted, and the seeds are sent to another location to be sold as biomass. The
local people normally consume the açaí pulp as part of their meal; therefore, they
usually buy and consume the fresh pulp.
The last company selected was Açaí Santa Helena (Company III), which
is focussed on the Brazilian and international market, shipping their product for
all of the regions of the country and abroad. They usually process the fruits that
were harvested the day before inside the Amazon forest. When the fruits arrive at
the plant, they are first selected and washed 3 times with filtered water, then a
chlorine solution and are again filtered with water to wash the remaining chlorine
from the pulp. The berries are softened, and the pulp is extracted. The seeds are
also sold as biomass and the product is finally packed and frozen. Furthermore,
the international market values more the bright purple colour of the berries than
the natural taste of açaí, therefore following their clients demand, they also could
add citric acid to ‘brighten’ the purple colour of the pulp.
204
Figure 5.23: Summary of the açaí processing steps and the differences between
companies, being Company I: Açaí Amazonas, Company II: Point
do açaí; and Company III: Açaí Santa Helena.
Açaí processing
Harvest
Transport toprocessing plant
Step 1: ozonedwater
Step 2: ozonedwater
Water tank at 45°Cuntil softening
Mechanical separation of pulpand seed
Seed
FertiliserBiomass
Pulp
Frozen pulp
Freezedrying
PowderFresh pulp
Selection
Step 1: Tap water Step 2: Hypochoride
solution Step 3: Tap water
Step 1: Water tank at80°C for 10 seconds Step 2: Water tank
room temperature untilsoftening
Company (I) Company (II) Company (III)
Step 1: Filtered water Step 2: Hypochoride
solution Step 3: Filtered water
Water tank at roomtemperature until
softening
Washing steps
Pulp softeningsteps
Addition of water
Addition of citric acid
205
Total polyphenol content: Materials and method
Total polyphenol content was analysed following the Folin-Ciocalteu assay
described in section 2.2. The freeze-dried açaí samples were extracted using a
0.5% (v/v) acetic acid solution, as presented in section 5.5.4 and analysed using
the micro-plate reader.
Total polyphenol content: Results and discussion
The total polyphenol (TP) content of non-commercial açaí (purple and
whole) samples collected during the field-trip study to the Amazon (April 2018)
are reported in Figure 5.23. The fruit and seed are related to the non-processed
berries acquired directly from the Amazon forest, whist the pulp is the processed
commercialised açaí, bought as frozen pulp.
It is clear that during the processing of the açaí berries, the total
polyphenol levels decrease suggesting that they are lost. This could be because
of the delay between harvest and the processing of the material, the washing or
pulp softening steps (refer to section 5.6.2), or even during the pasteurisation of
the material. The results for the processed samples (pulp in Figure 5.24) are in
agreement with the non-commercial, purple/whole samples reported in section
5.7.1 (Table 5.3) and the literature (Gordon et al., 2012, Augusti et al., 2016).
Interestingly, the non-commercial açaí seeds have detectable levels of total
polyphenols supporting the suggestion of the samples have an antioxidant
capacity, as previously reported in the literature (Rodrigues et al., 2006, Wycoff
et al., 2015, Melo et al., 2016). This information could be useful for the future
development of the non-commercial açaí seeds being an important by-product of
this ‘super-fruit’ industry.
206
Figure 5.24: Box plots representing the total polyphenol (TP) content (gallic acid
equivalent mg/kg f.w.) of açaí extractions using the Folin-Ciocalteu
assay (refer to section 2.2.). The samples relate to the type of
sample (non-commercial, purple/whole: fruit, n= 7; seed, n= 5; and
processed freeze-dried pulp, n= 8; where n is the number of
samples).
Furthermore, the data shown in Table 5.9 relates to the total polyphenol
(TP) levels of non-commercial purple/whole berries: fruit and seeds are non-
processed and the pulp is processed (with the moisture content indicated as
fluid, medium or thick).
These açaí berries, obtained from islands close to Belém (Ilhas), have
higher levels of total polyphenols (average of 39.90 mg/kg f.w.) compared with
similar berries from Genipauba (average of 13.67 mg/kg f.w.). Interestingly, the
local berries from the islands are sold at a higher price once the açaí has been
commercially processed. It has been suggested that this may be due to such
factors as the age of the palm tree, the maturation or the fruit exposure to
sunlight (Timmers et al., 2017).
Fruit
Seed
Pulp0
10
20
30
40
50
To
tal p
oly
ph
en
ol co
ncen
trati
on
mg
/kg
(w
et
weig
ht)
207
Table 5.9: Total polyphenol (TP) content of non-commercial açaí (purple/whole)
samples, (mg GAE / g) determined by Folin-Ciocalteu analysis (refer
to section 5.6.3). Fruit and seeds refer to non-processed berries and
the pulp is processed material (fluid, medium and thick relates to the
moisture content) (refer to section 5.6.2 for sample information).
Results are expressed as mean st dev in fresh weight, n is the
number of replicates, n = 3. Refer to Appendix 5.3 for code
information.
Code Origin* Type Total polyphenol GE-WB-P Genipauba Seed 5.07 ± 0.66
GE-WB-S Genipauba Fruit 13.64 ± 0.74
GE-PB-P Genipauba Seed 3.32 ± 0.22
GE-PB-S Genipauba Fruit 13.69 ± 1.47
IL-PA-P Ilhas Seed 3.62 ± 0.58
IL-PA-S Ilhas Fruit 40.51 ± 0.84
MA-PA-P Macapa Seed 4.48 ± 0.34
MA-PA-S Macapa Fruit 22.21 ± 0.32
AN-PA-P Anajas Seed 3.61 ± 0.18
AN-PA-S Anajas Fruit 25.05 ± 7.15
IC-PA-S Ilhas Fruit 39.28 ± 1.45
IM-PA-S Igarape-Miri Fruit 18.59 ± 2.83
PA-IC-PM Ilhas Pulp (medium) 2.93 ± 0.39
SH-AB-PF Abaetetuba Pulp (fluid) 1.63 ± 0.31
SH-IM-PF Igarape-Miri Pulp (fluid) 1.37 ± 0.06
SH-IM-PM Igarape-Miri Pulp (medium) 1.72 ± 0.10
SH-PA-PE Paragominas Pulp (thick) 3.56 ± 0.26
AA-OB-PM Obidos Pulp (medium) 2.09 ± 0.22
AA-OB-PE Obidos Pulp (thick) 2.77 ± 0.05
AA-OB-FD Obidos Freeze-dried 3.11 ± 0.29
*refer to Figure 5.22.
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The processed material (refer to section 5.6.2), described as non-
commercial pulp in Table 5.9, varies in the amount of water added according to
Brazilian legislation, as described in section 5.6.2, or if it was freeze-dried directly
in the processing plant (AA-OB-FD). The total polyphenol content increases in
the processed pulp which had less water added and a higher solid content.
Elemental composition: Materials and method
In order to evaluate the total elemental composition of the açaí pulp, the
samples were digested and analysed by ICP-MS, as described in section 2.3.
Elemental composition: Results and discussion
The elemental content (mean of 3 replicates) of the açaí (non-commercial
purple/whole) samples is summarised for Ca, Mg, Mn, Fe, Zn and Cu, as
previously outlined in section 5.7.9. The full dataset is in Appendices 5.4 to 5.7
(mean and standard deviations; mg/kg, fresh and dry weight). In general, the
data for Mg, Mn, Fe, Zn and Cu reported in Table 5.10, usually have higher
levels in the fruits when compared to the corresponded seed material.
Furthermore, the data in Appendices 5.6 and 5.7 also confirms this relationship
for Na, K, Cr and Ni. The toxic element levels for As, Cd and Pb are typically <
0.1 mg/kg fresh weight, which agrees with the values reported for other fruits
(Pendias-Kaabatas, 2010). Table 5.10 reports the mean value for the fruit and
seeds, whilst it should be also stated that there was a considerable degree of
variability in the mineral content at all sites (with standard deviations presented in
Appendices 5.4 and 5.5). It has already been reported that some plants tend to
accumulate various minerals in the flesh of the fruits (Kabata-Pendias, 2010).
Interestingly, the same pattern of higher polyphenol levels in fruit was reported in
section 5.8.1. It has been suggested that iron is ‘coordinated’ to polyphenols in
the açaí fruit (Yoshino and Murakami, 1998).
Comparison of this data for non-commercial purple/whole açaí samples
with literature values (Table 5.1) cannot be undertaken as only data for
209
processed samples have been published (Rogez, 2000, Menezes et al., 2008a,
Unicamp, 2011, Yuyama et al., 2011, Llorent-Martínez et al., 2013, da Silva
Santos et al., 2014, Moreda-Piñeiro et al., 2018).
Table 5.10: Total elemental concentration of açaí (non-commercial, purple/whole)
samples (mg/kg, fresh weight) analysed by ICP-MS (refer to section
5.6.4). Fruit and seeds refer to non-processed berries (section 5.6.2).
Results are expressed as a mean, n is the number of replicates, n =
3. Refer to Appendix 5.3 for code information.
Code Origin* Type Ca Mg Mn Fe Zn Cu GE-WB-P Genipauba Seed 71.66 133.94 7.02 4.81 5.15 4.52
GE-WB-S Genipauba Fruit 135.97 196.84 9.35 7.22 7.53 6.35
GE-PB-P Genipauba Seed 158.93 302.06 5.74 8.96 5.95 5.89
GE-PB-S Genipauba Fruit 158.1 334.07 6.34 8.53 6.33 4.27
IL-PA-P Ilhas Seed 673.38 343.63 55.15 6.32 5.94 4.96
IL-PA-S Ilhas Fruit 389.98 372.67 90.09 7.58 9.1 6.97
MA-PA-P Macapa Seed 848.72 407.38 83.95 5.75 5.5 4.4
MA-PA-S Macapa Fruit 480.75 343.59 156.17 7.2 7.6 5.36
AN-PA-P Anajas Seed 315.07 249.74 76.73 4.23 4.59 3.96
AN-PA-S Anajas Fruit 217.68 286.40 175.26 5.78 7.37 5.18
IC-PA-S Ilhas Fruit 378.57 297.16 105.97 6.5 5.31 4.6
IM-PA-S Igarape-Miri Fruit 395.12 312.00 45.24 45.94 7.49 6.99
*refer to Figure 5.22.
In this study, it was difficult to predict the effect the geographical origin of
the non-processed açaí has on the mineral content of the material. Yuyama et al.
(2011) suggested that several factors may influence the chemical composition,
such as: (i) the impact of tree growth in different environments, namely, aquatic,
terrestrial and floodable; (ii) climatic changes; (iii) soil composition; (iv) periodic
flooding in floodplain areas; or (v) cycles of agricultural production. This can be
confirmed by an inspection of the data in Table 5.10. As such, the fruit and seeds
collected from Genipauba have approximately 10-fold lower Mn (and Ca) levels
when compared with samples from the other locations. Furthermore, inspection
of the other elemental data in Appendices 5.6 and 5.7 shows variable patterns for
210
the other elements. In general, Genipauba is a community located next to the
Amazon river, as is Ilhas, Anajas and Igarape-Miri (refer to the map in Figure
5.22). Only Macapa is sited in the river estuary. Therefore, the factors reported
by Yuyama et al. (2011) support the fact that geographical effects are complex
and do not provide a clear guide on mineral uptake by açaí berries.
Table 5.11 reports the mineral levels for processed purple/whole açaí
(sampled from the same geographical location) and produced by three
commercial processing plants, namely, Point Açaí (code PA), Açaí Amazonas
(code AA) and Açaí Santa Helena (code SH) (refer to section 5.6.2 for details on
the different processing methods). In terms of the processing method the only
difference is the amount of water added during the pulp extraction step.
Inspection of this data shows that the amount of added water seems to have an
effect on the mineral content of the non-commercial açaí samples. The data
showed that materials with less water added are more concentrated (higher
solids content) and therefore have higher mineral or elemental levels (based on
fresh weight).
The manganese content of the açaí (pulp) samples ranged from 5.26 to
126.26 mg/kg (f.w.). These values are higher than the range of values reported in
the literature for açaí pulp (3.43 to 32.3 mg/kg f.w, refer to Table 5.1), although
the samples analysed in the literature are usually commercial samples with
additives (refer to Table 5.8).
The iron levels of the açaí (pulp) samples ranged from 2.29 to 8.96 mg/kg
(f.w.), with the exception of sample IM-PA-S, which had a much higher level of
45.94 0.56 mg/kg Fe. The values (when converted to a dry weight basis using
the conversion moisture values – refer to section 5.8) are similar to the range of
values presented in the literature (refer to Table 5.1), which have been reported
on a dry weight basis. The iron content did not show a significant trend between
the geographical origin or processing method. The same trend is found for
copper (1.19 – 6.99 mg/kg), zinc (1.93 – 9.1 mg/kg) and magnesium (133.94 –
407.38 mg/kg). The calcium concentration had a large variation ranging from
211
71.66 to 848.72 mg/kg (f.w.). Although samples from the same origin had similar
calcium levels. These range of concentrations were also reported in the literature
(refer to Table 5.1). Furthermore, as stated previously, the levels of arsenic,
cadmium and lead do not present a significant contribution to the chemical
composition of the açaí samples (typically <0.1 mg/kg f.w.).
Table 5.11: Total elemental concentration in commercially processed açaí
samples (mg/kg, fresh weight) analysed by ICP-MS (refer to section
5.6.4). Results expressed as a mean, n is the number of replicates,
n = 3. Refer to Appendix 5.3 for code information.
Code Origin* Type Water (%)
Ca Mg Mn Fe Zn Cu
PA-IC-PM Ilhas Pulp 89 276.45 186.31 82.67 3.85 2.54 1.51
SH-AB-PF Abaetetuba Pulp 92 178.76 148.10 31.46 2.29 1.94 1.36
SH-IM-PF Igarape-Miri Pulp 92 241.86 143.68 38.39 2.44 2.46 1.19
SH-IM-PM Igarape-Miri Pulp 89 402.81 212.23 93.03 3.33 3.11 1.73
SH-PA-PE Paragominas Pulp 86 402.54 265.47 25.41 4.73 3.47 2.59
AA-OB-PM Obidos Pulp 89 439.36 171.47 5.26 4.35 1.93 1.59
AA-OB-PE Obidos Pulp 86 562.68 216.03 15.01 4.90 2.15 1.67
AA-OB-FD Obidos FD** 86 525.45 246.16 46.74 5.71 3.24 2.44
*refer to Figure 5.22; **freeze-dried (FD) at the processing plant: PA- Point Açaí; SH - Açaí Santa Helena, and AA - Açaí Amazonas.
5.7. Link to Dietary Intake of Total Polyphenols and Minerals of Açaí
One of the aims of this research was to evaluate the contribution that the
consumption of açaí might make to the dietary intake of total polyphenols and
minerals. A previous study suggested that a 500 g (fresh weight) serving of açaí
would equate to the amount of the product that is consumed on a daily basis by
the Brazilian population (Heinrich et al., 2011). The results presented in this
study are related to the dry weight basis of the material which has been
converted to fresh weight using the conversion factor reported during the drying
process, i.e. 90% of the moisture weight is lost (Pavan et al., 2012). Therefore, in
212
order to calculate the daily intake of açaí, the fresh weight was taken into
consideration in the calculation.
The açaí pulp provided 50.9 to 424.3 mg GAE/100 g fresh weight of total
polyphenols. The content of typical berries is presented in Table 5.5. The range
of total polyphenol content of açaí pulp is similar to that reported for cranberries,
raspberries, blueberries and strawberries (Table 5.5). It is difficult to estimate a
recommended daily intake for total polyphenols, due to the variation in the levels
of the phenolic compounds in a particular foodstuff. This is due to the structural
diversity of the phenolic compounds or the lack of standardised analytical
methods (Scalbert and Williamson, 2000). Even though there may be variations
in the dietary intake of phenolic compounds between geographical regions and
consumption age groups, a selection of previous studies have proposed a range
of 1 g of total polyphenols per day (Kühnau, 1976, Faller and Fialho, 2009,
Landete, 2013). Most authors refer to Fukushima et al. (2009), who based on a
balanced Japanese diet, calculated a daily consumption of 1492 mg (fresh
weight) of polyphenols. In terms of the serving values reported by Unicamp
(2011), a 500 g serving of commercial açaí pulp (fresh weight) would contribute
17 to 142 % of the daily intake of total polyphenols.
The recommended daily intake (RDA) is defined by the World Health
Organisation or WHO (WHO, 1996) for males (M) and females (F) and is
presented in Tables 5.9 (total polyphenols and minor elements) and 5.10 (trace
elements). The WHO RDA guidelines are compared with the calculated % intake
of the chemicals for the consumption of açaí pulp; based on using the mg/day
level (reported in Appendix 5.8) calculated from the data in Tables 5.12 (total
polyphenols) and 5.13 (elements).
213
Table 5.12: Percentage intake (%) of total polyphenol and minor elements based
on the consumption of a 500 g serving (Heinrich et al., 2011) of the
commercial and non-commercial açaí pulp (reported on a fresh weight
basis). The data is compared with the World Health Organisation
recommended daily allowance (RDA) for males (M) and females (F).
Total polyphenol Ca Mg M F M F M F
WHO RDA (mg/day)* 1492 1300 400 310
Non-commercial
Purple whole 107.2 18.0 29.3 37.6
Purple de-fatted 132.0 20.3 34.1 43.9
White whole 31.5 16.0 30.9 39.8
White de-fatted 39.2 20.2 37.7 48.8
Commercial
Pulp SP 94.8 6.4 25.3 32.6
Powder SP 142.1 7.8 25.5 32.9
Powder UK 17.1 2.6 10.7 13.9
* World Health Organisation Recommended Daily Allowance (reported in mg/day) (WHO, 1996) and used to calculate the % intake from the data for total polyphenols and elements (Appendix 5.8; mg/day).
The açaí berries represent a potential source of Ca, Mg, Mn, Fe, Zn and
Cu (with the exception of the UK commercial açaí powder product). It has already
been noted that this product seems to have been adulterated, that is, has much
lower chemical levels than local commercial and non-commercial samples,
suggesting the addition of other non-nutrient ‘bulking’ material to the powder
(refer to sections 5.7.3 and 5.7.9).
It is important to highlight that even though açaí could be considered a
source of iron, recent studies have shown that the element is not bioavailable in
açaí. As reported by Toaiari et al. (2005), 40 anemic rats were fed with a non-
commecial sample of purple açaí and commercial feed for 7 days. The rats
developed anemia which was linked to a depletion of dietetic iron. The
concentration of hemoglobin was measured after the experiment and showed
214
that the rats fed with açaí did not show any significant increase when compared
with the commecial feed, since the recovery of hemoglobin concentration from
anemic rats was not observed. A clinical study based on children also proved the
low bioavailability of iron in açaí (Yuyama et al., 2002). In this study, 85 children
aged from 2 to 6 years old were fed for 120 days with açaí and iron aminoacid
chelate as a source of iron. The results demonstrated the impact of açai as an
energy source with a significant weight gain in the children (1.76 kg). However,
the recovery of the anemic children was higher in the group that received iron
aminoacid chelate (reduction of 34%) in comparison with açaí (reduction of 11%).
The low bioavailability of iron in açaí may be explained due to the high
concentration of tannins and fibers in the berries (Yuyama et al., 2002). However,
the consumption of açaí pulp or powder, along with other sources of vitamin C
(such as citrus), could potentially increase the bioavailability of iron (Silva et al.,
2004). The bioavailability of the chemicals present in açaí may be influenced by
the chelation of a metal-polyphenol complex. Moreover, the total polyphenol data
reported in Table 5.3 and the manganese data reported in Table 5.7(a) suggests
a possible correlation, where the respective concentrations potencially increase
together (Spearman’s correlation; R= 0.36, p=0.43 n= 7 and = 0.05). This
correlation could influence the bioavailability of both chemicals.
Also based on the low levels of the toxic elements, the daily intake of As
and Pb were not significant (refer to Appendix 5.2). The toxic element Cd
represented an average of 0.0035 mg/day, which does not provide a significant
contribution to the Brazilian average dietary intake of 1.31 g of Cd (Avegliano et
al., 2011).
As was stated in section 5.7.10, one of the aims of this research was to
evaluate the contribution that the consumption of açaí might make to the dietary
intake of total polyphenols and minerals. As was explained for the processed
samples, the same evaluation is presented for the non-processed material (fruits,
seeds and pulp) using a 500 g (fresh weight) serving of açaí that equates to the
amount of the product that is consumed on a daily basis by the Brazilian
215
population (Heinrich et al., 2011). Appendix 5.9 provides the calculated % dietary
intake of all the elements based on a daily consumption of 500 g of açaí (pulp,
purple/whole, fresh weight basis) relative to the recommended daily intake
defined by the World Health Organisation (WHO). The results, presented as box
plots in Figure 5.25, are for the minor elements, Ca, Mg, Fe and Zn. Figure 5.26
provides the data for Mn and Cu. Similarly, the daily intake of total polyphenols
are also presented in Figure 5.26, based on the recommendations of Fukushima
et al. (2009).
Table 5.13: Percentage intake (%) of selected trace elements based on a 500 g
serving (Heinrich et al., 2011) of non-commercial or commercial açaí
pulp or powder (fresh weight). The data is compared with the World
Health Organisation recommended daily allowance (RDA) for males
(M) and females (F).
Mn Fe Zn Cu M F M F M F M F
WHO RDA (mg/day)* 2.3 1.8 8 18 11 8 0.9
Non-commercial
Purple whole 1392.7 1779.5 18.8 8.4 11.3 15.6 100.6
Purple de-fatted 1759.2 2247.7 26.1 11.6 14.7 20.3 123.4
White whole 1329.2 1698.3 22.8 10.2 12.0 16.6 95.3
White de-fatted 1758.4 2246.8 26.9 12.0 16.4 22.6 138.7
Commercial
Pulp SP 581.8 743.4 18.8 8.3 12.3 16.9 84.6
Powder SP 1189.2 1519.6 13.7 6.1 10.6 14.5 83.2
Powder UK 35.3 45.1 1.4 0.6 5.8 7.9 19.5
* World Health Organisation Recommended Daily Allowance (reported in mg/day) (WHO, 1996) and used to calculate the % intake from the data for total polyphenols and elements (Appendix 5.8; mg/day).
The data in Figure 5.25 (and Table 5.13) clearly shows a variation in
the % intake of the minor elements relative to the RDA. This is true for both
males and females. This is not surprising as the elemental levels for this açaí
product also covered a wide range, as reported in section 5.8. In general, the
216
açaí pulp samples are potentially a good source of minor elements, with the
order of % intake based on the RDA being: magnesium (both male and female) >
iron (male) > zinc (both M and F) > calcium. In addition, the % intake of sodium
(1 to 5 % for both M and F) and potassium (~9% for M and F) are low. In terms of
the essential trace element % intake of Mn and Cu are very high, being > 1000 %
for both gender groups (Table 5.13). The levels for the other trace elements are
very low, with Cr (8% for M and 11% for F), and for Co, Se and Mo (having 0%).
Figure 5.25: Box plots representing the percentage intake (%) of minor elements
based on the consumption of a 500 g serving of açaí pulp
(purple/whole) (fresh weight). The data is compared with the World
Health Organisation (WHO) recommended daily allowance (RDA)
for males (M) and females (F). Note: WHO provide no gender data
for Ca.
Figure 5.26 confirms that the daily consumption of 500 g of fresh weight
açaí pulp (purple/whole) would contribute to about 350 % of the intake for total
polyphenols. When compared to other berries and typical fruits from Brazil the
total polyphenol content of açaí is similar to other well-known ‘super-fruits’, such
as, blueberries and acerola, contributes more than 100% to the recommended
daily intake of total polyphenols (refer to Tables 5.4 and 5.5).
Ca Mg (M) Mg (F) Fe (M) Fe (F) Zn (M) Zn (F)0
20
40
60
80
% In
take o
f R
DA
in
aça
í (5
00 g
serv
ing
)
217
It is important to highlight that even though this study reports the potential
intake of total polyphenols and elements for all açaí samples, only the processed
açaí is available for consumption due to food safety (refer to section 5.2.2).
Figure 5.26: Percentage intake (%) of total polyphenol (TP) and trace elements
of 500 g serving of açaí (fresh weight) in relationship to the
recommended daily allowance (RDA) for males (M) and females
(F).
5.8. Conclusion
This chapter reviewed the chemical characterisation, antioxidant and
biological activities of different samples of açaí. Furthermore, this study also
evaluated the potential contribution of the açaí pulp to the dietary intake of
polyphenols and elemental nutrients. This research provided for the first time a
comprehensive investigation of the polyphenol and trace element composition of
non-commercial and commercial; processed and non-processed; white and
purple; whole and de-fatted samples of açaí obtained from Brazil.
The total polyphenol content of non-commercial and commercial açaí
samples were analysed by Folin-Ciocalteu assay (refer to section 5.5.5) and
were found to range from 32.00 to 39.40 mg/g d.w. for non-commercial purple
TP Mn (M) Mn (F) Cu0
2000
4000
6000
% In
take o
f R
DA
in
aça
í (5
00 g
serv
ing
)
218
samples; 5.09 to 42.40 mg/g d.w. for commercial purple samples and 9.40 to
11.70 mg/g d.w. for non-commercial white samples (refer to section 5.7.1). The
purple non-commercial samples had a significantly higher concentration of total
polyphenols in comparison with the white samples. Moreover, the commercial
purple samples had a higher level of variability (standard error of the mean of
9.26 GAE mg/g) when compared with the non-commercial purple açaí berries
(2.90 GAE mg/g). The removal of the oil fraction also showed a significantly
higher concentration on the total polyphenol content for the non-commercial
samples as demonstrated on section 5.7.1. The total flavonoid content analysed
by the AlCl3 assay (refer to section 5.5.6) of açaí pulp extractions covered a
range from 6.39 to 8.05 mg/g d.w. for non-commercial purple samples; 1.88 to
6.07 mg/g d.w. for commercial purple samples and 2.12 to 2.38 mg/g d.w. for
non-commercial white samples. This flavonoid analysis shows a similar pattern to
the total polyphenol content data, as would be expected because the flavonoids
are a subclass of polyphenols (refer to section 5.7.2)
When comparing the total polyphenol content of non-processed açaí
samples (non-commercial, purple and whole), presented in section 5.8.1; it is
clear that during the processing of the açaí berries, the total polyphenol level
decreases, suggesting that they are lost. The non-processed açaí fruits ranged
from 13.64 to 40.51 mg/kg f.w.; the non-processed seed from 3.32 to 5.07 mg/kg
f.w. and the processed pulp from 1.37 to 3.56 mg/kg f.w.. The açaí samples from
the islands close to Belém (Ilhas), had higher levels (average of 39.90 mg/kg
f.w.) when compared to samples from Genipauba (average of 13.67 mg/kg f.w.).
Also, the total polyphenol content increased in the processed açaí pulp that has a
higher solids content.
The total anthocyanin profile and levels were analysed by HPLC (refer to
section 5.5.8). The results showed that cyandin-3-glucoside and cyandin-3-
rutinoside are the major anthocyanins in the purple samples and were not
present in the white açaí samples and one of the commercial samples (Powder
UK). This suggests that the Powder UK sample might be adulterated (refer to
section 5.7.3). The total anthocyanin content for the non-commercial purple açaí
219
samples ranges from 102.0 to 143.3 mg/100 g f.w. and for the commercial purple
açaí samples 35.9 – 47.0 mg/100 g f.w. The total proanthocyanidin (PAC)
content of the açaí samples were determined using the DMAC assay (refer to
section 5.7.4). This assay was the only one that presented a significant
difference for the extraction method. The aqueous extraction resulted in
significantly higher PAC concentrations in comparison with the methanolic
extraction, due to the oligomeric nature of the PAC compounds. The PAC for the
non-commercial purple açaí samples ranges from 5.06 to 6.10 mg/g d.w.; the
commercial purple samples from 4.47 to 5.48 mg/g d.w. and for non-commercial
white samples 2.60 – 3.96 mg/g d.w. (refer to section 5.7.4).
