comparative study of the effects of ultraviolet light and high hydrostatic pressure on the quality
TRANSCRIPT
Comparative Study of the Effects of Ultraviolet Light and High Hydrostatic
Pressure on the Quality and Health Related Constituents of Wheatgrass Juice
by
Nagwa Ali
A Thesis
presented to
The University of Guelph
In partial fulfilment of requirements
for the degree of
Master of Science
in
Food Science
Guelph, Ontario, Canada
©Nagwa Ali, November, 2016
ABSTRACT
COMPARATIVE STUDY OF THE EFFECT OF ULTRAVIOLET LIGHT AND HIGH
HYDROSTATIC PRESSURE ON THE QUALITY AND HEALTH RELATED
CONSTITUENTS OF WHEATGRASS JUICE
Nagwa Ali Advisory Committee:
University of Guelph, 2016 Dr. Keith Warriner
Dr. Tatiana Koutchma
The perceived health benefits of low acid juices have resulted in increased demands for vegetable
beverages such as wheatgrass. The following reports on a comparative study to evaluate
Ultraviolet Light UV-C or High Hydrostatic Pressure HHP as alternative non-thermal methods for
wheatgrass juice. A thermal treatment of wheatgrass at 75°C for 15s was included as control.
Pressure treatments of 500MPa and 600MPa for 60, 90 and 180s supported 5-log CFU reduction
of bacteria inoculated into wheatgrass juice. To achieve the same level of bacterial inactivation, a
UV dose of 25.4mJ/cm2 at 254 nm was required. The UV and HHP treatments significantly
increased the chlorophyll of juice. Both HTST and HHP treatments resulted in negligible losses in
antioxidants, but UV preserved TPC and antioxidants. HHP treatment did not have a significant
reduce in color and enzymes levels. The study illustrated that HHP would be preferred non-thermal
treatment for treating wheatgrass juice.
iii
ACKNOWLEDGMENTS
First, I wholeheartedly express my thanks to Allah, who helped me and was my guide in every
successful step in my life. I am indebted also to my parents, Dkhiel and Kadija, for their
unconditional and endless love. Their understanding, trust, and guidance are essential in my quest
for a science career. Warm thanks to my lovely husband, Ahmed, as he always supports me and
gives all kinds of encouragement during my studies and life. Big thanks to my only sister Ahlam
and my brothers, Fathi, Ali and Mohammed. Special thanks to my sisters in law Wafa, Hana and
Fadwa. Also I cannot forget my friend, Asma, who is more than sister to me.
I would like to acknowledge many people sincerely for helping and supporting me during my
research. Thanks and appreciations especially go to my advisors, Dr. Keith Warriner, who has
guided me on each step: research process, scientific thinking and writing. I am very grateful for
the opportunity to do my master’s degree under his tutelage. Without his encouragement, I do not
know if I could go further in academics. I would also like to thank Dr. Tatiana Koutchma from
Agriculture and Agri-Food Canada. Thank you for your invaluable suggestions and discussions
and your serving on my committee.
My many thanks must also go to Fan Wu and Vladimir Popovic, who have helped me so much
during my research processing and for their willingness to share extensive knowledge of research
with me and for their technical assistance.
I extend my thanks to Libyan government for providing me great opportunity to complete my
study in Canada.
iv
TABLE OF CONTENTS
ABSTRACT……………………………………………………………………………………………… ....ii
ACKNOWLEDGMENTS………………………………………………………………………………......iii
LIST OF TABLES……………………………………………………………………………………..........vi
LIST OF FIGURES……………………………………………………...……………………...………....viii
CHAPTER 1…………………………………………………………………...………………………….....1
1.1. Introduction………………………………………………………….………………………………....1
Hypothesis and Objectives……………………………………………….……………………………..….2
1.2. Literature Review…………………………………………………………………….…………...…...3
1.2.1. Canadian / USA vegetable and fruit juices market value…………………….……………….…...3
1.2.2. Wheatgrass juice…………………………………………………………….….……………........4
1.2.2.1. Wheatgrass juice properties and contents……………………………….….………………...5
1.2.3. Human Pathogens in low acid juices………………………………………….….………….........9
1.2.4. Outbreaks of foodborne illness associated with the consumption of unpasteurized and/or low acid
fruits and vegetables juice……………………………………………………..…………….…...10
1.2.5. Regulatory requirements: Hazards Analysis Critical Control Point of juice (HACCP)……........12
1.3. Juice Treatment Technologies……………………..…………………………………………..…....13
1.3.1. Thermal treatment……………….……………………….……………………………….…....13
1.3.1.1. Effects of pasteurization on liquid foods….…………………………………………….…..15
Effects of heat on microorganisms and enzymes of juices……………………………….…...15
Effect of heat on nutritional and sensory characteristics of juices…………………………….15
1.3.1.2. Advantages and disadvantages of thermal treatment………………………………………..16
1.3.2. Non-thermal technologies for food treatment…………………………………………….......16 1.3.2.1. High Hydrostatic Pressure technology……..……………………………………………......16
Main Components of HHP Units….……………………………………………………….......18
Batch operation…………………………………………………………………………….......19
Principles of HHP………………………………………………………………………...........20
1.3.2.1.1. Effects of High Hydrostatic Pressure treatment………………………………….......20
HHP Effects on microorganisms in fruit and vegetable juices……………………………20
Effect of high pressure on the physical and chemical characteristics of food systems…….22
1.3.2.1.2. Advantages and limitations of HHP treatment……………………………………….23
1.3.2.2. Ultraviolet light treatment……………………………………………………………….......24
Application and Sources of UV light………………………………………………………..26
Collimated Beam…………………………………………………………………………….27
UV Reactor designs………………………………………………………………………….28
1.3.2.2.1. Effects of UV-C light treatment on food…………………….……………………30
Effects on microorganisms in liquid foods………………………………………......30
Effects on nutritional quality and enzymes of juices…………………………………31
v
1.3.2.2.2. Advantages and disadvantages of UV-C treatment…………………………….......32
CHAPTER 2.……………………………………………………………………………………………..34
2. Materials and Methods………………………………………………………………………………..34
2.1. Chemicals…………………………………………………………………………………………......34
