impacts of water, extraction procedure and origin on anthocyanins and ... - virginia tech · 2020....

Impacts of Water, Extraction Procedure and Origin on Anthocyanins and Volatile

Compositions of Hibiscus Extracts and Freeze-Dried Hibiscus

Oumoule NDIAYE

Thesis submitted to the faculty of the Virginia Polytechnic Institute and State University

in partial fulfillment of the requirements for the degree of

Master of Science in Life Science

In

Food Science and Technology

Committee Members

Sean F. O’Keefe

Susan E. Duncan

Mady Cisse

August 18, 2016

Blacksburg, Virginia

Keywords: Hibiscus, Calyces, Anthocyanin, Hibiscin, freeze-dried, Volatiles

Copyright 2016 Oumoule Ndiaye



Oumoule NDIAYE

ABSTRACT

There has been a lot of interest in Roselle (Hibiscus sabdariffa L.), called Bissap in

Senegal, hibiscus recently because of consumer interest in nutraceutical products.

However, beverages made from hibiscus have a short self-life due to anthocyanin and

flavor degradation. The purpose of our study was first to assess the impacts of water,

extraction procedure and origin on the anthocyanins of hibiscus extracts and secondly, to

examine the impacts of freeze-drying on the anthocyanins and the volatiles compositions

of hibiscus extracts. For the first experiment, a 2x3 factorial design was used with

hibiscus calyces from Senegal and Egypt for the factor origin, distilled water and

reformulated Dakar (Senegal) water for the second factor water, and then cold and hot

extraction procedures were applied. For the second experiment, Senegalese hibiscus was

extracted with hot and cold water and one part of each extract was freeze-dried. For both

objectives, a ratio of 1:15 w/v (1 kilogram of calyces for 15 liters of water) were used.

The time-temperature was 98ºC / 30 min for hot and 22 ºC / 4 hours for cold extractions.

The anthocyanins were determined using high performance liquid chromatography

(HPLC). And the volatiles were measured using headspace-solid phase microextraction

and gas chromatography-mass spectrometry (HS-SPME-GCMS). Origin and temperature

as well as their interaction had significant effects on the anthocyanin contents, with

respective p-values of 0.0036 and 0.0025 and 0.0002. Freeze-drying showed no effect on

the anthocyanins in cold extracts. In contrast, a significant difference between the hot

iii

extract and its freeze-dried product was observed with a p-value of 0.0013. For the flavor

compounds, the aroma profiles were different between cold and hot extracts and their

instant powders. Globally the results of this study can help in the optimization when

processing hibiscus derivatives.

iii



Oumoule NDIAYE

GENERAL AUDIENCE ABSTRACT

Roselle (Hibiscus sabdariffa L.), called Bissap in Senegal, is an annual shrub of tropical

countries, locally used as a nutritious functional beverage. It has been associated with

many health benefits and is also used in many industrial applications, hence its growing

interest among entrepreneurs. However, beverages made from hibiscus have a short self-

life due to degradation of anthocyanins and flavors. The purpose of our study was first to

assess the impacts of water, extraction procedure and origin on the anthocyanins of

hibiscus extracts. Senegal and Egypt hibiscus were used to prepare beverages with

distilled water and reformulated Dakar (Senegal) water with cold and hot extraction

temperature. Secondly, we examined the impact of freeze-drying on the anthocyanins and

the volatile compositions of hibiscus extracts. Senegalese hibiscus was extracted in hot

and cold conditions one part of each extract was freeze-dried. The result of this

investigation show that origin and extraction temperature have significant effects on the

anthocyanins contents. As expected, freeze-drying as no effect on the anthocyanins for

cold extract. In contrast, significant differences were seen between the hot extract and its

freeze-dried products. The aromas profiles were different when comparing cold and hot

extracts to their respective instant powders as well as between the hot and cold extracts.

The results of this study show freeze-dried hibiscus has volatiles and anthocyanins

similar to non dried, suggesting that freeze drying is an option for stabilizing hibiscus.

iv

DEDICATION

To

Living memories of my mother Ndiéme DIOUF and my dad Aliou NDIAYE a strong and

humanitarian soul whose unconditional love and daily life inspires wherever I am and in

whatever I do and allows me to set higher targets.

My brother Amadou, I am really grateful to you, you have been my inspiration; Thank

you for your great support and continuous care.

My friend Bijieck Jiecnyal for your support and everything.

v

ACKNOWLEDGEMENTS

I would like to express the deepest appreciation to my advisor Professor Sean

O`Keefe who continuously and convincingly conveyed a spirit of adventure to reseach

and excitement in regards to teaching. His guidance and persistent help have made this

work easy for me.

I would like to thank my committee members Professor Susan E. Duncan and Dr.

Mady Cisse for their help, support guidance and understanding.

My special thanks to my sponsors: USAID/ERA (Education & Research in

Agriculture) & OIRED who funded my studies and research at Virginia Polytechnic

Institute and State University (Virginia Tech) in the USA and also the Senegalese

Institute of Food Technology for giving me the opportunity to be one of their candidates

applying for and getting the USAID/ERA scholarship.

I am sincerely grateful to the Senegalese Students of Blacksburg and the Food

Science and Technology (FST) Department staff and graduate students.

Finally, I acknowledge those who helped or supported me in finishing this thesis

specially Bijiek Jieknyal for your support, advice and encourage and everything you have

done for me.

vi

TABLE OF CONTENTS

ABSTRACT ....................................................................................................................... ii GENERAL AUDIENCE ABSTRACT ........................................................................... iii DEDICATION.................................................................................................................. iv ACKNOWLEDGEMENTS ............................................................................................. v

TABLE OF CONTENTS ................................................................................................ vi CHAPTER I ...................................................................................................................... 1

INTRODUCTION ..................................................................................................................... 1

REFRENCES ............................................................................................................................. 3

CHAPTER II ..................................................................................................................... 5 LITERATURE REVIEW ......................................................................................................... 5

2.1 Overview of hibiscus ............................................................................................................. 5

2.2 Origin and distribution .......................................................................................................... 5

2.3 Botanical characteristics and diversity .................................................................................. 6

2.4 Physico-chemical characteristics of Hibiscus sabdariffa calyces ......................................... 8

2.5 Anthocyanins in the calyces .................................................................................................. 9

2.5.1 Structure, composition, and properties of the anthocyanins ............................................... 9

2.5.4 Effect of water, oxygen and enzymes ............................................................................... 12

2.6. Aroma compounds ............................................................................................................. 12

2.7 Microbiological quality ....................................................................................................... 14

2.8 Production of hibiscus and commercialization. ................................................................... 14

2.9 Main uses, consumption, trends, and health benefits .......................................................... 15

REFERENCES .......................................................................................................................... 17

CHAPTER III ................................................................................................................... 24 MATERIALS AND METHODS .............................................................................................. 24

3.1 Plant material and water used for the extraction ................................................................. 24

3.2 Aqueous preparation and freeze-drying process. ................................................................ 25

3. 3 Experiment I: Effects of Origin Water and Temperature on the Anthocyanins and Volatile

of Hibiscus. ................................................................................................................................ 26

3. 4 Experiment II: Effects of freeze-drying on anthocyanins and volatiles of instant Hibiscus

powder obtained from Hot and Cold Extracted juice. ............................................................... 27

3.6 Brix measurement ................................................................................................................ 29

3.7 Total anthocyanins and hibiscidin measurement by HPLC analysis ................................... 29

3.8 Aroma compounds analyses by SPME and GC-MS analysis ............................................. 29

REFERENCE ............................................................................................................................ 31

CHAPTER IV.................................................................................................................. 32 RESULTS AND DISCUSSIONS............................................................................................ 32

vii

Results Experiment I ................................................................................................................. 32

4.1 Effects of Origin, Water and Temperature on Total Anthocyanin Contents ....................... 32

Results Experiment II ................................................................................................................ 41

4.3 Effect of Freeze-Drying on Total Anthocyanins and Hibiscin Contents using Hot Extraction

Procedure ................................................................................................................................... 41

4.4 Effect of Freeze-drying on Total Anthocyanins and Hibiscidin Contents using Cold

Extraction Procedure ................................................................................................................. 43

4.5 pH and the Brix in hibiscus extracts .................................................................................... 44

4.6 Effects of freeze-drying on the volatiles of Instant hibiscus powder obtained from cold

extraction. .................................................................................................................................. 45

4.7 Effects of freeze-drying on the volatiles of instant hibiscus powder obtained from hot

extracted juice ............................................................................................................................ 48

REFERENCES ........................................................................................................................ 53

CHAPTER V ................................................................................................................... 55 CONCLUSIONS ...................................................................................................................... 55

APPENDICES ........................................................................................................................... 57

APPENDIX A – 1 ................................................................................................................. 57

APPENDIX B– 1 ................................................................................................................... 60

APPENDIX C – 1 .................................................................................................................. 60

APPENDIX D – 1 ................................................................................................................. 61

APPENDIX E – 1 .................................................................................................................. 61

APPENDIX F – 1 .................................................................................................................. 61

1

CHAPTER I

INTRODUCTION

In tropical countries, research investments have focused mainly on agricultural production.

Because of this, product development and food processing are often not well developed (Nkem

Khumbah & Melvin, 2014); this has negative impacts, particularly on the economy. People who live

in large cities rely heavily on imported, processed food. According to Van Wyk 2015, in recent years

research has been conducted on many African medicinal plants in order to find the best ways to

promote growth in their use and sales. Among the targeted species, Hibiscus sabdariffa, also called

Roselle is considered as a functional foods (Van Wyk 2015). Hibiscus has numerous health benefits,

such as lowering blood pressure, anticancer activity, and bactericidal properties. Consequently,

consumer demand for this nutraceutical product has increased worldwide (Chiou, & Langrish, 2007;

Hopkins, Lamm, Funk, & Ritenbaugh; Patel 2014; Sindi, Marshall, & Morgan, 2014; Camelo-

Méndez, Ragazzo-Sánchez, Jimenez-Aparicio, Vanegas-Espinoza, Paredes-López, & Del Villar-

Martinez, 2013; Cisse, Dornier, Sakho, Ndiaye, Reynes, & Sock, 2009a; Rodrigues, 2010.

Similarly, studies conducted by Wong, Yusof, Ghazali, & Che Man, 2002; Gonzalez-Palomares,

Estarrón-Espinosa, Gómez-Leyva, & Andrade-González, 2009; and Duangmal, Saicheua, &

Sueeprasan, 2008 found Roselle to be valuable to pharmaceutical, cosmetic and food industries.

Some researchers have considered hibiscus to be one of the most important botanical products in the

global market (Cisse, Dornier, Sakho, Ndiaye, Reynes, & Sock, 2009a; FAO, 2004). Lastly, research

has shown that Roselle contributes to the development of the rural economy and strengthens food

security. Observers see Roselle as an alternative to some food crops like peanut in Senegal (Cisse,

Dornier, Sakho, Ndiaye, Reynes, & Sock, 2009a). However, some studies have suggested that

http://www.sciencedirect.com/science/article/pii/S0378874115301896

2

beverages made from hibiscus flowers main edible (the calyces) have a short self-life, due to

anthocyanin and flavor degradation (Sarni-Manchad & Cheynier V. 2006). This makes it very

difficult to ship, transport, and commercialize hibiscus products. Therefore, finding ways to obtain

ready-to-use and easy-to-export products from hibiscus extracts that are safe, have good qualities,

great consumer acceptability and acceptable shelf-life can improve product accessibility and

availability throughout the world. A lot of studies have been conducted on Hibiscus sabdariffa in

order to end up with products that are successful in the market place. For instance, Duangmal,

Saicheua, & Sueeprasan, (2008) have investigated the stability of anthocyanins in freeze-dried

Roselle using either maltodextrin or trehalose as a stabilizer. They found that the addition of these

products retarded total anthocyanin degradation. The effect of the temperature on spray drying of

Roselle extracts (using ethanol as solvent) has also been investigated by Gonzalez-Palomares,

Estarrón-Espinosa, Gómez-Leyva, & Andrade-González, (2009). They reported a significant effect

of spray drying temperature on the volatiles. However, further investigations are needed in order to

improve hibiscus calyces transformation, commercialization and consumption worldwide. The

purpose of this study is to investigate the effects of origin, water, and temperature on the

anthocyanin contents of hibiscus extracts and also to determine the effect of freeze-drying on

anthocyanin contents and aroma compositions of hibiscus instant powder obtained from hibiscus

extracts. The results of this current work will help determine factors relevant to processing hibiscus

instant powder.