The antioxidant activity levels were analysed by ABTS and DPPH assays
(refer to section 5.5.6). The non-commercial purple açaí was the most prominent
of all the antioxidant analysis when compared to the commercial purple and non-
commercial white samples, due to its higher levels of total polyphenols and total
anthocyanins. Furthermore, commercially purple sample (Powder UK) had a
significantly lower activity when compared to the other commercial purple
samples (refer to section 5.7.5). The strong antioxidant effect of the açaí samples
were confirmed on cells through the inhibition on the radical oxygen species
production (refer to section 5.7.7) where all the açaí samples confirmed a
statistically significant effect on the inhibition of the ROS generation. Although,
the samples did not show any inhibition of the NO production, contradicting the
literature (refer to section 5.7.7). Furthermore, neither of the açaí samples
demonstrated any toxicity effect on cells, even at the higher concentration of 250
µg/mL as demonstrated on section 5.7.7. The wound healing experiment was
performed on human fibroblast cells (refer to section 5.5.14). The visually
demonstrated migration effect of the açaí extracts on this cells proved açaí as a
potential wound healing agent. In addition, the samples where the oil was
present presented a positive effect on the wound healing.
The total elemental concentrations were determined in the açaí samples
(refer to section 5.6.4). The calcium, magnesium, manganese, iron, zinc and
copper; essential minor and trace elements found in significant levels in açaí
220
samples were highlighted in section 5.7.9. Overall, for both white and purple non-
commercial açaí samples, all of the elemental values for the de-fatted samples
are slightly more concentrated than the whole material due to the removal of the
oil fraction, as demonstrated before. The enhancement of total polyphenols in the
defatted samples is on average 28% for purple and 38% for white samples.
There was no statistically significant difference between purple and white
samples. In contrast, the commercial sample (Powder UK) had elemental levels
typically 30% lower than other commercial samples. The commercial processing
of açaí reduced the concentration of Ca and Mn by about 50% and the
commercial pulp had a significant variance of these elements. The manganese
levels were on average 703.25 mg/kg d.w. for non-commercial samples and
270.5 mg/kg d.w. for commercial purple samples. Similarly, calcium levels were
4.75 g/kg d.w. for non-commercial and 1.42 mg/kg d.w. for commercial purple
samples. Magnesium (2.59 and 1.60 mg/kg d.w.); iron (36.81 and 17.69 mg/kg
d.w.); zinc (29.4 and 20.47 mg/kg d.w.); and copper (20.20 and 10.99 mg/kg
d.w.). The toxic elements, arsenic, cadmium and lead were found to be at very
low levels in the commercial and non-commercial; purple and white açaí products
(typically <0.01 mg/kg f.w.).
This study also provides elemental data for non-processed açaí samples
(non-commercial, purple and whole), as presented in section 5.6. Overall, the
data for Mg, Mn, Fe, Zn, Cu, Na, K, Cr and Ni are higher in the non-processed
fruits when compared to the corresponding seed material. The fruit and seeds
collected from Genipauba presented approximately a 10-fold lower Mn (and Ca)
level when compared with samples from the other locations. The data showed
that materials with less water added (higher solids content) had higher mineral
levels (based on fresh weight). The manganese content of the açaí (pulp)
samples ranged from 5.26 to 126.26 mg/kg f.w., iron from 2.29 to 8.96 mg/kg
f.w.; copper (1.19 – 6.99 mg/kg), zinc (1.93 – 9.1 mg/kg) and magnesium (133.94
– 407.38 mg/kg) and calcium had a large variation ranging from 71.66 to 848.72
mg/kg (f.w.). The toxic element levels for As, Cd and Pb were typically < 0.1
mg/kg f.w (refer to section 5.8.2).
221
One of the aims of this research was also to evaluate the contribution that
regular consumption of açaí (500g) could make to the dietary intake of
polyphenols and minerals. The total polyphenol intake of purple açaí is similar to
other well-known ‘super-fruits’, such as blueberries and acerola, contributing to
the recommended daily intake of total polyphenols by more than 100% in a single
serving. In relation to the mineral levels, açaí may have a great potential as a
source of manganese (average of 1500%) and copper (average of 90%); along
with calcium (20%), magnesium (30%) and zinc (15%). However, it is known that
the form of iron present in açaí may not be bioavailable to humans (Yuyama et
al., 2002). Also, the low levels of Cd, As and Pb did not present a significant
contribution to the elemental daily intake (refer to sections 5.7.10 and 5.8.3).
222
Chapter 6. Conclusions and Future Work
223
6.1. Overview
Brazil is a major producer of special natural foods and beverages that are
commercialised and sold as natural and processed products. Many are marketed
as being good sources of nutrients, namely, elements (or in terms of nutrition are
referred to as minerals) and polyphenols, that play an important role in human
health (Torres and Farah, 2017). Figure 1.1 summerised many of the main
products that are being consumed, especially throughout Brazil. At present, very
few scientific and health-related studies have been reported on the chemical
composition of these natural foods or beverages using locally sourced materials
in Brazil. Moreover, Tables 3.1 (yerba mate), 4.1 (roasted coffee) and 5.1 (açaí)
listed the data that has been published for selected elements (primarily Mg, Ca,
Mn, Fe, Cu and Zn). In terms of a the critical analysis of these studies, it is
difficult to make an estimation of the daily intake of such chemicals when there is
a general lack of reported analytical details (including the sampling protocols,
weight basis of calculated data, lack of validation procedures and instrumental
details – limit of detection, linear dynamic range of the calibration standards and
dilution factors). Furthermore, many of the published studies focus on only a
particular food or beverage and provide data for specific chemicals (Santos et al.,
2014a, Stelmach et al., 2015, Rusinek-Prystupa et al., 2016).
The aim of this research was to determine the levels of chemical elements
(major, minor and trace) and polyphenols of yerba mate (from the southern
region of Brazil), roasted coffee (São Paulo state) and açaí berries (from the
Amazon region). This data was then used to assess the impact of consumption in
terms of the daily dietary intake in providing an adequate nutrient supply for
Brazilians. As was stated in chapter 1, section 1.5, a main feature of this
research was to establish an analytically robust method(s) that enabled an
evaluation of the impact of sample selection and treatment; the influence of
dilution in prepared sample solutions before instrumental analysis; and the
calculation, statistical analysis and reporting of data in relation to what is
traditionally consumed by individuals in Brazil.
224
In summary, the aim and objectives of this research (refer to section 1.5)
have been meet for the scientific evaluation of Brazilian and Argentinian yerba
mate (plant material, commercial products and infusions) grown in southern
Brazil or the Misiones region of Argentina (chapter 3); roasted coffee beans and
infusions from samples grown in Amparo, São Paulo State (chapter 4); and açaí
fruits (pulp and seeds) and processed pulp collected from the Amazonian region,
northern Brazil (chapter 5).
6.2. Yerba Mate
Chapter 3 presented the chemical analysis of different samples of yerba
mate. This research provided for the first time a comprehensive investigation of
the elemental and polyphenol composition of non-commercial, non-processed
and processed samples of yerba mate from Brazil and Argentina. Manganese
was an element highlighted in this study (section 1.2.5) due to its higher
concentration in the yerba mate samples and prepared infusions (Table 3.1). No
previous study has assessed what the potential intake of Mn would be through
the consumption of different yerba mate infusions in terms of the total dietary
intake of this element by the South American population (section 3.6.6).
The first study focussed on the elemental composition of non-commercial
yerba mate leaves from the Barão de Cotegipe plantation in southern Brazil -
with the field-trip undertaken in April 2017 (section 3.5). Overall, in terms of the
age of the leaves collected from traditional plantations (refer to section 3.5.3), the
general trend was for the elements to be at higher levels in the old leaves (new <
old; refer to Table 3.2). Statistical analysis using a two-tailed Student t-test
confirmed that there was a significant difference between the age of the leaves
(p<0.05) collected from the plantations for Mg, Ca, Fe and Cu (Table 3.3).
Moreover, the new yerba mate leaves grown on trees located in an organic
plantation had higher levels of most of the elements, especially for Mn, when
compared with other plantations treated with NPK fertilisers (new leaves; fert <
org, Table 3.2). Once again, statistical analysis using two-tailed Student t-test,
225
confirmed a significant difference (p<0.05) between the age of the leaves from
the same tree, for Ca and Mn. Interestingly, for the older leaves, all of the
elements were higher in plants grown in the fertilser-added plantation (old leaves;
fert > org; Table 3.2). A two-tailed Student t-test confirmed a significant difference
(p<0.05) for the Fe values reported in Table 3.2, which are higher in the fertilsers-
added plantation. An evaluation of the elemental levels of leaves collected at
different heights of a yerba mate tree grown in an organic plantation confirmed a
translocation of manganese to the upper parts of the tree; Mn concentrations in
the tree: bottom 1070 ± 4; middle 1009 ± 82 and 1229 ± 21 top mg/kg, dry weight
(d.w.); Table 3.5. Higher elemental levels were also found in yerba mate plants
grown in the traditional organic plantations rather than in the natural forests
(Table 3.6). In relation to the processing of the yerba mate material, the elements
showed higher levels after the sapeco stage (leaf exposed to an open fire for a
short period of time) of the process (refer to section 3.5.5, Table 3.8).
Secondly, the elemental composition of commercial yerba mate products
from Brazil and Argentina was evaluated in terms of the type of sample (green or
roasted), processing package (loose or tea bags) and origin (Brazil or Argentina)
(refer to section 3.6.3). In summary, the elemental levels were similar for the two
countries (Table 3.9), which is to be expected as yerba mate is primarily grown
between the Paraná and Paraguay river basins in South America (Figure 3.1).
The Mn levels for the green loose yerba mate samples from Brazil ranged from
486 to 834 and the Argentinian samples from 383 to 671 mg/kg (d.w.). In terms
of the method of packaging, all of the elements measured had higher elemental
levels for the tea bag products when compared to the green loose material
(Table 3.9). The Mn values were found to range from 483 to 1098 mg/kg (d.w.)
for the tea bag samples and 383 to 671 mg/kg (d.w.) for the green loose material.
The roasted (loose and tea bag) samples also had higher elemental levels when
compared with the Brazilian green loose material (Table 3.9). The Mn levels of
the roasted samples ranged from 397 to 889 mg/kg (d.w.) where for the green
loose samples the values were 486 to 834 mg/kg (d.w.).
226
In order to evaluate the chemical intake of yerba mate, different infusion
methods were prepared (refer to section 3.6.2). The regular infusions (prepared
using a tea-based method) of yerba mate (green loose) from Brazil had slightly
higher chemical levels (Tables 3.11, 3.16 and 3.19). All of the elements (Table
3.11), polyphenols and xanthines (section 3.6.5) had higher chemical levels for
the infusions prepared using tea bag samples when compared to the green loose
regular infusions. Moreover, regular infusions made with green loose yerba mate
had significantly higher levels of trace elements, polyphenols and xanthines in
comparison with the roasted samples. Statistical analysis using a two-tailed
Student t-test showed a very highly significant difference (p < 0.0001) between
the total polyphenol content of green and roasted yerba mate samples (Table
3.16).
Finally, an evaluation of the potential intake of minor or trace elements
and polyphenols through the consumption of yerba mate products was
undertaken in section 3.6.6. The trace elements analysed in this study
represented 0.1 to 5% of the recommended daily allowance (RDA) (WHO, 1996),
for all of the infusion methods (regular, Brazilian and bombilla). The exception
was found for manganese. A regular infusion serving (1 cup of 200 mL) would
provide 23.7 to 106.0 % for males and 30.3 to 135.5 % for females of the
manganese RDA, depending on the type of yerba mate product (refer to Table
3.22). Although, the bombilla method (traditional method of consumption in South
America – section 3.2.3) involved a serving of 1 litre of drink, this would provide
up to 1482 % for males and 1894 % for females of the manganese RDA. The
impact of consuming such high levels of manganese may be influenced by the
bioavailability of Mn in the various infusions (as discussed in section 6.5). In
terms of the total polyphenol intake, a regular infusion serving (200 mL) could
contribute 4.0 to 14.5 % of the daily intake and the bombilla method can provide
up to twice the amount of the adequate daily intake of total polyphenols (refer to
Table 3.23).
227
6.3. Chemical Composition of Roasting Brazilian Coffee
Chapter 4 investigated for the first time, the roasting process of Brazilian
coffee (based on samples and a process undertaken at a local plantation in
Brazil) and the impact on the chemical composition of the roasted beans and
related beverages. The effect of roasting different coffee varieties (Obatã, Catuaí,
Bourbon Amarelo and blend) collected from the Fazenda Palmares and Flor
plantations (Amparo, São Paulo State) resulted in a slight increase of the
elemental content of the beans during the roasting process (Tables 4.2 to 4.4).
The manganese values ranged from 18.32 (t = 0 minutes) to 26.60 (t = 0
minutes) mg/kg (d.w.) for the roasting process of a Bourbon Amarelo coffee
variety (Table 4.4). This suggests that a small decrease in the moisture content
may result in a ‘pre-concentration’ of the elements in the beans as a result of the
roasting process. The data also highlighted that there are no elemental losses
during the roasting process (Tables 4.2 to 4.4). The total polyphenol content of
coffee infusions, produced from beans collected at different times of the roasting
process, showed 7 to 52 % higher levels in the dark roast (10 minutes) when
compared to the green bean infusions (0 minutes, Table 4.6). The chlorogenic
acid and caffeine data showed a similar trend with an increase in the levels of the
infusions prepared using the medium roast coffee (Figures 4.4 to 4.7).
Furthermore, the effect of the particle size of the ground beans on the levels of
chlorogenic acids and caffeine in the coffee infusions was investigated. The data
showed that the levels were inversely proportional to the particle size, confirming
that the grinding stage influences the extraction of the chemicals during the
preparation of a coffee infusion (Figures 4.8 and 4.9). The manual selection of
coffee beans after the roasting process (10 minutes) was undertaken based on
quality factors (refer to section 4.3.2). This procedure is undertaken by the
manufacturer to enhance the chemical quality of the coffee product. The data
showed that manual selection does not result in any major difference in the
elemental composition or chlorogenic acid levels of the roasted coffee products
(Tables 4.5 and 4.7). However, a different finding was found for the coffee
228
infusions prepared using selected (higher quality) beans, namely, higher total
polyphenol levels (Table 4.7). The physical changes on the structure of coffee
beans during the roasting process were investigated and scanning electron
microscope images showed an expansion of the coffee bean and an increase in
the size of the bean micropores (Figures 4.10 (A) to (F)). Finally, an evaluation of
the potential intake of polyphenols from coffee infusions showed that a cup of
coffee (92 mL) can contribute up to 7 % of the estimated daily intake of
polyphenols. Elemental data was not obtained for this specific study due to the
coffee infusions causing instrument signal instability for the inductively coupled
plasma mass spectrometer (section 4.7).
6.4. Açaí
Chapter 5 reported the chemical characterisation, antioxidant and
biological activities of different samples of açaí (berries, seeds and processed
pulp) and provided for the first time a comprehensive investigation of the
polyphenol and minor or trace element composition of non-commercial and
commercial; processed and non-processed; white and purple; whole and de-
fatted samples of açaí (refer to section 5.5.2 for the classification of these terms).
In this specific study, priority was given to the polyphenol analysis
because of the investigation of the relationship between the chemical
composition and biological activity. This research was undertaken at North
Carolina State University (USA) (section 5.5). The non-commercial purple mature
berries (that differ from white ones which are a different variety) had a
significantly higher (two-tailed Student t-test, p<0.001) concentration of total
polyphenols in comparison with the white samples (Table 5.3 and Figure 5.8).
The non-commercial purple samples also had a higher range of the total
flavonoid content (Table 5.3). The total anthocyanin profile showed that cyandin-
3-glucoside and cyandin-3-rutinoside are the major anthocyanins in the purple
açaí pulp samples, although the white berries had inconsistent values (Figure
5.10). The total anthocyanin content was higher for the non-commercial
229
processed purple açaí pulp when compared to the commercial purple açaí
samples (Table 5.3). The total proanthocyanidin (PAC) analysis resulted in
significantly higher PAC concentrations in the aqueous extraction (paired two-
tailed t-test, p<0.005), due to the oligomeric nature of the PAC compounds
(Figure 5.11). The non-commercial purple açaí pulp was found to be very
important in terms of the antioxidant analysis (ABTS and DPPH assays, refer to
section 5.5.12). This is due to the higher levels of total polyphenols and total
anthocyanins when compared to the commercial purple and non-commercial
white pulp samples (section 5.5.12). The strong antioxidant effect of the açaí
samples was confirmed using mouse cells through the inhibition of the production
of radical oxygen species (refer to section 5.6.3). This study involved all of the
açaí pulp samples and confirmed a statistically significant effect on the inhibition
of radical oxygen species (ROS) generation (Figure 5.17). A wound healing
experiment was performed using human fibroblast cells. The data confirmed a
migration effect on human cells subjected to açaí pulp extracts. These results
are very important, as such an experiment has never been reported, and implies
that processed açaí pulp may have potential as a wound healing agent (Figures
5.19 and 5.20).
The calcium, magnesium, manganese, iron, zinc and copper levels in açaí
samples were found to be higher than that reported in the literature for other
typical Brazilian fruits (based on TACO values) (Table 5.7 (a) and Figures 5.25
and 5.26) (Unicamp, 2011). Overall, the elemental values for the de-fatted açaí
samples were found to be more concentrated than the whole material due to the
removal of the oil fraction. It is suggested that this results in a ‘pre-concentration’
of the elements in the processed pulp (Table 5.7 (a)). Interestingly, there was no
statistically significant difference in the elemental content of the purple and white
samples (Table 5.7 (a)). Conversely, the commercial açaí sample (Powder UK)
had elemental levels that were typically 30% lower than that measured in other
commercial fruit samples. (Table 5.7 (a)). For example, the manganese levels of
the non-commercial pulp were 64.06 ± 0.93 mg/kg Mn (f.w.) for purple and 61.14
± 0.60 mg/kg Mn (f.w.) for white; whilst the commercial pulp (purple) was 26.76 ±
230
1.51 mg/kg Mn (f.w.). This study also investigated the elemental analysis of non-
processed açaí samples (fruit and seeds) from the Amazonian region of Brazil.
Overall, the data for most of the elements had higher elemental levels for the
non-processed fruits, when compared to the corresponding seeds (Table 5.10).
The data showed that processed pulp, with less water added (higher solids
content), also had higher elemental levels (based on a fresh weight) (Table 5.11).
The manganese content of the açaí (pulp) samples ranged from 5.26 to 126.26
mg/kg (f.w.) depending on the origin and water content of the samples. The total
polyphenol content of non-processed açaí samples showed clearly that during
the processing of the açaí berries, the levels decrease, which suggests that they
are ‘lost’ (Table 5.9). Furthermore, the açaí samples collected from the islands
close to Belém (Ilhas) in northern Brazil, had higher total polyphenol levels when
compared to samples from Genipauba (which is inland within the State of Pará).
Also, the total polyphenol content increased in processed açaí pulp that also had
a higher solids content (Table 5.9).
This study was very important as açaí plays a major nutritional role in
terms of the diet of the local population of the Amazonian region, and also now
as the comercialised products are becoming popular throughout Brazil (section
5.2). An evaluation was made using the data produced in this study to determine
the contribution that a regular consumption of açaí (500g) would make to the
dietary intake of polyphenols and minerals by Brazilians. This study showed that
the total polyphenol intake of purple açaí would be similar to that obtained from
the consumption of other well-known ‘super-fruits’, such as, blueberries and
acerola (section 5.7). Moreover, the consumption of processed açaí pulp would
contribute, in a single serving, more than 100% of the recommended daily
allowance (RDA) intake of total polyphenols (Figure 5.26). In relation to the
elemental data, the consumption of açaí represents a good source of manganese
(with an average of 1500% of the RDA), copper (average of 90%), calcium
(20%), magnesium (30%) and zinc (15%) (Tables 5.9 and 5.10). As was
mentioned in chapter 3, Brazilian yerba mate infusions were also found to be
good sources of manganese, with the high % intake levels based on the daily
231
consumption of this beverage (section 3.6.6.1). Clearly, once again there may be
a concern about the high levels of Mn being consumed from these products,
which will be discussed in the next section in terms of the bioavailability of Mn in
foodstuffs and beverages.
6.5. Limitations
The main aim and objectives of this research have been achieved,
although, it is important to highlight the limitations of the presented study. During
the field-trips to the different plantations in Brazil, there were time contraints
imposed by the commercial owners and the costs involved in the collection and
transport of samples. Moreover, trips to Brazil had to be undertaken at specific
university (non-teaching times) and therefore the availability of samples was
strongly influenced by seasonality and the access to specific types and varieties
of yerba mate, coffee and acai berries. Also, in the processing plants, different
materials were being processed at the same time, therefore only a limited
number of samples were taken at each phase of the process for chemical
investigation. The polyphenol studies were carried-out in commercial laboratories
in London (UK) and North Carolina (USA) which enhanced the quality of the
research but became a limiting factor due to the time made available to use
these instruments. In summary, only a limited number of samples were obtained
and analysed in this research. As a result, the power of the statistical analysis
undertaken has focused mainly on parametric methods. If more samples could
have been collected and transported to the UK it would have been possible to do
more advanced analysis. Moreover, another limitation associated with the
sampling strategy is that the analyses were performed on usually a single sample
of plant or berry material. This results in a challenge to determine the
representativity of the reported levels of the compounds in the actual plant
species. There is also a variability in the different chemical levels due to seasonal
differences that should be further investigated (Timmers et al., 2017). Finally, the
232
limited availability of matrix-matched certified reference materials and standards
may have an impact on the method validation of the polyphenol analysis
6.6. Future Work
It is proposed that future research should evaluate the possible chemical
interactions between elements and organic compounds in the natural and related
processed products. These interactions may influence the bioavailability of not
only the elemental species, but also the polyphenols. One study has already
been reported in terms of an interaction between iron and polyphenols in tea
(Perron and Brumaghim, 2009). This study reported that polyphenols in tea
infusions decreased the levels of bioavailable iron due to complexation of the
chemicals. Moreover, the bioavailability of iron (and in this study for manganese
and other trace elements) would impact on the human intake of these chemicals
through the consumption of such products (yerba mate, roasted coffee and açaí
pulp), thereby increasing or decreasing the potential bioinorganic effect on the
human body (Fairweather-Tait and Hurrell, 1996). To this end, a continuation of
this research would be to study the complexation of metal(s)-polyphenol(s) in
these natural and commercial food products and to evaluate the bioavailabity of
these elements (minerals) using in vivo analysis.
Furthermore, an extensive evaluation of the potential human and mouse
biological effects of the chemicals present in the investigated products (yerba
mate, roasted coffee and açaí) could be undertaken (extending the pilot studies
carried-out in the USA on açaí reported in section 5.5). A series of in vitro
analyses on a range of different human or animal cell lines could be used in
order to assess the claimed health benefits of consuming these Brazilian
products.
233
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ZHISHEN, J., MENGCHENG, T. & JIANMING, W. 1999. The determination of flavonoid contents in mulberry and their scavenging effects on superoxide radicals. Food Chemistry, 64, 555-559.
ZIELINSKI, A. A. F., HAMINIUK, C. W. I., ALBERTI, A., NOGUEIRA, A., DEMIATE, I. M. & GRANATO, D. 2014. A comparative study of the phenolic compounds and the in vitro antioxidant activity of different Brazilian teas using multivariate statistical techniques. Food Research International, 60, 246-254.
Chemical Analysis of Typical Beverages and Açaí Berry
from South America
by Fernanda Vanoni Matta
APPENDICES
Faculty of Engineering and Physical Sciences
University of Surrey, Guildford, GU2 7XH
2019
i
List of Appendices
Page Appendix 3.1 Sample list of non-commercial samples from Barão de Cotegipe (Rio Grande do Sul State – Brazil). 1 Appendix 3.2 Na, Mg, K, Ca, V and Cr levels (mean ± standard deviation) of non-commercial samples yerba mate (mg/kg, dry
weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates. 3
Appendix 3.3 Mn, Fe, Co, Ni, Cu and Zn levels (mean ± standard deviation) of non-commercial yerba mate samples (mg/kg dry weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
6
Appendix 3.4 As, Se, Mo, Cd ans Pb levels (mean ± standard deviation) of non-commercial yerba mate samples (mg/kg dry weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
9
Appendix 3.5 Sample list of commercial yerba mate samples from Brazil and Argentina. 12 Appendix 3.6 Na, Mg, K, Ca, V and Cr levels (mean ± standard deviation) of commercial yerba mate samples (mg/kg dry weight)
determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates. 15
Appendix 3.7 Mn, Fe, Co, Ni, Cu ans Zn levels (mean ± standard deviation) of commercial yerba mate samples (mg/kg dry weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates
18
Appendix 3.8 As, Se, Mo, Cd and Pb levels (mean ± standard deviation) of commercial yerba mate samples (mg/kg dry weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates
21
Appendix 3.9 V, Cr, Mn, Fe, Co and Ni levels (mean ± standard deviation) of commercial yerba mate regular infusion samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
24
Appendix 3.10 Cu, Zn, As, Se, Mo, Cd and Pb levels (mean ± standard deviation) of commercial yerba mate regular infusion samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates
27
Appendix 3.11 V, Cr, Mn, Fe, Co and Ni levels (mean ± standard deviation) of commercial yerba mate Brazilian iced tea infusion samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of
30
Appendix 3.12 Cu, Zn, As, Se, Mo Cd and Pb levels (mean ± standard deviation) of commercial yerba mate Brazilian iced tea infusion samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
31
Appendix 3.13 Mn and Fe levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples (µg/200 mL) 32
ii
determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates. Appendix 3.14 Cu and Zn levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples (µg/200 mL)
determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates. 34
Appendix 3.15 Ni and Cr levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
35
Appendix 3.16 V and Co levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
37
Appendix 3.17 As and Se levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
38
Appendix 3.18 Mo and Cd levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
40
Appendix 3.19 Pb levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates
41
Appendix 3.20 Total polyphenol content of commercial green loose yerba mate regular and bombilla infusions (mg GAE/200 mL) determined using Folin-Ciocalteu assay (refer to section 2.4).
43
Appendix 3.21 Total polyphenol content of commercial green/roasted loose or tea bag yerba mate regular infusions (mg GAE/200mL) determined using Folin-Ciocalteu assay (refer to section 2.4
45
Appendix 3.22 Total polyphenol content of commercial green/roasted loose or tea bag yerba mate Brazilian iced tea infusions (mg GAE/200mL) determined using Folin-Ciocalteu assay (refer to section 2.4).
47
Appendix 3.23 Chlorogenic acid, theobromine and caffeine content of commercial green/roasted loose or tea bag yerba mate regular infusions (mg/200 mL) determined by UHPLC (refer to section 2.5).
48
Appendix 3.24 Chlorogenic acid, theobromine and caffeine content of commercial green/roasted loose or tea bag yerba mate bombilla infusions (mg/200 mL) determined by UHPLC (refer to section 2.5).
49
Appendix 3.25 Comparison of standard deviations using a F-test (Miller et al., 2018) to evaluate the relationship between the elemental levels of yerba mate leaves (based on age – new and old) for non-commercial samples collected from traditional plantations cultivated either using NPK fertilisers or non-chemical (organic).
51
iii
Appendix 3.26 Comparison of standard deviations using a F-test (Miller et al., 2018) to evaluate the relationship between the elemental levels of yerba mate leaves based on the use or non-use (organic) of NPK fertilsers during traditional cultivation.
52
Appendix 3.27 Comparison of standard deviations using a F-test (Miller et al., 2018) to evaluate the relationship between the elemental levels of yerba mate leaves (new and old) grown in traditional organic or native forest plantations (refer to Table 3.6).
53
Appendix 3.28 Comparison of standard deviations using a F-test (Miller et al., 2018) to evaluate the relationship between the origin (Brazil and Argentina); packaging (loose and tea bags) and roasting (green and roasted) of commercial yerba mate samples (refer to Table 3.9).
54
Appendix 3.29 Comparison of standard deviations using a F-test (Miller et al., 2018) to evaluate the relationship between the origin (Brazil and Argentina); packaging (loose and tea bags) and roasting process (green loose and roasted) of regular infusions of commercial yerba mate (refer to Table 3.11).
55
Appendix 4.1 Sample list of Brazilian coffee samples from Amparo, São Paulo State. 56 Appendix 4.2 Na, Mg, K, Ca, V and Cr levels (mean ± standard deviation) of Brazilian coffee samples (mg/kg dry weight) determined
using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates. 57
Appendix 4.3 Mn, Fe, Co, Ni, Cu and Zn levels (mean ± standard deviation) of Brazilian coffee samples (mg/kg dry weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
59
Appendix 4.4 As, Se, Mo, Cd and Pb levels (mean ± standard deviation) of Brazilian coffee samples (mg/kg dry weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
61
Appendix 5.1 Total elemental levels (mean ± standard deviation) of açaí pulp samples (mg/kg dry weight) determined using ICP-MS (refer to section 2.3). Data relates to the type of sample (non-commercial : purple n= 6; and white n= 4; and commercial: purple n= 4; n is the number of samples).