2.2. Wheatgrass Juice Extraction……………………………………………………………………….....34
2.2.1. Experimental materials……………………………………………………………………………..34
2.2.2. Juice Extraction…………………………………..…………………………………………………35
2.3. Physical and chemical analysis of untreated wheatgrass juice……………………………………….36
2.4. Thermal and Non-thermal treatments of wheatgrass juice……………..………………………...42
2.4.1. Thermal treatment (HTST)……………………………………………………………....42
2.4.2. Non-thermal treatment…………………………………………………………………...43
2.4.2.1. High Hydrostatic Pressure treatment…………………………………………………....43
2.4.2.2. UV-C Light Parameters for wheatgrass juice treatment………………………………...44
Collimated beam and experiment set up ……………………………………………………….....44
Dean Flow Reactor and experiment set up………………………………………………………..47
2.5. Microbes and cultivation methods……………………………………………………………….....51
2.5.1. Escherichia coli cultivation and enumeration……………………………………………...51
2.5.2. Salmonella Typhimurium WG49 cultivation and enumeration…………………..………...52
2.5.3. Listeria Innocua cultivation and enumeration……………………………………………..52
2.6. Preparation of wheatgrass juice sample for different treatments to analyze the nutrients…….53
2.6.1. Pasteurization HTST…………………………………………………………………….....53
2.6.2. Non-thermal treatment ………………………………………………………………….....53
2.6.2.1. High Hydrostatic Pressure …………………………………………………………….53
2.6.2.2. UV-C treatment ………………………………………………………………………..53
2.7. Experimental Design and Statistics ………………………………………………………………..54
2.7.1. Microbial counts……………………………………………………………………..…......54
2.7.2. Statistics of physiochemical analysis of wheatgrass juice nutrients ……………..……..54
CHAPTER 3………………………………………………………………………………………………..55
3. Results………………………………………….………………………………………………….…...55
CHAPTER 4…………………………………………………………………………………………….….80
4. Discussion…………………………………………..…………………………………………….……80
CHAPTER 5 Conclusion …………………………………………………………………………………98
Future work…………………………………………………………..…………………………………..100
References……………………………………………………………..………………………………….101
vi
LIST OF TABLES
Table 1.1- Levels of vitamins and minerals in 100 mL of wheatgrass juice and the contents of
amino acids in mL of wheatgrass juice……………………………………………………6
Table 1.2- Outbreaks linked to unpasteurized and/or low acid juices during the period of 1974–
2010 in the USA and Canada……………………………………………………………..11
Table 1.3- Some types of thermal treatment applications…………………..……………………14
Table 1.4- Heat resistance of selected pathogens…………………………………………………15
Table 2.1- Processing parameters for the wheatgrass juice extraction from the raw material…….35
Table 2.2- Parameters of the collimated beam during wheatgrass juice treatment……………….45
Table 2.3- Technical characteristic of the Dean Flow reactor…………………………………..48
Table 2.4- Overview of the different UV-C treatments at a flow rate of 2.6 cm3/s………………..51
Table 3.1- Physical and chemical properties of wheatgrass juice from Evergreen company (Don Mills
ON, Canada) ……………………………..………………………………………….…….56
Table 3.2- Microbial inactivation for inoculated wheatgrass juice with different bacteria after
thermal treatment……………………………………………………………………...…58
Table 3.3- D values (s) achieved 1-log reduction for different microbes in wheatgrass juice after
HHP treatment at 400 MPa, 500 MPa and 600MPa for 60, 90 and 180s…………………62
Table 3.4- D values (mJ/cm2) achieved 1 log reduction for different microbes in wheatgrass
juice………………………………………………………………………………………64
Table 3.5- Effect of UV dose on inactivation of different microbes in wheatgrass juice using
collimated beam…………………………………………………………………………65
Table 3.6- D values (mJ/cm2) achieved 1 log reduction for different microbes in wheatgrass juice
after Dean flow UV treatment…………………………………………………………….68
Table 3.7- Effects of pasteurization on the nutritional quality of wheatgrass juice……………..71
Table 3.8- Effects of HHP treatments on pH, TSS and TA of wheatgrass juice…………...……73
Table 3.9- Effects of HHP treatments vitamin C, Chlorophyll and protein of wheatgrass
juice…................................................................................................................................74
Table 3.10- Effects of HHP treatments on the TPC and antioxidants activity of wheatgrass
juice………………………………………………………………………………………75
Table 3.11- Effects of HHP treatments on color of wheatgrass juice…………………………...76
Table 3.12- Effects of UV-C treatment nutritional quality of wheatgrass juice………………….78
vii
Table 4.1- Summary of physical and chemical properties values of wheatgrass juice after heat,
HHP and UV treatments…………………………………………………………………97
viii
LIST OF FIGURES
Figure 1.1- Structure of hemoglobin and chlorophyll……………………………………….……7
Figure 1.2- HHP unit used for commercial operations……………………………………………18
Figure 1.3- Schematic of batch High-pressure processing system……………………………...19
Figure 1.4- Process of batch operation in HHP………………………………………………..…19
Figure 1.5- The different wavelengths of light and UV kinds…………………………………….26
Figure 1.6- Example of bench scale devices for conducting UV experiments……………………28
Figure 1.7- Schematics of (a) a laminar Taylor-Couette UV reactor and (b) a laminar thin film
reactor (Cider Sure)………………………………………………………………………29
Figure 1.8- Schematics of turbulent channel reactor (a) and Dean flow reactor (b)…………….29
Figure 1.9- The effectiveness of UVC on the DNA structure in the microorganisms……………..31
Figure 2.1- The juice fountain compact juicer used for wheatgrass juice extraction……………...36
Figure 2.2-Wheatgrass juice used in all experiments……………………………………………..36
Figure 2.3- Schematic graph of Collimated beam instrument……………………………….…..46
Figure 2.4- The quartz coil of UV-C reactor used for treatment of wheatgrass juice…………….48
Figure 2.5- Dean Flow reactor experiment set up……………………………………………..…49
Figure 3.1- Wheatgrass juice extraction from raw materials with and without pectinase enzyme at
different incubation times………………………………………………………………...55
Figure 3.2- Time/Temperature monitoring of wheatgrass juice during thermal treatment……….57
Figure 3.3- Microbial inactivation curve for inoculated wheatgrass juice with Listeria innocua