3

REFRENCES

Nkem Khumbah and Melvin P.Foote. (2014). Africa Needs Science, Not Aid. The New York

Time (http://www.nytimes.com/2014/08/01/opinion/africa-needs-Science-not-aid.html see on June

4th 2016).

Van Wyk, B.E. (2015). A review of commercially important African medicinal plants).

Chiou, D., & Langrish, T. A. G. (2007). Development and characterization of novel

nutraceuticals with spray drying technology. Journal of Food Engineering, 82(1), 84-91.

Hopkins, A. L., Lamm, M. G., Funk, J. L., & Ritenbaugh, C. (2013). Hibiscus sabdariffa L.

in the treatment of hypertension and hyperlipidemia: a comprehensive review of animal and human

studies. Fitoterapia, 85, 84-94.

Patel, S. (2014). Hibiscus sabdariffa: An ideal yet under-exploited candidate for nutraceutical

applications. Biomedicine & Preventive Nutrition, 4(1), 23-27.

Sindi, H. A., Marshall, L. J., & Morgan, M. R. (2014). Comparative chemical and

biochemical analysis of extracts of Hibiscus sabdariffa. Food chemistry, 164, 23-29.

Camelo-Méndez, G. A., Ragazzo-Sánchez, J. A., Jimenez-Aparicio, A. R., Vanegas-

Espinoza, P. E., Paredes-López, O., & Del Villar-Martinez, A. A. (2013). Comparative study of

anthocyanin and volatile compounds content of four varieties of Mexican roselle (Hibiscus

sabdariffa L.) by multivariable analysis. Plant foods for human nutrition, 68(3), 229-234.

Cisse, M., Dornier, M., Sakho, M., Ndiaye, A., Reynes, M., & Sock, O. (2009a). Le bissap

(Hibiscus sabdariffa L.): composition et principales utilisations. Fruits, 64(03), 179-193

Wong, P. K., Yusof, S., Ghazali, H. M., & Che Man, Y. B. (2002). Physico-chemical

characteristics of roselle (Hibiscus sabdariffa L.). Nutrition & Food Science, 32(2), 68-73.

http://www.nytimes.com/2014/08/01/opinion/africa-needs-Science-not-aid.html

http://www.sciencedirect.com/science/article/pii/S0378874115301896

4

Gonzalez-Palomares, S., Estarrón-Espinosa, M., Gómez-Leyva, J. F., & Andrade-González,

I. (2009). Effect of the temperature on the spray drying of roselle extracts (Hibiscus sabdariffa

L.). Plant foods for human nutrition,64(1), 62-67.

Duangmal, K., Saicheua, B., & Sueeprasan, S. (2008). Colour evaluation of freeze-dried

roselle extract as a natural food colorant in a model system of a drink. LWT-Food Science and

Technology, 41(8), 1437-1445.

Food and Agriculture Organization of the United Nations (FAO) HIBISCUS: Post-

Production Management for Improved Market (Access, 2004)

Sarni-Manchad P. & Cheynier V. 2006. Les polyphénols en agroalimentaire, Éd Tec & Doc.,

Coll. Sci. & Techn. Agroaliment., Lavoisier, Paris, 398 p. [ Cheynier, V., & Sarni-Manchado, P.

(2006). Les polyphénols en agroalimentaire. Paris: Lavoisier Tec & Doc, 50-59.]

5

CHAPTER II

LITERATURE REVIEW

2.1 Overview of hibiscus

Hibiscus sabdariffa, an herbaceous perennial shrub belonging to the Malvaceae family, is known by

different names. Some of its vernacular names include Roselle, Bissap in Senegal, Jamaican sorrel,

Indian sorrel, karkadeh or karkadé in North Africa, Oseille de Guinée, thé rose d’Abyssinie, Roselle

or sorrel sour tea in English, ngai ngai in Central Africa, currant Christmas in the Caribbean, and

Jamaica floras in Central America (Nyarko, Bayor, Craigon, & Suleimana, 2006; Cisse, Dornier,

Sakho, Diop, Reynes, Sock, 2009b; Juliani, Welch, Wu, Diouf, Malainy, & Simon, 2009;

McClintock, & El Tahir, 2011; Ali, Wabel, & Blunden, 2005; FAO 2004).

Hibiscus sabdariffa L is cultivated largely in tropical and subtropical areas of both hemispheres.

Hibiscus sabdariffa has several varieties and is used in various fields due to its biochemical

composition and growing consumers interest in nutraceutical products but also because it can be

used for many industrial applications and, as a consequence, generates income (Da-Costa-Rocha,

Bonnlaender, Sievers, Pischel, & Heinrich, 2014; Camelo-Méndez, Ragazzo-Sánchez, Jimenez-

Aparicio, Vanegas-Espinoza, Paredes-López, & Del Villar-Martinez, 2013; McClintock, & El

Tahir, 2011; Cissé, Bohuon, Sambe, Kane, Sakho, & Dornier, 2012). In Senegal, the production

and exportation of hibiscus has expanded due to an increased demand at local and international

levels. But certified seed supply and better management in production and processing would increase

interest in commercialization of hibiscus (Cissé, 2010 2012; McClintock, & El Tahir, 2011).

2.2 Origin and distribution

6

The origin of Roselle or Sorrel (Hibiscus sabdariffa) is not exactly known. According to

some authors, it is native to West Africa (El Naim, & Ahmed, 2010; Ataogye, 2012). It was

eventually brought to other parts of the world like Asia, Central America and the United States of

America during slave trade (El Naim, & Ahmed, 2010). However, according to other authors,

Hibiscus sabdariffa came from India (Jade Senegal http://www.jade.sn/bissap/botanique.htm;

Nzikou, Bouanga-Kalou, Matos, Ganongo-Po, Mboungou-Mboussi, Moutoula, & Desobry, 2011 ;

Atta, Sarr, Diallo, Bakasso, Lona, & Saadou, 2013). Roselle is now cultivated in many tropical and

subtropical regions of the world. But the variety altissama is rare in Africa. In Senegal, Hibiscus

sabdariffa was introduced in the 19th century (Kerharo, & Adam, 1974; Cisse, Dornier, Sakho,

Diop, Reynes, & Sock, 2009b; Ataogye, 2012, Atta, Sarr, Diallo, Bakasso, Lona, & Saadou, 2013;

FAO, 2004; McClintock & El Tahir, 2011; Akanbi, Olaniyan, Togun, Ilupeju, & Olaniran, 2009).

Currently in Senegal, hibiscus is grown in many regions (Kaolack, Fatick, Diourbel, Thies, Saint-

Louis, Louga and Ziguinchor). The varieties grown, including Vimto, Koor, Thai and CLT 92. This

helps farmers have an alternative to the production of traditional crops, especially peanut, which is

declining nowadays. (Cisse, Dornier, Sakho, Diop, Reynes, & Sock, 2009b; Reynes, & Sock,

2009b).

2.3 Botanical characteristics and diversity

According to some authors, more than 200–300 species of hibiscus can be found in both

tropical and subtropical areas. Hibiscus has simple leaves that can be regular or lobed. The two

botanical kinds based on the color of its fibers are: the red type and the green one (or white). The red

and green (or white) depends on the presence or absence of anthocyanins (McClintock, & El Tahir,

2011; Omobuwajo, Sanni, & Balami, 2000; Cisse, Dornier, Sakho, Diop, Reynes, & Sock, 2009 and

http://www.jade.sn/bissap/botanique.htm

7

Jade Senegal http://www.jade.sn/bissap/botanique.htm). Except for Hibiscus sabdariffa, the other

species are used for ornamental purposes and according to Kerharo, & Adam (1974) and Morton

(1987), it has two botanical varieties: the variety altissima and the variety sabdariffa L. The variety

altissima grows tallest; it can reach three up to four meters in height, is not much branched, without

fleshy fruits, and its use is justified by its fibers. Hibiscus sabdariffa L. variety sabdariffa is a

vascular herb, with alternated leaves and fleshy fruits. It belongs to the family of Malvaceae

(Morton, 1987; Cisse, Dornier, Sakho, Ndiaye, Reynes, & Sock, 2009a). The variety sabdariffa L

has two botanical types differing in the color of its fibers. The hibiscus flowers are solitary, axillary,

form loose fake cobs and are hermaphrodites. They self-pollinate with the reproduction cycle lasting

is about 120 to 165 days. (Cisse, Dornier, Sakho, Diop, Reynes, & Sock, (2009 b). Roselle can be

produced as a rain field crop, which requires the seed to be sown early in the rainy season, but also

in market gardening. Roselle is drought resistant and the production of the calyces requires

temperatures higher than 17°C, and at least 13 hours of daylight. The fruit is a globoid cap, covered

by the calyx and contains kidney-shaped brown to dark brown seeds. The fruits have two parts: the

calyx representing about 6g and the part that envelops the seeds weighing about 5g (McClintock, &

El Tahir, 2011; Cisse, Dornier, Sakho, Diop, Reynes, & Sock, 2009b;

http://www.jade.sn/bissap/botanique.htm; Wong, Yusof, Ghazali, & Che Man, 2002; Cisse, Dornier,

Sakho, Ndiaye, Reynes, & Sock, 2009a).

Some characteristics of certain varieties of hibiscus found in Senegal were given by Cisse, Dornier,

Sakho, Diop, Reynes, & Sock, 2009b. The Vimto characterized by dark red color, amount of

anthocyanin that can reach 10 to 15 grams per kilogram and the vitamin C content 0.5 gram per kilo.

The "Koor" more acidic than the Vimto variety produces less anthocyanin but might have more

organic acids. The Thaï variety first identified in Thailand has high fiber content. The Thaï have



8

been introduced Senegal by l’ASNAPP-Sénégal (l’antenne sénégalaise de l’Agribusiness in

Sustainable Natural African Plan Products) and fondation Éducation-Santé. The ''CLT 92 " variety is

characterized by blue-purple or dark red pigments and was brought to Senegal by VAPROVET

(Valorisation des Produits Végétaux). Other red varieties of Hibiscus sabdariffa include

"Bambara","Burkina", and "Violette," also called ordinary (Cisse, Dornier, Sakho, Diop, Reynes, &

Sock, 2009b).

2.4 Physico-chemical characteristics of Hibiscus sabdariffa calyces

The calyces constitute the main portion of the fruit with more than 54.11± 2.34% in terms of

percentages, and the rests consist of seeds (Wong, Yusof, Ghazali, & Che Man, 2002). Hibiscus

calyces are the main edible part of this plant. Their composition can vary, depending on several

factors such as the variety of hibiscus, its genetic composition, the area where the calyces are

produced, the harvesting conditions, the amount of rainfall, the nature of the soil, the temperature,

and so on (Da-Costa-Rocha, Bonnlaender, Sievers, Pischel, & Heinrich, 2014; Kerharo, & Adam,

1974; and Morton 1987).

Many authors have studied physicochemical characteristics of Roselle and its components and

Rodrigues 2010 has given a summary of nutritional composition of fresh hibiscus calyces.

Other components such as organic acids (succinic, oxalic, tartaric, and malic), with succinic and

oxalic representing the main ones (76%) (Wong, Yusof, Ghazali, & Che Man, 2002; Babalola,

Babalola, & Aworh, 2001; Dafallah, al-Mustafa 1996). Sugars, mainly glucose, fructose and sucrose,

were also reported, as were ascrobic acid, β-carotene, and lycopene (Wong, Yusof, Ghazali, & Che

9

Man, 2002) and protocatechuic acid (Dickel, Rates, & Ritter, 2007 and Herrera-Arellano, Flores-

Romero, Chavez-Soto, & Tortoriello, 2004).