63
Appendix 5.2 Total elemental levels (mean ± standard deviation) of açaí pulp samples (mg/kg fresh weight) determined using ICP-MS (refer to section 2.3). Data relates to the type of sample (non-commercial : purple n= 6; and white n= 4; and commercial: purple n= 4; n is the number of samples).
65
Appendix 5.3 Sample list for the evaluation of the Amazon geographical variability and industrial processing on açaí. 67
iv
Appendix 5.4 Total elemental and polyphenol levels (mean ± standard deviation) of açaí pulp samples (mg/kg, dry weight) determined using ICP-MS (refer to section 2.3). Data relates to the type of sample; n= 3; n is the number of replicates).
68
Appendix 5.5 Total elemental (mean ± standard deviation) of açaí pulp samples (mg/kg dry weight) determined using ICP-MS (refer to section 2.3). Data relates to the type of sample; n= 3; n is the number of replicates).
70
Appendix 5.6 Total elemental and polyphenol levels (mean ± standard deviation) of açaí pulp samples (mg/kg fresh weight) determined using ICP-MS (refer to section 2.3). Data relates to the type of sample; n= 3; n is the number of replicates).
71
Appendix 5.7 Total elemental and polyphenol levels (mean ± standard deviation) of açaí pulp samples (mg/kg fresh weight) determined using ICP-MS (refer to section 2.3). Data relates to the type of sample; n= 3; n is replicates).
73
Appendix 5.8 Total polyphenol (TP) and minor elements daily intake (mg/day) based on the consumpsion of a 500 g serving of the commercial and non-commercial açaí pulp (fresh weight).
74
Appendix 5.9 Percentage intake (%) of total polyphenol and minor elements based on the consumption of a 500 g serving of the commercial and non-commercial açaí pulp (fresh weight) when compared to the recommended daily allowance (RDA) for males (M) and females (F).
75
Appendix 5.10 Total polyphenol and minor elements daily intake (mg/day) based on a 500 g serving of açaí pulp. 77
1
Appendix 3.1: Sample list of non-commercial samples from Barão de Cotegipe (Rio Grande do Sul State – Brazil).
Code Plantation type Pesticides use Age (years) Sample type Cultivated (fertiliser)
EMC - 01 new Conventional cultivation non organic (NPK) 30 Plant - new leaves EMC - 01 old Conventional cultivation non organic (NPK) 30 Plant - old leaves EMC - 02 new Conventional cultivation non organic (NPK) 30 Plant - new leaves EMC - 02 old Conventional cultivation non organic (NPK) 30 Plant - old leaves EMC - 03 new Conventional cultivation non organic (NPK) 30 Plant - new leaves EMC - 03 old Conventional cultivation non organic (NPK) 30 Plant - old leaves EMC - 04 new Conventional cultivation non organic (NPK) 30 Plant - new leaves EMC - 04 old Conventional cultivation non organic (NPK) 30 Plant - old leaves Natural forests EMN - 01 new Natural forests organic 20 Plant - new leaves EMN - 01 old Natural forests organic 20 Plant - old leaves EMN - 02 new Natural forests organic 20 Plant - new leaves EMN - 02 old Natural forests organic 20 Plant - old leaves EMN - 03 new Natural forests organic 20 Plant - new leaves EMN - 03 old Natural forests organic 20 Plant - old leaves Cultivated (organic) EMO - 01 new Cultivated under sun organic 25 Plant - new leaves EMO - 01 old Cultivated under sun organic 25 Plant - old leaves EMO - 02 new Cultivated under sun organic 25 Plant - new leaves EMO - 02 old Cultivated under sun organic 25 Plant - old leaves
2
Code Plantation type Pesticides use Age (years) Sample type EMO - 03 new Cultivated under sun organic 25 Plant - new leaves EMO - 03 old Cultivated under sun organic 25 Plant - old leaves EMO - 04 new Cultivated under sun organic 25 Plant - new leaves EMO - 04 old Cultivated under sun organic 25 Plant - old leaves EMO - 05 bottom Cultivated under sun organic 25 Plant - bottom height EMO - 05 middle Cultivated under sun organic 25 Plant - middle height EMO - 05 top Cultivated under sun organic 25 Plant - top height EMO - 06 Cultivated under sun organic 25 Plant – leaves EMO - 07 new Cultivated under sun organic 25 Plant - new leaves EMO - 07 old Cultivated under sun organic 25 Plant - old leaves EMO - 08 leaves Cultivated under sun organic 2 Plant – old leaves EMO - 09 leaves Cultivated under sun organic 2 Plant – old leaves EMO - 10 new Cultivated under sun organic 25 Plant - new leaves EMO - 10 old Cultivated under sun organic 25 Plant - old leaves EMO - 11 new Cultivated under sun organic 25 Plant - new leaves EMO - 11 old Cultivated under sun organic 25 Plant - old leaves Processed* NA new Native organic n.r. Plant - new leaves NA old Native organic n.r. Plant - old leaves YM - Sap n.r. n.r. n.r. Plant – Sapeco* YM- CA n.r. n.r. n.r. Plant – dried*
*refer to section 3.5.1; n.r.: not reported.
3
Appendix 3.2: Na, Mg, K, Ca, V and Cr levels (mean ± standard deviation) of non-commercial samples yerba mate
(mg/kg, dry weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Code Na Mg K Ca V Cr
Cultivated (fertiliser)
EMC - 01 new 241 ± 11 2738 ± 64 8761 ± 150 2504 ± 46 0.15 ± 0.01 0.96 ± 0.02
EMC - 01 old 159 ± 22 5710 ± 75 5509 ± 168 6511 ± 158 0.20 ± 0.01 1.04 ± 0.01
EMC - 02 new 581 ± 15 3618 ± 1 8845 ± 98 3035 ± 86 0.07 ± 0.003 1.04 ± 0.05
EMC - 02 old 132 ± 5 8474 ± 35 4323 ± 13 9209 ± 9 0.11 ± 0.01 0.93 ± 0.17
EMC - 03 new 406 ± 18 2608 ± 14 8206 ± 37 2257 ± 83 0.09 ± 0.01 0.66 ± 0.10
EMC - 03 old 99 ± 19 7331 ± 171 3563 ± 79 10298 ± 488 0.24 ± 0.002 0.85 ± 0.09
EMC - 04 new 274 ± 26 3724 ± 129 8051 ± 154 4160 ± 305 0.08 ± 0.004 0.66 ± 0.002
EMC - 04 old 225 ± 19 6547 ± 51 5729 ± 64 8504 ± 12 0.19 ± 0.02 1.33 ± 0.06
Natural forest
EMN - 01 old 442 ± 26 3782 ± 48 9299 ± 66 3867 ± 3 0.22 ± 0.003 1.08 ± 0.16
EMN - 01 new 107 ± 6 7846 ± 155 3232 ± 60 11139 ± 547 0.21 ± 0.02 0.68 ± 0.11
EMN - 02 new 635 ± 30 2873 ± 85 9714 ± 373 3277 ± 60 0.32 ± 0.02 0.65 ± 0.10
EMN - 02 old 150 ± 26 6046 ± 49 5199 ± 50 8513 ± 96 0.49 ± 0.005 0.93 ± 0.06
EMN - 03 new 655 ± 53 2918 ± 21 10843 ± 224 3346 ± 7 0.33 ± 0.001 0.39 ± 0.10
EMN - 03 old 201 ± 54 4957 ± 15 4582 ± 45 8635 ± 91 0.22 ± 0.005 0.81 ± 0.01
4
Code Na Mg K Ca V Cr
Cultivated (organic)
EMO - 01 new 181 ± 25 3574 ± 10 6391 ± 29 5319 ± 344 0.07 ± 0.01 0.73 ± 0.06
EMO - 01 old 223 ± 1 2665 ± 46 7545 ± 193 2536 ± 172 0.04 ± 0.003 0.76 ± 0.16
EMO - 02 new 379 ± 23 2792 ± 60 8536 ± 182 2884 ± 77 0.05 ± 0.006 0.68 ± 0.08
EMO - 02 old 209 ± 4 4215 ± 38 5609 ± 85 6193 ± 22 0.06 ± 0.01 1.19 ± 0.25
EMO - 03 new 220 ± 8 2779 ± 7 7707 ± 46 3464 ± 6 0.06 ± 0.002 1.43 ± 0.03
EMO - 03 old 92 ± 41 2356 ± 1582 4031 ± 2673 3215 ± 2121 0.05 ± 0.03 1.32 ± 0.82
EMO - 04 new 358 ± 41 2807 ± 15 9156 ± 17 2588 ± 179 0.06 ± 0.006 1.00 ± 0.09
EMO - 04 old 115 ± 1 5604 ± 66 5780 ± 109 8193 ± 114 0.06 ± 0.003 1.32 ± 0.16
EMO - 05 bottom 259 ± 22 5830 ± 0.5 5854 ± 2 11383 ± 48 0.07 ± 0.004 1.15 ± 0.07
EMO - 05 middle 156 ± 14 3940 ± 30 5539 ± 99 5469 ± 181 0.05 ± 0.001 0.85 ± 0.08
EMO - 05 top 188 ± 22 4054 ± 24 6788 ± 15 5857 ± 12 0.12 ± 0.01 1.16 ± 0.24
EMO - 06 214 ± 16 4431 ± 40 6913 ± 58 5217 ± 5 0.10 ± 0.008 1.05 ± 0.07
EMO - 07 new 198 ± 53 5914 ± 40 6709 ± 31 5467 ± 117 0.07 ± 0.004 0.86 ± 0.06
EMO - 07 old 255 ± 22 6485 ± 80 5309 ± 55 7390 ± 197 0.07 ± 0.007 1.16 ± 0.22
EMO - 08 leaves 287 ± 129 5582 ± 80 6157 ± 9 10534 ± 3327 0.05 ± 0.003 0.77 ± 0.05
EMO - 09 leaves 287 ± 4 5270 ± 107 6045 ± 35 6437 ± 228 0.04 ± 0.002 0.34 ± 0.01
EMO - 10 new 652 ± 13 3566 ± 55 10246 ± 161 2839 ± 29 0.04 ± 0.005 0.31 ± 0.04
EMO - 10 old 190 ± 0.4 7466 ± 35 3508 ± 18 9723 ± 192 0.07 ± 0.01 0.68 ± 0.02
5
Code Na Mg K Ca V Cr
EMO - 11 new 224 ± 11 3719 ± 294 10536 ± 199 3515 ± 571 0.04 ± 0.002 0.25 ± 0.02
EMO - 11 old 298 ± 80 8218 ± 142 5595 ± 19 10904 ± 189 0.10 ± 0.02 0.79 ± 0.16
Processed*
NA new 164 ± 18 6193 ± 87 3233 ± 27 4853 ± 3 0.09 ± 0.005 0.31 ± 0.003
NA old 213 ± 7 7315 ± 166 3258 ± 103 6073 ± 118 0.07 ± 0.01 0.32 ± 0.02
YM - Sap 227 ± 38 7206 ± 21 4555 ± 173 7996 ± 424 0.34 ± 0.03 3.32 ± 0.10
YM- CA - NA 181 ± 19 4403 ± 40 4511 ± 58 6058 ± 244 0.12 ± 0.002 0.46 ± 0.15
YM- CA 156 ± 6 5962 ± 54 4644 ± 96 6771 ± 63 0.40 ± 0.03 0.83 ± 0.03 *refer to section 3.5.1
6
Appendix 3.3: Mn, Fe, Co, Ni, Cu and Zn levels (mean ± standard deviation) of non-commercial yerba mate samples
(mg/kg dry weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Code Mn Fe Co Ni Cu Zn
Cultivated (fertiliser) EMC - 01 new 610 ± 18 55.65 ± 3.11 0.39 ± 0.01 6.59 ± 0.05 37.19 ± 0.15 68.41 ± 0.13
EMC - 01 old 1394 ± 42 76.22 ± 3.91 0.07 ± 0.004 2.20 ± 0.12 14.68 ± 0.25 118.05 ± 3.89
EMC - 02 new 425 ± 11 45.08 ± 0.69 0.39 ± 0.01 4.83 ± 0.06 26.17 ± 0.43 76.92 ± 1.72
EMC - 02 old 560 ± 13 49.17 ± 0.57 0.10 ± 0.003 2.52 ± 0.002 14.18 ± 0.04 238.50 ± 11.42
EMC - 03 new 531 ± 9 36.87 ± 2.21 0.18 ± 0.002 3.67 ± 0.11 25.08 ± 0.39 67.71 ± 0.10
EMC - 03 old 715 ± 89 74.82 ± 0.09 0.03 ± 0.005 1.89 ± 0.15 16.35 ± 0.60 82.49 ± 1.99
EMC - 04 new 924 ± 3 50.75 ± 2.88 0.77 ± 0.02 6.45 ± 0.30 26.22 ± 0.68 104.95 ± 1.16
EMC - 04 old 1127 ± 13 64.32 ± 5.24 0.16 ± 0.003 3.13 ± 0.09 13.45 ± 0.04 225.51 ± 2.60
Natural forest
EMN - 01 old 1022 ± 16 69.63 ± 0.56 0.59 ± 0.05 7.93 ± 0.27 20.18 ± 0.19 71.86 ± 0.75
EMN - 01 new 982 ± 31 91.15 ± 2.80 0.37 ± 0.006 2.71 ± 0.06 9.35 ± 0.34 32.83 ± 0.60
EMN - 02 new 1085 ± 25 94.07 ± 4.48 0.71 ± 0.02 5.40 ± 0.02 22.09 ± 0.43 75.71 ± 0.13
EMN - 02 old 1365 ± 5 153.99 ± 0.92 0.26 ± 0.002 2.20 ± 0.33 11.62 ± 0.18 39.57 ± 0.30
EMN - 03 new 1159 ± 51 91.60 ± 2.09 0.27 ± 0.02 5.66 ± 0.05 22.87 ± 0.57 66.93 ± 0.85
EMN - 03 old 805 ± 1 89.10 ± 1.67 0.12 ± 0.005 2.97 ± 0.10 11.87 ± 0.41 49.39 ± 0.63
Cultivated (organic)
7
Code Mn Fe Co Ni Cu Zn
EMO - 01 new 871 ± 54 39.37 ± 0.14 0.25 ± 0.05 2.87 ± 0.003 8.87 ± 0.72 252.96 ± 14.68
EMO - 01 old 914 ± 17 43.22 ± 2.32 1.12 ± 0.03 4.62 ± 0.22 21.29 ± 0.59 80.18 ± 2.33
EMO - 02 new 924 ± 9 36.27 ± 0.10 0.62 ± 0.002 6.64 ± 0.51 17.93 ± 0.19 63.93 ± 0.71
EMO - 02 old 592 ± 3 37.13 ± 4.20 0.27 ± 0.01 2.95 ± 0.11 7.65 ± 0.46 48.82 ± 1.79
EMO - 03 new 775 ± 21 35.16 ± 3.85 0.24 ± 0.005 3.97 ± 0.004 12.00 ± 0.04 30.17 ± 0.34
EMO - 03 old 349 ± 192 28.22 ± 19.12 0.10 ± 0.07 3.52 ± 2.31 5.53 ± 3.66 15.74 ± 7.86
EMO - 04 new 757 ± 1 41.39 ± 1.73 1.27 ± 0.03 4.07 ± 0.12 26.90 ± 0.29 73.39 ± 0.22
EMO - 04 old 792 ± 29 37.87 ± 1.63 0.84 ± 0.002 5.44 ± 0.07 12.68 ± 0.43 119.23 ± 0.34
EMO - 05 bottom 1070 ± 41 39.10 ± 5.45 0.78 ± 0.01 1.37 ± 0.02 5.41 ± 0.40 81.79 ± 0.59
EMO - 05 middle 1009 ± 82 32.64 ± 5.05 0.46 ± 0.006 2.04 ± 0.01 7.68 ± 0.28 64.08 ± 1.09
EMO - 05 top 1229 ± 21 51.33 ± 1.92 0.43 ± 0.01 1.36 ± 0.05 5.21 ± 0.17 65.29 ± 1.28
EMO - 06 1189 ± 8 50.73 ± 1.22 0.22 ± 0.003 1.93 ± 0.17 9.54 ± 0.68 54.98 ± 2.36
EMO - 07 new 818 ± 0.8 31.58 ± 1.91 0.48 ± 0.01 3.92 ± 0.07 9.92 ± 0.84 148.31 ± 12.24
EMO - 07 old 609 ± 16 36.19 ± 4.13 0.37 ± 0.006 2.70 ± 0.26 7.08 ± 1.09 195.76 ± 5.52
EMO - 08 leaves 3395 ± 68 32.00 ± 2.88 0.34 ± 0.004 1.67 ± 0.23 11.62 ± 0.92 73.06 ± 0.15
EMO - 09 leaves 1569 ± 52 27.83 ± 0.12 1.45 ± 0.11 1.16 ± 0.09 11.45 ± 0.02 41.86 ± 1.38
EMO - 10 new 865 ± 25 33.15 ± 1.52 0.12 ± 0.003 5.29 ± 0.03 21.35 ± 0.24 48.19 ± 0.02
EMO - 10 old 613 ± 2 47.64 ± 1.26 0.24 ± 0.01 3.43 ± 0.05 8.90 ± 0.28 93.35 ± 2.67
EMO - 11 new 781 ± 16 31.76 ± 2.63 0.86 ± 0.05 4.68 ± 0.22 15.86 ± 0.21 79.48 ± 4.89
8
Code Mn Fe Co Ni Cu Zn
EMO - 11 old 641 ± 10 48.29 ± 8.02 0.72 ± 0.04 2.30 ± 0.05 6.55 ± 0.22 251.73 ± 5.53
Processed*
NA new 349 ± 2 51.26 ± 1.01 0.19 ± 0.01 1.95 ± 0.06 12.12 ± 0.07 33.73 ± 1.16
NA old 388 ± 4 57.14 ± 3.22 0.09 ± 0.01 2.46 ± 0.01 10.57 ± 0.05 25.86 ± 0.29
YM - Sap 632 ± 22 132.95 ± 10.85 0.27 ± 0.01 3.19 ± 0.08 10.20 ± 0.22 69.30 ± 2.72
YM- CA - NA 445 ± 1 68.52 ± 4.72 0.07 ± 0.002 2.13 ± 0.18 11.08 ± 0.33 34.78 ± 3.00
YM- CA 729 ± 53 121.61 ± 6.02 0.37 ± 0.005 4.10 ± 0.03 11.55 ± 0.52 75.75 ± 5.60 *refer to section 3.5.1
9
Appendix 3.4: As, Se, Mo, Cd ans Pb levels (mean ± standard deviation) of non-commercial yerba mate samples (mg/kg
dry weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Code As Se Mo Cd Pb
Cultivated (fertiliser)
EMC - 01 new 0.05 ± 0.003 0.02 ± 0.01 <LOD* 0.53 ± 0.02 0.34 ± 0.02
EMC - 01 old 0.03 ± 0.001 0.03 ± 0.02 <LOD* 0.44 ± 0.03 0.18 ± 0.02
EMC - 02 new 0.01 ± 0.0003 0.01 ± 0.001 0.01 ± 0.001 0.47 ± 0.002 0.12 ± 0.01
EMC - 02 old 0.02 ± 0.0004 0.13 ± 0.01 <LOD* 0.45 ± 0.01 0.09 ± 0.01
EMC - 03 new 0.02 ± 0.002 <LOD* <LOD* 0.35 ± 0.01 0.07 ± 0.002
EMC - 03 old 0.02 ± 0.001 0.05 ± 0.002 <LOD* 0.22 ± 0.03 0.03 ± 0.01
EMC - 04 new 0.02 ± 0.0003 0.01 ± 0.001 <LOD* 0.59 ± 0.02 0.08 ± 0.02
EMC - 04 old 0.02 ± 0.001 0.04 ± 0.01 <LOD* 0.35 ± 0.01 0.06 ± 0.001
Natural forest
EMN - 01 old 0.02 ± 0.001 0.03 ± 0.003 0.01 ± 0.002 0.51 ± 0.02 0.05 ± 0.002
EMN - 01 new 0.01 ± 0.001 0.08 ± 0.01 <LOD* 0.38 ± 0.02 0.03 ± 0.01
EMN - 02 new 0.02 ± 0.0001 0.02 ± 0.01 <LOD* 0.53 ± 0.04 0.02 ± 0.002
EMN - 02 old 0.02 ± 0.0001 0.12 ± 0.002 <LOD* 0.41 ± 0.00 0.01 ± 0.002
EMN - 03 new 0.02 ± 0.0001 0.01 ± 0.01 <LOD* 0.93 ± 0.04 0.02 ± 0.01
EMN - 03 old 0.01 ± 0.004 0.05 ± 0.01 <LOD* 0.78 ± 0.02 <LOD*
10
Code As Se Mo Cd Pb
Cultivated (organic)
EMO - 01 new 0.03 ± 0.01 0.04 ± 0.002 0.02 ± 0.002 0.25 ± 0.05 0.01 ± 0.001
EMO - 01 old 0.02 ± 0.003 0.01 ± 0.001 0.72 ± 0.70 0.33 ± 0.01 0.02 ± 0.002
EMO - 02 new 0.03 ± 0.0002 0.01 ± 0.01 0.03 ± 0.002 0.27 ± 0.01 0.01 ± 0.001
EMO - 02 old 0.01 ± 0.001 0.03 ± 0.002 0.01 ± 0.001 0.30 ± 0.01 0.01 ± 0.003
EMO - 03 new 0.02 ± 0.001 0.01 ± 0.001 0.01 ± 0.001 0.28 ± 0.01 0.01 ± 0.001
EMO - 03 old 0.01 ± 0.01 0.03 ± 0.03 0.01 ± 0.01 0.07 ± 0.05 <LOD*
EMO - 04 new 0.01 ± 0.007 0.01 ± 0.01 0.02 ± 0.001 0.53 ± 0.02 0.05 ± 0.01
EMO - 04 old 0.02 ± 0.004 0.09 ± 0.01 <LOD* 0.40 ± 0.02 0.02 ± 0.001
EMO - 05 bottom 0.02 ± 0.002 0.08 ± 0.02 0.01 ± 0.002 0.42 ± 0.01 0.01 ± 0.001
EMO - 05 middle 0.02 ± 0.001 0.09 ± 0.02 <LOD* 0.34 ± 0.02 0.02 ± 0.003
EMO - 05 top 0.02 ± 0.001 0.05 ± 0.001 0.02 ± 0.002 0.23 ± 0.02 0.05 ± 0.002
EMO - 06 0.02 ± 0.005 0.04 ± 0.002 0.02 ± 0.001 0.25 ± 0.01 0.08 ± 0.001
EMO - 07 new 0.02 ± 0.002 0.03 ± 0.002 0.03 ± 0.001 0.44 ± 0.04 0.02 ± 0.002
EMO - 07 old 0.01 ± 0.001 0.04 ± 0.01 0.01 ± 0.001 0.48 ± 0.06 0.07 ± 0.06
EMO - 08 leaves 0.01 ± 0.004 0.04 ± 0.001 0.01 ± 0.002 0.41 ± 0.01 0.05 ± 0.01
EMO - 09 leaves 0.01 ± 0.002 0.04 ± 0.002 <LOD* 0.45 ± 0.02 0.01 ± 0.002
EMO - 10 new 0.01 ± 0.001 0.03 ± 0.003 0.02 ± 0.002 1.17 ± 0.02 0.05 ± 0.02
EMO - 10 old 0.01 ± 0.003 0.14 ± 0.01 <LOD* 0.47 ± 0.01 0.04 ± 0.001
11
Code As Se Mo Cd Pb
EMO - 11 new 0.01 ± 0.002 0.02 ± 0.01 0.02 ± 0.001 0.99 ± 0.05 0.05 ± 0.01
EMO - 11 old 0.02 ± 0.001 0.20 ± 0.02 <LOD* 0.45 ± 0.002 0.04 ± 0.002
Processed**
NA new 0.05 ± 0.004 0.04 ± 0.02 0.05 ± 0.01 0.15 ± 0.01 0.08 ± 0.002
NA old 0.02 ± 0.005 0.05 ± 0.01 <LOD* 0.08 ± 0.01 0.08 ± 0.02
YM - Sap 0.02 ± 0.002 0.04 ± 0.002 0.01 ± 0.001 0.35 ± 0.03 0.06 ± 0.001
YM- CA - NA 0.02 ± 0.003 0.06 ± 0.01 0.01 ± 0.001 0.18 ± 0.04 0.04 ± 0.01
YM- CA 0.03 ± 0.001 0.03 ± 0.02 0.01 ± 0.002 0.48 ± 0.02 0.17 ± 0.11 *refer to section 3.5.1; **refer to Table 2.2
12
Appendix 3.5: Sample list of commercial yerba mate samples from Brazil and Argentina.