after HHP Treatment…………………………………………………………………..…59
Figure 3.4- Microbial inactivation curve for inoculated wheatgrass juice with Salmonella WG 49
after HHP Treatment……………………………………………………..………………60
Figure 3.5- Microbial inactivation curve for inoculated wheatgrass juice with E. coli P36 after
HHP treatment…………………………………………………………………………...61
Figure 3.6- Pressure and time response of E.coli P36, listeria innocua and salmonella WG49 in
wheatgrass juice after HHP treatment at 600MPa………………………………………..62
Figure 3.7- Pressure and time response of E.coli P36, listeria innocua and salmonella WG49 in
wheatgrass juice after HHP treatment at 500MPa………………………………………63
ix
Figure 3.8- Pressure and time response of E.coli P36, listeria innocua and salmonella WG49 in
wheatgrass juice after HHP treatment at 400MPa………………………………………..63
Figure 3.9- D value calculation for E.coli in wheatgrass juice with different UV doses using
collimated beam……………………………………………………………….…………65
Figure 3.10- D value calculation for Listeria innocua in wheatgrass juice with different UV doses
using collimated beam……………………………………………………………………66
Figure 3.11- D value calculation for Salmonella WG49 in wheatgrass juice with different UV doses
using collimated beam…………………………………………………..……………….66
Figure 3.12- UV-Dose response of E.coli P36, listeria innocua and salmonella WG49 in
wheatgrass juice using collimated beam……………………………………………...…67
Figure 3.13- D values calculation for E. coli after Dean flow UV treatment (Quartz coli
reactor)…………………………………………………………………………………...68
Figure 3.14- D values calculation for salmonella after Dean flow UV treatment (Quartz coli
reactor)………………………………………………………………………………...…68
Figure 3.15- D values calculation for Listeria innocua after Dean flow UV treatment (Quartz coli
reactor)……………………………………………………………………………….…..69
Figure: 3.16- Effects of UV reactor upon inactivation of Listeria innocua, Salmonella WG 49 and
E. coli P36…………………………………………………………………………..……69
Figure 3.17- Residual activity percentage of POD and PPO enzymes after thermal
treatment………………………………………………………………………………....72
Figure 3.18- Residual activity percentage of PPO enzyme in wheatgrass juice after HHP
treatment……………………………………………………………………………...….77
Figure 3.19- Residual activity percentage of POD enzyme in wheatgrass juice after HHP
treatment………………………………………………………………………….…… ..77
Figure 3.20- Residual activity percentage of POD and PPO enzymes in wheatgrass juice after UV
treatment………………………………………………………………………………....79
Figure 4.1- Microbial inactivation of three tested bacteria after heat, HHP and UV treatments...84
Figure 4.2- Comparison of the residual contents of vitamin C, chlorophyll and protein in
wheatgrass juice after different treatments………………………………………………89
Figure 4.3- Comparison of the residual contents of TPC and antioxidants in wheatgrass juice after
different treatments……………………………………………………………………...92
Figure 4.4-Comparision of the residual contents of color values (L*, a* and b*) in wheatgrass juice
after different treatments………………………………………………………………....94
Figure 4.5- Comparison of the residual contents of POD and PPO enzymes in wheatgrass juice
after different treatments…………………………………………………………………96
1
CHAPTER 1
1.1 Introduction
Health organizations continue to highlight the importance of increasing the intake of fresh fruits
and vegetables in the daily diet to prevent chronic conditions (WHO, 2015). This in part has been
the main driver for stimulating the growth of the fruit and vegetable juice sector that has
significantly expanded over the last decade. The U.S. Food and Drug Administration defines juice
as “the aqueous liquid expressed or extracted from one or more fruits or vegetables, purees of the
edible portions of one or more fruits or vegetables, or any concentrates of such liquid or puree”
(US FDA, 2004). Juices are widely recognized as rich sources of vitamins and a variety of other
nutrients. Consumers’ demands have required minimally processed juices that have retained their
raw qualities, but also assurance that the product is both microbiologically safe and stable. To be
compliant with US regulations, all juices with a shelf-life beyond 5 days should be treated with a
process that ensures a 5-log10 CFU reduction in levels of the relevant pathogens (21 CFR 120.24).
The primary method that the juice industry uses to achieve this reduction is through thermal
processing, which can affect nutrient levels and flavor. Consequently, there is interest in
developing alternative, non-thermal, methods for pasteurizing juices with High Pressure
Processing (HPP) and Ultraviolet-Light (UV) being the main technologies applied (Agriculture
and Agri-Food Canada, 2016).
Previously, the juice market was dominated by high acid juices although through changing
consumer preferences, there is an increasing demand for low-acid products. One such juice is
wheatgrass juice that is essentially an extract of immature wheat that is characterized as being low
in acid but high in chlorophyll. The perceived benefits derived from wheatgrass include high
2
antioxidant activity and increased blood oxygen levels due to the chlorophyll content (Luo, Wang
and Zhang, 2013). Although not studied in detail, thermal processing of wheatgrass is not
considered an option due the sensitivity of oxidants and chlorophyll to heat. Therefore, there is
interest in applying non-thermal processing methods to achieve the 5-log10 CFU reduction of
relevant pathogens. The purpose of this study is to reduce microorganisms in, and measure the fate
of nutrients and enzymes of wheatgrass juice after its treatment by various non-thermal
technologies (Ultraviolet light [UV-C] and High Hydrostatic Pressure [HHP]) and thermal
pasteurization.
Hypothesis and Objectives
The hypothesis of this research is that non-thermal pasteurization of defrosted wheatgrass juice by
Ultraviolet light or High Hydrostatic Pressure preserves the nutrient content of the wheatgrass
juice to a greater extent compared to when thermal processing is applied while also achieving an
equivalent decrease in bacterial pathogen levels.