Tableau 1. Amino acid composition (mg/g of dried mater) of Hibiscus sabdariffa calyces. Calices

Morton 1987

Ala Arg Asp Cys Gln Gly His Ile Leu Lys Met Phe Pro Ser Thr Trp Tyr Val

3.70 3.60 16.30 1.30 7.20 3.80 1.50 3.00 5.00 3.90 1.00 3.20 5.60 3.50 3.00 - 2.20 3.80

2.5 Anthocyanins in the calyces

2.5.1 Structure, composition, and properties of the anthocyanins

The richness in anthocyanins is one of the major characteristics of hibiscus calyces. Anthocyanins

are molecules with a C15 skeleton, classified as flavonoid derivates, and are water soluble. The

chromophores of the anthocyanins, the aglycones, constitute their basic structure. The molecule has

two aromatic rings and one heterocycle which consists of carbon and oxygen. Anthocyanins are able

to absorb visible light and therefore can be quantified by measuring their visible spectra. They are

also natural pigments present in fruits, vegetables flowers, etc. They are responsible for a wide range

of colors in many species ranging from blue, red, purple to pink. This coloring power makes them a

good substitute to the artificial dyes. They are used in food industries for color ranging from red to

violet (Delgado-Vargas, Jiménez, & Paredes-López, 2000; Gradinaru, Biliaderis, Kallithraka,

Kefalas, & Garcia-Viguera, 2003; Ramirez‐Rodrigues, Plaza, Azeredo, Balaban, & Marshall, 2011;

Castaneda-Ovando, de Lourdes Pacheco-Hernández, Páez-Hernández, Rodríguez, & Galán-Vidal,

2009; Cisse, Dornier, Sakho, Ndiaye, Reynes, & Sock, 2009a; Wang, Wang, Lin, Chu, Chou, &

Tseng, 2000).

The epidermal cell of the plant contains the vacuole where these pigments are localized (Harborne,

1967). Four types of anthocyanins were identified by Francis (1973) Many researchers have shown

10

that anthocyanins are present at high concentrations in Hibiscus sabdariffa calyces and, according to

Wong and others 2002, its content is 2.52 gram per 100g expressed as delphinidin-3-glucoside. The

delphinidin-3-sambubioside (also known as delphinidin-3-xylosylglucoside or hibiscin) was 71.40%

of the total anthocyanins and the rest was constituted by the cyanidin-3-sambubioside or cyanidin-3-

xylosylglucoside or gossypicyanin and delphinidin-3-glucoside (Wong, Yusof, Ghazali, & Che Man,

2002).

Researchers have shown these two main anthocyanin component are quickly absorbed in rats’

gastrointestinal tract, hence their good bioavailability (Wang, Wang, Lin, Chu, Chou, Tseng, T. H.

(2000).

Figure 1. Chemical structures of main anthocyanins.

https://www.researchgate.net/profile/Michael_Heinrich/publication/262937428/figure/fig1/Chemical

-structures-of-main-anthocyanins.png.

2.5.2. Effect of pH on the anthocyanins

At very low pH, the anthocyanin color is red, but this decreases strongly at higher pH. Color

changes in the anthocyanins are due to chemical equilibrium that occurs depending on the four

https://www.researchgate.net/profile/Michael_Heinrich/publication/262937428/figure/fig1/Chemical-structures-of-main-anthocyanins.png

https://www.researchgate.net/profile/Michael_Heinrich/publication/262937428/figure/fig1/Chemical-structures-of-main-anthocyanins.png

11

various forms that anthocyanin/aglycones present. According to Gradinaru, the copigmentation

(association of an anthocyanin chromophore with the planar electronically saturated part of the

copigments) is responsible for the greater absorbance that can be observed at visible range. In many

papers, the hibiscus pH extracts turn color around 2.4. According to Gradinaru, at neutral or less

acidic pH, the anthocyanins are colorless; therefore, their degradation is pH dependent (Gradinaru,

Biliaderis, Kallithraka, Kefalas, & Garcia-Viguera, 2003; Wong, Yusof, Ghazali, & Che Man, 2002;

Da-Costa-Rocha, Bonnlaender, Sievers, Pischel, & Heinrich, 2014; Cisse, Dornier, Sakho, Diop,

Reynes, & Sock, 2009b).

2.5.3 Effect of temperature on the anthocyanins

Many studies have shown that high temperature can affect the anthocyanins color, lifespan,

etc. As stated by Gradinaru et al temperature had a major influence on the degradation kinetics”.

Treatments by heat and storage at high temperature lead to anthocyanin degradation and color

changes going from red to brown. The only positive effect that hot extraction can have on hibiscus

calyces are that an increase from 30 to 100◦C can sxtract more anthocyanins and sugar but also

contribute to neutralization of microorganisms such as total microorganisms’ flora, fecal coliforms

and yeasts/molds. Thermal treatment such hot extraction and pasteurization (90◦C for 5 min)

negatively impacts the physicochemical characteristics of hibiscus calyces. At high temperature, the

pigment–copigments interaction is weakened (Cissé, Bohuon, Sambe, Kane, Sakho, & Dornier,

2012; Gradinaru, Biliaderis, Kallithraka, Kefalas, & Garcia-Viguera, 2003; Sarni-Manchad &

Cheynier 2006; Brouillard, Dangles, & Harborne, 1994.

Light has two opposite effects on anthocyanins: in vivo within the plant itself, light promotes the

biosynthesis of the anthocyanins but in vitro in the extracts or other derivatives of hibiscus, light

12

accelerates the degradation of the anthocyanins. Generally, the anthocyanins are unstable when

exposed to UV radiation or visible light or to other ionizing radiation. Their photo-oxidation gives

the same decomposition as those obtained by heating.

2.5.4 Effect of water, oxygen and enzymes

Some authors have shown that anthocyanin breakdown occurs from contact with water.

Anthocyanins degradation requires water availability. When anthocyanin degradation due to high

water activity occurs, it can lead to the formation of aglycones or chalcone (Erlandson, & Wrolstad,

1972; Gradinaru, Biliaderis, Kallithraka, Kefalas, & Garcia-Viguera, 2003; Markakis, Jurd, (1974).

Oxygen is believed to be one of the main agents that destroy anthocyanins. Under its effect, whether

directly (oxidation) or through oxidized elements, phenolic pigments are altered. This result in

formation of colorless or brown pigments (Gradinaru, Biliaderis, Kallithraka, Kefalas, & Garcia-

Viguera, 2003) have shown that when exposed to high relative humidity without a good barrier that

is impermeable to oxygen, faster anthocyanin degradation is observed.

The phenolic pigments can be degraded by proteins that have enzymatic activities such as

galactosidases and peroxidases (Cisse, Dornier, Sakho, Diop, Reynes, & Sock, 2009b)

2.6. Aroma compounds

Several studies have revealed that hibiscus contains volatile compounds, but the findings

differ a lot in term of amount and also in term of characteristics of those volatile profiles. Many of

the authors have mentioned that difference can be related to factors such as heat treatment, variety,

and extraction methods used to isolate the volatile (solvent used, time, solute concentration)

(Ramirez‐Rodrigues, Plaza, Azeredo, Balaban, & Marshall, 2011; Ramírez‐Rodrigues, 2010; Chen,

13

Huang, Ho, & Tsai, 1998; Gonzalez-Palomares, Estarrón-Espinosa, Gómez-Leyva, & Andrade-

González, 2009. By using cold (22 ◦C for 4 h) and hot (98 ◦C for 16 min) extraction on both fresh

and dried calyces and using GC-MS, Ramirez‐Rodrigues, Plaza, Azeredo, Balaban, & Marshall,

(2011) identified a total of 32 aroma compounds. They found that each of the extracts presented a

different volatile profile even though in all four extracts, thirteen volatiles were found identical. The

32 compounds were divided into two groups; one constituted by 15 volatiles compounds never

determined in in earlier studies and the second group by components such as limonene, linalool, α-

terpinol, eugenol, and furfural that were previously reported by Chen, Huang, Ho, & Tsai in 1998

and Salvador Gonzalez-Palomares in 2009.

In 1998, Chen, Huang, Ho, & Tsai reported the presence of 37 compounds including fatty acid

derivatives, sugar derivatives, phenolic derivatives and terpenes. They found lipid derivative (C6)

and 5-methyl 2-furfural, respectively, dominant in fresh and dried Roselle. The α-terpineol, linalool

oxide, and limonene were significantly reduced by drying. According to the authors, terpenes

derivative with fragrance are responsible of the Roselle flavor (Chen, Huang, Ho, & Tsai, 1998).

Salvador Gonzalez-Palomares et al have used SPME-GC-MS to identify volatiles in hibiscus

products. In their experiment, they have used fresh Roselle, ethanol as an extracting solvent and have

applied seven spray drying temperatures (150, 160, 170, 180, 190, 200 and 210 °C). In the Roselle

extract, 20 volatiles (terpene, esters, aldehydes and phenolic derivatives) compounds were reported.

In the powder, 14 volatile compounds were reported; ten were identical to those found in the extract

but with smaller amounts. The appearance of 4 other volatiles (furfural, cis-linalool oxide, furanic

linalool oxide Z and E, and eugenol) were justified by spray drying temperature, which causes

chemical degradations during the process, leading to their formation. They reported that the

alteration of sugar was responsible for the furfural, chemical destruction of and fatty acid generated

14

the cis-linalool oxide and the furanic linalool oxide Z and E and that the spray drying process

temperature leads to the eugenol (Gonzalez-Palomares, Estarrón-Espinosa, Gómez-Leyva, &

Andrade-González, 2009).

2.7 Microbiological quality

According to Cisse et al, microbiological problems are, in general, not encountered in

Hibiscus sabdariffa calyces or its derivatives such as the juices. This is due to the lower pH (Cisse,

Dornier, Sakho, Diop, Reynes, & Sock, 2009b)

2.8 Production of hibiscus and commercialization.

Pre-harvest operations include planting seeds at the beginning of the rainy season. The

calyces are considered ripe when the flowers have dropped but before the seed pods is dried and

opened. This stage determined the harvesting period of the fresh calyces. Calyces and seedpods are

separated by hand, or by using a metal tool. Drying under the sun is the mostly used, but also drying

tunnels or using air dryers can be used but should not be conducted under temperature higher than

43OC. To avoid contamination (insects, birds, rodents and dust) during harvesting, drying and

storage, special care is required (http://www.fao.org/3/a-av006e.pdf).

Studies have shown that countries around the world such as China, Thailand, Mexico, Egypt,

Senegal, Mali, Chad, Sudan, Tanzania and Jamaica produce and commercialize hibiscus; but for

most, the production is for local consumption. Market prices of hibiscus fluctuate a lot depending on

factors such as the oversupply, online transactions that occur with increases in demand of calyces,

amount of rainfall but also to climate and quality control problems. The main importers of hibiscus

calyces are Germany and the United States. Hibiscus calyces of best quality has red color with a

http://www.fao.org/3/a-av006e.pdf

15

good sour-fruity taste and are clean. For commercial purposes, the calyces can be dried or frozen and

packaged. Senegal dried hibiscus calyces sold in Europe are filled in balls that can weighing 175 lbs

(80 kg). Senegalese farmers also supply Adina for Life the producer of Hibiscus Lemon Bissap

located in California, USA McClintock, & El Tahir, 2011.; FAO 2004; Ramírez‐Rodrigues, 2010).