Brand Origin Code
Green loose Baldo Uruguay/Brazil* BA Barão de Cotegipe - Cambona Brazil BC- Ca Barão de Cotegipe - Moída Grossa Brazil BC-Mg Barão de Cotegipe - Native Brazil BC- Na Barão de Cotegipe - Premium Brazil BC-PR Barão de Cotegipe - Terere Brazil BC-Te Barão de Cotegipe- tipo Uruguay Uruguay/Brazil* BC-Ex Canarias Uruguay/Brazil* CAN Sara Uruguay/Brazil* SAR Amanda Argentina AM Barão de Cotegipe - Traditional Brazil BC - Try Flor Verde Brazil FV Foller Brazil FO Jerper Argentina JE Ka-a Argentina KA Kraus Organic Argentina KR Pipore Argentina PI Porto Vitoria Brazil PV Roapipo Organic Argentina RO
13
Brand Origin Code Rosamonte Argentina RM Santo Antonio Brazil SA Yerba Uruguaia Uruguay/Brazil* YU
Green tea bag Amanda – Mate cocido Argentina AT Asis Organic Argentina AO Cachamate Argentina CA Carrefour Argentina CR Cruz de Malta – mate cocido Argentina CM Don Lucas Argentina DL Jumala Argentina JU La Anonima Argentina LA La Hoja Argentina LH La Posadena Argentina LP La Tranquera Argentina LT Litoral (La Virginia) Argentina LI Mate Tucangua Argentina MT Playadito Argentina PL Suave Union Argentina SU Taragui Argentina TA Taragui Ninos Argentina TN Vea Argentina VE
14
Brand Origin Code Yer-vita Argentina YVI
Roasted loose Mate Leao Natural Brazil MN Barão de Cotegipe - Mate Tostado Brazil BC-Cm Mate Leao Organic Roasted Brazil MO
Roasted tea bag Dr. Oetker Brazil DO Lin Tea Brazil LIT Qualita Brazil QU Leao – Cha Mate Tostado Brazil ML
*refer to section 3.6.1
15
Appendix 3.6: Na, Mg, K, Ca, V and Cr levels (mean ± standard deviation) of commercial yerba mate samples (mg/kg dry
weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Code Na Mg K Ca V Cr
Green loose BA 134 ± 16 5503 ± 23 3913 ± 120 7747 ± 65 0.35 ± 0.08 0.53 ± 0.05 BC- Ca 135 ± 11 4199 ± 317 4072 ± 17 6855 ± 730 0.18 ± 0.001 0.61 ± 0.01 BC-Mg 192 ± 18 5933 ± 166 4174 ± 272 8639 ± 280 0.14 ± 0.02 0.50 ± 0.03 BC- Na 176 ± 27 3987 ± 105 4539 ± 173 6306 ± 481 0.21 ± 0.06 0.54 ± 0.002 BC-Pr 132 ± 21 4181 ± 251 4503 ± 95 8280 ± 251 0.15 ± 0.002 0.37 ± 0.03 BC-Te 148 ± 4 4042 ± 98 4868 ± 82 6517 ± 98 0.09 ± 0.002 0.52 ± 0.07 BC-Ex 92 ± 2 3493 ± 266 4010 ± 259 6026 ± 15 0.34 ± 0.02 0.67 ± 0.04 CAN 141 ± 14 4958 ± 372 4288 ± 172 6874 ± 471 0.30 ± 0.03 0.62 ± 0.12 SAR 171 ± 2 5872 ± 13 4221 ± 229 7684 ± 14 0.23 ± 0.01 0.57 ± 0.02 AM 177 ± 34 5417 ± 161 4090 ± 99 7216 ± 217 0.40 ± 0.09 0.76 ± 0.002 BC - Tr 202 ± 42 5894 ± 121 4258 ± 104 8633 ± 96 0.36 ± 0.05 0.69 ± 0.10 FV 162 ± 5 2786 ± 41 3895 ± 10 6348 ± 38 0.35 ± 0.02 1.18 ± 0.04 FO 154 ± 12 3741 ± 168 3935 ± 19 7111 ± 375 0.27 ± 0.01 0.36 ± 0.08 JE 160 ± 16 5449 ± 299 3271 ± 52 8418 ± 651 0.42 ± 0.01 0.89 ± 0.08 KA 137 ± 20 5177 ± 534 3729 ± 249 7960 ± 760 0.17 ± 0.01 1.18 ± 0.02 KR 201 ± 26 4649 ± 65 3720 ± 228 7894 ± 551 0.26 ± 0.03 1.42 ± 0.03 PI 239 ± 39 5053 ± 306 3605 ± 27 8091 ± 193 0.20 ± 0.01 0.93 ± 0.001 PV 128 ± 9 3725 ± 256 3520 ± 67 5971 ± 560 0.21 ± 0.01 0.30 ± 0.03
16
Code Na Mg K Ca V Cr RO 170 ± 28 5494 ± 424 2888 ± 105 6926 ± 1551 0.23 ± 0.01 0.81 ± 0.02 RM 164 ± 9 3466 ± 110 3200 ± 57 6501 ± 58 0.22 ± 0.03 0.98 ± 0.02 SA 243 ± 27 5243 ± 118 3763 ± 182 8090 ± 59 0.35 ± 0.01 0.61 ± 0.05 YU 158 ± 12 5095 ± 268 3686 ± 133 7080 ± 110 0.22 ± 0.02 0.52 ± 0.04
Green tea bag AT 211 ± 30 5502 ± 135 4542 ± 174 7518 ± 421 0.86 ± 0.06 0.98 ± 0.01 AO 209 ± 9 5585 ± 52 4169 ± 165 6739 ± 100 0.25 ± 0.01 1.44 ± 0.16 CA 151 ± 3 4614 ± 36 4552 ± 40 7279 ± 194 0.26 ± 0.002 0.95 ± 0.002 CR 228 ± 27 5582 ± 66 4052 ± 36 7978 ± 171 1.21 ± 0.05 0.98 ± 0.001 CM 162 ± 25 5160 ± 154 4052 ± 3 8734 ± 50 0.70 ± 0.02 1.01 ± 0.04 DL 116 ± 12 5227 ± 26 5005 ± 50 7030 ± 35 1.01 ± 0.20 1.05 ± 0.04 JU 158 ± 0.4 6100 ± 231 3380 ± 364 8743 ± 520 0.41 ± 0.04 1.16 ± 0.02 LA 146 ± 3 5705 ± 114 4453 ± 229 8650 ± 295 1.61 ± 0.02 1.36 ± 0.01 LH 195 ± 34 5277 ± 445 5211 ± 559 7537 ± 733 0.37 ± 0.01 0.90 ± 0.02 LP 211 ± 39 5259 ± 99 4438 ± 49 7720 ± 102 1.45 ± 0.23 1.25 ± 0.01 LT 190 ± 21 6131 ± 309 4913 ± 181 8971 ± 504 0.42 ± 0.07 1.23 ± 0.03 LI 152 ± 6 5631 ± 117 4968 ± 27 8422 ± 13 1.31 ± 0.23 1.13 ± 0.09 MT 175 ± 5 5570 ± 11 4286 ± 39 7218 ± 34 0.26 ± 0.02 1.26 ± 0.09 PL 216 ± 6 6196 ± 78 3681 ± 72 7007 ± 377 0.28 ± 0.04 1.16 ± 0.04 SU 112 ± 6 5778 ± 133 4513 ± 54 6801 ± 99 0.44 ± 0.01 1.14 ± 0.02 TA 124 ± 12 5781 ± 92 4285 ± 48 6924 ± 141 0.37 ± 0.01 0.96 ± 0.08 TN 156 ± 13 5259 ± 16 4207 ± 16 6301 ± 85 0.31 ± 0.03 1.12 ± 0.16
17
Code Na Mg K Ca V Cr VE 137 ± 6 5414 ± 103 4084 ± 226 9072 ± 531 1.20 ± 0.37 1.15 ± 0.04 YVI 142 ± 2 6190 ± 66 3624 ± 24 7154 ± 16 0.94 ± 0.08 1.15 ± 0.07
Roasted loose MN 129 ± 12 5188 ± 311 4837 ± 49 9254 ± 127 0.17 ± 0.02 0.67 ± 0.002 BC-Cm 127 ± 2 6933 ± 171 4065 ± 160 9970 ± 334 0.13 ± 0.003 0.61 ± 0.09 MO 151 ± 20 6297 ± 80 4804 ± 42 10121 ± 490 0.30 ± 0.05 1.00 ± 0.20
Roasted tea bag DO 134 ± 26 4557 ± 19 3412 ± 60 8461 ± 44 0.37 ± 0.01 1.05 ± 0.08 LIT 194 ± 36 5176 ± 44 3637 ± 32 9011 ± 126 0.47 ± 0.01 0.81 ± 0.05 QU 165 ± 13 5028 ± 49 3394 ± 94 8179 ± 335 0.35 ± 0.04 0.91 ± 0.11 ML 215 ± 14 7488 ± 18 4263 ± 97 8290 ± 95 0.50 ± 0.09 0.93 ± 0.02
18
Appendix 3.7: Mn, Fe, Co, Ni, Cu ans Zn levels (mean ± standard deviation) of commercial yerba mate samples (mg/kg
dry weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Code Mn Fe Co Ni Cu Zn
Green loose BA 565 ± 44 42.34 ± 1.06 0.12 ± 0.002 3.22 ± 0.13 9.63 ± 0.22 50.76 ± 2.77
BC- Ca 781 ± 54 35.94 ± 0.88 0.13 ± 0.003 1.94 ± 0.01 8.37 ± 0.38 49.62 ± 1.96 BC-Mg 652 ± 16 14.85 ± 9.47 0.14 ± 0.002 1.84 ± 0.26 9.89 ± 0.37 67.16 ± 6.76 BC- Na 538 ± 33 23.17 ± 4.23 0.08 ± 0.01 2.28 ± 0.50 11.20 ± 2.20 43.31 ± 2.24 BC-Pr 663 ± 58 27.76 ± 1.69 0.07 ± 0.01 2.74 ± 0.09 11.76 ± 0.49 43.67 ± 4.33 BC-Te 485 ± 25 30.92 ± 1.16 0.16 ± 0.02 1.00 ± 0.10 7.67 ± 0.47 49.13 ± 6.67 BC-Ex 632 ± 2 64.76 ± 4.74 0.13 ± 0.002 2.81 ± 0.04 9.21 ± 0.01 66.52 ± 7.68 CAN 597 ± 40 45.65 ± 2.11 0.10 ± 0.001 3.58 ± 0.08 11.20 ± 1.39 44.57 ± 0.47 SAR 543 ± 1 44.49 ± 2.84 0.10 ± 0.001 3.33 ± 0.04 9.50 ± 0.31 51.58 ± 1.72 AM 800 ± 96 12.16 ± 4.80 0.25 ± 0.002 3.06 ± 0.23 7.33 ± 0.29 98.96 ± 1.61
BC - Tr 834 ± 0.3 36.82 ± 10.27 0.16 ± 0.02 2.44 ± 0.29 9.39 ± 0.39 62.43 ± 0.41 FV 755 ± 22 83.49 ± 5.32 0.19 ± 0.002 2.22 ± 0.10 9.33 ± 0.23 86.74 ± 2.53 FO 644 ± 33 34.30 ± 4.34 0.10 ± 0.01 3.29 ± 0.07 8.31 ± 0.01 29.42 ± 0.82 JE 661 ± 4 52.33 ± 2.52 0.25 ± 0.01 2.96 ± 0.13 8.41 ± 0.03 68.25 ± 1.55 KA 532 ± 4 10.62 ± 4.05 0.23 ± 0.01 3.2 ± 0.10 7.28 ± 0.13 71.19 ± 4.26 KR 612 ± 58 20.90 ± 4.58 0.49 ± 0.02 4.02 ± 0.13 7.33 ± 0.31 67.44 ± 1.73 PI 671 ± 54 10.58 ± 0.41 0.32 ± 0.01 3.66 ± 0.22 7.15 ± 0.03 80.88 ± 3.53 PV 631 ± 40 34.04 ± 4.85 0.11 ± 0.01 3.84 ± 0.11 10.81 ± 0.16 36.83 ± 2.28
19
Code Mn Fe Co Ni Cu Zn RO 419 ± 9 44.01 ± 25.86 0.23 ± 0.01 3.40 ± 0.004 9.02 ± 1.62 104.92 ± 7.79 RM 606 ± 88 17.63 ± 5.80 0.52 ± 0.08 4.38 ± 0.51 7.92 ± 0.88 60.01 ± 2.38 SA 383 ± 40 61.18 ± 4.22 0.21 ± 0.01 1.62 ± 0.01 7.65 ± 0.03 73.04 ± 5.02 YU 489 ± 31 30.05 ± 9.95 0.09 ± 0.001 3.52 ± 0.17 8.87 ± 0.53 46.48 ± 4.62
Green tea bag AT 675 ± 22 73.39 ± 3.46 0.35 ± 0.03 5.41 ± 0.002 10.70 ± 1.44 97.31 ± 4.81 AO 603 ± 18 28.95 ± 1.50 0.32 ± 0.02 4.54 ± 0.08 8.45 ± 0.14 123.79 ± 2.28 CA 662 ± 2 31.51 ± 7.43 0.18 ± 0.002 3.93 ± 0.10 8.79 ± 0.05 85.90 ± 2.14 CR 634 ± 20 154.51 ± 21.96 0.42 ± 0.001 4.93 ± 0.02 10.82 ± 0.89 121.04 ± 16.66 CM 530 ± 29 95.20 ± 5.67 0.34 ± 0.01 4.65 ± 0.20 15.00 ± 5.15 115.77 ± 2.05 DL 739 ± 10 108.95 ± 15.2 0.34 ± 0.001 4.84 ± 0.08 9.33 ± 0.07 80.98 ± 1.16 JU 741 ± 72 43.79 ± 6.62 0.51 ± 0.01 4.80 ± 0.01 7.97 ± 0.12 54.24 ± 0.55 LA 807 ± 204 164.31 ± 4.25 0.43 ± 0.02 6.06 ± 1.28 9.39 ± 0.10 102.88 ± 2.20 LH 834 ± 15 50.60 ± 0.75 0.23 ± 0.002 4.28 ± 0.06 8.59 ± 0.03 80.86 ± 2.13 LP 539 ± 75 228.13 ± 9.63 0.41 ± 0.01 4.54 ± 0.29 11.60 ± 0.55 117.48 ± 8.32 LT 646 ± 0.5 57.26 ± 2.07 0.28 ± 0.002 4.20 ± 0.09 8.08 ± 0.01 105.38 ± 2.53 LI 642 ± 4 151.70 ± 6.29 0.35 ± 0.002 5.09 ± 0.19 10.15 ± 0.21 113.42 ± 6.01
MT 514 ± 12 22.76 ± 0.03 0.26 ± 0.001 4.65 ± 0.17 8.32 ± 0.17 100.60 ± 2.04 PL 989 ± 11 42.6 ± 7.00 0.46 ± 0.01 5.80 ± 0.22 9.26 ± 0.05 63.30 ± 1.75 SU 1065 ± 6 50.04 ± 1.19 0.44 ± 0.01 5.74 ± 0.05 8.66 ± 0.02 59.27 ± 1.84 TA 1029 ± 19 18.21 ± 2.98 0.45 ± 0.01 6.03 ± 0.16 8.89 ± 0.02 65.03 ± 0.73 TN 1098 ± 35 33.06 ± 1.01 0.55 ± 0.01 5.32 ± 0.03 8.26 ± 0.10 601.58 ± 88.38
20
Code Mn Fe Co Ni Cu Zn VE 482 ± 8 72.82 ± 2.33 0.37 ± 0.002 4.11 ± 0.06 10.14 ± 1.08 80.28 ± 0.41 YVI 651 ± 10 96.65 ± 0.11 0.44 ± 0.01 6.60 ± 0.13 9.26 ± 0.12 54.78 ± 0.50
Roasted loose MN 692 ± 25 20.70 ± 3.32 0.24 ± 0.002 2.41 ± 0.21 9.07 ± 0.08 102.77 ± 23.81
BC-Cm 657 ± 27 21.23 ± 1.40 0.16 ± 0.001 1.54 ± 0.20 9.30 ± 0.08 65.14 ± 2.75 MO 888 ± 22 53.03 ± 11.07 0.35 ± 0.02 2.96 ± 0.07 9.61 ± 0.04 164.38 ± 4.94
Roasted tea bag DO 413 ± 1 83.55 ± 2.34 0.18 ± 0.01 3.37 ± 0.01 11.90 ± 0.18 98.31 ± 18.69 LIT 454 ± 14 109.63 ± 0.31 0.18 ± 0.002 3.82 ± 0.03 12.32 ± 0.73 81.12 ± 4.83 QU 397 ± 8 79.31 ± 11.20 0.18 ± 0.03 3.06 ± 0.05 11.44 ± 0.33 78.04 ± 8.82 ML 879 ± 8 47.77 ± 5.61 0.36 ± 0.07 4.41 ± 0.04 11.06 ± 0.52 81.65 ± 1.15
21
Appendix 3.8: As, Se, Mo, Cd and Pb levels (mean ± standard deviation) of commercial yerba mate samples (mg/kg dry
weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Code As Se Mo Cd Pb
Green loose BA 0.08 ± 0.01 0.02 ± 0.002 <LOD* 0.25 ± 0.001 0.12 ± 0.02
BC- Ca 0.03 ± 0.001 <LOD* <LOD* 0.21 ± 0.002 0.15 ± 0.01 BC-Mg 0.03 ± 0.002 0.02 ± 0.001 <LOD* 0.35 ± 0.04 0.05 ± 0.001 BC- Na 0.02 ± 0.001 <LOD* <LOD* 0.23 ± 0.05 0.03 ± 0.001 BC-Pr 0.02 ± 0.001 <LOD* <LOD* 0.17 ± 0.002 0.05 ± 0.01 BC-Te 0.01 ± 0.001 0.02 ± 0.001 0.09 ± 0.01 0.25 ± 0.02 0.13 ± 0.03 BC-Ex 0.03 ± 0.01 0.02 ± 0.001 <LOD* 0.29 ± 0.01 0.10 ± 0.01 CAN 0.03 ± 0.002 0.02 ± 0.001 <LOD* 0.24 ± 0.002 0.20 ± 0.03 SAR 0.03 ± 0.001 <LOD* <LOD* 0.22 ± 0.01 0.14 ± 0.002 AM 0.04 ± 0.01 0.03 ± 0.002 <LOD* 0.35 ± 0.01 0.06 ± 0.02
BC - Tr 0.50 ± 0.46 0.02 ± 0.001 <LOD* 0.26 ± 0.06 0.07 ± 0.01 FV 0.03 ± 0.002 0.02 ± 0.001 <LOD* 0.43 ± 0.002 0.12 ± 0.01 FO 0.08 ± 0.05 <LOD* <LOD* 0.13 ± 0.01 0.04 ± 0.01 JE 0.06 ± 0.02 0.03 ± 0.002 <LOD* 0.21 ± 0.001 0.05 ± 0.002 KA 0.11 ± 0.08 0.04 ± 0.001 <LOD* 0.20 ± 0.03 0.07 ± 0.01 KR 0.32 ± 0.28 0.03 ± 0.001 <LOD* 0.12 ± 0.01 0.07 ± 0.01 PI 0.04 ± 0.001 0.04 ± 0.002 <LOD* 0.18 ± 0.01 0.11 ± 0.01 PV 0.41 ± 0.38 <LOD* <LOD* 0.17 ± 0.002 0.08 ± 0.002
22
Code As Se Mo Cd Pb RO 0.05 ± 0.01 0.06 ± 0.02 <LOD* 0.22 ± 0.03 0.10 ± 0.01 RM 0.04 ± 0.01 0.03 ± 0.002 <LOD* 0.18 ± 0.01 0.08 ± 0.01 SA 0.35 ± 0.32 0.02 ± 0.001 <LOD* 0.34 ± 0.02 0.14 ± 0.01 YU 0.11 ± 0.06 0.02 ± 0.003 <LOD* 0.20 ± 0.02 0.11 ± 0.02
Green tea bag AT 0.09 ± 0.01 0.04 ± 0.002 <LOD* 0.31 ± 0.01 0.12 ± 0.04 AO 0.05 ± 0.01 0.04 ± 0.003 <LOD* 0.23 ± 0.002 0.03 ± 0.003 CA 0.06 ± 0.01 0.04 ± 0.003 <LOD* 0.25 ± 0.01 0.08 ± 0.01 CR 0.11 ± 0.03 0.05 ± 0.002 <LOD* 0.29 ± 0.02 0.13 ± 0.02 CM 0.14 ± 0.07 0.04 ± 0.001 <LOD* 0.34 ± 0.02 <LOD* DL 0.10 ± 0.01 0.05 ± 0.003 <LOD* 0.24 ± 0.001 0.20. ± 0.07 JU 0.06 ± 0.01 0.03 ± 0.002 <LOD* 0.18 ± 0.002 0.06 ± 0.003 LA 0.14 ± 0.03 0.04 ± 0.01 <LOD* 0.31 ± 0.01 0.12 ± 0.02 LH 0.09 ± 0.03 0.03 ± 0.002 <LOD* 0.23 ± 0.01 0.06 ± 0.003 LP 0.08 ± 0.01 0.05 ± 0.001 <LOD* 0.43 ± 0.02 0.07 ± 0.01 LT 0.05 ± 0.002 0.03 ± 0.001 <LOD* 0.31 ± 0.01 0.06 ± 0.002 LI 0.12 ± 0.002 0.04 ± 0.001 <LOD* 0.35 ± 0.01 0.10 ± 0.01
MT 0.05 ± 0.01 0.05 ± 0.003 <LOD* 0.25 ± 0.01 0.04 ± 0.002 PL 0.04 ± 0.001 0.03 ± 0.002 <LOD* 0.19 ± 0.002 0.04 ± 0.002 SU 0.06 ± 0.001 0.03 ± 0.003 <LOD* 0.16 ± 0.003 0.07 ± 0.003 TA 0.04 ± 0.002 0.03 ± 0.002 <LOD* 0.18 ± 0.01 0.04 ± 0.002 TN 0.04 ± 0.002 0.03 ± 0.002 <LOD* 0.13 ± 0.002 0.05 ± 0.002
23
Code As Se Mo Cd Pb VE 0.10 ± 0.002 0.03 ± 0.003 <LOD* 0.31 ± 0.001 0.09 ± 0.02 YVI 0.07 ± 0.001 0.04 ± 0.002 <LOD* 0.15 ± 0.003 0.10 ± 0.04
Roasted loose MN 0.03 ± 0.01 0.02 ± 0.003 <LOD* 0.41 ± 0.06 0.36 ± 0.21
BC-Cm 0.02 ± 0.001 0.02 ± 0.002 0.11 ± 0.06 0.25 ± 0.02 0.13 ± 0.05 MO 0.02 ± 0.001 0.02 ± 0.002 <LOD* 0.83 ± 0.10 0.08 ± 0.01
Roasted tea bag DO 0.05 ± 0.002 0.03 ± 0.001 <LOD* 0.39 ± 0.003 0.18 ± 0.02 LIT 0.05 ± 0.01 0.03 ± 0.002 <LOD* 0.39 ± 0.002 0.20 ± 0.02 QU 0.05 ± 0.01 0.02 ± 0.001 <LOD* 0.34 ± 0.01 0.28 ± 0.01 ML 0.03 ± 0.002 0.03 ± 0.001 <LOD* 0.30 ± 0.02 0.10 ± 0.01
*refer to Table 2.2; <LOD less than the limit of detection.
24
Appendix 3.9: V, Cr, Mn, Fe, Co and Ni levels (mean ± standard deviation) of commercial yerba mate regular infusion
samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of
replicates.
Code V Cr Mn Fe Co Ni
Green loose BA 0.07 ± 0.002 0.25 ± 0.02 1322 ± 97 4.58 ± 0.44 0.18 ± 0.01 5.43 ± 0.43
BC- Ca 0.07 ± 0.002 0.37 ± 0.01 1408 ± 56 4.73 ± 0.17 0.24 ± 0.002 3.54 ± 0.16 BC-Mg 0.07 ± 0.001 0.18 ± 0.01 1063 ± 44 3.99 ± 0.23 0.22 ± 0.02 3.50 ± 0.13 BC- Na 0.07 ± 0.001 0.10 ± 0.002 1718 ± 16 3.58 ± 0.06 0.12 ± 0.001 4.01 ± 0.03 BC-Pr 0.07 ± 0.002 <LOD* 1178 ± 71 4.15 ± 0.36 0.11 ± 0.001 4.92 ± 0.26 BC-Te 0.07 ± 0.001 0.09 ± 0.002 473 ± 24 2.53 ± 0.27 0.18 ± 0.01 1.40 ± 0.09 BC-Ex 0.03 ± 0.003 0.49 ± 0.02 1308 ± 101 9.66 ± 0.24 0.29 ± 0.02 5.82 ± 0.26 CAN 0.02 ± 0.003 0.28 ± 0.01 1499 ± 41 6.36 ± 0.07 0.26 ± 0.04 4.60 ± 0.06 SAR 0.07 ± 0.001 0.19 ± 0.001 1239 ± 18 5.31 ± 0.11 0.18 ± 0.003 6.47 ± 0.01 AM 0.08 ± 0.001 0.25 ± 0.01 1647 ± 104 5.98 ± 0.13 0.19 ± 0.01 6.17 ± 0.18
BC - Tr 0.03 ± 0.002 0.43 ± 0.02 1258 ± 17 4.28 ± 0.14 0.42 ± 0.01 5.45 ± 0.22 FV 0.02 ± 0.003 1.39 ± 0.10 951 ± 85 9.41 ± 0.52 0.34 ± 0.02 3.88 ± 0.31 FO 0.02 ± 0.001 0.13 ± 0.002 3612 ± 10 7.68 ± 0.07 0.25 ± 0.002 8.32 ± 0.14 JE 0.03 ± 0.01 0.42 ± 0.07 1218 ± 189 5.77 ± 1.28 0.41 ± 0.08 5.32 ± 1.03 KA 0.08 ± 0.003 0.63 ± 0.01 1239 ± 18 2.72 ± 0.22 0.29 ± 0.01 3.76 ± 0.14 KR 0.05 ± 0.02 1.09 ± 0.05 681 ± 43 4.63 ± 0.02 0.70 ± 0.04 5.88 ± 0.27 PI 0.07 ± 0.002 0.43 ± 0.04 585 ± 57 1.91 ± 0.28 0.28 ± 0.03 3.85 ± 0.38
25
Code V Cr Mn Fe Co Ni PV 0.01 ± 0.003 0.16 ± 0.003 4255 ± 83 5.98 ± 0.14 0.24 ± 0.01 7.72 ± 0.25 RO 0.03 ± 0.001 0.41 ± 0.06 518 ± 14 5.40 ± 0.17 0.29 ± 0.01 5.16 ± 0.10 RM 0.03 ± 0.003 0.76 ± 0.06 1239 ± 112 4.15 ± 0.42 0.77 ± 0.04 6.81 ± 0.55 SA 0.02 ± 0.004 0.27 ± 0.01 614 ± 12 8.21 ± 0.54 0.30 ± 0.002 2.48 ± 0.13 YU 0.08 ± 0.002 0.23 ± 0.002 1421 ± 10 4.95 ± 0.19 0.16 ± 0.001 6.64 ± 0.27
Green tea bag AT 0.09 ± 0.01 1.32 ± 0.14 2734 ± 300 19.70 ±1.67 1.03 ± 0.12 18.05 ± 1.99 AO 0.02 ± 0.002 1.32 ± 0.09 1504 ± 223 9.55 ± 0.28 0.65 ± 0.05 8.97 ± 0.66 CA 0.03 ± 0.002 1.09 ± 0.21 1508 ± 167 8.74 ± 1.60 0.31 ± 0.01 8.04 ± 1.54 CR 0.16 ± 0.01 1.15 ± 0.08 2298 ± 209 18.61 ± 1.38 0.36 ± 0.07 14.18 ± 1.69 CM 0.13 ± 0.003 0.89 ± 0.003 1682 ± 14 11.96 ± 0.09 0.77 ± 0.01 9.50 ± 0.04 DL 0.14 ± 0.002 0.97 ± 0.01 1626 ± 30 20.30 ± 0.53 0.97 ± 0.01 16.33 ± 0.13 JU 0.05 ± 0.002 1.45 ± 0.01 2834 ± 97 10.43 ± 0.21 1.32 ± 0.02 11.74 ± 0.17 LA 0.14 ± 0.001 1.37 ± 0.01 2285 ± 68 20.44 ± 0.13 1.11 ± 0.01 14.44 ± 0.05 LH 0.06 ± 0.002 1.16 ± 0.03 2354 ± 34 20.31 ± 0.28 0.82 ± 0.01 16.29 ± 0.003 LP 0.13 ± 0.002 0.86 ± 0.002 1886 ± 4 17.450 ± 0.49 0.72 ± 0.001 6.91 ± 0.04 LT 0.05 ± 0.01 1.78 ± 0.20 2289 ± 370 12.97 ± 1.52 0.81 ± 0.09 12.44 ± 1.93 LI 0.14 ± 0.01 1.26 ± 0.01 2235 ± 31 20.48 ± 1.30 1.03 ± 0.002 16.04 ± 0.13
MT 0.05 ± 0.002 1.98 ± 0.002 2837 ± 0.1 16.25 ± 0.16 0.83 ± 0.01 14.33 ± 0.05 PL 0.09 ± 0.003 1.69 ± 0.04 3937 ± 15 10.54 ± 0.56 1.29 ± 0.03 14.91 ± 0.01 SU 0.05 ± 0.003 1.71 ± 0.08 3025 ± 147 17.28 ± 0.08 1.42 ± 0.07 19.63 ± 0.79 TA 0.05 ± 0.003 2.01 ± 0.12 3687 ± 53 17.90 ± 0.13 1.93 ± 0.11 20.88 ± 1.10
26
Code V Cr Mn Fe Co Ni TN 0.03 ± 0.003 1.23 ± 0.01 2733 ± 53 10.83 ± 0.01 1.35 ± 0.02 15.54 ± 0.25 VE 0.06 ± 0.003 0.77 ± 0.001 1282 ± 9 10.18 ± 0.19 0.69 ± 0.002 7.89 ± 0.02 YVI 0.12 ± 0.003 1.77 ± 0.03 3587 ± 3 19.22 ± 0.12 1.53 ± 0.03 22.48 ± 0.44
Roasted loose MN 0.02 ± 0.002 0.23 ± 0.002 425 ± 71 3.37 ± 0.19 0.11 ± 0.001 2.58 ± 0.16
BC-Cm 0.07 ± 0.003 0.23 ± 0.01 372 ± 7 2.22 ± 0.13 0.09 ± 0.001 1.14 ± 0.02 MO 0.03 ± 0.003 0.32 ± 0.02 495 ± 35 4.38 ± 0.15 0.15 ± 0.01 2.67 ± 0.34
Roasted tea bag DO 0.03 ± 0.003 0.29 ± 0.01 513 ± 5 7.49 ± 0.30 0.10 ± 0.002 2.87 ± 0.11 LIT 0.05 ± 0.003 0.30 ± 0.01 663 ± 18 10.65 ± 0.20 0.12 ± 0.01 3.66 ± 0.16 QU 0.04 ± 0.002 0.25 ± 0.002 490 ± 20 7.35 ± 0.42 0.09 ± 0.001 2.55 ± 0.17 ML 0.04 ± 0.001 0.56 ± 0.03 861 ± 25 5.85 ± 0.21 0.26 ± 0.01 4.51 ± 0.21
*refer to Table 2.2; <LOD less than the limit of detection.
27
Appendix 3.10: Cu, Zn, As, Se, Mo, Cd and Pb levels (mean ± standard deviation) of commercial yerba mate regular
infusion samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of
replicates.