The sequence of achieving the project’s objectives from optimizing the juice extraction to assess
the juice’s nutrients after the treatment is to:
1) Evaluate extraction of juice from wheatgrass raw material
2) Verify assays for determining polyphenol oxidase, peroxidase, vitamin C, phenolic
compounds, protein, chlorophyll, overall antioxidant content of wheatgrass juice
3) Identify processing parameters of UV, HHP and thermal processing to achieve 5-log
reduction of model pathogens in wheatgrass juice
4) Determine the changes in nutrients’ composition of wheatgrass juice by UV, HHP and
thermal processing applying conditions identified in objective 3
3
1.2 Literature Review
Wheatgrass is an example of an opaque low acid juice similar to those produced from juicing
leafy greens. Despite the popularity of wheatgrass there has been little research performed with
respect to non-thermal processing principally because wheatgrass was not considered to represent
a significant food safety risk. There have been outbreaks/recalls of E. coli O145 (Food Quality
news.com, 2016) and Listeria monocytogenes (Food safety news, 2013) associated with sprouted
wheatgrass (micro-greens) and dried extracts although these tend to be rare events.
In the following review, the general structure of trends in the fruit and vegetable beverage sector
will be provided along with a description of the characteristics of wheatgrass. An overview of non-
thermal pasteurization technologies will be provided along with a description of the pathogens of
concern such as E. coli and Listeria.
1.2.1 Canadian / USA vegetable and fruit juice market value
Diversity is a key feature of the North American juice market with a wide range of juice types
available. This marketing traditionally was mainly comprised of high acid juices although
consumer trends are going towards low acid products (Celik, 2012). The low acid juice sector was
typically confined to food service outlets due to their short-self life given that there are a few
hurdles to prevent microbial growth. However, given the popularity of low-acid juices, an
increasing number of retail chains are marketing such products. In this case, the short shelf-life is
addressed by freezing the product during distribution and retail. However, this does not exempt
the product from needing to go through a pasteurization process (Health Canada, 2007; Luo, Wang
and Zhang, 2013).
4
In 2010, in the United States (U.S.), the market of fruit and vegetable juice was valued at
US$16.2 billion. In retail markets, the most popular fruit/vegetable juice section was 100% juice,
was valued at US$8.8 billion. However, citrus juice consumption decreased by 29.7% between
2000 and 2009, while non-citrus juice consumption increased by 13.5% in the same period of time
(Agriculture and Agri-Food Canada, 2012). It is projected that the global juice market will reach
$21 billion by 2017 (Fruit and Vegetable Juices: U.S. Market Trends, 2013, para.5).
To date, the majority of focus has been on high acid juices that are relatively stable and compatible
with extended shelf-life following pasteurization. However, many nutrients and enzymes
associated with raw juices are pH sensitive with the consequence that acidification is not an option.
For example, wheatgrass is an example of a low acid juice that is sold based on its nutritive
qualities and requires alternative hurdles to pH to ensure safety along with shelf-stability.
1.2.2 Wheatgrass juice
Wheatgrass is a nutrient-rich type of young grass (micro-green) in the wheat family Triticum
aestivum. Gandhi, Kamboj and Rana (2011) show that wheatgrass juice is an aqueous extract and
is the pressed young shoots of the plant Triticum aestivum, a member of the Poaceae family. In
recent years, wheatgrass juice has been sold as a dietary supplement in tablets, capsules and liquid
forms, in some European countries, USA and India (Acharya et al, 2006). In general, wheat
germinated over a period of 6–10 days is called ‘wheatgrass’. During germination, enzymes are
synthesized and the emerging seedling synthesizes antioxidants and chlorophyll as the plant
develops (Acharya et al, 2006). Benincasa et al. (2014) stated that “The use of sprouts (i.e.
seedlings just after germination) and micro-greens (i.e. young plant of 1–2 weeks’ age) in
preparing wheatgrass juice is increasing”. Benincasa et al. stated that this rise in drinking of
5
wheatgrass juice is because the sprouts and micro-greens are known as healthy foods. The sprouts
and micro-greens have a high nutrient concentration and positive impacts on human health such
as prevention of cancer and heart diseases (Benincasa et al., 2014, p.1). In the current study, the
wheatgrass used to prepare the juice was cultivated outdoors for approximately 6 weeks. The
wheatgrass is more developed than micro-greens and has varying moisture content depending on
conditions at the time of harvest.
1.2.2.1 Wheatgrass juice properties and contents
Wheatgrass juice is a low acid juice that has significant quantities of vitamins and minerals such
as calcium and magnesium, chlorophyll, and enzymes. Wheatgrass juice contains at least 13
vitamins such as vitamin B12, A, C and E (Acharya et al., 2006). Chakraborty, Das and
Raychaudhuri (2012) have reported that ascorbic acid in wheatgrass is high, but it becomes
damaged because of chemical degradation and/or heat processing. In addition, wheatgrass has
different amounts of minerals such as potassium, sodium, calcium and magnesium, which are
listed in Table 1.1. The mineral content of wheatgrass increases as the plant develops although
zinc and iron reach a maximum after 8 days of growth (Acharya, Kulkarni, Nair, Rajurkar and
Reddy, 2005).
6
Table 1.1-Levels of vitamins and minerals in 100 mL of wheatgrass juice and the contents of amino
acids in mL of wheatgrass juice (Bar-Sela, Goldberg, Fried and Tsalic, 2007).
Vitamins and minerals Amount (mg/100 mL) Amino acids Amount (µg/mL)
Ascorbic acid
Dehydroascorbic acid
Vitamin E
Carotene
Potassium
Phosphorus
Calcium
Sulfur
Magnesium
Sodium
Aluminum
Zinc
Copper
25.2
7.6
8.5
2.43
57
8.2
2.4
2.37
1.7
1.42
0.31
0.02
0.007
Aspartic acid
Threonine
Serine
Asparagine
Glutamine
Proline
Glycine
Alanin
Valine
Methionine
Isoleucine
Leucine
Tyrosine
Phenylalanine
Lysine
Histidine
Tryptophan
Arginine
510.3
105.8
201.8
3039.6
200.6
33.6
20.6
166.4
272.1
14.0
145.1
101.0
121.8
200.9
174.5
232.2
160.1
252.9
Chlorophyll is considered the most important quality metric of wheatgrass juice (Meyerowitz,
1999). Chlorophyll content of wheatgrass is about 70% water-soluble and is released from
chloroplasts during the juicing process (Gandhi, Kamboj and Rana, 2011). The structure of
chlorophyll and hemoglobin is similar in having a tetra pyrrole ring structure, but the difference
between the two is the nature of central metal atom. The central metal atom is magnesium (Mg) in
chlorophyll and iron (Fe) in hemoglobin as shown in Figure 1.1. As a result, it is believed that
7
chlorophyll replenishes human blood (Gandhi, Kamboj and Rana, 2011; Meyerowitz, 1999).