2.9 Main uses, consumption, trends, and health benefits

Hibiscus is used for domestic consumption and in traditional medicine. Due to some of its

characteristic such as red color, refreshing properties, nutritious functional beverage with health

benefits and its applications in food, pharmaceutical, cosmetic and wine industries, interest has

grown in its production. It is used as a non-alcoholic beverage (hot, cold and with sugar), tea,

vegetable infusion, for jellies, jam, syrup, cocktails, etc. The refreshing sour tasting juice called

“Bissap” in Senegal (mainly used during hot weather and the month of Ramadan), da bilenni’ (Côte

d’Ivoire, Mali, Burkina Faso) is sold in urban markets. In African countries and in West India, it is

used mainly as a vegetable, but also as a beverage whereas in Europe, the calyces are utilized to

make extracts for flavoring liqueurs. The Food and Drug Administration in the United States allows

hibiscus extracts in alcoholic beverages. In the market place, ready to drink beverages using hibiscus

as a main ingredient are being manufactured and some of them are Hibiscus Lemon Bissap, Cnita

Aguas Fresca, Squish Hibiscus Presse and Simply Hibi. The leaves and green calyces are more used

as vegetable in sauces mixed with some ingredients and in Senegal a condiment called ‘bëkëj’ made

for the leaves or green calyces is eaten with a rice dishes. (Da-Costa-Rocha, Bonnlaender, Sievers,

Pischel, & Heinrich, 2014; McClintock, & El Tahir, 2011; FAO 2004; Cisse, Dornier, Sakho,

Ndiaye, Reynes, & Sock, 2009a; Wong, Yusof, Ghazali, & Che Man, 2002; Chiou, & Langrish,

2007., Ramírez‐Rodrigues, et al 2010). Many researchers have shown that the growing interest in

16

hibiscus and its derivatives is due to its health or medical benefits and its basic nutritional value. The

health benefits associated with hibiscus are multiple. It can lower the blood pressure; Herrera-

Arellano, Flores-Romero, Chavez-Soto, & Tortoriello, (2004) found that Hibiscus sabdariffa can

lower the systolic blood pressure on average from 139. to 124 mm Hg and the diastolic from 91 to

80 mm Hg. Other scientists have also reported that daily consumption of hibiscus tea like in diet may

lower the blood pressure (McKay, Chen, Saltzman, & Blumberg, 2010).

Some researchers have also found that many plants are popularly associated with weight loss. They

reported this “Seven species present pre-clinical data that indicate a potential role in the control of

certain conditions which are associated with obesity, such as hyperlipidemia”. Among those species

figured Hibiscus sabdariffa. It is believed to have no side effects. (Dickel, Rates, & Ritter, 2007 and

Hopkins, Lamm, Funk, & Ritenbaugh (2013) reported that antioxidant properties of hibiscus might

be related to Tocopherol or Vitamin E and acid ascorbic or Vitamin C. Much more health benefits

were reviewed by Da-Costa-Rocha, Bonnlaender, Sievers, Pischel, & Heinrich, (2014) and authors

such as Hopkins, Lamm, Funk, & Ritenbaugh, (2013) reported properties such as anticancer activity,

antiaging, antiparasitic and bactericidal, anti-inflammatory properties, but also effects, on diabetes,

nephro- and hepato-protective, renal/diuretic effect, anti-cholesterol, antispasmodic effect on uterus

and intestine smooth muscles etc. Some of these effects can be related to strong antioxidant

activities, inhibition of α-glucosidase and α-amylase, inhibition of angiotensin-converting enzymes,

and direct vaso-relaxant effect or calcium channel modulation (Da-Costa-Rocha, Bonnlaender,

Sievers, Pischel, & Heinrich 2014). Hibiscus bioactive elements such as organic acids, anthocyanins,

polysaccharides and flavonoids provide it with all these pharmacological properties (Eggensperger

& Wilker, 1996; Müller & Franz, 1990).

17

REFERENCES

Nyarko, G., Bayor, H., Craigon, J., & Suleimana, I. A. (2006). The effect of container types,

seed dressings and desicants on the viability and vigour of roselle (Hibiscus sabdariffa L. var.

saddariffa) seeds.

Cisse M, Dornier M., Sakho M., Diop C.M., Reynes M., Sock O. La

production de bissap (Hibiscus sabdariffa) au Sénégal (2009 b) Fruits, vol. 64, 111–124

Cisse, M., Dornier, M., Sakho, M., Ndiaye, A., Reynes, M., & Sock, O. (2009a). Le bissap

(Hibiscus sabdariffa L.): composition et principales utilisations. Fruits, 64(3), 179-193.

Juliani, H. R., Welch, C. R., Wu, Q., Diouf, B., Malainy, D., & Simon, J. E. (2009).

Chemistry and Quality of Hibiscus (Hibiscus sabdariffa) for Developing the Natural‐Product

Industry in Senegal. Journal of Food Science, 74(2), S113-S121.

McClintock, N.C. & El Tahir, I.M., 2011. Hibiscus sabdariffa L. [Internet] Record from

PROTA4U. Brink, M. & Achigan-Dako, E.G. (Editors). PROTA (Plant Resources of Tropical

Africa / Ressources végétales de l’Afrique tropicale), Wageningen, Netherlands.

<http://www.prota4u.org/search.asp>. Accessed 8 June 20164 June 2016.

Ali, B. H., Wabel, N. A., & Blunden, G. (2005). Phytochemical, pharmacological and

toxicological aspects of Hibiscus sabdariffa L.: a review.Phytotherapy research, 19(5), 369-375.

Food and Agriculture Organization of the United Nations (FAO) HIBISCUS: Post-

Production Management for Improved Market (Access, 2004).

Da-Costa-Rocha, I., Bonnlaender, B., Sievers, H., Pischel, I., & Heinrich, M. (2014).

Hibiscus sabdariffa L.–A phytochemical and pharmacological review.Food chemistry, 165, 424-443.

http://www.prota4u.org/search.asp

18

Camelo-Méndez, G. A., Ragazzo-Sánchez, J. A., Jimenez-Aparicio, A. R., Vanegas-

Espinoza, P. E., Paredes-López, O., & Del Villar-Martinez, A. A. (2013). Comparative study of

anthocyanin and volatile compounds content of four varieties of Mexican roselle (Hibiscus

sabdariffa L.) by multivariable analysis. Plant Foods for Human Nutrition, 68(3), 229-234.

Cissé, M., Bohuon, P., Sambe, F., Kane, C., Sakho, M., & Dornier, M. (2012). Aqueous

extraction of anthocyanins from Hibiscus sabdariffa: Experimental kinetics and modeling. Journal

of Food Engineering, 109(1), 16-21.

El Naim, A. M., & Ahmed, S. E. (2010). Effect of weeding frequencies on growth and yield

of two roselle (Hibiscus sabdariffa L) Varieties under rain fed. Australian Journal of Basic and

Applied Sciences, 4(9), 4250-4255.

Ataogye, G. (2012). Microbial Contamination of an Indigenous Leafy Vegetable, Roselle

(Hibiscus Sabdariffa L.) and Associated Risk Factors on Farm and Market Samples in the Kasena-

Nankana East Municipality of the Upper East Region (Doctoral dissertation).

(http://www.jade.sn/bissap/botanique.htm JADE SENEGAL membre du réseau SYFIA:

Nourritures inconnus d’Afrique le BISSAP Le vin sénégalais consulted le 02/ 23/ 2015.

Nzikou, J. M., Bouanga-Kalou, G., Matos, L., Ganongo-Po, F. B., Mboungou-Mboussi, P. S.,

Moutoula, F. E., & Desobry, S. (2011). Characteristics and nutritional evaluation of seed oil from

Roselle (Hibiscus sabdariffa L.) in Congo-Brazzaville. Current Research Journal of Biological

Sciences, 3(2), 141-146.


19

Atta, S., Sarr, B., Diallo, A. B., Bakasso, Y., Lona, I., & Saadou, M. (2013). Nutrients

composition of calyces and seeds of three Roselle (Hibiscus sabdariffa L.) ecotypes from

Niger. African Journal of Biotechnology, 12(26).

Kerharo, J., & Adam, J. G. (1974). La pharmacopée sénégalaise traditionnelle: plantes

médicinales et toxiques. http://agris.fao.org/agris-search/search.do?recordID=XF2015040378

Cisse, M., Dornier, M., Sakho, M., Diop, C. M., Reynes, M., & Sock, O. (2009 b). Bissap

(Hibiscus sabdariffa L.) production in Senegal Production area. Fruits (Paris), 64(2), 111-124.

Akanbi, W. B., Olaniyan, A. B., Togun, A. O., Ilupeju, A. E. O., & Olaniran, O. A. (2009).

The effect of organic and inorganic fertilizer on growth, calyx yield and quality of Roselle (Hibiscus

sabdariffa L.). American-Eurasian Journal of Sustainable Agriculture, 3(4), 652-657.

Omobuwajo, T. O., Sanni, L. A., & Balami, Y. A. (2000). Physical properties of sorrel

(Hibiscus sabdariffa) seeds. Journal of Food Engineering, 45(1), 37-41

Morton, J.F., (1987) Morton J.F., Roselle, in: Dowling C.F. (Ed.), Fruits of warm climates,

Media, Inc., Green-sboro, NC, USA, 1987, pp. 281–286.

Wong, P. K., Yusof, S., Ghazali, H. M., & Che Man, Y. B. (2002). Physico-chemical

characteristics of roselle (Hibiscus sabdariffa L.). Nutrition & Food Science, 32(2), 68-73.

Rodrigues, M. M. R. (2010). Processing Hibiscus Beverage Using Dense Phase Carbon

Dioxide. University of Florida.

Babalola, S. O., Babalola, A. O., & Aworh, O. C. (2001). Compositional attributes of the

calyces of roselle (Hibiscus sabdariffa L.). J. Food Tech-nol. Afr. 6 (4) (2001) 133-134

http://agris.fao.org/agris-search/search.do?recordID=XF2015040378

20

Dafallah, A. A., & Al-Mustafa, Z. (1996). Investigation of the anti-inflammatory activity of

Acacia nilotica and Hibiscus sabdariffa. The American Journal of Chinese Medicine, 24(03n04),

263-269.

Delgado-Vargas, F., Jiménez, A. R., & Paredes-López, O. (2000). Natural pigments:

carotenoids, anthocyanins, and betalains—characteristics, biosynthesis, processing, and

stability. Critical Reviews in Food Science and Nutrition, 40(3), 173-289.

Gradinaru, G., Biliaderis, C. G., Kallithraka, S., Kefalas, P., & Garcia-Viguera, C. (2003).

Thermal stability of Hibiscus sabdariffa L. anthocyanins in solution and in solid state: effects of

copigmentation and glass transition. Food Chemistry, 83(3), 423-436.

Ramirez‐Rodrigues, M. M., Plaza, M. L., Azeredo, A., Balaban, M. O., & Marshall, M. R. (2011).

Physicochemical and phytochemical properties of cold and hot water extraction from Hibiscus

sabdariffa. Journal of food science, 76(3), C428-C435.

Castaneda-Ovando, A., de Lourdes Pacheco-Hernández, M., Páez-Hernández, M. E.,

Rodríguez, J. A., & Galán-Vidal, C. A. (2009). Chemical studies of anthocyanins: A review. Food

Chemistry, 113(4), 859-871.

Wang, C. J., Wang, J. M., Lin, W. L., Chu, C. Y., Chou, F. P., & Tseng, T. H. (2000).

Protective effect of Hibiscus anthocyanins against tert-butyl hydroperoxide-induced hepatic toxicity

in rats. Food and Chemical Toxicology, 38(5), 411-416.

Harborne, J. B. (1967). Comparative biochemistry of the flavonoids.

Brouillard, R., Dangles, O., & Harborne, J. B. (1994). The Flavonoids: Advances in Research

Since 1986. by Harborne JB, Chapman & Hall, London, 565-588.

21

Erlandson, J. A., & Wrolstad, R. E. (1972). Degradation of anthocyanins at limited water

concentration. Journal of Food Science, 37(4), 592-595.

Markakis, P., & Jurd, L. (1974). Anthocyanins and their stability in foods. Critical Reviews

in Food Science & Nutrition, 4(4), 437-456.

Markakis, P. (1982). Stability of anthocyanins in foods. Anthocyanins as food colors, 163-180.

Chen, S. H., Huang, T. C., Ho, C. T., & Tsai, P. J. (1998). Extraction, analysis, and study on

the volatiles in roselle tea. Journal of Agricultural and Food Chemistry, 46(3), 1101-1105

Herrera-Arellano, A., Flores-Romero, S., Chavez-Soto, M. A., & Tortoriello, J. (2004).