Code Cu Zn As Se Mo Cd Pb
Green loose
BA 10.84 ± 0.59 50.55 ± 4.85 <LOD* <LOD* <LOD* 0.09 ± 0.01 0.05 ± 0.001 BC- Ca 6.68 ± 0.14 54.92 ± 2.53 <LOD* <LOD* 0.14 ± 0.002 0.06 ± 0.002 0.05 ± 0.001 BC-Mg 8.69 ± 0.13 58.81 ± 4.13 <LOD* 0.03 ± 0.002 0.05 ± 0.001 0.09 ± 0.003 0.03 ± 0.002 BC- Na 8.68 ± 0.06 45.51 ± 0.99 <LOD* <LOD* <LOD* 0.06 ± 0.003 <LOD* BC-Pr 10.81 ± 0.34 45.38 ± 2.67 <LOD* <LOD* <LOD* 0.04 ± 0.01 <LOD* BC-Te 4.69 ± 0.07 33.67 ± 1.70 <LOD* <LOD* <LOD* 0.10 ± 0.04 <LOD* BC-Ex 13.12 ± 0.81 76.24 ± 5.23 0.03 ± 0.002 0.08 ± 0.002 <LOD* 0.13 ± 0.01 <LOD* CAN 9.16 ± 0.11 61.90 ± 0.13 0.03 ± 0.002 0.06 ± 0.001 0.13 ± 0.01 0.08 ± 0.003 <LOD* SAR 12.90 ± 0.30 52.13 ± 1.98 <LOD* <LOD* <LOD* 0.08 ± 0.003 0.07 ± 0.002 AM 11.71 ± 0.15 56.32 ± 3.68 <LOD* <LOD* <LOD* 0.08 ± 0.01 0.08 ± 0.002
BC - Tr 6.01 ± 0.11 78.24 ± 4.32 0.03 ± 0.001 0.06 ± 0.01 <LOD* 0.12 ± 0.003 <LOD* FV 8.21 ± 0.23 82.01 ± 7.43 0.03 ± 0.002 0.07 ± 0.002 <LOD* 0.15 ± 0.01 <LOD* FO 11.06 ± 0.23 46.83 ± 1.65 0.02 ± 0.001 0.03 ± 0.001 0.35 ± 0.14 0.06 ± 0.001 <LOD* JE 8.41 ± 1.49 69.58 ± 13.30 0.03 ± 0.01 0.08 ± 0.01 <LOD* 0.07 ± 0.01 <LOD* KA 5.65 ± 0.02 51.24 ± 2.24 0.02 ± 0.002 0.03 ± 0.001 <LOD* 0.06 ± 0.001 0.04 ± 0.01 KR 6.47 ± 0.30 61.34 ± 1.43 0.03 ± 0.001 0.07 ± 0.01 <LOD* 0.04 ± 0.002 <LOD* PI 4.15 ± 0.29 44.85 ± 3.66 <LOD* 0.03 ± 0.002 <LOD* 0.04 ± 0.002 <LOD*
28
Code Cu Zn As Se Mo Cd Pb PV 11.23 ± 0.05 52.96 ± 3.30 0.03 ± 0.001 <LOD* 0.62 ± 0.13 0.07 ± 0.001 <LOD* RO 5.32 ± 0.18 93.13 ± 1.39 0.05 ± 0.002 0.08 ± 0.003 <LOD* 0.07 ± 0.002 <LOD* RM 5.61 ± 0.33 53.72 ± 7.00 0.05 ± 0.002 0.08 ± 0.003 <LOD* 0.01 ± 0.002 <LOD* SA 7.50 ± 0.60 59.70 ± 3.92 0.02 ± 0.001 0.04 ± 0.002 <LOD* 0.11 ± 0.01 <LOD* YU 11.53 ± 0.30 47.06 ± 3.33 <LOD* <LOD* <LOD* 0.13 ± 0.07 0.07 ± 0.01
Green tea bag AT 12.47 ± 0.94 184.91 ± 17.29 0.13 ± 0.01 0.15 ± 0.02 <LOD* 0.21 ± 0.01 <LOD* AO 8.61 ± 0.40 136.18 ± 8.97 0.04 ± 0.001 0.12 ± 0.01 <LOD* 0.12 ± 0.01 <LOD* CA 7.60 ± 1.13 109.59 ± 22.55 0.07 ± 0.01 0.08 ± 0.01 <LOD* 0.14 ± 0.03 <LOD* CR 14.00 ± 0.31 157.68 ± 14.40 0.11 ± 0.01 0.15 ± 0.02 <LOD* 0.18 ± 0.01 <LOD* CM 11.70 ± 0.57 123.32 ± 0.31 0.07 ± 0.002 0.07 ± 0.003 <LOD* 0.20 ± 0.002 0.05 ± 0.002 DL 15.33 ± 0.06 161.73 ± 3.51 0.10 ± 0.002 0.18 ± 0.01 <LOD* 0.13 ± 0.002 <LOD* JU 8.46 ± 0.48 100.84 ± 1.79 0.09 ± 0.001 0.10 ± 0.002 <LOD* 0.10 ± 0.001 <LOD* LA 14.72 ± 0.86 167.69 ± 0.50 0.13 ± 0.01 0.15 ± 0.002 <LOD* 0.20 ± 0.001 <LOD* LH 19.06 ± 0.60 160.00 ± 3.81 0.11 ± 0.002 0.17 ± 0.001 <LOD* 0.14 ± 0.002 <LOD* LP 5.96 ± 0.55 122.11 ± 0.17 0.06 ± 0.001 0.07 ± 0.001 <LOD* 0.19 ± 0.01 <LOD* LT 10.98 ± 2.54 165.00 ± 19.31 0.09 ± 0.01 0.13 ± 0.01 <LOD* 0.18 ± 0.03 <LOD* LI 15.65 ± 0.68 191.92 ± 2.97 0.16 ± 0.01 0.15 ± 0.003 <LOD* 0.20 ± 0.002 0.05 ± 0.01
MT 14.90 ± 1.15 174.67 ± 2.52 0.06 ± 0.001 0.19 ± 0.003 0.12 ± 0.01 0.18 ± 0.01 0.04 ± 0.02 PL 11.84 ± 1.06 102.29 ± 3.69 0.06 ± 0.002 0.07 ± 0.01 0.10 ± 0.003 0.12 ± 0.01 0.06 ± 0.002 SU 17.28 ± 0.09 133.95 ± 6.07 0.08 ± 0.002 0.14 ± 0.002 <LOD* 0.10 ± 0.003 <LOD* TA 17.26 ± 0.12 133.79 ± 7.69 0.09 ± 0.001 0.15 ± 0.001 <LOD* 0.13 ± 0.003 0.04 ± 0.001
29
Code Cu Zn As Se Mo Cd Pb TN 3.62 ± 0.59 162.53 ± 16.96 0.07 ± 0.001 0.12 ± 0.001 <LOD* 0.08 ± 0.002 <LOD* VE 8.27 ± 0.43 104.92 ± 2.84 0.12 ± 0.002 0.10 ± 0.002 <LOD* 0.12 ± 0.01 <LOD* YVI 20.37 ± 0.06 130.68 ± 5.31 0.11 ± 0.003 0.16 ± 0.003 0.10 ± 0.002 0.11 ± 0.002 0.04 ± 0.001
Roasted loose MN 0.47 ± 0.03 25.69 ± 0.20 0.02 ± 0.002 0.07 ± 0.003 <LOD* 0.07 ± 0.002 <LOD*
BC-Cm 0.30 ± 0.01 9.74 ± 0.56 <LOD* <LOD* <LOD* 0.02 ± 0.01 <LOD* MO 0.86 ± 0.04 35.76 ± 1.68 0.03 ± 0.001 0.07 ± 0.002 <LOD* 0.13 ± 0.01 <LOD*
Roasted tea bag DO 0.67 ± 0.03 22.15 ± 1.22 0.04 ± 0.002 0.08 ± 0.01 <LOD* 0.04 ± 0.002 <LOD* LIT <LOD* 24.14 ± 2.16 0.04 ± 0.001 0.07 ± 0.002 <LOD* 0.05 ± 0.001 <LOD* QU 0.16 ± 0.48 18.97 ± 1.14 0.04 ± 0.001 0.07 ± 0.01 <LOD* 0.04 ± 0.001 0.06 ± 0.02 ML 0.24 ± 0.00 30.05 ± 2.10 0.03 ± 0.003 0.08 ± 0.003 <LOD* 0.07 ± 0.002 <LOD*
*refer to Table 2.2; <LOD less than the limit of detection.
30
Appendix 3.11: V, Cr, Mn, Fe, Co and Ni levels (mean ± standard deviation) of commercial yerba mate Brazilian iced tea
infusion samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of
replicates.
Code V Cr Mn Fe Co Ni
Green loose
AM 0.03 ± 0.002 0.58 ± 0.02 1295 ± 5 6.14 ± 0.08 0.56 ± 0.002 8.21 ± 0.26 BC - Tr 0.02 ± 0.001 0.38 ± 0.002 1721 ± 37 9.12 ± 0.15 0.44 ± 0.003 6.76 ± 0.02
FO 0.03 ± 0.001 0.18 ± 0.01 2659 ± 63 13.04 ± 1.33 0.43 ± 0.03 16.30 ± 1.22 JE 0.04 ± 0.002 0.56 ± 0.02 1527 ± 23 8.11 ± 0.14 0.54 ± 0.01 7.14 ± 0.07 PV 0.02 ± 0.002 0.24 ± 0.01 2446 ± 55 9.82 ± 0.38 0.42 ± 0.03 14.03 ± 1.95 RM 0.04 ± 0.001 1.29 ± 0.03 1964 ± 4 6.41 ± 0.01 1.19 ± 0.04 10.56 ± 0.33
Green tea bag AT 0.16 ± 0.01 2.40 ± 0.11 3226 ± 364 40.88 ± 1.73 1.85 ± 0.10 32.07 ± 1.21 DL 0.23 ± 0.01 1.65 ± 0.03 2061 ± 18 38.93 ± 0.34 1.63 ± 0.01 27.76 ± 0.12 JU 0.07 ± 0.02 2.25 ± 0.59 2860 ± 883 17.77 ± 6.20 1.99 ± 0.53 19.69 ± 4.85
Roasted loose MN 0.03 ± 0.003 0.47 ± 0.02 899 ± 106 6.94 ± 0.41 0.22 ± 0.01 4.48 ± 0.41
BC-Cm 0.02 ± 0.003 0.53 ± 0.01 671 ± 3 6.88 ± 0.63 0.17 ± 0.02 2.19 ± 0.10 MO 0.05 ± 0.01 0.54 ± 0.08 890 ± 165 6.98 ± 0.94 0.26 ± 0.03 3.78 ± 0.60
Roasted tea bag DO 0.05 ± 0.002 0.42 ± 0.01 541 ± 35 13.67 ± 0.18 0.17 ± 0.001 4.91 ± 0.34 LIT 0.08 ± 0.01 0.47 ± 0.002 811 ± 42 20.21 ± 0.41 0.20 ± 0.001 6.16 ± 0.15
31
Code V Cr Mn Fe Co Ni QU 0.06 ± 0.001 0.37 ± 0.01 596 ± 17 14.80 ± 0.07 0.16 ± 0.002 4.41 ± 0.02
Appendix 3.12: Cu, Zn, As, Se, Mo Cd and Pb levels (mean ± standard deviation) of commercial yerba mate Brazilian iced
tea infusion samples (µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number
of replicates.
Code Cu Zn As Se Mo Cd Pb
Green loose AM 10.97 ± 0.09 109.22 ± 2.86 0.04 ± 0.003 0.11 ± 0.003 <LOD* 0.13 ± 0.004 0.01 ± 0.001
BC - Tr 16.38 ± 0.71 89.24 ± 0.24 0.04 ± 0.002 0.10 ± 0.01 0.07 ± 0.002 0.10 ± 0.004 <LOD* FO 23.62 ± 0.91 69.91 ± 6.04 0.04 ± 0.002 0.09 ± 0.002 <LOD* 0.08 ± 0.01 0.01 ± 0.002 JE 14.45 ± 0.01 91.47 ± 1.98 0.03 ± 0.002 0.13 ± 0.002 <LOD* 0.08 ± 0.003 0.01 ± 0.002 PV 22.55 ± 0.40 80.13 ± 10.32 0.05 ± 0.001 0.09 ± 0.002 0.04 ± 0.01 0.08 ± 0.001 0.01 ± 0.002 RM 10.95 ± 0.74 78.43 ± 0.75 0.05 ± 0.001 0.11 ± 0.002 <LOD* 0.08 ± 0.002 0.02 ± 0.002
Green tea bag AT 23.96 ± 1.60 316.00 ± 11.34 0.23 ± 0.01 0.28 ± 0.01 0.16 ± 0.01 0.37 ± 0.01 0.05 ± 0.01 DL 28.44 ± 1.52 254.30 ± 1.55 0.19 ± 0.01 0.28 ± 0.01 0.15 ± 0.002 0.20 ± 0.002 0.03 ± 0.001 JU 12.47 ± 3.97 139.64 ± 33.23 0.14 ± 0.04 0.16 ± 0.02 0.04 ± 0.03 0.14 ± 0.04 0.04 ± 0.01
Roasted loose MN <LOD* 39.66 ± 1.45 0.03 ± 0.002 0.09 ± 0.001 0.02 ± 0.003 0.08 ± 0.002 0.02 ± 0.002
BC-Cm <LOD* 15.87 ± 0.46 0.03 ± 0.002 0.08 ± 0.001 0.03 ± 0.01 0.03 ± 0.001 <LOD*
32
Code Cu Zn As Se Mo Cd Pb MO 0.05 ± 0.03 59.64 ± 6.05 0.04 ± 0.01 0.09 ± 0.002 0.00 ± 0.01 0.13 ± 0.01 <LOD*
Roasted tea bag DO 0.17 ± 0.13 30.17 ± 2.22 0.07 ± 0.01 0.10 ± 0.003 0.01 ± 0.003 0.06 ± 0.002 0.02 ± 0.002 LIT 0.29 ± 0.05 34.89 ± 0.58 0.06 ± 0.001 0.10 ± 0.002 0.02 ± 0.002 0.09 ± 0.01 0.04 ± 0.001 QU <LOD* 26.18 ± 1.62 0.07 ± 0.001 0.10 ± 0.002 0.02 ± 0.003 0.06 ± 0.002 0.07 ± 0.001
*refer to Table 2.2; <LOD lower than the limit of detection.
Appendix 3.13: Mn and Fe levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples
(µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Mn Fe
Code F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
BA 9421 ± 1106 10376 ± 152 6910 ± 11 4407 ± 75 2781 ± 347 26.32 ± 3.63 49.08 ± 3.82 35.72 ± 0.92 20.57 ± 1.32 11.38 ± 1.45
BC- Ca 18579 ± 1080 9863 ± 1043 4614 ± 453 2583 ± 289 1905 ± 436 72.07 ± 7.34 18.14 ± 12.98 23.83 ± 3.02 13.97 ± 0.92 8.41 ± 1.48
BC-Mg 11650 ± 319 9443 ± 394 4267 ± 88 2694 ± 68 1711 ± 123 48.68 ± 0.86 29.21 ± 4.44 18.42 ± 1.17 16.04 ± 0.73 9.95 ± 0.25
BC- Na 18988 ± 256 12538 ± 229 6274 ± 501 3586 ± 461 2315 ± 498 36.70 ± 0.16 16.15 ± 1.49 22.27 ± 3.01 12.89 ± 1.74 7.44 ± 1.04
BC-Pr 14610 ± 171 7908 ± 70 3309 ± 137 1034 ± 856 1099 ± 34 49.23 ± 8.00 17.36 ± 1.82 18.6 ± 0.87 11.40 ± 0.26 6.61 ± 0.16
BC-Te 3119 ± 252 3025 ± 138 2430 ± 510 1491 ± 44 1175 ± 15 16.25 ± 0.90 11.88 ± 0.88 14.42 ± 0.27 11.15 ± 0.15 8.53 ± 0.17
BC-Ex 12081 ± 671 10606 ± 385 5813 ± 474 3478 ± 263 2280 ± 189 57.48 ± 5.89 64.73 ± 12.52 45.22 ± 5.09 23.96 ± 1.92 14.29 ± 1.21
CAN 7756 ± 418 9618 ± 94 6359 ± 2251 3954 ± 115 2578 ± 35 21.45 ± 4.21 26.54 ± 5.60 22.43 ± 1.07 22.00 ± 1.33 12.94 ± 0.46
SAR 13205 ± 1229 12121 ± 136 7448 ± 165 4599 ± 225 2957 ± 263 42.81 ± 5.26 53.80 ± 4.58 41.04 ± 2.32 22.54 ± 1.71 12.61 ± 0.62
AM 6808 ± 827 5466 ± 24 4398 ± 559 2822 ± 84 4310 ± 68 13.71 ± 1.75 21.61 ± 3.26 24.35 ± 2.38 13.98 ± 0.04 8.59 ± 0.02
BC - Tr 19459 ± 827 9965 ± 134 5091 ± 977 2643 ± 286 1889 ± 61 67.85 ± 11.76 41.8 ± 11.64 27.44 ± 6.73 13.33 ± 2.50 8.40 ± 0.73
33
Mn Fe
Code F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
FV 7647 ± 7 6909 ± 419 3737 ± 203 2341 ± 84 1596 ± 164 59.41 ± 17.02 32.95 ± 8.66 21.41 ± 1.08 21.68 ± 0.94 13.10 ± 0.59
FO 30591 ± 2142 16964 ± 1180 7275 ± 649 4073 ± 19 2418 ± 0.9 74.13 ± 8.58 35.78 ± 0.78 25.47 ± 2.65 14.17 ± 0.61 8.87 ± 0.33
JE 6236 ± 732 7105 ± 885 5482 ± 563 3606 ± 165 2566 ± 76 22.68 ± 5.75 39.12 ± 0.55 33.01 ± 2.41 20.04 ± 2.11 12.11 ± 1.53
KA 6232 ± 369 6898 ± 432 6903 ± 275 4952 ± 285 3723 ± 143 10.82 ± 0.96 17.80 ± 1.87 21.09 ± 1.92 14.13 ± 1.04 9.73 ± 0.44
KR 4508 ± 179 4831 ± 613 3480 ± 339 2537 ± 5 1931 ± 106 13.27 ± 0.66 14.64 ± 0.10 23.48 ± 1.41 13.79 ± 1.20 11.20 ± 0.11
PI 3678 ± 394 4930 ± 115 3753 ± 109 2893 ± 83 2065 ± 24 9.38 ± 0.50 12.86 ± 0.22 20.45 ± 0.13 13.77 ± 0.60 9.84 ± 0.27
PV 28403 ± 1606 13986 ± 834 5953 ± 175 2835 ± 3 1808 ± 42 61.59 ± 2.04 30.48 ± 4.37 20.68 ± 0.47 9.68 ± 0.12 6.07 ± 0.11
RO 2554 ± 26 2563 ± 126 1803 ± 78 13747 ± 30 1102 ± 38 13.58 ± 0.43 13.89 ± 3.88 16.30 ± 0.51 11.69 ± 0.38 9.21 ± 0.50
RM 7165 ± 738 9003 ± 708 6353 ± 302 4714 ± 37 3550 ± 43 12.20 ± 1.80 23.77 ± 3.32 22.95 ± 2.01 14.88 ± 0.59 9.18 ± 0.03
SA 5975 ± 820 3094 ± 211 1449 ± 186 741 ± 71 436 ± 47 66.79 ± 18.73 43.55 ± 8.75 28.54 ± 4.33 15.05 ± 1.01 9.94 ± 0.69
YU 7092 ± 522 10596 ± 1241 7806 ± 462 5869 ± 98 3755 ± 45 13.46 ± 0.06 23.42 ± 8.84 31.09 ± 5.61 27.92 ± 1.39 15.88 ± 0.28
34
Appendix 3.14: Cu and Zn levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples
(µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Cu Zn
Code F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
BA 92.98 ± 4.31 134.90 ± 5.79 76.31 ± 1.48 49.37 ± 2.40 29.47 ± 2.97 360.53 ± 52.72 494.66 ± 20.56 315.55 ± 3.67 198.54 ± 9.44 112.16 ± 13.89
BC- Ca 114.06 ± 5.37 44.27 ± 43.20 38.86 ± 3.12 25.47 ± 3.01 16.99 ± 4.13 646.91 ± 32.29 230.33 ± 228.19 183.68 ± 15.23 101.26 ± 11.58 56.24 ± 11.19
BC-Mg 131.38 ± 2.00 108.67 ± 6.81 58.98 ± 4.79 37.39 ± 2.02 23.84 ± 0.82 673.53 ± 17.11 564.90 ± 0.92 282.45 ± 54.09 176.85 ± 9.86 102.04 ± 6.09
BC- Na 115.83 ± 0.09 87.50 ± 3.56 48.75 ± 2.80 32.12 ± 3.42 20.98 ± 4.32 508.36 ± 2.62 388.72 ± 14.22 195.26 ± 16.07 115.37 ± 15.94 59.22 ± 13.31
BC-Pr 157.09 ± 0.66 97.75 ± 4.33 48.32 ± 2.67 29.72 ± 1.51 16.83 ± 0.13 549.44 ± 21.49 318.09 ± 31.12 130.46 ± 2.99 66.16 ± 3.41 31.37 ± 0.74
BC-Te 48.46 ± 4.13 43.92 ± 1.81 31.36 ± 0.99 24.19 ± 0.18 19.26 ± 0.11 188.16 ± 6.10 196.26 ± 9.73 118.41 ± 0.07 113.32 ± 4.44 80.31 ± 4.14
BC-Ex 142.81 ± 2.21 153.30 ± 3.25 74.42 ± 7.25 44.72 ± 3.77 28.28 ± 2.35 593.09 ± 33.86 652.37 ± 9.14 336.17 ± 25.19 201.13 ± 7.10 124.23 ± 4.33
CAN 103.26 ± 11.87 127.08 ± 8.41 111.68 ± 10.22 52.60 ± 2.34 34.36 ± 0.14 305.47 ± 51.88 391.33 ± 1.55 268.69 ± 2.93 186.99 ± 11.16 116.26 ± 3.70
SAR 113.95 ± 12.04 141.12 ± 8.95 75.31 ± 2.98 46.68 ± 2.33 28.69 ± 2.05 472.53 ± 53.81 546.32 ± 67.06 314.66 ± 17.96 187.71 ± 13.27 111.79 ± 4.70
AM 45.20 ± 6.43 61.62 ± 0.23 42.48 ± 3.00 27.95 ± 0.16 18.28 ± 0.50 478.34 ± 70.28 504.38 ± 2.94 347.13 ± 27.22 211.77 ± 2.93 131.50 ± 3.35
BC - Tr 133.34 ± 11.45 96.62 ± 1.01 46.00 ± 6.79 26.65 ± 2.50 16.95 ± 0.73 690.77 ± 70.38 428.24 ± 5.66 206.65 ± 38.58 97.80 ± 18.42 54.77 ± 4.29
FV 94.00 ± 0.49 76.73 ± 4.76 54.04 ± 0.18 33.08 ± 1.00 21.67 ± 1.61 570.02 ± 2.89 477.61 ± 20.20 288.20 ± 0.56 195.87 ± 6.65 117.87 ± 8.85
FO 183.22 ± 16.31 137.62 ± 9.62 54.73 ± 3.97 30.76 ± 0.25 17.54 ± 0.08 495.81 ± 30.67 361.88 ± 17.61 139.41 ± 12.27 65.00 ± 2.33 32.69 ± 0.02
JE 67.73 ± 20.12 88.57 ± 14.21 54.22 ± 6.04 36.03 ± 1.70 24.02 ± 0.01 306.25 ± 53.86 411.84 ± 24.90 277.16 ± 25.37 190.76 ± 9.00 122.10 ± 1.06
KA 43.54 ± 1.61 54.52 ± 4.70 45.81 ± 2.42 32.14 ± 1.67 23.08 ± 0.55 265.03 ± 13.46 347.11 ± 39.03 308.53 ± 21.15 214.27 ± 15.82 146.37 ± 5.26
KR 52.61 ± 0.29 53.48 ± 5.67 48.99 ± 6.11 30.64 ± 0.43 23.11 ± 1.50 303.49 ± 11.53 338.23 ± 50.64 287.69 ± 32.73 186.13 ± 2.16 132.01 ± 4.61
PI 32.60 ± 2.92 53.98 ± 1.46 44.40 ± 0.43 29.61 ± 1.08 22.21 ± 0.35 250.79 ± 10.63 395.04 ± 11.63 311.90 ± 5.50 218.73 ± 14.67 152.52 ± 8.78
PV 174.98 ± 24.77 110.29 ± 7.40 48.38 ± 0.04 23.24 ± 0.12 12.46 ± 0.07 511.90 ± 43.32 261.28 ± 16.83 130.01 ± 1.92 47.46 ± 0.06 24.25 ± 0.83
RO 35.12 ± 0.06 34.90 ± 3.16 30.80 ± 0.52 21.92 ± 1.07 18.29 ± 1.70 362.57 ± 18.09 347.48 ± 38.09 286.83 ± 5.04 199.91 ± 10.11 150.67 ± 14.61
35
Cu Zn
Code F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
RM 39.97 ± 0.66 65.13 ± 3.45 39.40 ± 2.54 29.21 ± 0.71 19.64 ± 0.14 253.92 ± 10.25 348.39 ± 2.07 232.15 ± 20.70 164.58 ± 7.34 103.49 ± 1.08
SA 121.35 ± 19.89 76.96 ± 2.28 36.45 ± 3.03 20.33 ± 1.13 11.78 ± 0.42 695.14 ± 101.79 426.60 ± 4.84 197.84 ± 17.45 107.02 ± 4.71 59.06 ± 2.36
YU 77.04 ± 2.08 145.81 ± 43.41 97.22 ± 6.86 64.94 ± 0.49 41.39 ± 0.35 241.18 ± 23.46 410.75 ± 58.79 306.40 ± 13.05 232.73 ± 3.61 142.03 ± 1.51
Appendix 3.15: Ni and Cr levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples (µg/200
mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Ni Cr
Code F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
BA 41.78 ± 5.03 58.42 ± 0.10 33.41 ± 0.16 21.45 ± 0.87 11.71 ± 1.31 1.75 ± 0.18 2.55 ± 0.03 1.40 ± 0.02 0.97 ± 0.05 0.58 ± 0.06
BC- Ca 55.36 ± 2.81 19.55 ± 18.29 10.88 ± 0.87 6.16 ± 0.67 3.59 ± 0.78 5.23 ± 0.26 2.34 ± 1.50 1.30 ± 0.09 0.84 ± 0.09 0.54 ± 0.11
BC-Mg 43.59 ± 0.51 38.56 ± 2.23 20.55 ± 1.76 9.46 ± 0.61 5.62 ± 0.17 2.08 ± 0.01 2.05 ± 0.25 1.33 ± 0.06 0.56 ± 0.03 0.35 ± 0.01
BC- Na 48.51 ± 0.74 34.48 ± 1.72 13.71 ± 1.08 8.00 ± 1.15 4.34 ± 0.88 1.10 ± 0.03 1.03 ± 0.05 0.34 ± 0.03 0.22 ± 0.03 0.13 ± 0.02
BC-Pr 72.80 ± 2.40 41.91 ± 1.03 13.00 ± 0.47 6.94 ± 0.52 3.34 ± 0.17 0.81 ± 0.003 0.63 ± 0.02 0.17 ± 0.01 0.11 ± 0.003 0.07 ± 0.002
BC-Te 10.61 ± 0.18 10.50 ± 0.42 5.97 ± 0.35 4.93 ± 0.08 3.95 ± 0.01 0.66 ± 0.07 0.71 ± 0.02 0.37 ± 0.002 0.32 ± 0.01 0.26 ± 0.01
BC-Ex 49.98 ± 1.51 57.54 ± 2.14 27.96 ± 2.96 16.27 ± 1.94 9.93 ± 0.81 3.16 ± 0.10 3.95 ± 0.16 1.92 ± 0.22 1.23 ± 0.10 0.79 ± 0.06
CAN 46.01 ± 5.60 59.18 ± 1.67 47.34 ± 2.05 24.26 ± 1.03 14.51 ± 0.13 1.27 ± 0.15 1.74 ± 0.05 1.43 ± 0.11 0.68 ± 0.04 0.45 ± 0.01
SAR 55.43 ± 6.15 65.50 ± 3.51 34.99 ± 1.08 21.56 ± 0.75 12.36 ± 0.93 2.04 ± 0.21 2.60 ± 0.06 1.34 ± 0.03 0.88 ± 0.05 0.54 ± 0.03