Although such a theory has not been proven it should be noted that as with many juices, the
marketing of perceptions is more important than clinical data supporting health claims.
Figure 1.1-Structure of hemoglobin and chlorophyll (Gandhi, Kamboj & Rana, 2011, p.447)
In addition to the nutritional elements, herbs and sprouts like wheatgrass contain antioxidants.
The phenolic compounds class including flavonoids and their derivatives as well as carotenoids
and tocopherols are responsible for the most part of antioxidant effect (Acharya et al, 2006). In
fact, the antioxidant level in sprouts is higher than that of non-sprouted seeds, wheat germ or young
wheat plants (Bonfili et al., 2009). In addition, the growth time and growth conditions affect the
antioxidants activity and their amounts. However, Acharya et al (2006) show that the total amounts
of phenolic and flavonoid in wheatgrass increase with the wheatgrass during growth. Flavonoids
and phenolic concentrations within wheatgrass can be concentrated by drying although typically
the produce is minimally processed (Chakraborty, Das & Raychaudhuri, 2012). Moreover,
Acharya et al, (2006) reported that after 15 days of growth, wheatgrass is measured in the highest
amount of FRAP (Ferric reducing antioxidants power). Nevertheless, ORAC (Oxygen radical
absorbance capacity) is measured in the largest amount after 10 days into the wheatgrass growth.
8
Furthermore, the amount of DPPH (1, 1-diphenyl-2-picrylhdrazyl) increases with the growth
period. As a result, the amount of antioxidants in wheatgrass depends on the growth period and
prevailing conditions.
Antioxidants have also shown anti-mutagenicity property because wheatgrass was reported to
be helpful in treatment certain diseases such as thalassemia and distal ulcerative colitis (Acharya
et al., 2005). It is reported that wheatgrass extracts inhibit the DNA oxidative damage and are
efficient in suppressing the superoxide radical that can result in various diseases. Antioxidant
molecules isolated from wheat sprouts can also protect DNA against oxidative stress caused by
reactive oxygen species and as a result, the aqueous wheat sprout extract has anti-mutagenic
properties (Bonfili et al, 2009). In addition, because of the high content inorganic phosphates,
enzymes, reducing glycosides and polyphenols, wheat sprouts possess radical scavenging activity
(Bonfili et al, 2009). As a result, the nutrient content of wheatgrass increases its popularity as a
healthy beverage.
Various enzymes responsible for wheatgrass pharmacological actions are protease, amylase,
lipase, cytochrome oxidase, transhydrogenase and superoxide dismutase (SOD) (Gandhi et al,
2011; Dhamija et al. 2010). SOD converts two superoxide anions into a hydrogen peroxide
molecule, which has an extra oxygen molecule to kill cancer cells. Moreover, Chang et al. (2006)
add that peroxidase is an oxidoreductase that catalyzes different electron donor substrates
oxidation. Peroxidase is broadly found in fruits and vegetables, and often contributes to
degenerative differences in color, flavor and texture in juices. Thus, knowing more about enzyme
activity of wheatgrass juice will help juice processors to better process the juice.
9
1.2.3 Human Pathogens in low acid juices
Most commercial juices are acidic (pH < 4.6) and in general are stable but have been
occasionally implicated in foodborne illness cases (Ray and Bhunia, 2008). In contrast, low acid
juices (pH > 4.6) are considered to represent a greater food safety risk due to the ability to support
the growth of pathogens (Çelik, 2012). The main pathogen of concern in most low-acid juices is
Clostridium botulinum that can potentially germinate and produce neurotoxins. Given the
resistance of spores to thermal and non-thermal inactivation, control measures for C. botulinum
are mainly based on temperature control. In the case of wheatgrass juice production in distribution,
freezing the final product is normal practice to prevent spore germination along with controlling
spoilage microbes (U.S FDA, 2004).
It is considered that the "pertinent microorganism" is the most resistant microorganism of public
health significance. Pertinent microorganisms are likely to occur in the juice and is the
microorganisms that must be the target for the 5-log pathogen reduction treatment (21 CFR
120.24(a). The pertinent microorganisms are one of the pathogens that should be targeted during
the processing treatments because those pathogens have been demonstrated to be potential
contaminants in certain juices through outbreaks. Which of these pathogens is determined to be
the pertinent bacteria will depend upon which is most resistant to the means of treatment such as
pasteurization and UV radiation. Those treatments are used to reduce the pathogens to 5-log CFU
reduction that is required under the juice Hazards Analysis Critical Control Point HACCP
regulation (U.S FDA, 2004).
10
1.2.4 Outbreaks of foodborne illness associated with the consumption of unpasteurized
and/or low acid fruits and vegetables juice
Susceptible people within the population that includes children, the elderly and people with the
low immunity are advised not to consume unpasteurized products (Health Canada, 2007). About
2% of all juices sold in the United States are unpasteurized (U.S. FDA, 2015). This means it has
not been treated to eliminate disease-causing bacteria and could therefore be contaminated with E.
coli O157:H7, Salmonella and Cryptosporidium (Health Canada, 2007).
While pathogen contamination routes have not been definitively confirmed in many juice
outbreaks, the use of dropped fruit, the use of non-potable water, and the presence of cattle and
deer near the orchards are some of the causes of contamination. History of juice-related outbreaks
have been relatively uncommon and were generally related to very small commercial processors
or home-prepared products (U.S. FDA, 2015). In USA and Canada, unpasteurized juices
contaminated with pathogenic bacteria such as E. coli O157:H7 and Salmonella have caused
numerous illnesses and some fatalities as shown in Table 1.2.