Effectiveness and tolerability of a standardized extract from Hibiscus sabdariffa in patients with

mild to moderate hypertension: a controlled and randomized clinical trial. Phytomedicine, 11(5),

375-382

McKay, D. L., Chen, C. O., Saltzman, E., & Blumberg, J. B. (2010). Hibiscus sabdariffa L.

tea (tisane) lowers blood pressure in prehypertensive and mildly hypertensive adults. The Journal of

nutrition, 140(2), 298-303.

Dickel, M. L., Rates, S. M. K., & Ritter, M. R. (2007). Plants popularly used for losing

weight purposes in Porto Alegre, South Brazil. Journal of Ethnopharmacology, 109(1), 60-71.

Bako, I. G., Mabrouk, M. A., & Abubakar, A. (2009). Antioxidant effect of ethanolic seed

extract of Hibiscus sabdariffa Linn (Malvaceae) alleviate the toxicity induced by chronic

administration of sodium nitrate on some haematological parameters in wistars rats. Advance

Journal of Food Science and Technology, 1(1), 39-42.

22

Ismail, A., Ikram, E. H. K., & Nazri, H. S. M. (2008). Roselle (Hibiscus sabdariffa L.)

seeds-nutritional composition, protein quality and health benefits. Food, 2(1), 1-16.

Francis, F. J., & Markakis, P. C. (1989). Food colorants: anthocyanins. Critical Reviews in

Food Science & Nutrition, 28(4), 273-314.

Wang, L. S., & Stoner, G. D. (2008). Anthocyanins and their role in cancer

prevention. Cancer Letters, 269(2), 281-290.

Patras, A., Brunton, N. P., O'Donnell, C., & Tiwari, B. K. (2010). Effect of thermal

processing on anthocyanin stability in foods; mechanisms and kinetics of degradation. Trends in

Food Science & Technology, 21(1), 3-11.

Cisse, M., Vaillant, F., Acosta, O., Dhuique-Mayer, C., & Dornier, M. (2009). Thermal

degradation kinetics of anthocyanins from blood orange, blackberry, and roselle using the arrhenius,

eyring, and ball models. Journal of Agricultural and Food Chemistry, 57(14), 6285-6291.

Brouillard, R. (1988). Flavonoids and flower colour. In The flavonoids (pp. 525-538).

Springer US.

Introduction to SAS. UCLA: Statistical Consulting Group.

from http://www.ats.ucla.edu/stat/sas/notes2/ (accessed November 24, 2007)

Da-Costa-Rocha, I., Bonnlaender, B., Sievers, H., Pischel, I., & Heinrich, M. (2014).

Hibiscus sabdariffa L.–A phytochemical and pharmacological review.Food chemistry, 165, 424-443.




23

Da-Costa-Rocha, Bonnlaender, Sievers, Pischel, & Heinrich, (2014). Hibiscus

sabdariffa L.–A phytochemical and pharmacological review.Food chemistry, 165, 424-443.

Hopkins, A. L., Lamm, M. G., Funk, J. L., & Ritenbaugh, C. (2013). Hibiscus sabdariffa L. in the

treatment of hypertension and hyperlipidemia: a comprehensive review of animal and human

studies. Fitoterapia, 85, 84-94.

24

CHAPTER III

MATERIALS AND METHODS

This work was done in the FST and HABB1 laboratories in Food Science and Technology

Department of Virginia Polytechnic Institute and State University, Virginia, United States of

America.

3.1 Plant material and water used for the extraction

The raw materials used for this study are dried calyx of Hibiscus sabdariffa L "Vimto"

variety purchased from Senegal, West Africa. The calyces were produced in some villages located in

the Department of Nioro, region of Kaolack in Senegal. The production of the calyces is done under

the supervision of qualified persons or non-governmental organizations. They were dried and packed

under the direction of APROVET, that helps the local producers of hibiscus. The samples were

purchased in May 2015. The packaging material used are large plastic bags designed so that

moisture, light, and insects cannot easily penetrate and alter calices. Another sample of Hibiscus

calyces was obtained from Egypt in March 2016. The samples were stored at room temperature in

Senegal and also in the USA. Hibiscus samples were cleaned twice. One at the Institute of Food

Technology lab in Dakar to remove contaminants and seeds. A second time at the laboratory of Food

Science and Technology Department in HABBI (Human and Agricultural Biosciences Building I),

Virginia Tech.

Distilled water (purchased in Kroger store) and formulated Dakar (Senegal) were used for juice

extraction. To formulate Dakar Water, major elements such as NaCl, MgSO4, CaCl2, NaHCO3, and

CaCO3 were added to distilled water based on their respective proportion given by OMS (OMS or

Human Health Organization water quality bulletin of the urban area of Dakar). Thus the following

25

proportions were added to volume of 6L of distilled water. Table salt (NaCl) 1.9g; epsom salt

(MgSO4) 0.7g; calcium (chloride CaCl2) 0.5g; baking soda (NaHCO3) 0.1g and chalk (CaCO3) 0.5g.

After addition of these elements, the pH was adjusted to the pH of Dakar water given by the same

source (OMS or Human Health Organization water quality bulletin of the urban area of Dakar).

3.2 Aqueous preparation and freeze-drying process.

As the calices are quiet large, before extraction they were ground in order to increase their

surface area with the water facilitating the juice extraction process. For this purpose, a Ninja brand

grinder was used. A total of two tests was performed and for both types of extraction carried out in

this study, a mass ratio of 1:15 (one kilogram of calyces per 15 liters of water) was used. Cold

extractions were done at room temperature (around 22º C) and the extraction time was set for 4

hours. A time temperature of 30 minutes for 98 º C was applied for all hot extractions. For the latter,

the Roselle calyces were always added at the boiling water and the temperature was monitored using

a thermometer.

Pots, bucket and jars and tanks were used when extracting higher volumes but to carry out smaller

extractions just lab materials such as beakers of one liter or more and funnel filter were used. Then,

all macerates were filtered using gravity filtration. In some cases, the macerate was filtered using a

stainless steel sieve of 1mm diameter in order to remove the bigger residues before performing the

finest filtration which was done using a cloth filter of 25 µm pore size.

For each test, after filtration, the parameters pH, Brix, anthocyanin content and aroma compounds of

the extract solution were immediately measured. Then the extracts solution was frozen in a -80º C

freezer (model 88600A Thermo Fisher Scientific Ashville, NC USA). The frozen extracts were then

freeze-dried for 72 hours in a Freeze-dryer (LABCONCO Kansas City, Missouri USA). At the end

26

of the freezing-drying process, the dried and instant hibiscus powder was immediately collected in

plastic bags then completely covered with aluminum foil in order to avoid the effect of the of

moisture, and the light. Anthocyanins and aroma compounds of this freeze-dried hibiscus were later

determined.

3. 3 Experiment I: Effects of Origin Water and Temperature on the Anthocyanins and Volatile of

Hibiscus.

This test consisted of the evaluation the effect of origin of hibiscus, the water used and the

extraction temperature as well as their interactions with anthocyanins and aroma compound of

hibiscus extracts and their freeze-dried products.

To do so, a 23 (two, x three) factorial experimental design was conducted in triplicate. The two levels

of each parameter being respectively for origin Egyptian vs. Senegalese; distilled water vs. Dakar

water for water and cold (22º C / 4 hours) vs. hot (98 º C / 30 mn) for temperature. The Table 6

below gives a summary of the different combinations of the three variables (source, water and

temperature). After filtration, pH, brix, anthocyanins and volatiles were measured and the rest of the

extracts was used for freeze-drying for process. After this step, anthocyanins and volatiles in the

instant powder were determined in order to compare these values to those of the fresh product.

Analyses of the experimental results were performed using Analysis of Variance (ANOVA), mean

comparisons using Tukey’s HSD and a Slice test with a level of significance of 5% (α=0.05. The

software JMP® Pro 11.0.0 statistical analysis software (SAS, Cary, NC, USA) used was.

27

Table 6. summary of the different combinations of the three variables (source, water and

temperature) for test 2.

Source Water Temperature

Egyptian Hibiscus Distilled Cold ( 22 ºC / 4 hours)



Egyptian Hibiscus Distilled Hot (98ºC / 30 mn)



Egyptian Hibiscus Dakar Water Cold ( 22 ºC / 4 hours)



Egyptian Hibiscus Dakar Water Hot (98ºC / 30 mn)



Senegalese Hibiscus Distilled Water Cold ( 22 ºC / 4 hours)



Senegalese Hibiscus Distilled Water Hot (98ºC / 30 mn)



Senegalese Hibiscus Dakar Water Cold ( 22 ºC / 4 hours)



Senegalese Hibiscus Dakar Water Hot (98ºC / 30 mn)



3. 4 Experiment II: Effects of freeze-drying on anthocyanins and volatiles of instant Hibiscus

powder obtained from Hot and Cold Extracted juice.

28

To investigate the effect of freeze-drying on hibiscus derivatives, hot and cold extraction

were conducted in three replicates each. Senegalese Hibiscus” Vimto” variety and distilled water

was used for this purpose and a ratio of 1:15 as mentioned above was applied. The following table is

a summary of the two processes.

Table 5. Extraction mode, ratio and time/temperature for test 1.

Factors Cold Extraction Hot Extraction

Samples Senegalese Hibiscus “Vimto” Senegalese Hibiscus “Vimto”

Ratio 1:15 1:15

Time/ Temperature 4 hours / 22 º C 30 mn / 98 º C

For each of the processes, after extraction, filtration was performed and then pH, brix, anthocyanins,

and volatiles measured immediately and then the process of freeze-drying initiated. The

anthocyanins and volatiles were determined on both instant powders obtained from freezing drying.

To evaluate the effect of freeze-drying on the dried products, comparison of the results from each

fresh and those from its instant powder (or freeze-drying product) was made. High-performance

liquid chromatography (HPLC) and gas chromatography–mass spectrometry (GC–MS) were used

respectively, for anthocyanins and volatile compounds measurements. All data were analyzed by

performing Analysis of Variance (ANOVA) and a level of 5% (α = 0.05) as a criterion of statistical

significance was taken into account to determine whether a variable or variables interaction have

effect or not. JMP® Pro 11.0.0 statistical analysis software (SAS, Cary, NC, USA) was used to

perform the analyses. Follow up statistical analyses were conducted on all significant effects by

performing mean comparisons using Tukey’s HSD and Slice tests.

29

3.5 pH measurement

For both tests, after extraction the pH values was measured (Fisher Scientific™ accumet™

Research AR25, Fisher Scientific, Pittsburgh, PA, US). The pH meter was calibrated using Buffer

solutions of pH 4.00 and pH 7.00.

3.6 Brix measurement

Brix measurement was performed using a Refractometer ATAGO portable.

3.7 Total anthocyanins and hibiscidin measurement by HPLC analysis

Anthocyanins are some of the compounds dissolved in n hibiscus solutio extracts. They are

constituted of elements but here we have only measured the total anthocyanins and the hibiscidin.

This was analyzed using high-performance liquid chromatography (HPLC). The specifications of

HPLC are: Agilent Technologies model 1260 series HPLC system (Santa Clara, CA), a column

25cm x 0.45cm i.d. 5µ C – C18C2 Luna column was obtained from Pheromenex (Torrex, CA) was

used and the mobile phases were composed of two solutions: solution A (99% distilled water, 1%

acetonitrile) and mobile phase B (99% distilled water, 1% acetic acid) with a flow rate of 1.0ml/min

and the Column temperature was set and maintained at 40°C.

3.8 Aroma compounds analyses by SPME and GC-MS analysis

The aroma compounds of the hibiscus juice were extracted by headspace solid-phase micro-

extraction (HS-SPME) and analyzed with gas chromatography-mass spectrometry (GC-MS). The

method described by Sheibani was used. For the extraction of the volatiles, a Shimadzu GCMS-

QP2010 Ultra equipped with an AOC-5000 Plus SPME auto-sampler (Shimadzu Scientific,

Columbia, MD, USA) was used as well as for injection into the GC-MS and identification. The

30

extraction injection and identification procedure followed the method described by Sheibani,

Duncan, Kuhn, Dietrich, Newkirk, & O'Keefe, (2015). The only difference being the column

specification. A Shimadzu SH Rxi-5MS column with 30.0m of length, 0.25µm of thickness and a

diameter of 0.25mm was used here.