AM 33.66 ± 3.28 42.10 ± 0.03 27.05 ± 2.25 16.68 ± 0.51 10.47 ± 0.23 2.14 ± 0.27 2.88 ± 0.02 1.73 ± 0.12 1.18 ± 0.02 0.77 ± 0.03
BC - Tr 65.88 ± 5.50 41.07 ± 0.40 15.88 ± 4.00 7.36 ± 1.18 4.15 ± 0.43 2.79 ± 0.13 1.96 ± 0.02 0.80 ± 0.14 0.46 ± 0.06 0.29 ± 0.02
FV 29.90 ± 0.24 27.13 ± 0.62 18.75 ± 0.16 9.35 ± 0.31 6.08 ± 0.44 9.29 ± 0.11 9.01 ± 0.16 6.59 ± 0.14 3.59 ± 0.13 2.40 ± 0.15
36
Ni Cr
Code F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
FO 117.75 ± 9.17 81.20 ± 6.68 27.56 ± 2.11 12.20 ± 0.10 6.05 ± 0.09 0.78 ± 0.05 0.62 ± 0.03 0.18 ± 0.01 0.11 ± 0.002 0.06 ± 0.002
JE 26.78 ± 4.54 36.68 ± 3.64 23.20 ± 2.81 14.73 ± 0.42 9.74 ± 0.12 1.76 ± 0.27 2.43 ± 0.26 1.52 ± 0.18 1.10 ± 0.06 0.74 ± 0.002
KA 22.09 ± 0.26 29.54 ± 1.82 25.18 ± 1.54 18.03 ± 1.32 11.99 ± 0.4 3.69 ± 0.05 5.14 ± 0.34 4.25 ± 0.23 3.17 ± 0.20 2.27 ± 0.06
KR 31.54 ± 2.16 35.41 ± 3.56 32.74 ± 3.45 20.54 ± 0.56 14.31 ± 0.70 4.96 ± 0.21 5.84 ± 0.50 5.53 ± 0.65 3.51 ± 0.08 2.70 ± 0.16
PI 24.64 ± 1.69 37.98 ± 0.21 33.20 ± 0.14 21.74 ± 0.55 14.89 ± 0.20 2.88 ± 0.26 4.46 ± 0.06 4.07 ± 0.06 2.66 ± 0.06 2.05 ± 0.01
PV 103.2 ± 8.69 58.66 ± 2.86 20.25 ± 0.37 7.40 ± 0.11 3.65 ± 0.11 1.48 ± 0.22 0.82 ± 0.02 0.29 ± 0.01 0.15 ± 0.00 0.09 ± 0.002
RO 21.02 ± 0.05 22.29 ± 1.21 19.96 ± 0.25 12.11 ± 0.18 10.04 ± 0.46 1.49 ± 0.05 1.59 ± 0.06 1.48 ± 0.01 0.89 ± 0.00 0.74 ± 0.02
RM 35.77 ± 2.12 53.99 ± 2.06 32.23 ± 2.16 23.68 ± 0.66 14.91 ± 0.14 3.77 ± 0.41 5.82 ± 0.34 3.47 ± 0.38 2.68 ± 0.19 1.78 ± 0.08
SA 31.36 ± 4.49 20.43 ± 1.09 7.76 ± 0.78 4.41 ± 0.31 2.61 ± 0.10 2.46 ± 0.30 1.78 ± 0.06 0.75 ± 0.07 0.45 ± 0.03 0.28 ± 0.02
YU 35.18 ± 1.43 58.26 ± 5.93 49.82 ± 3.11 32.56 ± 0.57 19.96 ± 0.07 1.16 ± 0.03 1.84 ± 0.24 1.61 ± 0.13 0.99 ± 0.03 0.64 ± 0.01
37
Appendix 3.16: V and Co levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples (µg/200
mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
V Co
Code F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
BA 0.09 ± 0.02 0.09 ± 0.01 0.04 ± 0.003 0.03 ± 0.001 0.02 ± 0.002 1.48 ± 0.19 1.89 ± 0.02 1.04 ± 0.02 0.67 ± 0.002 0.40 ± 0.03 BC- Ca 0.17 ± 0.02 0.19 ± 0.06 0.43 ± 0.28 0.34 ± 0.11 0.14 ± 0.02 3.23 ± 0.08 1.14 ± 1.24 0.87 ± 0.06 0.55 ± 0.05 0.37 ± 0.07 BC-Mg 0.08 ± 0.01 0.19 ± 0.11 0.30 ± 0.01 0.03 ± 0.002 0.04 ± 0.01 2.57 ± 0.03 2.28 ± 0.20 1.21 ± 0.04 0.64 ± 0.03 0.40 ± 0.02 BC- Na 0.10 ± 0.01 0.22 ± 0.002 0.07 ± 0.04 0.11 ± 0.06 0.12 ± 0.08 1.41 ± 0.03 1.10 ± 0.03 0.49 ± 0.03 0.31 ± 0.03 0.18 ± 0.03 BC-Pr 0.10 ± 0.01 0.11 ± 0.01 0.04 ± 0.02 0.05 ± 0.02 0.04 ± 0.01 1.37 ± 0.08 0.85 ± 0.003 0.33 ± 0.002 0.20 ± 0.02 0.11 ± 0.01 BC-Te 0.06 ± 0.01 0.06 ± 0.01 0.01 ± 0.003 0.01 ± 0.002 0.01 ± 0.002 1.34 ± 0.08 1.49 ± 0.11 0.84 ± 0.02 0.69 ± 0.01 0.56 ± 0.01 BC-Ex 0.24 ± 0.03 0.25 ± 0.08 0.24 ± 0.18 0.34 ± 0.30 0.32 ± 0.29 2.69 ± 0.15 2.62 ± 0.03 1.22 ± 0.10 0.76 ± 0.02 0.48 ± 0.02 CAN 0.11 ± 0.05 0.09 ± 0.05 0.13 ± 0.07 0.03 ± 0.01 0.02 ± 0.01 1.32 ± 0.08 1.63 ± 0.01 1.26 ± 0.09 0.62 ± 0.05 0.41 ± 0.01 SAR 0.14 ± 0.03 0.15 ± 0.01 0.06 ± 0.01 0.04 ± 0.003 0.02 ± 0.001 1.77 ± 0.22 1.97 ± 0.12 0.99 ± 0.05 0.61 ± 0.04 0.37 ± 0.02 AM 0.51 ± 0.25 0.28 ± 0.09 0.18 ± 0.04 0.09 ± 0.002 0.07 ± 0.002 2.94 ± 0.50 3.36 ± 0.01 1.90 ± 0.10 1.24 ± 0.04 0.81 ± 0.03
BC - Tr 0.18 ± 0.03 0.09 ± 0.01 0.05 ± 0.002 0.05 ± 0.01 0.03 ± 0.002 4.34 ± 0.39 2.63 ± 0.15 1.09 ± 0.19 0.58 ± 0.09 0.35 ± 0.03 FV 0.11 ± 0.01 0.11 ± 0.003 0.16 ± 0.002 0.04 ± 0.01 0.03 ± 0.001 2.77 ± 0.01 2.48 ± 0.10 1.60 ± 0.04 0.82 ± 0.02 0.53 ± 0.04 FO 0.14 ± 0.01 0.08 ± 0.01 0.02 ± 0.001 0.02 ± 0.002 0.02 ± 0.001 3.21 ± 0.21 2.26 ± 0.11 0.82 ± 0.04 0.44 ± 0.001 0.24 ± 0.01 JE 0.15 ± 0.03 0.16 ± 0.03 0.07 ± 0.003 0.04 ± 0.001 0.03 ± 0.002 2.15 ± 0.36 2.65 ± 0.18 1.65 ± 0.16 1.16 ± 0.04 0.78 ± 0.02 KA 0.06 ± 0.002 0.06 ± 0.002 0.03 ± 0.01 0.02 ± 0.002 0.02 ± 0.002 1.59 ± 0.02 1.96 ± 0.10 1.71 ± 0.11 1.26 ± 0.08 0.92 ± 0.02 KR 0.10 ± 0.004 0.11 ± 0.04 0.10 ± 0.02 0.02 ± 0.001 0.02 ± 0.002 3.69 ± 0.27 4.08 ± 0.30 3.62 ± 0.50 2.26 ± 0.08 1.72 ± 0.09 PI 0.05 ± 0.01 0.12 ± 0.01 0.11 ± 0.01 0.02 ± 0.002 0.02 ± 0.001 1.83 ± 0.14 2.67 ± 0.08 2.39 ± 0.04 1.47 ± 0.06 1.13 ± 0.01 PV 0.15 ± 0.01 0.07 ± 0.001 0.03 ± 0.002 0.02 ± 0.003 0.02 ± 0.002 3.12 ± 0.13 1.84 ± 0.03 0.68 ± 0.003 0.32 ± 0.01 0.18 ± 0.01 RO 0.08 ± 0.006 0.14 ± 0.02 0.09 ± 0.03 0.03 ± 0.001 0.03 ± 0.01 1.29 ± 0.03 1.30 ± 0.01 0.89 ± 0.01 0.66 ± 0.02 0.54 ± 0.04
38
V Co
Code F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
RM 0.29 ± 0.11 0.32 ± 0.13 0.22 ± 0.12 0.16 ± 0.07 0.09 ± 0.04 4.21 ± 0.28 6.00 ± 0.32 3.35 ± 0.25 2.50 ± 0.07 1.66 ± 0.01 SA 0.15 ± 0.02 0.09 ± 0.02 0.04 ± 0.01 0.03 ± 0.002 0.02 ± 0.002 3.79 ± 0.47 2.52 ± 0.16 1.06 ± 0.09 0.61 ± 0.03 0.37 ± 0.01 YU 0.08 ± 0.01 0.12 ± 0.04 0.10 ± 0.002 0.03 ± 0.001 0.02 ± 0.001 0.81 ± 0.07 1.24 ± 0.14 1.01 ± 0.10 0.67 ± 0.02 0.43 ± 0.002
Appendix 3.17: As and Se levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples
(µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
As Se
Code F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
BA 0.12 ± 0.02 0.09 ± 0.03 0.17 ± 0.01 0.14 ± 0.01 0.10 ± 0.01 0.12 ± 0.02 0.04 ± 0.002 0.18 ± 0.002 0.15 ± 0.002 0.07 ± 0.001 BC- Ca 0.20 ± 0.06 0.02 ± 0.07 0.15 ± 0.01 0.14 ± 0.01 0.12 ± 0.002 0.20 ± 0.06 0.03 ± 0.01 0.11 ± 0.002 0.11 ± 0.01 0.07 ± 0.002 BC-Mg 0.11 ± 0.02 0.33 ± 0.11 0.22 ± 0.05 0.15 ± 0.01 0.14 ± 0.01 0.11 ± 0.02 0.33 ± 0.11 0.04 ± 0.001 0.10 ± 0.002 0.08 ± 0.003 BC- Na 0.08 ± 0.03 0.24 ± 0.03 0.09 ± 0.002 0.08 ± 0.01 0.06 ± 0.01 0.08 ± 0.03 0.24 ± 0.03 0.10 ± 0.001 0.09 ± 0.01 0.07 ± 0.003 BC-Pr 0.10 ± 0.03 0.17 ± 0.03 0.11 ± 0.01 0.12 ± 0.01 0.10 ± 0.01 0.10 ± 0.03 0.17 ± 0.03 0.10 ± 0.01 0.08 ± 0.002 0.06 ± 0.002 BC-Te 0.06 ± 0.01 0.16 ± 0.03 0.11 ± 0.02 0.10 ± 0.002 0.09 ± 0.001 0.06 ± 0.01 0.16 ± 0.03 0.11 ± 0.01 0.10 ± 0.002 0.07 ± 0.003 BC-Ex 0.15 ± 0.02 0.06 ± 0.03 0.11 ± 0.01 0.09 ± 0.02 0.07 ± 0.02 0.15 ± 0.02 0.04 ± 0.002 0.20 ± 0.01 0.15 ± 0.002 0.09 ± 0.002 CAN 0.13 ± 0.08 0.15 ± 0.01 0.07 ± 0.04 0.08 ± 0.02 0.06 ± 0.01 0.13 ± 0.08 0.15 ± 0.01 0.03 ± 0.002 0.14 ± 0.01 0.13 ± 0.001 SAR 0.22 ± 0.14 0.06 ± 0.03 0.13 ± 0.02 0.08 ± 0.02 0.06 ± 0.01 0.22 ± 0.14 0.04 ± 0.001 0.15 ± 0.01 0.11 ± 0.01 0.06 ± 0.002 AM 0.12 ± 0.01 0.10 ± 0.002 0.17 ± 0.01 0.13 ± 0.02 0.10 ± 0.02 0.12 ± 0.01 0.04 ± 0.001 0.20 ± 0.01 0.14 ± 0.003 0.05 ± 0.01
BC - Tr 0.28 ± 0.06 0.16 ± 0.03 0.24 ± 0.02 0.18 ± 0.02 0.15 ± 0.03 0.28 ± 0.06 0.04 ± 0.002 0.15 ± 0.02 0.10 ± 0.01 0.04 ± 0.001 FV 0.14 ± 0.06 0.29 ± 0.06 0.16 ± 0.01 0.11 ± 0.002 0.09 ± 0.01 0.14 ± 0.06 0.29 ± 0.06 0.04 ± 0.003 0.12 ± 0.002 0.09 ± 0.01
39
As Se
Code F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
FO 0.14 ± 0.01 0.07 ± 0.02 0.15 ± 0.01 0.13 ± 0.01 0.11 ± 0.01 0.14 ± 0.01 0.04 ± 0.003 0.10 ± 0.002 0.09 ± 0.001 0.06 ± 0.002 JE 0.07 ± 0.04 0.05 ± 0.001 0.08 ± 0.001 0.06 ± 0.01 0.05 ± 0.01 0.07 ± 0.04 0.03 ± 0.003 0.27 ± 0.03 0.21 ± 0.02 0.13 ± 0.002 KA 0.11 ± 0.003 0.04 ± 0.02 0.16 ± 0.02 0.13 ± 0.02 0.11 ± 0.01 0.11 ± 0.002 0.03 ± 0.002 0.24 ± 0.02 0.20 ± 0.01 0.13 ± 0.003 KR 0.03 ± 0.03 0.11 ± 0.01 0.08 ± 0.01 0.11 ± 0.01 0.09 ± 0.01 0.03 ± 0.03 0.11 ± 0.01 0.03 ± 0.001 0.15 ± 0.001 0.12 ± 0.01 PI 0.05 ± 0.003 0.15 ± 0.05 0.11 ± 0.002 0.16 ± 0.01 0.14 ± 0.01 0.05 ± 0.001 0.15 ± 0.05 0.03 ± 0.002 0.22 ± 0.02 0.18 ± 0.002 PV 0.28 ± 0.002 0.20 ± 0.05 0.22 ± 0.01 0.16 ± 0.01 0.13 ± 0.01 0.28 ± 0.001 0.03 ± 0.002 0.11 ± 0.002 0.08 ± 0.002 0.03 ± 0.004 RO 0.17 ± 0.03 0.19 ± 0.002 0.14 ± 0.01 0.17 ± 0.01 0.16 ± 0.01 0.17 ± 0.03 0.19 ± 0.002 0.04 ± 0.002 0.12 ± 0.003 0.11 ± 0.003 RM 0.28 ± 0.03 0.41 ± 0.12 0.30 ± 0.04 0.24 ± 0.02 0.18 ± 0.01 0.28 ± 0.03 0.04 ± 0.003 0.24 ± 0.02 0.17 ± 0.01 0.08 ± 0.01 SA 0.04 ± 0.002 0.02 ± 0.02 0.06 ± 0.01 0.05 ± 0.01 0.05 ± 0.01 0.04 ± 0.002 0.04 ± 0.003 0.13 ± 0.01 0.10 ± 0.001 0.08 ± 0.002 YU 0.08 ± 0.02 0.23 ± 0.01 0.13 ± 0.01 0.21 ± 0.01 0.16 ± 0.01 0.08 ± 0.02 0.23 ± 0.01 0.03 ± 0.002 0.16 ± 0.001 0.12 ± 0.01
40
Appendix 3.18: Mo and Cd levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples
(µg/200 mL) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Mo Cd
Code F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
BA 0.16 ± 0.07 0.25 ± 0.002 0.37 ± 0.01 0.24 ± 0.01 0.16 ± 0.02 1.19 ± 0.13 1.18 ± 0.01 0.62 ± 0.01 0.38 ± 0.01 0.23 ± 0.02 BC- Ca 0.30 ± 0.01 0.46 ± 0.14 0.36 ± 0.03 0.22 ± 0.02 0.14 ± 0.02 1.22 ± 0.07 0.36 ± 0.27 0.24 ± 0.01 0.16 ± 0.01 0.11 ± 0.01 BC-Mg 0.16 ± 0.03 0.13 ± 0.05 0.22 ± 0.01 0.13 ± 0.002 0.08 ± 0.002 1.17 ± 0.04 1.09 ± 0.10 0.57 ± 0.05 0.26 ± 0.02 0.18 ± 0.01 BC- Na 0.12 ± 0.002 0.32 ± 0.03 0.21 ± 0.01 0.13 ± 0.01 0.08 ± 0.01 0.77 ± 0.06 0.62 ± 0.03 0.23 ± 0.03 0.15 ± 0.03 0.09 ± 0.02 BC-Pr 0.16 ± 0.06 0.20 ± 0.03 0.19 ± 0.004 0.11 ± 0.01 0.07 ± 0.002 0.60 ± 0.04 0.39 ± 0.02 0.11 ± 0.002 0.07 ± 0.01 0.05 ± 0.001 BC-Te 0.02 ± 0.02 0.04 ± 0.05 0.16 ± 0.003 0.13 ± 0.004 0.10 ± 0.003 0.55 ± 0.05 0.59 ± 0.03 0.33 ± 0.001 0.28 ± 0.003 0.21 ± 0.003 BC-Ex 0.23 ± 0.03 0.24 ± 0.04 0.37 ± 0.04 0.22 ± 0.01 0.15 ± 0.01 1.75 ± 0.05 1.40 ± 0.01 0.61 ± 0.03 0.38 ± 0.01 0.25 ± 0.002 CAN 0.05 ± 0.07 0.14 ± 0.06 0.29 ± 0.08 0.20 ± 0.01 0.13 ± 0.002 0.94 ± 0.10 1.05 ± 0.03 0.74 ± 0.07 0.33 ± 0.04 0.21 ± 0.01 SAR 0.17 ± 0.06 0.14 ± 0.04 0.25 ± 0.01 0.15 ± 0.01 0.11 ± 0.001 1.28 ± 0.06 1.10 ± 0.07 0.55 ± 0.02 0.34 ± 0.02 0.21 ± 0.003 AM 0.08 ± 0.02 0.11 ± 0.09 0.14 ± 0.01 0.09 ± 0.003 0.08 ± 0.002 1.34 ± 0.12 1.33 ± 0.32 0.70 ± 0.002 0.47 ± 0.03 0.38 ± 0.002
BC - Tr 0.41 ± 0.07 0.24 ± 0.07 0.34 ± 0.07 0.19 ± 0.02 0.13 ± 0.01 1.26 ± 0.03 0.68 ± 0.02 0.28 ± 0.05 0.17 ± 0.02 0.12 ± 0.01 FV 0.12 ± 0.08 0.21 ± 0.08 0.17 ± 0.04 0.23 ± 0.002 0.16 ± 0.01 1.72 ± 0.11 1.35 ± 0.11 0.80 ± 0.003 0.41 ± 0.01 0.27 ± 0.02 FO 0.08 ± 0.06 0.04 ± 0.01 0.09 ± 0.01 0.05 ± 0.002 0.03 ± 0.002 0.92 ± 0.05 0.59 ± 0.05 0.20 ± 0.01 0.13 ± 0.001 0.07 ± 0.002 JE 0.05 ± 0.05 0.03 ± 0.01 0.10 ± 0.01 0.07 ± 0.001 0.04 ± 0.003 0.54 ± 0.10 0.59 ± 0.06 0.35 ± 0.02 0.27 ± 0.003 0.18 ± 0.003 KA 0.02 ± 0.03 0.01 ± 0.01 0.14 ± 0.01 0.10 ± 0.01 0.07 ± 0.001 0.47 ± 0.04 0.55 ± 0.04 0.40 ± 0.02 0.28 ± 0.02 0.20 ± 0.002 KR 0.003 ± 0.001 0.05 ± 0.04 0.05 ± 0.03 0.07 ± 0.001 0.05 ± 0.01 0.50 ± 0.06 0.49 ± 0.002 0.34 ± 0.02 0.18 ± 0.002 0.13 ± 0.01 PI 0.02 ± 0.01 0.03 ± 0.01 0.02 ± 0.002 0.09 ± 0.002 0.06 ± 0.001 0.55 ± 0.13 0.65 ± 0.10 0.47 ± 0.02 0.27 ± 0.001 0.19 ± 0.001 PV 0.39 ± 0.01 0.10 ± 0.04 0.18 ± 0.001 0.09 ± 0.001 0.07 ± 0.002 0.93 ± 0.02 0.52 ± 0.02 0.19 ± 0.006 0.11 ± 0.003 0.08 ± 0.005 RO 0.01 ± 0.005 0.04 ± 0.02 0.02 ± 0.01 0.08 ± 0.002 0.06 ± 0.002 0.51 ± 0.01 0.49 ± 0.06 0.37 ± 0.01 0.21 ± 0.01 0.16 ± 0.02
41
Mo Cd
Code F1 F2 F3 F4 F5 F1 F2 F3 F4 F5
RM 0.05 ± 0.03 0.01 ± 0.03 0.09 ± 0.01 0.06 ± 0.002 0.06 ± 0.001 0.93 ± 0.01 0.78 ± 0.02 0.45 ± 0.01 0.31 ± 0.004 0.22 ± 0.00 SA 0.05 ± 0.02 0.06 ± 0.05 0.09 ± 0.01 0.06 ± 0.01 0.05 ± 0.002 1.76 ± 0.25 1.05 ± 0.002 0.43 ± 0.03 0.25 ± 0.005 0.14 ± 0.003 YU 0.02 ± 0.01 0.12 ± 0.07 0.20 ± 0.06 0.21 ± 0.01 0.12 ± 0.001 0.72 ± 0.07 1.07 ± 0.11 0.74 ± 0.01 0.44 ± 0.01 0.27 ± 0.002
Appendix 3.19: Pb levels (mean ± standard deviation) of commercial yerba mate bombilla infusion samples (µg/200 mL)
determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Pb Code F1 F2 F3 F4 F5
BA 0.54 ± 0.04 0.42 ± 0.04 0.19 ± 0.01 0.12 ± 0.002 0.09 ± 0.01 BC- Ca 0.66 ± 0.01 0.22 ± 0.11 0.13 ± 0.01 0.09 ± 0.01 0.07 ± 0.01 BC-Mg 0.43 ± 0.03 0.36 ± 0.05 0.26 ± 0.02 0.06 ± 0.002 0.05 ± 0.003 BC- Na 0.43 ± 0.002 0.36 ± 0.002 0.08 ± 0.01 0.06 ± 0.01 0.04 ± 0.01 BC-Pr 0.51 ± 0.05 0.39 ± 0.05 0.05 ± 0.01 0.05 ± 0.01 0.04 ± 0.01 BC-Te 0.23 ± 0.01 0.22 ± 0.01 0.14 ± 0.01 0.12 ± 0.002 0.09 ± 0.002 BC-Ex 0.77 ± 0.07 0.45 ± 0.03 0.17 ± 0.01 0.10 ± 0.01 0.08 ± 0.001 CAN 1.01 ± 0.35 0.70 ± 0.06 0.88 ± 0.43 0.13 ± 0.04 0.09 ± 0.002 SAR 0.72 ± 0.01 0.54 ± 0.01 0.23 ± 0.01 0.15 ± 0.01 0.11 ± 0.001 AM 0.70 ± 0.46 0.27 ± 0.08 0.10 ± 0.03 0.07 ± 0.002 0.06 ± 0.001
BC - Tr 0.32 ± 0.01 0.16 ± 0.01 0.05 ± 0.01 0.03 ± 0.003 0.03 ± 0.002 FV 0.59 ± 0.01 0.38 ± 0.04 0.18 ± 0.01 0.08 ± 0.001 0.06 ± 0.01
42
Pb Code F1 F2 F3 F4 F5 FO 0.52 ± 0.03 0.23 ± 0.04 0.07 ± 0.001 0.04 ± 0.002 0.03 ± 0.003 JE 0.05 ± 0.05 0.03 ± 0.01 0.10 ± 0.01 0.07 ± 0.00 0.04 ± 0.003 KA 0.02 ± 0.03 0.01 ± 0.01 0.14 ± 0.01 0.10 ± 0.01 0.07 ± 0.002 KR 0.003 ± 0.001 0.05 ± 0.04 0.05 ± 0.03 0.07 ± 0.002 0.05 ± 0.01 PI 0.02 ± 0.01 0.03 ± 0.01 0.02 ± 0.001 0.09 ± 0.002 0.06 ± 0.001 PV 0.39 ± 0.01 0.10 ± 0.04 0.18 ± 0.002 0.09 ± 0.001 0.07 ± 0.001 RO 0.01 ± 0.005 0.04 ± 0.02 0.02 ± 0.01 0.08 ± 0.001 0.06 ± 0.002 RM 0.05 ± 0.03 0.01 ± 0.03 0.09 ± 0.01 0.06 ± 0.002 0.06 ± 0.001 SA 0.05 ± 0.02 0.06 ± 0.05 0.09 ± 0.01 0.06 ± 0.01 0.05 ± 0.003 YU 0.02 ± 0.01 0.12 ± 0.07 0.20 ± 0.06 0.21 ± 0.01 0.12 ± 0.001
43
Appendix 3.20: Total polyphenol content of commercial green loose yerba mate regular and bombilla infusions (mg
GAE/200 mL) determined using Folin-Ciocalteu assay (refer to section 2.4).
Code Regular infusions Bombilla F1 F2 F3 F4 F5
BA 151.74 1370.27 1062.52 731.10 499.10 338.92 BC- Ca 163.61 2088.29 1090.70 584.18 343.50 230.34 BC-Mg 120.53 1204.56 904.17 548.82 373.90 239.49 BC- Na 156.69 1674.07 968.62 583.80 419.93 291.05 BC-Pr 135.36 2236.81 1020.65 487.19 314.76 228.54 BC-Te 79.73 475.43 421.77 320.51 265.27 232.13 BC-Ex 161.78 1679.59 1105.13 646.40 396.00 305.78 CAN 148.62 911.26 1162.11 807.14 552.80 386.77 SAR 154.48 1087.11 1088.90 711.71 487.19 329.13 AM 159.04 886.37 875.67 652.85 454.98 321.29
BC - Tr 152.20 2259.58 1215.61 554.34 366.54 318.66 FV 155.76 1818.74 966.76 639.86 427.91 282.43 FO 169.09 2055.28 1044.00 544.67 286.02 255.48 JE 149.46 939.82 894.92 548.26 440.49 312.96 KA 106.54 990.74 969.63 717.06 459.18 340.85 KR 125.26 1011.94 999.66 676.43 473.31 319.65 PI 135.76 868.87 701.16 551.03 445.05 319.65 PV 149.91 2451.35 1083.52 593.16 302.18 172.86 RO 81.89 869.77 790.74 462.04 302.18 226.74
44
Code Regular infusions Bombilla F1 F2 F3 F4 F5
RM 117.04 610.07 565.50 444.29 314.16 282.07 SA 103.80 1108.66 638.07 300.39 194.41 129.75 YU 135.76 974.85 1193.91 824.80 528.07 369.11
45
Appendix 3.21: Total polyphenol content of commercial green/roasted loose or tea bag yerba mate regular infusions (mg
GAE/200mL) determined using Folin-Ciocalteu assay (refer to section 2.4).
Code Total polyphenol Green tea bag
AT 196.48 AO 147.17 CA 174.11 CR 235.87 CM 231.33 DL 256.49 JU 181.01 LA 250.35 LH 261.03 LP 166.57 LT 246.17 LI 216.89
MT 252.78 PL 228.03 SU 228.85 TA 237.10 TN 213.59 VE 134.81 YVI 239.58
46
Code Total polyphenol
Roasted loose MN 44.91
BC-Cm 44.03 MO 46.74
Roasted tea bag DO 56.33 LIT 71.39 QU 59.52 ML 93.64
47
Appendix 3.22: Total polyphenol content of commercial green/roasted loose or tea bag yerba mate Brazilian iced tea
infusions (mg GAE/200mL) determined using Folin-Ciocalteu assay (refer to section 2.4).
Code Total polyphenol
Green loose BC - Tr 112.18
AT 312.46 Green tea bag
DL 297.63 JU 216.50
Roasted loose MN 41.71
BC-Cm 29.20 MO 35.69
Roasted tea bag DO 63.97 LIT 82.98 QU 67.68
48
Appendix 3.23: Chlorogenic acid, theobromine and caffeine content of commercial green/roasted loose or tea bag yerba
mate regular infusions (mg/200 mL) determined by UHPLC (refer to section 2.5).