11
Table 1.2-Outbreaks linked to unpasteurized and/or low acid juices during the period of 1974–2010
in the USA and Canada (Danyluk, Goodrich-Schneider, Schneider, Harris, and Worobo, 2012)
Type Product Pathogen Year Location Venue Cases
(deaths)
Apple Unpasteurized S. Typhimurium 1974 USA (NJ) Farm, small retail
outlets
296 (0)
Unpasteurized E. coli O157:H7 1980 Canada (ON) Local market 14 (1)
Unpasteurized E. coli O157:H7 1991 USA (MA) Small cider mill 23 (0)
Unpasteurized Cryptosporidium 1993 USA (ME) School 213 (0)
Unpasteurized C. parvum 1996 USA (NY) Small cider mill 31 (0)
Unpasteurized E. coli O157:H7 1996 USA (CT) Small cider mill 14 (0)
Unpasteurized E. coli O157:H7 1996 USA (WA) Small cider mill 6 (0)
Unpasteurized E. coli O157:H7 1996 Canada (BC), USA (CA,
CO, WA)
Retail 70 (1)
Unpasteurized E. coli O157:H7 1997 USA (IN) Farm 6
Unpasteurized E. coli O157:H7 1998 Canada (ON) Farm/Home 14 (0)
Unpasteurized E. coli O157:H7 1999 USA (OK) NR 25
Unpasteurized C. parvum 2003 USA (OH) Farm/Retail 144
Unpasteurized E. coli O111 and
C. Parvum
2004 USA (NY) Farm/Home 212
Unpasteurized E. coli O157:H7 2005 Canada (ON) NR 4
Unpasteurized E. coli O157:H7 2007 USA (MA) NR 9
Unpasteurized E. coli O157:H7 2008 USA (IA) Fair 7
Unpasteurized E. coli O157:H7 2010 USA (MD) Retail 7
Carrot Homemade C. botulinum 1993 USA (WA) Home 1 (0)
Pasteurized C. botulinum 2006 USA Retail 4
Coconut Milk Vibrio cholerae 1991 USA (MD) Home/picnic 4
Mamey Frozen Puree S. Typhi 1999 USA NR 19
Frozen Pulp S. Typhi 2010 USA Retail 9
Mixed Fruit Unspecified Shigella sonnei 2002 Canada, USA Resort 78
Acai, banana,
strawberry, sugar cane
Hepatitis A 2007 USA (FL) Food Service 3
Watermelon Homemade
Salmonella spp
1993 USA (FL)
Home 18 (0)
12
1.2.5 Regulatory requirements: Hazards Analysis Critical Control Point of juice HACCP
The FDA recommends all processors of fruit and vegetable juice to follow HACCP rules to assure
the safety of the juice. The most important points of the juice HACCP Regulation are:
Juice processors must evaluate their processing operations using HACCP principles.
Juice processing operations must follow the Current Good Manufacturing Practice (CGMP)
regulations.
The HACCP plan and other Sanitation Standard Operating Procedures records (SSOPs) and
HACCP operations must be available for inspection and auditing.
Employees, involved in a HACCP plan, must be trained in HACCP principles.
The 5-log pathogen reduction must be accomplished for the most resistant microorganism of
public health concern
Cleaned and uninjured tree-picked fruit treatment must be verified by tests products regularly
for generic E. coli.
Shelf stable juices must be made using a single thermal processing step and juice
concentrates must be made using a thermal concentration process
Low-acid canned juice and juice subject to the acidified foods regulation is exempt from the
requirement to include control measures in HACCP plan to achieve the 5-log pathogen
reduction,
Juice processors who sell juice directly to consumers and do not sell juice to other businesses
are exempt from the juice HACCP regulation, but must use the needed warning label,
"Warning labels will soon be compulsory for untreated juices” (U.S. FDA, 2004).
13
1.3 Juice Treatment Technologies
1.3.1 Thermal treatment
Pasteurization is a process, named after scientist Louis Pasteur, was originally conceived as a
method of preventing wine and beer from souring as cited from Carlisle (2004) (Koutchma, 2012).
Pasteurization is a thermal treatment that aims to achieve a 5-log CFU reduction of the pertinent
vegetative pathogen and decrease the general microflora. For example, in the case of milk a process
is designed to support a 5-log CFU reduction of Coxiella burnetii given the bacterium represents
the most thermal resistant pathogen encountered in raw milk. The thermal process can be
undertaken under different time and temperature conditions provided the overall lethality achieves
the 5-log10 CFU reduction. For example, High-Temperature-Short-Time Treatment (HTST) uses
a temperature of 72°C for 15 seconds, whereas Low-Temperature-Long-Time Treatment (LTLT)
heats at 63°C for 30 minutes. Typically, the HTST treatment results in less detrimental sensory
changes although this depends on consumer preference (Rupasinghe & Juan, 2012) (Table 1.3).
Osaili (2012) correlated the efficiency of thermal processing to the product parameters, such as
pH, fat level and water activity along with the intrinsic resistance of the microbe. Thermal
resistance of a microbe within a defined matrix is referred to as the D value that is defined as the
time at constant temperature to achieve a 1-log CFU reduction.
Equation 1-1. 𝐃 = 𝐭𝟐 − 𝐭𝟏/Log 10(A) - Log10 (B) (1.1)
Where A and B represent the survivor counts following heating for times t1 and t2 in minutes.
Second, the Z value reflects the temperature dependence of the reaction. It is defined as the
temperature change required to change the D value by a factor of 10 using Equation 1-2 (Osaili,
2012). 𝐙 = 𝐓𝟐 − 𝐓𝟏/Log 10(D1)-log 10(D2) (1.2)
14
Where D1 and D2 are D values at temperatures T1 and T2, respectively.
Table 1.3-Some types of thermal treatment applications
The main Applications of thermal treatment in fruit
and vegetables products (Ahmed & Shivhare, 2012,
p.390)
Applications of thermal processing in packaging
(Fellows, 2009)
1- Blanching: is to inhabit oxidative enzymes as
polyphenol oxidase (PPO) and peroxidase (POD),
vegetables are treated by steam, Ohmic, High Pressure
or hot water.
1-Pasteurization of packaged foods: after dressing
some liquid foods as fruit juices into packages, liquid
foods are pasteurized by using hot water if the foods
are in glass containers, but if foods are in plastic or
metal packages, they are pasteurized by a mixture of
steam air or hot water (p.387).
2- Pasteurization: is to inactivate microbes and
enzymes, pasteurization uses mild heat treatment with
low effects on product characteristics. For example,
fruit juices are heated for 30 min at 60 - 75 °C, then
filled and pasteurized for 15-45 min at 84- 88°C
depending on the containers’ size.
2-Pasteurization of unpackaged liquids: “for small-
scale batch liquid products pasteurization, open
jacketed boiling pans are used. Nevertheless, large-
scale pasteurization of low-viscosity liquids as fruit
juices and milk usually employs continuous
equipment, and plate or tube heat exchangers are
commonly used " (p. 388).