31

REFERENCE

Sheibani, E. (2014). Effects of water chemistry and panning on flavor volatiles and catechins in teas

(Camelia Sinensis). Ph.D Dissertation (Advisor: Sean F. O’Keefe). Virginia Polytechnic

Institute and State University, Blacksburg, VA. 152 pp.

Sheibani, E., Duncan, S. E., Kuhn, D. D., Dietrich, A. M., Newkirk, J. J., & O'Keefe, Sean F. (2015).

Changes in flavor volatile composition of oolong tea after panning during tea processing.

Food Science & Nutrition, n/a-n/a.

OMS or Human Health Organization water quality bulletin of the urban area of Dakar

32

CHAPTER IV

RESULTS AND DISCUSSIONS

Results Experiment I

4.1 Effects of Origin, Water and Temperature on Total Anthocyanin Contents

A 2x3 factorial experimental design was applied to evaluate the effects of origin, water, and

temperature on hibiscus anthocyanins. Based on the standard curve, the determination of total

anthocyanins (in µg/mL) is shown in Table1.

33

Table 1. Total Anthocyanin contents of the samples

Origin Water Temperature

(°C)

Total Anthocyanins

(mg/mL)

Egypt Hib Distilled water Cold Ext 1659.21



Egypt Hib Distilled water Hot Ext 1247.09



Egypt Hib Dakar water Cold Ext 1622.59



Egypt Hib Dakar water Hot Ext 1208.51



Senegal Hib Distilled water Cold Ext 1293.64



Senegal Hib Distilled water Hot Ext 1292.71



Senegal Hib Dakar water Cold Ext 1422.99



Senegal Hib Dakar water Hot Ext 1391.51



A 0.05 criterion of statistical significance was used to determine whether a variable or variable

interaction has an effect or not. As shown in the JMP output in Appendix A1, the factors Origin and

Temperature were statistically significant with respective p-values of 0.0036 and 0.0025 as well as

their interaction Origin*Temperature with a p-value of 0.0002. The null hypotheses test regarding

Origin and Temperature as well as their interaction total anthocyanin contents were that there are no

treatment effects and that all treatment means were equal. As there is evidence of significant effect,

those null hypotheses were rejected. Therefore, Multiple Comparisons (post hoc tests) were

34

performed to make inferences about the individual pairs of treatment means to see which pairs of

treatment means do and do not differ significantly. A useful graphic, the LS Means Plot provides a

good way to illustrate how the group mean varies and how this interaction between Origin and

Temperature evolved. Least Squares Means being defined as a linear combination from a linear

model; (http://webpages.uidaho.edu/calsstatprog/sas/workshops/glm/lsmeans.htm). The LS Means

Plots for each main effect (Water, Origin and Temperature) as well as the interaction

Origin*Temperature plot are shown in Figure 4.1 below for a better understanding of factors

affecting the anthocyanin extraction.

Figure 1. LS Mean plot for Water (A), Origin (B), Temperature (C) and the interaction

Origin*Temperature plot (D).

As can be noticed, the plot A shows an almost straight line indicating that the amount of anthocyanin

stayed the same whether distilled water or Dakar water was used. This means that the quality of the

water did not affect the total anthocyanin contents in this case. In contrast, the plots B and C

A B

C D

http://webpages.uidaho.edu/calsstatprog/sas/workshops/glm/lsmeans.htm

35

respectively for the variable source and temperature display lines that have clear trend. A decrease of

the amount of total anthocyanins is noticed in hot extraction samples. In figure D, the two-lines of

the LS Means Plot cross over each other illustrating the interaction between Source*Temperature.

The printed line trend shows a reduction in the total anthocyanins content in Egyptian hibiscus from

cold to hot conditions but for the Senegalese hibiscus, a slight difference was noticed between cold

and hot conditions. Globally, among the overall effects, the only evidence of significant two-way

interactions was between Source and Temperature. Thus, the follow up analysis focused on that

interaction. For more details on this interaction, a pairwise comparisons on the marginal means was

performed. A Tukey's HSD “honest significant difference” procedure was used for this purpose, a

considering an error rate at a maximum of 0.05. A Tukey’s HSD is defined as a “pairwise

comparisons technique that uses the Studentized range distribution to construct simultaneous

confidence intervals for differences of all pairs of means”. In this test, different letters stand for

significant difference, (Chapter3, Lecture Analysis of Variance, Stat 5606: Biometry II, Spring 2015

Yili Hong).

Table 2. Results of the Tukey HSD for the interaction Source*Temperature on total anthocyanins of

the Egyptian and Senegalese hibiscus

Level Connecting

letter

Least Sq Mean

Egypt Hib,Cold Ext A 1561.88

Senegal Hib,Hot Ext B 1319.28

Senegal Hib,Cold Ext B 1277.20

Egypt Hib,Hot Ext B 1270.77

(Levels not connected by same letter are significantly different.)

The outcome of Tukey`s HSD test shows two different groups that are significantly different because

they are not connected by the same letter. On one hand, it shows the total anthocyanins obtained

36

with the Egyptian hibiscus juice cold extract (letter A) is different from the other samples (Senegal

Hib,Hot Ext, Senegal Hib,Cold Ext, and Egypt Hib,Hot Ext). These samples are connected by the

same letter B and with slight difference in their means indicating that they are not significantly

different from one another. The Least Square Mean of Egypt Hib,Cold Ext (1561.9 mg/L) is much

higher compared to the Least Square Mean obtained while carrying out hot extraction on the same

product (1270.8 mg/L); thus the variable temperature really has an effect on the anthocyanin

contents. Both time and temperature are important and we chose times for extractions based on

previous work. As the factors source and temperature were statistically significant as well as their

interaction Source*Temperature, a Slice test was performed in order to be able to see how each

factor evolves in this interaction upon the level the other factor. An interpretation given of a Slice

test is: ‘Comparisons among the levels of A depend on the level of B they are performed at; or

Comparisons among the levels of B depend on the level of A they are performed at”. The technique

of slicing is a procedure for which the correct terms are simple effects or simple main effects.

(http://www.ats.ucla.edu/stat/sas/library/SASSlice_os.htm accessed on July 28th 2016).

The JMP output in Appendix X shows the results of the different Slice tests indicating as can be

noticed that only Egyptian Origin has a significant effect (p-value<.0001*) and also only cold

temperature (p-value <.0001*) has significant effect on the total anthocyanin contents. However, as

we do not have a wide range of samples from Egypt to test, firm conclusion on the concentration in

Egyptian hibiscus is not possible with our data.

http://www.ats.ucla.edu/stat/sas/library/SASSlice_os.htm

37

4.2 Effects Origin, Water and Temperature on Hibiscidin contents

Hibiscus contains many kinds of anthocyanins and among them we have the delphinidin-3-

sambubioside (also known as delphinidin-3-xylosylglucoside or Hibiscidin), which constitutes the

majority (around 70%) of the anthocyanins of hibiscus. (Francis, 1973; Wang et al., 2000). That is

why its amount was evaluated in this study in order to see how it would evolve in regard to the

experiments we have carried out. The results are displayed in the following Table 3.

38

Table 3. Hibiscidin content of the samples based on the different combinations

Source Water Temperature(°C) Hibiscidin mg/L

























In Appendix B1, it can be noticed that at significance level of α = 0.05, only the interaction

Origin*Temperature has a significant effect on the hibiscin content of the samples with a p-value of

0.0001*. As for the total anthocyanins, the only significant two-way interaction was between Origin

and Temperature. Therefore, the next part of the analysis focused on that interaction between those

two variables. Here also, a Least Squares Means Plot was used for the visualization

Origin*Temperature interaction.

39

Figure 2. LS Means Plot of the interaction Source*Temperature

As can be observed in this plot, the interaction between the two variables is demonstrated by the two

lines that are not parallel, they overlap. For pairwise comparisons on the marginal means, a Tukey's

HSD test was applied as well considering an error rate at a maximum of 0.05.

Table 4. Outcome of Tukey HSD test for the interaction Source*Temperature effect on hibiscin

contents

As for Tukey`s HSD, levels not connected by same letter are significantly different; it can be noticed

that there are three different groups from this Table. Similarly to the results for the total anthocyanin

contents, the amount of hibiscin obtained in the combination Egypt Hib,Cold Ext (group A)

826.39±21.68 µg/mL and in Egypt Hib,Hot Ext (group C) 688.69±21.68 µg/mL are significantly

different. For the Senegalese hibiscus variety, the reverse situation was observed; there was no

significant difference between the amount of hibiscin got from the combination Senegal Hib,Hot Ext

and Senegal Hib,Cold (level being connected by the same letter).

Globally, for both components total anthocyanin and hibiscin, the interaction between Source and

Temperature has a significant effect on their amounts, characterized by a decrease. Additionally, the

Level (Combinations) Connecting letters Least Squares Mean

Egypt Hib,Cold Ext A 826.39

Senegal Hib,Hot Ext A B 781.13

Senegal Hib,Cold Ext B C 699.18

Egypt Hib,Hot Ext C 688.69

40

variable Temperature has also an effect on total anthocyanin contents whether hibiscus form Egypt

or hibiscus from Senegal was used and also whether distilled water or reformulated water was used

for the extraction. This decrease in the amount of total anthocyanins observed for hot extraction was

expected based on the results from previous studies. Earlier studies have shown that the anthocyanin

contents in hibiscus extracts varies depending on factors such as variety, extraction temperature,

production area, and rainfall. According to Gradinaru et al (2003), temperature contributes to the

degradation kinetics. This is in agreement Ramirez Rodrigues 2010 who, while evaluating the hue

tint (HT), (measurement of color degradation in anthocyanin containing products) in cold and hot

extracts of hibiscus, noticed that extracts obtained with cold water had lower HT values than those

obtained with hot water. This finding allowed her to conclude “temperature had an effect on hibiscus

extract color, and thus anthocyanins”. A lower HT is desirable as it indicates less anthocyanin

degradation.

Contrary to the temperature, the water chemistry did not affect the amounts of anthocyanins. The

results were almost the same whether distilled water or Dakar water was used and whatever was the

combination with the other parameters. This indicates that the hibiscus juice or concentrates

produced by small producers, mainly groups of female entrepreneurs are of good quality in terms of

eventual risk or concerns coming from the water. Hence purchasing hibiscus concentrate from those

individuals for exportation or to make hibiscus derivatives such as juice instant powder other

functional foods is a better way to promote activities around this tropical plant and as a consequence,

generate more income and reduce poverty in some rural areas.

41

Results Experiment II

4.3 Effect of Freeze-Drying on Total Anthocyanins and Hibiscin Contents using Hot Extraction

Procedure

To assess the effect of freeze-drying on total anthocyanins and hibiscin contents, Senegal

hibiscus (Vimto variety) was used and a hot extraction was conducted using three replicates. Each

extract was then divided into two parts and one part was freeze-dried. Anthocyanin contents (total

anthocyanins and hibiscin contents) were quantified by using high performance liquid

chromatography (HPLC) and the visible absorbance wavelength of 520nm on the extracts and also

on the powder obtained by freeze-drying the extract. Each experimental unit was analyzed three

times and the three replications were averaged. The Tables 5 and 6 below show, respectively, the

results for the total anthocyanins and the hibiscin contents of the samples.

Table 5. Total anthocyanin in hot and Table 6. Hibiscin contents in hot freeze-

dried hibiscus extracts and freeze-dried hibiscus extracts

determined by HPLC determined by HPLC

The data were analyzed using a One-Way ANOVA. The results showed a significant difference

between the hot extract and its freeze-dried product (p-value = 0.0013) in term of anthocyanin

content. The following plot illustrates better this significant difference between the fresh extract and

the freeze-dried extract.

Product Total Anthocyanins (mg/L)

E1 1462.0 ± 20.1

E2 1425.4 ± 20.1

E3 1458.1 ± 20.1

F E1 1333.9 ± 14.3

F E2 1348.1 ± 14.3

F E3 1319.5 ± 14.3

Product Hibiscin(mg/L)

E1 815.9 ± 15.4

E2 790.2 ± 15.4

E3 788.4 ± 15.4

F E1 732.6 ± 10.4

F E2 746.1± 10.4

F E3 753.0 ± 10.4

42

Figure 4.3 Normal Quantile Plot illustrating the difference between the hot extract and its freeze-

dried powder.