Code 3-Caffeoylquinic acid Theobromine 4-Caffeoylquinic acid 5-Caffeoylquinic acid Caffeine
Green loose BC- Ca 42.95 3.46 15.00 26.95 18.57 BC-Pr 55.62 4.27 13.44 20.56 34.58
PV 47.36 5.03 15.73 29.09 26.94 Green tea bag
CA 41.44 3.31 12.99 19.43 21.87 Roasted loose
BC-Cm 2.86 1.13 2.03 2.79 5.14 Roasted tea bag
QU 3.98 1.53 3.16 3.62 7.58
49
Appendix 3.24: Chlorogenic acid, theobromine and caffeine content of commercial green/roasted loose or tea bag yerba
mate bombilla infusions (mg/200 mL) determined by UHPLC (refer to section 2.5).
Compound Fraction BC-Ca BC Pr PV CAN
3-Caffeoylquinic acid
F1 506.60 761.37 493.38 305.08
F2 415.41 473.00 395.68 483.18
F3 224.83 235.04 240.00 269.40
F4 82.47 103.77 100.27 198.35
F5 47.12 57.84 49.85 133.68
Theobromine
F1 38.49 52.43 51.29 23.80
F2 30.66 33.81 40.50 33.10
F3 17.31 17.38 25.46 19.39
F4 6.87 8.42 10.92 14.68
F5 4.25 5.11 5.52 10.51
4-Caffeoylquinic acid
F1 166.85 174.31 150.84 91.04
F2 150.77 116.46 135.08 160.69
F3 83.72 62.11 85.84 87.45
F4 31.25 29.25 36.24 64.17
F5 17.90 17.04 19.42 42.76
5-Caffeoylquinic acid F1 284.63 261.07 275.84 136.81
F2 263.42 184.65 246.48 235.69
50
Compound Fraction BC-Ca BC Pr PV CAN
F3 150.05 98.18 159.57 130.75
F4 56.75 46.75 67.97 96.41
F5 34.18 27.16 36.61 64.82
Caffeine
F1 203.95 425.62 261.35 144.13
F2 163.09 267.67 209.76 206.73
F3 92.96 144.85 135.23 124.15
F4 37.30 72.28 61.70 97.79
F5 23.88 45.77 34.18 70.90
51
Appendix 3.25: Comparison of standard deviations using a F-test (Miller et al., 2018) to evaluate the relationship between
the elemental levels of yerba mate leaves (based on age – new and old) for non-commercial samples
collected from traditional plantations cultivated either using NPK fertilisers or non-chemical (organic).
Fertiliser Organic DFn, DFd 3,3 6,6
Fcrit 9.28 4.28
Fcalc Level of significance Fcalc Level of
significance Mg 4.11 ns 4.28 ns Ca 3.56 ns 6.97 * Mn 3.14 ns 8.29 * Fe 2.40 ns 3.56 ns Cu 21.4 * 1.40 ns Zn 19.7 * 1.15 ns
n is the number of samples; Fcalc is the calculated value (refer to section 2.6.2): Fcrit is the critical value obtained for n-1 degrees of freedom, p is the level of probability; ns is that there is no statistically significant difference at p>0.05; * statistically significant at probability p<0.05.
52
Appendix 3.26: Comparison of standard deviations using a F-test (Miller et al., 2018) to evaluate the relationship between
the elemental levels of yerba mate leaves based on the use or non-use (organic) of NPK fertilsers during
traditional cultivation.
New leaves Old leaves DFn, DFd 3,6 6,3
Fcrit 4.76 8.94
Fcalc Level of significance Fcalc Level of
significance Mg 6.63 ns 3.80 ns Ca 1.98 ns 3.87 ns Mn 12.7 * 4.65 ns Fe 4.58 ns 3.09 ns Cu 1.30 ns 20.0 * Zn 19.5 * 1.14 ns
n is the number of samples; Fcalc is the calculated value (refer to section 2.6.2): Fcrit is the critical value obtained for n-1 degrees of freedom, p is the level of probability; ns is that there is no statistically significant difference at p>0.05; * statistically significant at probability p<0.05.
53
Appendix 3.27: Comparison of standard deviations using a F-test (Miller et al., 2018) to evaluate the relationship between
the elemental levels of yerba mate leaves (new and old) grown in traditional organic or native forest
plantations (refer to Table 3.6).
New leaves Old leaves DFn, DFd 2,6 6,2
Fcrit 5.14 19.3
Fcalc Level of significance Fcalc Level of
significance Mg 5.11 ns 2.01 ns Ca 11.4 * 1.21 ns Mn 5.08 ns 1.05 ns Fe 2.28 ns 37.9 *** Cu 1.07 ns 1.14 ns Zn 1.27 ns 22.7 *
n is the number of samples; Fcalc is the calculated value (refer to section 2.6.2): Fcrit is the critical value obtained for n-1 degrees of freedom, p is the level of probability; ns is that there is no statistically significant difference at p>0.05; * statistically significant at probability p<0.05; and *** very highly significant p<0.001.
54
Appendix 3.28: Comparison of standard deviations using a F-test (Miller et al., 2018) to evaluate the relationship between
the origin (Brazil and Argentina); packaging (loose and tea bags) and roasting (green and roasted) of
commercial yerba mate samples (refer to Table 3.9).
Origin (Brazil and Argentina)
Packaging (Argentina)
Roasting (Brazil)
DFn, DFd 14,6 18,6 14,2 Fcrit 2.88 2.66 3.74
Fcalc Level of significance Fcalc Level of
significance Fcalc Level of significance
Mg 1.83 ns 2.13 ns 1.26 ns Ca 1.71 ns 1.49 ns 3.71 ns Mn 1.01 ns 2.60 ns 1.08 ns Fe 1.06 ns 7.52 * 1.04 ns Cu 2.24 ns 2.15 ns 2.23 ns Zn 1.29 ns 1.97 ns 11.1 **
n is the number of samples; Fcalc is the calculated value (refer to section 2.6.2): Fcrit is the critical value obtained for n-1 degrees of freedom, p is the level of probability; ns is that there is no statistically significant difference at p>0.05; * statistically significant at probability p<0.05; ** highly significant at probability p<0.01.
55
Appendix 3.29: Comparison of standard deviations using a F-test (Miller et al., 2018) to evaluate the relationship between
the origin (Brazil and Argentina); packaging (loose and tea bags) and roasting process (green loose and
roasted) of regular infusions of commercial yerba mate (refer to Table 3.11).
Origin (Brazil and Argentina)
Packaging (Argentina)
Roasting (Brazil)
DFn, DFd 14,6 18,6 14,2 Fcrit 2.88 2.66 3.74
Fcalc Level of significance Fcalc Level of
significance Fcalc Level of significance
Mn 1.51 ns 2.28 ns 3.15 ns Fe 1.92 ns 8.05 * 3.97 ns Cu 1.03 ns 2.26 ns 7.62 * Zn 1.39 ns 2.18 ns 1.07 ns
n is the number of samples; Fcalc is the calculated value (refer to section 2.6.2): Fcrit is the critical value obtained for n-1 degrees of freedom, p is the level of probability; ns is that there is no statistically significant difference at p>0.05; * statistically significant at probability p<0.05.
56
Appendix 4.1: Sample list of Brazilian coffee samples from Amparo, São Paulo State.
Code Variety Roasting time (min) Origin OB-FL-t0 Obatã 0 Fazenda Flor OB-FL-t2 Obatã 2 Fazenda Flor OB-FL-t4 Obatã 4 Fazenda Flor OB-FL-t6 Obatã 6 Fazenda Flor OB-FL-t8 Obatã 8 Fazenda Flor OB-FL-t10 Obatã 10 Fazenda Flor OB-FL-DE Obatã 10 (defected bean) Fazenda Flor CA-FL-DE Catuaí 10 (defected bean) Fazenda Flor CA-PA-t0 Catuaí 0 Fazenda Palmares CA-PA-t2 Catuaí 2 Fazenda Palmares CA-PA-t4 Catuaí 4 Fazenda Palmares CA-PA-t6 Catuaí 6 Fazenda Palmares CA-PA-t8 Catuaí 8 Fazenda Palmares CA-PA-t10 Catuaí 10 Fazenda Palmares CA-PA-t10def Catuaí 10 (defected bean) Fazenda Palmares BA-PA-t0 Bourbon Amarelo 0 Fazenda Palmares BA-PA-t2 Bourbon Amarelo 2 Fazenda Palmares BA-PA-t4 Bourbon Amarelo 4 Fazenda Palmares BA-PA-t6 Bourbon Amarelo 6 Fazenda Palmares BA-PA-t8 Bourbon Amarelo 8 Fazenda Palmares BA-PA-t10 Bourbon Amarelo 10 Fazenda Palmares
57
Code Variety Roasting time (min) Origin BA-PA-t10def Bourbon Amarelo 10 (defected bean) Fazenda Palmares
* def is defected beans.
Appendix 4.2: Na, Mg, K, Ca, V and Cr levels (mean ± standard deviation) of Brazilian coffee samples (mg/kg dry weight)
determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Code Na Mg K Ca V Cr
OB-FL-t0 35 ± 5 1827 ± 38 14937 ± 278 1025 ± 65 0.03 ± 0.01 0.59 ± 0.02
OB-FL-t2 94 ± 27 1925 ± 18 14145 ± 61 1262 ± 179 0.05 ± 0.01 0.96 ± 0.17
OB-FL-t4 62 ± 22 1734 ± 20 15232 ± 923 789 ± 66 0.04 ± 0.02 0.54 ± 0.09
OB-FL-t6 92 ± 30 1760 ± 5 14937 ± 38 1011 ± 24 0.04 ± 0.02 0.57 ± 0.12
OB-FL-t8 90 ± 1 1832 ± 13 15886 ± 192 779 ± 47 0.01 ± 0.002 0.46 ± 0.11
OB-FL-t10 96 ± 21 2012 ± 94 16761 ± 884 1017 ± 11 0.02 ± 0.01 0.59 ± 0.02
OB-FL-DE 118 ± 36 1698 ± 15 14193 ± 100 1140 ± 45 0.02 ± 0.01 0.51 ± 0.04
CA-FL-DE 100 ± 7 1791 ± 64 15097 ± 416 1310 ± 88 0.01 ± 0.003 0.60 ± 0.02
CA-PA-t0 85 ± 3 1715 ± 10 16655 ± 137 1262 ± 41 0.01 ± 0.004 0.56 ± 0.25
CA-PA-t2 99 ± 31 1937 ± 24 17141 ± 47 1776 ± 113 0.01 ± 0.003 0.50 ± 0.01
CA-PA-t4 133 ± 48 1972 ± 34 18099 ± 26 1409 ± 121 0.03 ± 0.01 0.41 ± 0.02
CA-PA-t6 106 ± 23 1940 ± 53 18597 ± 260 1512 ± 59 0.04 ± 0.01 0.33 ± 0.02
CA-PA-t8 105 ± 43 1847 ± 18 17874 ± 164 1324 ± 10 0.05 ± 0.01 0.27 ± 0.02
58
Code Na Mg K Ca V Cr
CA-PA-t10 177 ± 117 2009 ± 0 18366 ± 272 1402 ± 9 0.04 ± 0.01 0.19 ± 0.03
CA-PA-t10def 182 ± 69 2257 ± 33 20877 ± 276 1552 ± 64 0.05 ± 0.04 0.35 ± 0.19
BA-PA-t0 213 ± 119 2282 ± 68 21415 ± 93 1628 ± 11 0.05 ± 0.03 0.30 ± 0.01
BA-PA-t2 135 ± 87 1842 ± 23 18562 ± 460 1653 ± 18 0.06 ± 0.03 0.31 ± 0.03
BA-PA-t4 155 ± 80 1915 ± 4 18130 ± 511 1429 ± 164 0.09 ± 0.04 0.35 ± 0.03
BA-PA-t6 243 ± 30 1925 ± 29 16538 ± 426 1522 ± 17 0.05 ± 0.005 0.31 ± 0.05
BA-PA-t8 246 ± 17 1908 ± 79 19791 ± 415 1255 ± 78 0.06 ± 0.01 0.28 ± 0.04
BA-PA-t10 236 ± 11 1888 ± 10 18670 ± 267 1517 ± 18 0.06 ± 0.01 0.39 ± 0.03
BA-PA-t10def 235 ± 204 2142 ± 22 19548 ± 320 1620 ± 56 0.07 ± 0.07 0.54 ± 0.22 * def is defected beans.
59
Appendix 4.3: Mn, Fe, Co, Ni, Cu and Zn levels (mean ± standard deviation) of Brazilian coffee samples (mg/kg dry
weight) determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Code Mn Fe Co Ni Cu Zn
OB-FL-t0 31 ± 2 27.88 ± 0.79 0.46 ± 0.02 1.11 ± 0.13 14.21 ± 0.07 10.65 ± 5.3
OB-FL-t2 39 ± 5 38.08 ± 5.34 1.87 ± 0.54 1.52 ± 0.24 13.82 ± 0.07 7.04 ± 1.97
OB-FL-t4 29 ± 2 25.94 ± 1.17 0.43 ± 0.07 0.92 ± 0.1 13.08 ± 0.25 8 ± 2.59
OB-FL-t6 34 ± 2 24.47 ± 0.79 0.31 ± 0.04 0.89 ± 0.1 13.21 ± 0.16 8.42 ± 1.51
OB-FL-t8 31 ± 1 30.19 ± 0.1 0.33 ± 0.01 1.71 ± 0.32 16.36 ± 0.12 8.56 ± 0.05
OB-FL-t10 31 ± 1 30.41 ± 1.16 0.37 ± 0.03 0.91 ± 0.14 14.43 ± 0.82 6.21 ± 0.24
OB-FL-DE 31 ± 2 34.05 ± 6.43 0.19 ± 0 0.92 ± 0.37 14.14 ± 1.43 5.95 ± 0.25
CA-FL-DE 36 ± 0.8 25.34 ± 0.68 0.22 ± 0.01 0.81 ± 0.06 14.74 ± 1.9 6.61 ± 0.07
CA-PA-t0 44 ± 2 23.5 ± 1.09 0.3 ± 0.01 0.78 ± 0.21 15.16 ± 0.79 6.42 ± 1.17
CA-PA-t2 27 ± 1 23.04 ± 0.15 0.37 ± 0.02 0.97 ± 0.2 12.65 ± 0.06 6.21 ± 0.44
CA-PA-t4 25 ± 1 22.06 ± 0.52 0.39 ± 0.01 1.26 ± 0.21 13.01 ± 0.05 6.88 ± 1.35
CA-PA-t6 27 ± 1 22.02 ± 0.19 0.27 ± 0.03 1.13 ± 0.23 12.17 ± 0.55 7.29 ± 2
CA-PA-t8 25 ± 0.6 22.11 ± 0.6 0.42 ± 0.01 0.96 ± 0.03 11.67 ± 0.04 11.77 ± 2.84
CA-PA-t10 32 ± 0.2 26.81 ± 6.96 0.38 ± 0 1.04 ± 0.05 13.27 ± 0.76 8.83 ± 0.31
CA-PA-t10def 26 ± 0.7 27.71 ± 0.46 0.44 ± 0.01 0.99 ± 0.09 13.61 ± 0.1 7.56 ± 0.41
BA-PA-t0 23 ± 1 29 ± 6.22 0.44 ± 0.01 0.98 ± 0.2 14.74 ± 1.14 7.83 ± 0.19
BA-PA-t2 18 ± 0.4 24.88 ± 4.86 0.28 ± 0.01 0.74 ± 0.19 15.23 ± 2.36 7.88 ± 0.29
60
Code Mn Fe Co Ni Cu Zn
BA-PA-t4 26 ± 0.3 24.69 ± 4.3 0.23 ± 0 0.61 ± 0.25 14.14 ± 3.11 7.99 ± 1.87
BA-PA-t6 23 ± 0.4 26.67 ± 0.57 0.41 ± 0.02 0.76 ± 0.05 17.96 ± 0.86 6.59 ± 0.56
BA-PA-t8 20 ± 1 27.12 ± 1.02 0.24 ± 0.01 0.37 ± 0.05 17.87 ± 0.15 6.09 ± 0.48
BA-PA-t10 24 ± 0.02 25.45 ± 0.46 0.44 ± 0.01 0.54 ± 0.01 17.48 ± 0.48 5.68 ± 0.33
BA-PA-t10def 27 ± 1 29.95 ± 7.15 0.32 ± 0.01 0.96 ± 0.19 16.47 ± 3.78 10.56 ± 2.3 * def is defected beans.
61
Appendix 4.4: As, Se, Mo, Cd and Pb levels (mean ± standard deviation) of Brazilian coffee samples (mg/kg dry weight)
determined using ICP-MS (refer to section 2.3) and n= 2; n is the number of replicates.
Code As Se Mo Cd Pb
OB-FL-t0 <LOD* <LOD* 0.09 ± 0.03 <LOD* 0.05 ± 0.01
OB-FL-t2 0.01 ± 0.002 <LOD* 0.09 ± 0.02 <LOD* 0.04 ± 0.01
OB-FL-t4 <LOD* <LOD* 0.06 ± 0.01 0.01 ± 0.001 0.01 ± 0.01
OB-FL-t6 <LOD* <LOD* 0.04 ± 0.002 <LOD* <LOD*
OB-FL-t8 <LOD* <LOD* 0.04 ± 0.02 <LOD* <LOD*
OB-FL-t10 0.01 ± 0.001 <LOD* 0.29 ± 0.07 <LOD* 0.06 ± 0.01
OB-FL-DE <LOD* 0.01 ± 0.001 0.07 ± 0.002 <LOD* 0.06 ± 0.01
CA-FL-DE 0.01 ± 0.002 <LOD* 0.1 ± 0.002 0.03 ± 0.001 0.01 ± 0.001
CA-PA-t0 <LOD* <LOD* 0.11 ± 0.01 0.04 ± 0.01 0.07 ± 0.02
CA-PA-t2 0.01 ± 0.002 <LOD* 0.16 ± 0.04 0.06 ± 0.001 0.04 ± 0.01
CA-PA-t4 <LOD* <LOD* 0.11 ± 0.02 <LOD* 0.08 ± 0.02
CA-PA-t6 0.01 ± 0.002 <LOD* 0.05 ± 0.001 <LOD* 0.06 ± 0.001
CA-PA-t8 0.01 ± 0.003 <LOD* 0.09 ± 0.02 <LOD* 0.06 ± 0.002
CA-PA-t10 0.01 ± 0.001 <LOD* 0.05 ± 0.01 <LOD* 0.01 ± 0.001
CA-PA-t10def 0.01 ± 0.002 <LOD* 0.07 ± 0.02 0.03 ± 0.001 0.04 ± 0.01
BA-PA-t0 0.01 ± 0.001 0.01 ± 0.001 0.12 ± 0.02 <LOD* 0.05 ± 0.009
BA-PA-t2 0.01 ± 0.002 <LOD* 0.14 ± 0.02 0.01 ± 0.001 0.07 ± 0.005
62
Code As Se Mo Cd Pb
BA-PA-t4 0.02 ± 0.02 <LOD* 0.09 ± 0.02 <LOD* 0.03 ± 0.006
BA-PA-t6 0.02 ± 0.001 <LOD* 0.09 ± 0.01 <LOD* 0.08 ± 0.002
BA-PA-t8 0.02 ± 0.001 <LOD* 0.09 ± 0.01 <LOD* 0.01 ± 0.002
BA-PA-t10 0.02 ± 0.01 <LOD* 0.09 ± 0.02 <LOD* 0.04 ± 0.008
BA-PA-t10def 0.03 ± 0.002 <LOD* 0.15 ± 0.04 0.01 ± 0.001 0.03 ± 0.006 *refer to Table 2.2; def is defected beans; <LOD is below the limit of detection.
63
Appendix 5.1: Total elemental levels (mean ± standard deviation) of açaí pulp samples (mg/kg dry weight) determined
using ICP-MS (refer to section 2.3). Data relates to the type of sample (non-commercial : purple n= 6; and
white n= 4; and commercial: purple n= 4; n is the number of samples).
Non-commercial Commercial
Purple Açaí whole Purple Açaí de-fatted White Açaí whole White Açaí de-fatted Pulp SP Powder SP Powder UK
Na 238.74 ± 12.24 295.14 ± 9.43 493.38 ± 35.04 725.26 ± 38.25 894.91 ± 11.7 449.25 ± 43.56 107.53 ± 4.85
Mg 2332.29 ± 21.62 2723.64 ± 41.5 2469.16 ± 121.25 3017.68 ± 90.03 2022.1 ± 27.53 2036.86 ± 21.41 859.21 ± 11.3
K 12118.41 ± 1548.83 13529.26 ± 1024.94 11941.46 ± 78.7 14737.9 ± 443.56 8219.66 ± 335.61 8321.86 ± 297.01 2382.14 ± 0.86
Ca 4688.24 ± 141.5 5273.38 ± 580.66 4162.31 ± 22.03 5251.08 ± 132.77 1650.41 ± 161.18 2029.33 ± 172.47 661.73 ± 156.47
V 0.02 ± 0.002 0.03 ± 0.01 0.03 ± 0.005 0.04 ± 0.003 0.05 ± 0.02 0.02 ± 0.004 0.01 ± 0.002
Cr 4.84 ± 0.35 5.22 ± 0.09 5.26 ± 0.002 7.15 ± 0.94 4.36 ± 0.15 4.08 ± 0.61 2.55 ± 0.13
Mn 640.63 ± 9.33 809.18 ± 7.67 611.38 ± 5.98 808.86 ± 15.69 267.62 ± 15.07 547.04 ± 17.78 16.23 ± 0.06
Fe 30.09 ± 0.04 41.74 ± 1.18 36.54 ± 7.48 43.04 ± 0.96 30.01 ± 0.44 21.92 ± 0.28 2.29 ± 0.14
64
Non-commercial Commercial
Purple Açaí whole Purple Açaí de-fatted White Açaí whole White Açaí de-fatted Pulp SP Powder SP Powder UK
Co 0.08 ± 0.003 0.10 ± 0.004 0.08 ± 0.002 0.11 ± 0.002 0.10 ± 0.004 0.08 ± 0.005 0.01 ± 0.002
Ni 1.71 ± 0.22 1.89 ± 0.09 1.76 ± 0.02 2.63 ± 0.17 1.96 ± 0.02 1.69 ± 0.05 0.70 ± 0.04
Cu 18.11 ± 0.26 22.22 ± 0.09 17.16 ± 0.65 24.96 ± 0.97 15.23 ± 0.52 14.97 ± 0.90 3.51 ± 0.17
Zn 24.93 ± 0.74 32.40 ± 1.68 26.49 ± 0.26 36.18 ± 1.67 26.98 ± 6.47 23.23 ± 3.10 12.70 ± 1.18
As <LOD* <LOD* <LOD* <LOD* <LOD* <LOD* <LOD*
Se <LOD* <LOD* <LOD* 0.05 ± 0.02 0.06 ± 0.004 <LOD* <LOD*
Mo 0.09 ± 0.00 0.13 ± 0.01 0.07 ± 0.003 0.10 ± 0.005 0.22 ± 0.003 0.04 ± 0.005 0.18 ± 0.007
Cd 0.07 ± 0.003 0.10 ± 0.01 0.09 ± 0.005 0.18 ± 0.01 0.04 ± 0.01 0.06 ± 0.002 0.01 ± 0.005
Pb 0.52 ± 0.04 0.72 ± 0.01 0.31 ± 0.02 0.46 ± 0.02 0.07 ± 0.01 0.03 ± 0.003 0.02 ± 0.003
*refer to Table 2.2; <LOD lower than the limit of detection.
65
Appendix 5.2: Total elemental levels (mean ± standard deviation) of açaí pulp samples (mg/kg fresh weight) determined
using ICP-MS (refer to section 2.3). Data relates to the type of sample (non-commercial : purple n= 6; and
white n= 4; and commercial: purple n= 4; n is the number of samples).
Non-commercial Commercial
Purple Açaí whole Purple Açaí de-fatted White Açaí whole White Açaí de-fatted Pulp SP Powder SP Powder UK
Na 23.87 ± 1.22 29.51 ± 0.94 49.34 ± 3.50 72.53 ± 3.82 89.49 ± 1.17 44.93 ± 4.36 10.75 ± 0.48
Mg 233.23 ± 2.16 272.36 ± 4.15 246.92 ± 12.13 301.77 ± 9.00 202.21 ± 2.75 203.69 ± 2.14 85.92 ± 1.13
K 1211.84 ± 154.88 1352.93 ± 102.49 1194.15 ± 7.87 1473.79 ± 44.36 821.97 ± 33.56 832.19 ± 29.70 238.21 ± 0.09
Ca 468.82 ± 14.15 527.34 ± 58.07 416.23 ± 2.20 525.11 ± 13.28 165.04 ± 16.12 202.93 ± 17.25 66.17 ± 15.65
V 0.002 ± 0.002 0.003 ± 0.001 0.003 ± 0.0001 0.004 ± 0.0002 0.005 ± 0.020 0.002 ± 0.0002 0.001 ± 0.0001
Cr 0.48 ± 0.04 0.52 ± 0.01 0.53 ± 0.002 0.71 ± 0.09 0.44 ± 0.01 0.41 ± 0.06 0.25 ± 0.01
Mn 64.06 ± 0.93 80.92 ± 0.77 61.14 ± 0.60 80.89 ± 1.57 26.76 ± 1.51 54.70 ± 1.78 1.62 ± 0.01
Fe 3.01 ± 0.002 4.17 ± 0.12 3.65 ± 0.75 4.30 ± 0.10 3.00 ± 0.04 2.19 ± 0.03 0.23 ± 0.01
Co 0.01 ± 0.003 0.01 ± 0.002 0.01 ± 0.003 0.01 ± 0.004 0.01 ± 0.002 0.01 ± 0.005 0.001 ± 0.0002
66
Non-commercial Commercial Purple Açaí whole Purple Açaí de-fatted White Açaí whole White Açaí de-fatted Pulp SP Powder SP Powder UK
Ni 0.17 ± 0.02 0.19 ± 0.01 0.18 ± 0.004 0.26 ± 0.02 0.20 ± 0.003 0.17 ± 0.002 0.07 ± 0.006
Cu 1.81 ± 0.03 2.22 ± 0.01 1.72 ± 0.06 2.50 ± 0.10 1.52 ± 0.05 1.50 ± 0.09 0.35 ± 0.02
Zn 2.49 ± 0.07 3.24 ± 0.17 2.65 ± 0.03 3.62 ± 0.17 2.70 ± 0.65 2.32 ± 0.31 1.27 ± 0.12
As <LOD* <LOD* <LOD* <LOD* <LOD* <LOD* <LOD*
Se <LOD* <LOD* <LOD* 0.005 ± 0.002 0.006 ± 0.0003 <LOD* <LOD*
Mo 0.009 ± 0.0002 0.013 ± 0.001 0.007 ± 0.0002 0.010 ± 0.0001 0.022 ± 0.0003 0.004 ± 0.0003 0.018 ± 0.0002
Cd 0.007 ± 0.0003 0.010 ± 0.001 0.009 ± 0.0001 0.018 ± 0.001 0.004 ± 0.001 0.006 ± 0.0002 0.001 ± 0.0003
Pb 0.05 ± 0.001 0.07 ± 0.002 0.03 ± 0.003 0.05 ± 0.004 0.01 ± 0.001 0.003 ± 0.000 0.002 ± 0.0003
*refer to Table 2.2; <LOD lower than the limit of detection.
67
Appendix 5.3: Sample list for the evaluation of the Amazon geographical variability and industrial processing on açaí.