3-Sterilization: it describes the absence or destruction
of all viable microorganisms. Some products are
referred to as commercially sterile which means a
system of continuous flow or enclosed vessel is used for
preserving.
15
1.3.1.1 Effects of pasteurization on liquid foods
Effects of heat on microorganisms and enzymes of juices
Thermal treatment of fruits juice is typically applied using Ultra-High-Temperature (UHT) to
achieve a shelf stable product. Thermal pasteurization is also performed but the product requires
refrigeration during distribution and retail (Alzamora, Char, Guerrero & Mitilinaki, 2010). The
target pathogen of concern depends on the food type and acidity as shown in Table 1.4. Most
enzymes have D and Z-values within same range of microorganisms. As a result, enzymes are
destroyed during normal heat processing, but some enzymes are very heat resistant such as
peroxidase in vegetables and alkaline phosphatase in milk (Fellows, 2009).
Table 1.4- Heat resistance of selected pathogens (Fellows, 2009)
Microorganisms D-value (Min) Z-value Temperature (°C) Substrate/typical food
Escherichia coli O111:B4 5.5±6.6 - 55 Skim/whole milk
Listeria monocytogenes 0.22-0.58 5.5 63.3 Milk
Staphylococcus aureus 0.9 9.5 60 Milk
Salmonella. typhimurium 396-1050 17.7 70-71 Milk chocolate
Clostridium botulinum B 0.49-12.42 7.4-10.8 110 Vegetable products
Effect of heat on nutritional and sensory characteristics of juices
In addition to ensuring the destruction of microorganisms and inhibition of enzymes, the heat
treatment of fruit and vegetable juices also causes a number of other nutritional property changes.
Pasteurization causes changes to physicochemical properties such as pH, and total soluble solids
but significant juice browning occurred during storage. It is demonstrated that the enzymatic
browning by polyphenol oxidase is the main color deterioration cause in fruit juices. In addition,
in terms of nutritional properties, aroma compounds and pigments are destroyed by heat following
16
a similar first-order reaction to microbial destruction. Loss of volatiles during pasteurization can
lead to changes in product quality and flavor (Fellows, 2009; Chen et al., 2009).
1.3.1.2 Advantages and disadvantages of thermal treatment
Thermal treatment is the most widely used method to treat fruit juices to attain the 5-log
reduction of relevant pathogens (Alzamora et al., 2010). The popularity of thermal processing
stems from its historic effectiveness, along with low cost, consistency and consumer acceptability
(Koutchma, 2012; Fellows, 2009). However, the concerns of thermally processed juice are low
quality and loss of the “raw” sensory characteristics (Fellows, 2009). Moreover, Guerzoni et al.
(2010) reported that thermal processing has negative impacts on the nutritional qualities of juice
such as aroma, volatile and flavor components, and also some vitamins such as vitamin C are
sensitive to heat.
1.3.2 Non-thermal technologies for food treatment
1.3.2.1 High Hydrostatic Pressure technology HHP
High hydrostatic pressure also referred to as “High pressure processing (HPP), or ultra-high
pressure (UHP) processing, subjects to liquid and solid foods with or without packaging to
pressures between 100 and 800 MPa” (U.S FDA, 2014). High Hydrostatic Pressure is one of the
non-thermal physical techniques that was first described in the 19th century by Hite (1899). The
equipment used in these early studies was unreliable and hazardous and that explains why the
technology was not considered commercially viable. There were a small number of studies
performed to evaluate high pressure as a processing technique although the studies were relatively
few and far between. For example, Bridgman (1914) evaluated pressure to preserve egg albumin
with additional studies investigating meat and milk by Payens and Hermens (1969) and Macfarle
17
(1973) respectively. In 1990, a major revolution in HPP came in Japan by releasing the first high
pressure processed product onto the market. Over the past 30 years, high pressure processing has
received considerable attention in the food industry. Many studies have been performed to
understand the significant advances of HPP technology, which produces safe, fresh, nutritious, and
innovative food products. In 2009, the FDA approved the application of high pressure to a
preheated sample for commercial sterilization of low-acid foods (Balasubramaniam, Martínez-
Monteagudo & Gupta, 2015). However, Health Canada’s Guidance for Industry reported that
HHP-treated food is novel and it needs more assessments before it is sold in the Canadian market
(Koutchma, 2014a, p58).
In the HHP technology, pressures between 300 and 1000 MPa are used in commercial
applications of food treatment as in Figure 1.2 to treat foods in the pressure unit vessel with or
without additional heat (Koutchma, 2014a, p1). Fellows (2009) explains that the temperature of
foods rises at high pressures because of adiabatic heating, which is generated by the compression
of water and other food components even though HHP is considered to be a non-thermal process.
The temperature increase is about 3°C/100MPa depending on the food contents. Vessels are
specially designed to combat these pressures safely over several cycles. Commercial exposure time
of pressure can range from a millisecond pulse to a treatment time of over 1200s (U.S. FDA, 2014).
Thus, HHP treatment has been investigated as an attractive non-thermal technology for producing
minimally processed high quality foods (Koutchma, 2014a).
18
Figure 1.2-HHP unit used for commercial operations (source: http://www.hiperbaric.com/en/
hiperbaric55)
Main Components of HHP Units
As cited from (Anon 2000, Mertens 1995), the following are typical components of batch high-
pressure equipment as shown in Figure 1.3:
1) Pressure vessel
2) Two end closures to cover the cylindrical pressure vessel
3) Yoke (a structure for restraining end closures while under pressure)
4) High pressure pump and intensifier for generating target pressures
5) Process control and instrumentation
6) A handling system for loading and removing the product (Fellows, 2009)
19
Figure 1.3-Schematic of batch High-pressure processing system (source:
http://www.fnbnews.com/Beverage/impact-of-hpp-on-antioxidant-capacity-of-fruit-beverages-37935)
Batch operation
In high pressure processing, the pressure vessel is filled with a food product and pressure is applied
for the desired time after which it is depressurized (Fellows, 2009). A simplified flow-sheet is
given below in Figure 1.4.