Likewise, as for the total anthocyanins, evidence of significant difference was also noticed in

hibiscidin contents between the hot extract and its freeze-dried powder. The One-Way ANOVA gave

a p-value of 0.0013*. This result, (1448.5 ± 20.1 for the fresh extract and 1333.9 ± 14.3 for the freeze-

dried powder was not expected because among the steps (freezing and freeze-drying) applied to get

the freeze-dried powder from the extract, none is known to have an effect on the anthocyanins of

hibiscus. This result might be due to the fact that hot extraction makes extracts less stable over the

time. Or perhaps, parts of the total anthocyanins was lost during the time it takes to obtain the freeze-

dried product. Earlier studies have also reported this. Cisse, Vaillant, Kane, Ndiaye, & Dornier, 2012

have demonstrated that procedures including heat and/or pasteurization make the extract less stable

and induced color changes during storage. Duangmal, Saicheua, & Sueeprasan 2008 have also

demonstrated, using CIELAB system to evaluate the color stability of freeze-dried Roselle

anthocyanins, that the total anthocyanins were responsible for the changes in Chroma. When

maltodextrin and trehalose were used as stabilizers, they noticed that anthocyanin degradation was

slowed.

43

4.4 Effect of Freeze-drying on Total Anthocyanins and Hibiscidin Contents using Cold Extraction

Procedure

The effect of freeze-drying on total anthocyanins and hibiscin content was also investigated on

Senegal hibiscus (Vimto variety). The same process (extraction, freeze-drying and measurements

using HPLC) were used except that cold extraction was realized in two replicates instead of three.

Total anthocyanins and hibiscin contents were quantified by using a HPLC with visible absorbance

spectra at 520nm. The results of the total anthocyanins and the hibiscin content are displayed in

Table 4.7 and Table 4.8

Table 5. Total anthocyanin in cold and Table 6. Hibiscin contents in cold freeze-

dried hibiscus extracts and freeze-dried hibiscus extracts

using HPLC using HPLC

As expected, results show no significant effect of freeze-drying on the anthocyanins in the cold

extract. The p-value (0.0564) is slightly greater than the criterion (0.05) taken as reference here. The

average amount of anthocyanins in the fresh extract (1466.4 ± 04) did not differ much from the

amount in the freeze-dried powder (1278.8 ± 65.8).

Also for the hibiscin content, a comparison was also made and no significant effect was observed

(p-value = 0.4457). The amount of hibiscin (848.4 ± 31.6) was close to that of the freeze-dried

powder (819.7± 29.2). These results can be justified by the fact that no heat was applied during all

the processes of extracting the juice and its as freeze-drying, which was conducted in cool

conditions.

Samples Total Anthocyanins (µg/mL)

C 1466.1 ± 0.4

C 1466.7 ± 0.4

F C 1232.2 ± 65.8

F C 1325.3 ± 65.8

Products Hibiscin (µg/mL)

C 826.1 ± 31.6

C 870.7 ± 31.6

F C 799.1 ± 29.2

F C 840.4 ± 29.2

44

4.5 pH and the Brix in hibiscus extracts

For all experiments carried out in this study, pH and Brix measurements were recorded just

after extraction. The pH values were constant for all extracts (cold, hot and the 2 x 3 factorial design

combining water, source and temperature) with values around 2.4. This can be explained by the fact

that that hibiscus calyces are very acidic. Also the ratio 1:15 used for all the extraction procedure,

did not provide much water that could have made easy some chemical reactions such as protonation

that could affect the pH. For the Senegalese hibiscus extracts, the Brix results, on average were 4.1

for the hot and 3.4 for the cold. In the 2 x 3 factorial experiment also, the same phenomenon was

observed. As can be seen in the Table 4.9 below, the soluble extract was less when cold extract was

made and higher in hot, despite the presence of the two other variables (temperature and source). So

whatever the combined variables were, still the highest were for hot and the lowest values for the

cold. The highest amount of dry matter in the hot extract can be explained by the fact that when

extraction is carried using high temperatures, the heat makes easy the disintegration of some soluble

particles and this increase their quantity in the juice.

45

Table 4.9 Brix in the 2 x 3 factorial experiment on Egyptian and Senegalese hibiscus

Source Water Temperature Brix

























4.6 Effects of freeze-drying on the volatiles of Instant hibiscus powder obtained from cold

extraction.

Gas Chromatography - Mass Spectrometry (GC-MS) was used to identify the aroma

compounds present in the cold hibiscus extract and its freeze-dried powder. Mass spectra were

compared to NIST and Wiley libraries. Also, hydrocarbon standards (C5, C6, C7, C8, C10, C12,

C14, C16, C18, C20, C22) were run to calculate Kovats Indexes, also known as LRI, Linear

46

Retention Index, to facilitate identification of the aroma compounds. The identification of the

different flavor compounds was facilitated by consulting online databases Flavornet at

http://www.flavornet.org/flavornet.html and the Pherobase at http://www.pherobase.com/. Twenty

components were identified in the fresh, cold extract while only eight were identified in its freeze-

dried powder as can be observed in Table 4.10 and Table 4.11. This big difference in aromas

composition content between the two products can be justify by the different in soluble solids

present in the both product while running the GC-MS analyses. After freeze-drying the amount of

instant powder weighed to reconstituted solution did not generate the same amount of soluble solid

content as that of the fresh extract. This limiting factor may be the reason of the small amount of

aroma in the instant powder.

http://www.pherobase.com/

47

Table 10. Volatiles in cold Table 10. Volatiles in cold and

hibiscus extracts freeze-dried hibiscus extracts

determined by using GC-MS determined by using GC-MS Aroma compounds identified in

Cold Extract

alpha-terpineol

1-Octen-3-one

5-Methyl-2-Furancarboxaldehyde

(E)-2-Nonenal

(E-)-2-Octenal

6,10-dimethyl-5,9-Undecadien-2-one

5-6-methyl-Hepten-2-one

5-Hydroxymethylfurfural

Decanal

Eucalyptol

Furfural

Glycerol 1,2-diacetate

Heptanal

Hexanal

Acetic acid

Nonanal

Octanal

Oxime-, methoxy-phenyl-

2-methoxy-3-(2-propenyl)-Phenol

2-Decanone

Among the volatiles, three (furfural, hexanal and nonanal) were found in the cold extract and its

instant powder. The comparison of their amount of these three volatiles present in each product was

realized by comparing their peak areas. From the One Way ANOVA analysis, a significant

difference was noticed between the two products. Their comparison showed p-values that are lower

than 0.05 for all of the three volatiles allowing to conclude there is evidence of significant difference

of their concentration in the cold extract and its freeze-dried powder. The following Table 4.12

combines the different volatiles in both fresh cold extract and its instant powder as well as the p-

values of the JMP output.

Aroma in Freeze-dried Cold Extract


Furfural

Hexanal

Nonanal

Acetic acid

2-Methyl-Benzaldehyde

Dodecane

Hexanoic acid, hexyl ester

48

Table 10. Volatiles in cold and freeze-dried hibiscus extracts determined by using GC-MS

Volatiles names Cold Extract

Freeze-dried

Cold Extract p-value

alpha-Terpineol X

1-Octen-3-one X

5-methyl-2-Furancarboxaldehyde X X

(E)-2-Nonenal X

(E)-2-Octenal X

6,10-dimethyl-5,9-Undecadien-2-one X

6-methyl-5-Hepten-2-one X

5-Hydroxymethylfurfural X

Decanal X

Eucalyptol X

Furfural X X 6.13E-05

Glycerol 1,2-diacetate X

Heptanal X

Hexanal X X 0.0037

Nonanal X X 0.0047

Octanal X

Oxime-, methoxy-phenyl-_ X

Phenol, 2-methoxy-3-(2-propenyl)- X

2-Decanone X

Acetic acid X X

2-Methyl-Benzaldehyde X X

Dodecane X X

Hexanoic acid, hexyl ester X X

4.7 Effects of freeze-drying on the volatiles of instant hibiscus powder obtained from hot extracted

juice

Similar to the cold extracts, aroma compounds present in the hot hibiscus extracted juice and

its freeze-dried powder were identified using a GC-MS. Twenty-seven compounds were identified in

the fresh hot extract and twenty-three in its instant powder. Tables 4.12 and Table 4.13 below show

those different aroma components.

49

Table 12. Volatiles in hot Table 13. Volatiles in hot

hibiscus extracts determined freeze-dried hibiscus extracts

by using GC-MS determined by using GC-MS

Aroma in Hot Extract

alpha-Terpineol


(E)-2-Nonenal

(E)-2-Octenal


6-methyl-5-Hepten-2-one


Decanal

Eucalyptol

Furfural

Heptanal

Hexanal

Nonanal

Octanal

Oxime-, methoxy-phenyl-

2-methoxy-3-(2-propenyl)-Phenol

Acetic acid

Hexanoic acid, hexyl ester

1-Nonanol

1-Octen-3-ol

(E,Z)-2,6-Nonadienal

(E)-2-Heptenal

(E)-2-Tridecenal

tert-butyl-Benzene

Benzeneacetaldehyde

D-Limonene

Furyl hydroxymethyl ketone

The results show that many all the aroma compounds detected in the hot extract were also detected

in the instant powder obtained by from freeze-drying the same extract. Statistical analysis was

performed by a One Way ANOVA to do the comparison of their respective areas in each product.

The results are shown in the Table 4.14 below.

Aroma compounds in freeze-dried Hot

Extract

alpha-Terpineol


(E)-2-Nonenal

(E)-2-Octenal


6-methyl-5-Hepten-2-one


Decanal

Furfural

Hexanal

Nonanal

Octanal

Oxime-, methoxy-phenyl-_

Phenol, 2-methoxy-3-(2-propenyl)-

Acetic acid

(E,Z)-2,6-Nonadienal

(E)-2-Heptenal

Benzene acetaldehyde

(E,E)-2,4-Nonadienal

Dodecanal

Nonanoic acid

Butanoic acid

Dimethyl phosphine

50

Table 10. Volatiles in hot and freeze-dried hibiscus extracts determined by using GC-MS and the p-

values of the Volatiles present in both of the products

Volatiles names Hot Extract

Freeze-dried

Hot Extract p- values

alpha-Terpineol X X 0.0276

5-Methyl-2-Furancarboxaldehyde X X 0.5598

(E)-2-Nonenal X X 0.0199

(E)-2-Octenal X X 0.0109

6,10-dimethyl-5,9-Undecadien-2-one X X 0.5345

6-methyl-5-Hepten-2-one X X 0.38785

5-Hydroxymethylfurfural X X 0.2160

Decanal X X 0.5269

Eucalyptol X

Furfural X X 0.9153

Heptanal X

Hexanal X X 0.0563

Nonanal X X 0.0103

Octanal X X 0.0093

Oxime-, methoxy-phenyl- X X 0.00689

2-methoxy-3-(2-propenyl)-Phenol X X 0.2140

Acetic acid X X 0.0249

Hexanoic acid, hexyl ester X

1-Nonanol X

1-Octen-3-ol X

5-methyl-2-Furanone X

(E,Z)-2,6-Nonadienal X X 0.8452498

(E)-2-Heptenal X X

(E)-2-Tridecenal X

tert-butyl-Benzene X

Benzeneacetaldehyde X X 0.3440548

D-Limonene X

Furyl hydroxymethyl ketone X

2-ethyl-1-Hexanol

X

(E,E)-2,4-Nonadienal

X

2-Furancarboxylic acid

X

Dodecanal

X

Nonanoic acid

X

51

Many of the aroma components detected in the samples and the procedure used in this study have

been identified in previous studies. Among the aroma compounds we observed, the following were

previously reported (hexanal, heptanal, limonene, octanal, nonanal, 1-octen-3-ol, acetic acid,

decanal, (E)-2-nonenal (Ramirez‐Rodrigues & others, 2011; Gonzalez-Palomares & others- 2009

and Chen, Huang, Ho, & Tsai, 1998).