Code Origin Variety Type Moisture content (%) Processing place GE-WB-P Genipauba White bacapa Seed 46.4 Lab GE-WB-S Genipauba White bacapa Fruit 27.9 Lab GE-PB-P Genipauba Purple bacapa Seed 39.4 Lab GE-PB-S Genipauba Purple bacapa Fruit 32.2 Lab IL-PA-P Ilhas Purple Acai Seed 47.5 Lab IL-PA-S Ilhas Purple Acai Fruit 29.6 Lab
MA-PA-P Macapa Purple Acai Seed 54.9 Lab MA-PA-S Macapa Purple Acai Fruit 30.6 Lab AN-PA-P Anajas Purple Acai Seed 50.2 Lab AN-PA-S Anajas Purple Acai Fruit 31.7 Lab IC-PA-S Ilhas Purple Acai Fruit 33.2 Point do Açaí IM-PA-S Igarape-Miri Purple Acai Fruit 35.5 Açaí Santa Helena
PA-IC-PM Ilhas Purple Acai Pulp (medium) 89.0 Point do Açaí SH-AB-PF Abaetetuba Purple Acai Pulp (fluid) 92.0 Açaí Santa Helena SH-IM-PF Igarape-Miri Purple Acai Pulp (fluid) 92.0 Açaí Santa Helena SH-IM-PM Igarape-Miri Purple Acai Pulp (medium) 89.0 Açaí Santa Helena SH-PA-PE Paragominas Purple Acai Pulp (thick) 86.0 Açaí Santa Helena AA-OB-PM Obidos Purple Acai Pulp (medium) 89.0 Açaí Amazona AA-OB-PE Obidos Purple Acai Pulp (thick) 86.0 Açaí Amazona AA-OB-FD Obidos Purple Acai Freeze-dryed 86.0 Açaí Amazona
68
Appendix 5.4: Total elemental and polyphenol levels (mean ± standard deviation) of açaí pulp samples (mg/kg, dry
weight) determined using ICP-MS (refer to section 2.3). Data relates to the type of sample; n= 3; n is the
number of replicates).
Code Total polyphenol Na Mg K Ca V Mn Fe Cu Zn
GE-WB-P 9.45 ± 1.23 63.41 ± 32.88
249.84 ± 47.31
3719.68 ± 796.28
133.66 ± 12.12
0.04 ± 0.002
13.10 ± 1.72 8.97 ± 0.57 8.44 ± 1.38 9.61 ± 1.66
GE-WB-S 18.91 ± 1.02
210.13 ± 42.18
272.86 ± 69.82
4001.92 ± 288.76
188.48 ± 77.70 0.06 ± 0.01 12.96 ±
1.04 10.01 ±
0.28 8.80 ± 1.04 10.44 ± 2.26
GE-PB-P 5.47 ± 0.36 353.68 ± 30.94
498.29 ± 12.26
2509.03 ± 38.51
262.17 ± 4.73 0.06 ± 0.02 9.46 ± 0.28 14.78 ±
0.27 9.72 ± 0.15 9.82 ± 0.87
GE-PB-S 20.2 ± 2.17 530.63 ± 42.21
493.08 ± 14.75
2359.96 ± 113.75
233.36 ± 33.83 0.04 ± 0.02 9.36 ± 0.02 12.58 ±
0.40 6.30 ± 0.07 9.34 ± 0.85
IL-PA-P 6.88 ± 1.11 624.63 ± 44.49
654.22 ± 39.89
3528.97 ± 179.01
1282.02 ± 85.48
0.02 ± 0.003
104.99 ± 5.52
12.04 ± 0.72 9.45 ± 0.64 11.30 ±
1.45
IL-PA-S 57.57 ± 1.19
779.00 ± 27.17
529.64 ± 4.75
3284.90 ± 43.01
554.23 ± 58.71 0.05 ± 0.01 128.03 ±
5.05 10.78 ±
0.38 9.91 ± 0.02 12.93 ± 0.27
MA-PA-P 9.94 ± 0.76 891.39 ± 17.66
904.23 ± 74.00
4298.91 ± 315.64
1883.82 ± 307.40
0.01 ± 0.002
186.33 ± 12.15
12.76 ± 0.39 9.78 ± 0.53 12.20 ±
0.88
MA-PA-S 31.99 ± 0.47
1075.45 ± 21.88
494.98 ± 13.56
2573.05 ± 20.49
692.58 ± 39.44 0.02 ± 0.01 224.98 ±
21.55 10.37 ±
0.01 7.73 ± 0.19 10.96 ± 0.69
AN-PA-P 7.25 ± 0.36 1191.57 ± 37.24
501.26 ± 7.35
2630.92 ± 55.04
632.37 ± 124.02 0.01 ± 0.01 154.01 ±
26.27 8.49 ± 0.43 7.95 ± 0.09 9.20 ± 0.45
AN-PA-S 36.66 ± 10.47
1355.07 ± 77.08
419.17 ± 2.96
2966.18 ± 89.33
318.60 ± 22.55 0.02 ± 0.01 256.51 ±
5.89 8.47 ± 0.08 7.58 ± 0.19 10.79 ± 1.33
69
Code Total polyphenol Na Mg K Ca V Mn Fe Cu Zn
IC-PA-S 58.83 ± 2.18
1456.94 ± 72.83
445.08 ± 12.38
3016.99 ± 9.24
567.01 ± 204.00 0.01 ± 0.01 158.72 ±
14.45 9.74 ± 0.78 6.89 ± 0.42 7.95 ± 0.79
IM-PA-S 28.80 ± 4.39
1588.14 ± 18.34
483.49 ± 10.57
3209.86 ± 48.38
612.29 ± 42.31 0.04 ± 0.02 70.11 ±
2.37 71.19 ±
0.87 10.83 ±
0.23 11.61 ±
0.26
PA-IC-PM 26.60 ± 3.53
1611.51 ± 71.58
1693.77 ± 33.30
10870.64 ± 23.55
2513.16 ± 195.77 0.05 ± 0.01 751.58 ±
11.07 35.04 ±
0.52 13.77 ±
0.51 23.05 ±
0.10
SH-AB-PF 20.40 ± 3.92
1878.26 ± 24.56
1851.27 ± 42.43
10714.62 ± 252.60
2234.51 ± 43.97 0.06 ± 0.02 393.27 ±
13.01 28.56 ±
0.20 17.05 ±
0.10 24.27 ±
0.80
SH-IM-PF 17.15 ± 0.81
2031.44 ± 43.47
1795.99 ± 39.52
11688.08 ± 1541.24
3023.21 ± 131.22 0.05 ± 0.02 479.92 ±
303.87 30.56 ±
0.54 14.88 ±
0.24 30.78 ±
2.47
SH-IM-PM 15.63 ± 0.93
2128.45 ± 50.69
1929.36 ± 7.67
12275.75 ± 189.99
3661.91 ± 192.34 0.06 ± 0.02 845.72 ±
4.58 30.24 ±
0.90 15.71 ±
0.73 28.31 ±
0.22
SH-PA-PE 25.43 ± 1.83
2376.19 ± 14.22
1896.2 ± 34.91
10186.08 ± 208.59
2875.25 ± 44.28
0.04 ± 0.002
181.49 ± 0.62
33.78 ± 2.23
18.49 ± 0.47
24.78 ± 0.31
AA-OB-PM
18.97 ± 2.03
2471.43 ± 44.63
1558.81 ± 45.29
13554.15 ± 123.86
3994.21 ± 48.99 0.09 ± 0.02 47.84 ±
2.36 39.59 ±
4.62 14.41 ±
0.66 17.54 ±
0.71
AA-OB-PE 19.78 ± 0.39
2624.86 ± 57.95
1543.10 ± 5.00
14804.64 ± 16.44
4019.14 ± 243.10
0.04 ± 0.003
107.22 ± 0.23
34.98 ± 0.22
11.96 ± 0.08
15.38 ± 0.14
AA-OB-FD 22.19 ± 2.08
2673.07 ± 52.16
1758.29 ± 48.36
13756.60 ± 370.32
3753.20 ± 201.92
0.07 ± 0.002
333.82 ± 7.95
40.82 ± 0.62
17.42 ± 0.43
23.17 ± 1.06
70
Appendix 5.5: Total elemental (mean ± standard deviation) of açaí pulp samples (mg/kg dry weight) determined using
ICP-MS (refer to section 2.3). Data relates to the type of sample; n= 3; n is the number of replicates).
Code Cr Co Ni As Se Mo Cd Pb GE-WB-P 1.07 ± 0.47 <LOD* 0.95 ± 0.39 <0.01 0.03 ± 0.01 <LOD* 0.03 ± 0.01 0.07 ± 0.01 GE-WB-S 1.49 ± 0.47 <LOD* 0.89 ± 0.26 <0.01 0.03 ± 0.003 <LOD* 0.20 ± 0.06 0.07 ± 0.01 GE-PB-P 0.73 ± 0.14 <LOD* 0.66 ± 0.09 <0.01 0.02 ± 0.01 <LOD* 0.06 ± 0.04 <LOD* GE-PB-S 1.32 ± 0.13 <LOD* 0.62 ± 0.04 <LOD* 0.01 ± 0.01 <LOD* 0.09 ± 0.05 <LOD* IL-PA-P 0.84 ± 0.10 <LOD* 1.07 ± 0.24 <LOD* 0.02 ± 0.002 <LOD* 0.17 ± 0.002 0.13 ± 0.01 IL-PA-S 1.61 ± 0.05 <LOD* 1.36 ± 0.31 <LOD* 0.01 ± 0.01 0.23 ± 0.08 0.12 ± 0.01 0.11 ± 0.02 MA-PA-P 1.07 ± 0.01 <LOD* 1.38 ± 0.14 0.01 ± 0.003 0.02 ± 0.01 <LOD* 0.05 ± 0.01 <LOD* MA-PA-S 2.05 ± 0.32 <LOD* 1.62 ± 0.29 <LOD* 0.01 ± 0.003 <LOD* 0.03 ± 0.01 <LOD* AN-PA-P 0.97 ± 0.23 <LOD* 1.32 ± 0.11 <LOD* 0.02 ± 0.002 <LOD* 0.03 ± 0.004 <LOD* AN-PA-S 1.31 ± 0.07 <LOD* 1.79 ± 0.26 <LOD* 0.02 ± 0.002 <LOD* 0.02 ± 0.002 0.04 ± 0.01 IC-PA-S 1.36 ± 0.01 <LOD* 1.61 ± 0.33 0.01 ± 0.003 0.01 ± 0.002 <LOD* 0.04 ± 0.01 0.21 ± 0.05 IM-PA-S 1.65 ± 0.03 <LOD* 1.42 ± 0.04 <LOD* 0.07 ± 0.01 <LOD* 0.01 ± 0.003 <LOD* PA-IC-PM 3.91 ± 0.11 0.19 ± 0.003 2.83 ± 0.04 0.02 ± 0.002 0.02 ± 0.02 <LOD* 0.12 ± 0.04 <LOD* SH-AB-PF 2.12 ± 0.01 0.12 ± 0.002 1.83 ± 0.12 0.01 ± 0.003 0.02 ± 0.01 <LOD* 0.05 ± 0.02 <LOD* SH-IM-PF 2.43 ± 0.10 0.10 ± 0.003 2.15 ± 0.34 0.01 ± 0.001 0.02 ± 0.005 <LOD* 0.10 ± 0.01 <LOD* SH-IM-PM 2.17 ± 0.01 0.10 ± 0.001 1.92 ± 0.14 0.01 ± 0.005 0.02 ± 0.003 <LOD* 0.14 ± 0.07 <LOD* SH-PA-PE 2.18 ± 0.03 <LOD* 1.44 ± 0.12 <LOD* 0.07 ± 0.01 0.10 ± 0.00 0.01 ± 0.002 <LOD* AA-OB-PM 6.17 ± 0.14 <LOD* 1.80 ± 0.14 0.01 ± 0.004 0.02 ± 0.01 0.38 ± 0.05 0.03 ± 0.001 0.07 ± 0.01 AA-OB-PE 6.29 ± 0.002 <LOD* 1.89 ± 0.10 0.01 ± 0.003 0.02 ± 0.001 0.40 ± 0.01 0.03 ± 0.002 <LOD* AA-OB-FD 4.80 ± 0.13 <LOD* 2.58 ± 0.15 0.01 ± 0.005 0.02 ± 0.002 0.28 ± 0.03 0.07 ± 0.003 <LOD*
*refer to Table 2.2; <LOD lower than the limit of detection.
71
Appendix 5.6: Total elemental and polyphenol levels (mean ± standard deviation) of açaí pulp samples (mg/kg fresh
weight) determined using ICP-MS (refer to section 2.3). Data relates to the type of sample; n= 3; n is the
number of replicates).
Code Total polyphenol Na Mg K Ca V Mn Fe Cu Zn
GE-WB-P 5.07 ± 0.66 34 00± 17.63
133.94 ± 25.36
1994.19 ± 426.90
71.66 ± 6.50
0.02 ± 0.002 7.02 ± 0.92 4.81 ± 0.30 4.52 ± 0.74 5.15 ± 0.89
GE-WB-S 13.64 ± 0.74
151.59 ± 30.43
196.84 ± 50.37
2886.96 ± 208.31
135.97 ± 56.05 0.04 ± 0.01 9.35 ± 0.75 7.22 ± 0.20 6.35 ± 0.75 7.53 ± 1.63
GE-PB-P 3.32 ± 0.22 214.40 ± 18.76
302.06 ± 7.43
1520.99 ± 23.34
158.93 ± 2.87 0.03 ± 0.01 5.74 ± 0.17 8.96 ± 0.16 5.89 ± 0.09 5.95 ± 0.53
GE-PB-S 13.69 ± 1.47
359.51 ± 28.60
334.07 ± 9.99
1598.90 ± 77.06
158.10 ± 22.92 0.03 ± 0.01 6.34 ± 0.01 8.53 ± 0.27 4.27 ± 0.04 6.33 ± 0.58
IL-PA-P 3.62 ± 0.58 328.09 ± 23.37
343.63 ± 20.95
1853.59 ± 94.03
673.38 ± 44.90
0.01 ± 0.002
55.15 ± 2.90 6.32 ± 0.38 4.96 ± 0.34 5.94 ± 0.76
IL-PA-S 40.51 ± 0.84
548.12 ± 19.12
372.67 ± 3.34
2311.35 ± 30.26
389.98 ± 41.31 0.03 ± 0.01 90.09 ±
3.55 7.58 ± 0.27 6.97 ± 0.01 9.10 ± 0.19
MA-PA-P 4.48 ± 0.34 401.60± 7.96
407.38 ± 33.34
1936.80 ± 142.2
848.72 ± 138.49
0.01 ± 0.003
83.95 ± 5.47 5.75 ± 0.17 4.40 ± 0.24 5.50 ± 0.4
MA-PA-S 22.21 ± 0.32
746.51 ± 15.19
343.59 ± 9.41
1786.06 ± 14.23
480.75 ± 27.37 0.02 ± 0.01 156.17 ±
14.96 7.20 ± 0.00 5.36 ± 0.13 7.60 ± 0.48
AN-PA-P 3.61 ± 0.18 593.67 ± 18.56
249.74 ± 3.66
1310.80 ± 27.42
315.07 ± 61.79
0.01 ± 0.004
76.73 ± 13.09 4.23 ± 0.21 3.96 ± 0.05 4.59 ± 0.22
AN-PA-S 25.05 ± 7.15
925.85 ± 52.66
286.40 ± 2.02
2026.63 ± 61.03
217.68 ± 15.41 0.01 ± 0.01 175.26 ±
4.02 5.78 ± 0.06 5.18 ± 0.13 7.37 ± 0.91
72
Code Total polyphenol Na Mg K Ca V Mn Fe Cu Zn
IC-PA-S 39.28 ± 1.45
972.75 ± 48.62
297.16 ± 8.26
2014.33 ± 6.17
378.57 ± 136.20
0.01 ± 0.002
105.97 ± 9.64 6.50 ± 0.52 4.60 ± 0.28 5.31 ± 0.53
IM-PA-S 18.59 ± 2.83
1024.85 ± 11.83
312.00 ± 6.82
2071.39 ± 31.22
395.12 ± 27.30 0.03 ± 0.01 45.24 ±
1.53 45.94 ±
0.56 6.99 ± 0.15 7.49 ± 0.16
PA-IC-PM 2.93 ± 0.39 177.27 ± 7.87
186.31 ± 3.66
1195.77 ± 2.59
276.45 ± 21.54
0.01 ± 0.002
82.67 ± 1.22 3.85 ± 0.06 1.51 ± 0.06 2.54 ± 0.01
SH-AB-PF 1.63 ± 0.31 150.26 ± 1.96
148.10 ± 3.39
857.17 ± 20.21
178.76 ± 3.52
0.005 ± 0.001
31.46 ± 1.04 2.29 ± 0.02 1.36 ± 0.01 1.94 ± 0.06
SH-IM-PF 1.37 ± 0.06 162.52 ± 3.48
143.68 ± 3.16
935.05 ± 123.30
241.86 ± 10.50
0.004 ± 0.001
38.39 ± 24.31 2.44 ± 0.04 1.19 ± 0.02 2.46 ± 0.20
SH-IM-PM 1.72 ± 0.10 234.13 ± 5.58
212.23 ± 0.84
1350.33 ± 20.90
402.81 ± 21.16
0.01 ± 0.003
93.03 ± 0.50 3.33 ± 0.10 1.73 ± 0.08 3.11 ± 0.02
SH-PA-PE 3.56 ± 0.26 332.67 ± 1.99
265.47 ± 4.89
1426.05 ± 29.20
402.54 ± 6.20
0.005 ± 0.0001
25.41 ± 0.09 4.73 ± 0.31 2.59 ± 0.07 3.47 ± 0.04
AA-OB-PM 2.09 ± 0.22 271.86 ±
4.91 171.47 ±
4.98 1490.96 ±
13.62 439.36 ±
5.39 0.01 ± 0.004 5.26 ± 0.26 4.35 ± 0.51 1.59 ± 0.07 1.93 ± 0.08
AA-OB-PE 2.77 ± 0.05 367.48 ± 8.11
216.03 ± 0.70
2072.65 ± 2.30
562.68 ± 34.03
0.01 ± 0.003
15.01 ± 0.03 4.90 ± 0.03 1.67 ± 0.01 2.15 ± 0.02
AA-OB-FD 3.11 ± 0.29 374.23 ± 7.30
246.16 ± 6.77
1925.92 ± 51.85
525.45 ± 28.27
0.01 ± 0.003
46.74 ± 1.11 5.71 ± 0.09 2.44 ± 0.06 3.24 ± 0.15
73
Appendix 5.7: Total elemental and polyphenol levels (mean ± standard deviation) of açaí pulp samples (mg/kg fresh
weight) determined using ICP-MS (refer to section 2.3). Data relates to the type of sample; n= 3; n is
replicates).
Code Cr Co Ni As Se Mo Cd Pb
GE-WB-P 0.57 ± 0.25 <LOD* 0.51 ± 0.21 <LOD* 0.02 ± 0.002 <LOD* 0.01 ± 0.002 0.04 ± 0.001 GE-WB-S 1.07 ± 0.34 <LOD* 0.64 ± 0.19 <LOD* 0.02 ± 0.002 <LOD* 0.14 ± 0.05 0.05 ± 0.01 GE-PB-P 0.45 ± 0.09 <LOD* 0.40 ± 0.06 <LOD* 0.01 ± 0.01 <LOD* 0.03 ± 0.02 <LOD* GE-PB-S 0.89 ± 0.09 <LOD* 0.42 ± 0.03 <LOD* 0.01 ± 0.001 <LOD* 0.06 ± 0.03 <LOD* IL-PA-P 0.44 ± 0.05 <LOD* 0.56 ± 0.12 <LOD* 0.01 ± 0.001 <LOD* 0.09 ± 0.003 0.07 ± 0.01 IL-PA-S 1.13 ± 0.03 <LOD* 0.96 ± 0.21 <LOD* 0.01 ± 0.002 0.16 ± 0.06 0.09 ± 0.01 0.08 ± 0.01
MA-PA-P 0.48 ± 0.01 <LOD* 0.62 ± 0.06 0.003 ± 0.0002 0.01 ± 0.001 <LOD* 0.02 ± 0.005 <LOD* MA-PA-S 1.42 ± 0.22 <LOD* 1.13 ± 0.20 <LOD* 0.01 ± 0.002 <LOD* 0.02 ± 0.003 <LOD* AN-PA-P 0.48 ± 0.12 <LOD* 0.66 ± 0.05 <LOD* 0.01 ± 0.003 <LOD* 0.01 ± 0.006 <LOD* AN-PA-S 0.89 ± 0.05 <LOD* 1.23 ± 0.17 <LOD* 0.02 ± 0.001 <LOD* 0.01 ± 0.01 0.03 ± 0.01 IC-PA-S 0.91 ± 0.01 <LOD* 1.07 ± 0.22 0.004 ± 0.0004 0.01 ± 0.001 <LOD* 0.02 ± 0.01 0.14 ± 0.03 IM-PA-S 1.06 ± 0.02 <LOD* 0.92 ± 0.02 <LOD* 0.05 ± 0.01 <LOD* 0.01 ± 0.006 <LOD*
PA-IC-PM 0.43 ± 0.01 0.02 ± 0.004 0.31 ± 0.00 0.002 ± 0.0003 0.003 ± 0.002 <LOD* 0.01 ± 0.003 <LOD* SH-AB-PF 0.17 ± 0.003 0.01 ± 0.002 0.15 ± 0.01 0.001 ± 0.0002 0.002 ± 0.0002 <LOD* 0.004 ± 0.002 <LOD* SH-IM-PF 0.19 ± 0.01 0.01 ± 0.004 0.17 ± 0.03 0.001 ± 0.0005 0.001 ± 0.0001 <LOD* 0.01 ± 0.005 <LOD* SH-IM-PM 0.24 ± 0.004 0.01 ± 0.002 0.21 ± 0.02 0.002 ± 0.0002 0.002 ± 0.001 <LOD* 0.02 ± 0.01 <LOD* SH-PA-PE 0.31 ± 0.002 <LOD* 0.20 ± 0.02 <LOD* 0.01 ± 0.001 0.01 ± 0.003 0.002 ± 0.0003 <LOD* AA-OB-PM 0.68 ± 0.02 <LOD* 0.20 ± 0.02 0.001 ± 0.0005 0.003 ± 0.001 0.04 ± 0.01 0.003 ± 0.0002 0.01 ± 0.003
74
Code Cr Co Ni As Se Mo Cd Pb AA-OB-PE 0.88 ± 0.005 <LOD* 0.27 ± 0.01 0.001 ± 0.001 0.003 ± 0.0001 0.06 ± 0.006 0.004 ± 0.0002 <LOD* AA-OB-FD 0.67 ± 0.02 <LOD* 0.36 ± 0.02 0.001 ± 0.002 0.003 ± 0.00002 0.04 ± 0.002 0.01 ± 0.004 <LOD*
*refer to Table 2.2; <LOD lower than the limit of detection.
Appendix 5.8: Total polyphenol (TP) and minor elements daily intake (mg/day) based on the consumpsion of a 500 g
serving of the commercial and non-commercial açaí pulp (fresh weight).
Purple Açaí
whole Purple Açaí de-fatted White Açaí whole White Açaí de-fatted Pulp SP Commercial SP
Commercial UK
TP 1600.0 1970.0 470.0 585.0 1415.0 2120.0 254.5 Ca 234.4 263.7 208.1 262.6 82.5 101.5 33.1 Mg 116.6 136.2 123.5 150.9 101.1 101.8 43.0 Mn 32.0 40.5 30.6 40.4 13.4 27.4 0.8 Fe 1.5 2.1 1.8 2.2 1.5 1.1 0.1 Zn 1.2 1.6 1.3 1.8 1.3 1.2 0.6 Cu 0.9 1.1 0.9 1.2 0.8 0.7 0.2
75
Appendix 5.9: Percentage intake (%) of total polyphenol and minor elements based on the consumption of a 500 g serving
of the commercial and non-commercial açaí pulp (fresh weight) when compared to the recommended daily
allowance (RDA) for males (M) and females (F).
Total polyphenol Ca Mg Mn Fe Zn Cu Male Female Male Female Male Female Male Female Male Female Male Female Male Female
RDA 1492 1492 1300 1300 400 310 2.3 1.8 8 18 11 8 0.9
GE-WB-P 169.9 169.9 2.8 2.8 16.7 21.6 152.6 195.0 30.1 13.4 23.4 32.2 251.1 GE-WB-S 457.1 457.1 5.2 5.2 24.6 31.7 203.3 259.7 45.1 20.1 34.2 47.1 352.8 GE-PB-P 111.3 111.3 6.1 6.1 37.8 48.7 124.8 159.4 56.0 24.9 27.0 37.2 327.2 GE-PB-S 458.8 458.8 6.1 6.1 41.8 53.9 137.8 176.1 53.3 23.7 28.8 39.6 237.2 IL-PA-P 121.3 121.3 25.9 25.9 43.0 55.4 1198.9 1531.9 39.5 17.6 27.0 37.1 275.6 IL-PA-S 1357.6 1357.6 15.0 15.0 46.6 60.1 1958.5 2502.5 47.4 21.1 41.4 56.9 387.2
MA-PA-P 150.1 150.1 32.6 32.6 50.9 65.7 1825.0 2331.9 35.9 16.0 25.0 34.4 244.4 MA-PA-S 744.3 744.3 18.5 18.5 42.9 55.4 3395.0 4338.1 45.0 20.0 34.5 47.5 297.8 AN-PA-P 121.0 121.0 12.1 12.1 31.2 40.3 1668.0 2131.4 26.4 11.8 20.9 28.7 220.0 AN-PA-S 839.5 839.5 8.4 8.4 35.8 46.2 3810.0 4868.3 36.1 16.1 33.5 46.1 287.8 IC-PA-S 1316.4 1316.4 14.6 14.6 37.1 47.9 2303.7 2943.6 40.6 18.1 24.1 33.2 255.6 IM-PA-S 623.0 623.0 15.2 15.2 39.0 50.3 983.5 1256.7 287.1 127.6 34.0 46.8 388.3
PA-IC-PM 98.2 98.2 10.6 10.6 23.3 30.1 1797.2 2296.4 24.1 10.7 11.5 15.9 83.9 SH-AB-PF 54.6 54.6 6.9 6.9 18.5 23.9 683.9 873.9 14.3 6.4 8.8 12.1 75.6 SH-IM-PF 45.9 45.9 9.3 9.3 18.0 23.2 834.6 1066.4 15.3 6.8 11.2 15.4 66.1
76
Total polyphenol Ca Mg Mn Fe Zn Cu Male Female Male Female Male Female Male Female Male Female Male Female Male Female
RDA 1492 1492 1300 1300 400 310 2.3 1.8 8 18 11 8 0.9
SH-IM-PM 57.6 57.6 15.5 15.5 26.5 34.2 2022.4 2584.2 20.8 9.3 14.1 19.4 96.1 SH-PA-PE 119.3 119.3 15.5 15.5 33.2 42.8 552.4 705.8 29.6 13.1 15.8 21.7 143.9 AA-OB-PM 70.0 70.0 16.9 16.9 21.4 27.7 114.3 146.1 27.2 12.1 8.8 12.1 88.3 AA-OB-PE 92.8 92.8 21.6 21.6 27.0 34.8 326.3 416.9 30.6 13.6 9.8 13.4 92.8 AA-OB-FD 104.2 104.2 20.2 20.2 30.8 39.7 1016.1 1298.3 35.7 15.9 14.7 20.3 135.6
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Appendix 5.10: Total polyphenol and minor elements daily intake (mg/day) based on a 500 g serving of açaí pulp.
Total polyphenol Ca Mg Mn Fe Zn Cu
GE-WB-P 2535.0 35.8 67.0 3.5 2.4 2.6 2.3 GE-WB-S 6820.0 68.0 98.4 4.7 3.6 3.8 3.2 GE-PB-P 1660.0 79.5 151.0 2.9 4.5 3.0 2.9 GE-PB-S 6845.0 79.1 167.0 3.2 4.3 3.2 2.1 IL-PA-P 1810.0 336.7 171.8 27.6 3.2 3.0 2.5 IL-PA-S 20255.0 195.0 186.3 45.0 3.8 4.6 3.5
MA-PA-P 2240.0 424.4 203.7 42.0 2.9 2.8 2.2 MA-PA-S 11105.0 240.4 171.8 78.1 3.6 3.8 2.7 AN-PA-P 1805.0 157.5 124.9 38.4 2.1 2.3 2.0 AN-PA-S 12525.0 108.8 143.2 87.6 2.9 3.7 2.6 IC-PA-S 19640.0 189.3 148.6 53.0 3.3 2.7 2.3 IM-PA-S 9295.0 197.6 156.0 22.6 23.0 3.7 3.5
PA-IC-PM 1465.0 138.2 93.2 41.3 1.9 1.3 0.8 SH-AB-PF 815.0 89.4 74.1 15.7 1.1 1.0 0.7 SH-IM-PF 685.0 120.9 71.8 19.2 1.2 1.2 0.6 SH-IM-PM 860.0 201.4 106.1 46.5 1.7 1.6 0.9 SH-PA-PE 1780.0 201.3 132.7 12.7 2.4 1.7 1.3 AA-OB-PM 1045.0 219.7 85.7 2.6 2.2 1.0 0.8 AA-OB-PE 1385.0 281.3 108.0 7.5 2.5 1.1 0.8 AA-OB-FD 1555.0 262.7 123.1 23.4 2.9 1.6 1.2