As mentioned earlier in the literature review, many authors have found that differences in the

number of aroma compounds in the hibiscus or dried hibiscus can be related to factors such as heat

treatment, variety, and extraction methods used to isolate the volatile (solvent used, time, solute

concentration) (Ramirez‐Rodrigues, Plaza, Azeredo, Balaban, & Marshall, 2011;

Ramírez‐Rodrigues, 2010; Chen, Huang, Ho, & Tsai, 1998; Gonzalez-Palomares, Estarrón-Espinosa,

Gómez-Leyva, & Andrade-González, 2009).

Table 4.15 summary of all the aroma compounds found in the four different samples, the calculated

Kovats Indexes and similarity in the literature

Compound Cold Extract

Freeze-dried cold Extract

Hot Extract

Freeze-dried hot Extract

LRI (DB5) Odor description

Identied in the literature

Acetic acid X X X X 660 alkaline C

Hexanal X X X X 822 truffle, earth, nut AC

Furfural X X X X 838 ABC

Oxime-, methoxy-phenyl- X X X 898

sulfur, onion, meat

Heptanal X X 903 fat, citrus, rancid AC

(E)-2-Heptenal X X 959 AC 5-Methyl-2-furancarboxaldehyde X X X X 967 cooked meat

1-Octen-3-ol X 980 phenol C

1-Octen-3-one X 981

6-Methyl-5-hepten-2-one X X X 988 earth, herb C

Octanal X X X 1005 AC

52

tert-butyl-Benzene X 1030 citrus, mint

D-Limonene X 1035 Citrus, lemon

Benzeneacetaldehyde X X 1048 sweat

(E)-2-Octenal X X X 1060 green, nut, fat C

2-Methylbenzaldehyde X X 1086 roast Furyl hydroxymethyl ketone X 1086 roast

Nonanal X X X X 1106 AC

Hexanoic acid, hexyl ester X X X 1109 green, flower

(E,Z)-2,6-Nonadienal X X 1156

(E)-2-Nonenal X X X 1162

cucumber, fat, green C

1-Nonanol X 1172 flower C

Dodecane X X 1192 fruit, phenol

alpha-Terpineol X X X 1198 fruit, fat ABC

Decanal X X X 1207 BC

(E,E)-2,4-Nonadienal X

1217 fat, wax, green C

5-Hydroxymethylfurfural X X X 1226

Nonanoic acid X X 1263 mint, sweet

(E)-2-Tridecenal X 1264

Glycerol 1,2-diacetate X 1347 2-Methoxy-3-(2-propenyl)-phenol (eugenol) X X 1365 Turpentine, cloves

Dodecanal X 1411 green, spearmint 6,10-Dimethyl-5,9-undecadien-2-one X X X 1457

KI = Kovats Index.

A = Components earlier identified by Chen, Huang, Ho, & Tsai, 1998

B = Components earlier identified by Gonzalez-Palomares, Estarrón-Espinosa, Gómez-Leyva, &

Andrade-González, 2009

C = Components earlier identified by Ramirez‐Rodrigues, Plaza, Azeredo, Balaban, & Marshall,

2011

53

REFERENCES

http://webpages.uidaho.edu/calsstatprog/sas/workshops/glm/lsmeans.htm accessed on July

2016

Chapter3, Lecture Analysis of Variance, Stat 5606: Biometry II, Spring 2015 Yili Hong).

http://www.ats.ucla.edu/stat/sas/library/SASSlice_os.htm accessed on July 28th 2016.




Rodrigues, M. M. R. (2010). Processing Hibiscus Beverage Using Dense Phase Carbon

Dioxide. University of Florida

Cissé, M., Bohuon, P., Sambe, F., Kane, C., Sakho, M., & Dornier, M. (2012). Aqueous

extraction of anthocyanins from Hibiscus sabdariffa: Experimental kinetics and modeling. Journal of

Food Engineering, 109(1), 16-21.

Duangmal, K., Saicheua, B., & Sueeprasan, S. (2008). Colour evaluation of freeze-dried

roselle extract as a natural food colorant in a model system of a drink. LWT-Food Science and

Technology, 41(8), 1437-1445.

online databases Flavornet at http://www.flavornet.org/flavornet.html accessed on July 8th

2016

Pherobase at http://www.pherobase.com/. on July 8th 2016

Ramirez‐Rodrigues, M. M., Plaza, M. L., Azeredo, A., Balaban, M. O., & Marshall, M. R.

(2011). Physicochemical and phytochemical properties of cold and hot water extraction from

Hibiscus sabdariffa. Journal of food science, 76(3), C428-C435.

http://webpages.uidaho.edu/calsstatprog/sas/workshops/glm/lsmeans.htm

http://www.ats.ucla.edu/stat/sas/library/SASSlice_os.htm

http://www.pherobase.com/

54

Gonzalez-Palomares, S., Estarrón-Espinosa, M., Gómez-Leyva, J. F., & Andrade-González,

I. (2009). Effect of the temperature on the spray drying of roselle extracts (Hibiscus sabdariffa

L.). Plant foods for human nutrition,64(1), 62-67.

Chen, S. H., Huang, T. C., Ho, C. T., & Tsai, P. J. (1998). Extraction, analysis, and study on

the volatiles in roselle tea. Journal of Agricultural and Food Chemistry, 46(3), 1101-1105

55

CHAPTER V

CONCLUSIONS

This work focused on evaluating the effect of parameters such water, heat and origin on

hibiscus extracts and on instant powder obtained by freeze-drying part of the extracts. Parameters

such as anthocyanins as well as volatile components were determined. For the anthocyanin contents,

the variable temperature has a significant effect whether hibiscus was from Egypt or from Senegal

and also whether distilled water or reformulated water was used for the extraction. The anthocyanin

amount was higher when hot extraction was carried out. The variable Origin also impacted the

anthocyanin contents which was much more present in the Egyptian hibiscus. Although we do not

have evidence that this is true for all Egyptian hibiscus. Evidence of significant interaction effect

was noticed between these two variables on the anthocyanin content showing higher amount when

the combination Egyptian variety and cold extraction was applied. Contrary to the variables

temperature and source, the water chemistry did not affect the amounts of anthocyanins. The results

were almost the same whether distilled water or Dakar water (reformulated) was used and whatever

was the combination with the other parameters. There was no significant effect of freeze-drying on

the anthocyanins in the Cold Extract as expected. But in contrast to this, a difference was noticed in

anthocyanin contents between the hot extract and its freeze-dried powder. This result, was not

expected because among the steps (freezing and freeze-drying) applied to get the freeze-dried

powder from the extract, none is known to have an effect on the anthocyanins of hibiscus. This result

might be due to the fact that hot extraction makes extracts less stable over the time. Earlier studies

have also reported that anthocyanin were less stable Cisse et al 2012, Duangmal, Saicheua, &

Sueeprasan 2008). The pH was almost the same in all extracts. The soluble extract was less when

56

cold extract was made and higher in hot despite the presence of the two other variables (temperature

and source). This can be explained by the fact that heat makes easy the disintegration of some

soluble particles and this increase their quantity in the juice. GC-MS analysis of the volatiles showed

different profiles when comparing cold and hot extracts to their respective instant powder as well as

when comparing hot to dry extract. But many of the aroma compounds were found in both hot and

cold extraction. Globally, the results of this study can help in the optimization when processing

hibiscus derivatives such as juices, concentrates and instant powder in regards of the growing

interest worldwide in functional food product like hibiscus. The latter is a ready to use and easy to

commercialize and would be a good alternative for industrial as far as hibiscus has many potential

applications in the food, pharmaceutical, cosmetic industries, etc.

Expanding these investigations on hibiscus powder obtained from spray-drying and monitor the self-

life of both dried products over the time when stored at specific temperatures in future work can help

in activities carried around hibiscus. Likewise, the evaluation of consumer acceptability on the

different products through sensory evaluation tests also need to be done.

57

APPENDICES

APPENDIX A – 1

Figure A1 – Effect Tests of Effects of Source, Water and Temperature on the total anthocyanins content of

the samples

Least Squares Means Table Level Least Sq

Mean

Std Error

Egypt Hib,Cold Ext 1561.8828 34.717462

Egypt Hib,Hot Ext 1270.7760 34.717462

Senegal Hib,Cold Ext 1277.2046 34.717462

Senegal Hib,Hot Ext 1319.2830 34.717462

LS Means Plot

Appendix A – 2 Figure A2 – Least Squares Means Table and least square means plots of the

interaction Source*Temperature on total anthocyanins content

58

Appendix A – 3 Figure A3 – Slice source =Egyptian Hib

Appendix A – 4 Figure A4 – Slice source =Senegal Hib

Appendix A – 5 Figure A5 – Slice Temperature (°C)=Cold Ext

59

Appendix A – 6 Figure A6 Slice Temperature (°C)=Hot Ext

60

APPENDIX B– 1

Source Nparm DF Sum of

Squares

F Ratio Prob > F

Source 1 1 1813.124 0.6429 0.4344

Water 1 1 743.056 0.2635 0.6148

Temperature 1 1 4660.211 1.6523 0.2169

Source*Water 1 1 5343.255 1.8945 0.1877

Source*Temperature 1 1 72369.683 25.6591 0.0001*

Water*Temperature 1 1 950.796 0.3371 0.5696

Source*Water*Temperature 1 1 2401.854 0.8516 0.3698

Figure B1 – Effect Tests of Effects of Source, Water and Temperature on the Hibiscidin content

Least Squares Means Table Level Least Sq

Mean

Std Error

Egypt Hib,Cold Ext 826.39169 21.681117

Egypt Hib,Hot Ext 688.69694 21.681117

Senegal Hib,Cold Ext 699.18278 21.681117

Senegal Hib,Hot Ext 781.13879 21.681117

LS Means Plot

Figure B2 – Least Squares Means Table and least square means plots of the interaction

Source*Temperature on Hibiscidin content

APPENDIX C – 1 Analysis of Variance

Source DF Sum of

Squares

Mean Square F Ratio Prob > F

Product 1 19723.813 19723.8 64.7981 0.0013*

Error 4 1217.555 304.4

C. Total 5 20941.368

Means for Oneway Anova

Level Number Mean Std Error Lower 95% Upper 95%

E 3 1448.54 10.073 1420.6 1476.5

F E 3 1333.87 10.073 1305.9 1361.8

Std Error uses a pooled estimate of error variance

Figure C1 – Results of Total Anthocyanin the Senegalese Hot extraction and its freeze-dried powder.

61

APPENDIX D – 1

Analysis of Variance Source DF Sum of

Squares


Product 1 4414.9735 4414.97 25.7004 0.0071*

Error 4 687.1460 171.79

C. Total 5 5102.1195

Means for Oneway Anova Level Number Mean Std Error Lower 95% Upper 95%

E 3 798.148 7.5672 777.14 819.16

F E 3 743.896 7.5672 722.89 764.91 Std Error uses a pooled estimate of error variance

Figure D1 – Results of Hibiscidin of the Senegalese Hot extraction and its freeze-dried powder

APPENDIX E – 1


Squares


Samples 1 35211.772 35211.8 16.2452 0.0564

Error 2 4335.026 2167.5

C. Total 3 39546.798


C 2 1466.43 32.920 1324.8 1608.1

F C 2 1278.79 32.920 1137.1 1420.4

Std Error uses a pooled estimate of error variance

Figure E1 – Results of Total Anthocyanin the Senegalese Cold extraction and its freeze-dried powder

APPENDIX F – 1


Squares


Products 1 822.0836 822.084 0.8872 0.4457

Error 2 1853.2054 926.603

C. Total 3 2675.2890


C 2 848.420 21.524 755.81 941.03

F C 2 819.748 21.524 727.14 912.36 Std Error uses a pooled estimate of error variance

Figure F1 – Results of Hibiscin of the Senegalese Cold extraction and its freeze-dried powder

impacts of water, extraction procedure and origin on anthocyanins and ... - virginia tech · 2020....

Documents