Figure 1.4-Process of batch operation in HHP
Pack food in sterilized containers
Load in a pressure chamber
Fill chamber with water
Pressurize chamber
Hold under pressure
De-pressurize the chamber
Remove the treated foods
20
Principles of HHP
HHP has three variable components depending on what is being processed: pressure, holding
time, and temperature. According to Le Chaterlier's principle, the application of pressure shifts an
equilibrium to the state that occupies the smallest volume. Le Chaterlier's principle is that reactions
that produce an increase in volume are inhibited whereas reactions resulting in a decrease in
volume are accelerated. HHP can destroy the non-covalent bonds, but it cannot break covalent
bonds. Foods treated by HHP can retain their color, flavor, and nutrition (Koutchma, 2014a, p6).
Another principle of HHP is the “isostatic principle”. Pressure that is instantaneous and uniform
is transmitted through the food samples, and it is independent of the shape and size of the samples.
A uniform pressure will be applied in all directions of the sample, and this pressure will not change
the sample. As a result, the treated sample will return to its basic form when the pressure has been
released (Chen & Neetoo, 2012)
The Microscopic Ordering principle can also be applied to HHP. An increase in pressure
increases the ordering degree of a given substance molecules, at constant temperature. Therefore,
pressure and temperature apply combative forces on chemical reactions and molecular structure.
Based on Arrhenius relationship principle, thermal effects also affect different reaction rates during
pressure treatment, as with thermal processing. The net pressure-thermal effects can be
cooperative, additive, or combative (Koutchma, 2014a).
1.3.2.1.1 Effects of High Hydrostatic Pressure treatment
HHP effects on microorganisms in fruit and vegetable juices
The process is capable of inactivating microorganisms effectively with less heating. The groups
of microorganisms have a decreasing sequence of HHP sensitivity: “yeasts > Gram-negative
21
bacteria > complex viruses > molds > Gram-positive bacteria > bacterial endospores (most
resistant)” (Fellows, 2009). Generally, some bacterial spores can combat 1000MPa at room
temperatures, so they are the most pressure resistant. HHP can kill microorganisms by inactivating
key enzymes, lowering pH, and altering membrane functionalities such as changes to the cell
permeability and morphology, and physiological reactions inhibition (Hu, Liu, Zhao & Zou, 2012).
HHP results in microorganisms’ inactivation by changing non- covalent bonds in proteins,
which are responsible for cellular integrity, replication, and metabolism in their active forms. The
inevitable denaturation of one or more critical proteins (e.g. membrane-bound ATPase and
enzymes involved in DNA replication) causes cell injury or death (Fellows, 2009). High-pressure
effects on bacterial cell morphology and intracellular damage have also been observed in high-
pressure-exposed cells examined by scanning electron microscopy. Some research studies show
that high pressure causes the destabilization and reduction in the number of functional ribosomes
which prevent impaired cells from recovering and lead to inevitable cell death (Chen & Neetoo,
2012).
In addition, a research study describes apparent hypotheses for disruption to metabolic processes
caused by the effects of high pressures on cellular enzymes (Fellows, 2009). Pressurization at high
levels may result in partial or complete, and reversible or irreversible, enzymatic activity loss
depending on the enzyme molecular structure, conditions affecting the enzyme microenvironment
(e.g. pH), as well as level and exposure time of pressure and temperature. Therefore, in terms of
effects of high pressure, as cited from (Hoover et al. (1989); Palou et al. (2002), it was reported
that pressure affects biochemical and enzymatic processes in microorganisms in two possible
ways:
1- Change intra-molecular structures and decrease the available molecular space; and
22
2- Increase inter-chain reactions at substrate interfaces (Chen & Neetoo, 2012).
Effects of HHP on the physical and chemical characteristics of food systems
Minimum negative impacts on the sensorial, functional, and nutritional properties of juices is
one of the main advantages of HHP. The sensory and nutritional properties of juices are essential
quality characteristics affecting consumers’ acceptance of foods. Chen and Neetoo (2012) mention
that HHP processing could keep the food sensory attributes and nutritional value because of its
minimal effect on the covalent high moisture content bonds of low-molecular-mass compounds
such as flavor molecules and micronutrients. The physical structure of high moisture content foods
is unchanged after pressurization, so HHP has limited impact on the liquids textural characteristics
since no cut forces are produced by pressure.
High-pressure treatment typically also keeps the fresh color in food products. There are many
examples in the literature illustrating the ability of high pressure to retain the color parameters of
pressure-treated fruit and vegetable juices. For example, in watermelon juice, which is a low acid
juice treated by HHP, viscosity and cloudiness increased. Color values a* and b* were both
unchanged, while L*value increased, and browning degree decreased. Those changes occurred
when watermelon was treated by HHP at pressure values 200, 400, and 600MPa for 5, 15, 30, 45,
and 60 min at room temperature (Hu et al, 2012). Although food-quality characteristics such as
flavor, color, texture, and nutritional value are unaffected or only minimally altered by HHP,
enzymes related to food quality can be typically influenced by pressure. The effect of HHP on
enzymatic activity occurs in two systems:
1. Enzyme activation at low pressures in monomeric enzymes, and
23
2. Enzyme inactivation at high pressures stimulated by the loss of tertiary and quaternary
structures in oligomeric enzymes (Chen & Neetoo, 2012).
It has been established that high pressures only affect weak linkages such as electrostatic and
hydrophobic bonds, which causes protein molecules to unfold and aggregate. The effects of High
pressures on proteins differs widely because of changes in the proteins hydrophobicity. Up to
100MPa, hydrophobic interactions mostly cause a volume gain, but at higher pressures, they cause
a volume loss. Pressure does not change the small macromolecules, which produce flavor or odor
in a food, and foods that are subjected to HPP at ambient or chill temperatures do not undergo
substantial differences to flavor or color. Likewise, the molecular structure of vitamins and
availability of minerals is largely unaffected (Fellows, 2009).
1.3.2.1.2 Advantages and limitations of HHP treatment
HPP is gaining in popularity although the volume of production is relatively small. This increase
is not only because of its preservative action and minimal impacts on food quality, but also because
of its potential for high-pressure freezing or thawing, and its ability to change the functional
properties of foods. Fellows (2009) states that other major advantages of HPP are its ability to
inactivate pathogenic microorganisms in foods at room temperature and its potential to extend the
shelf-life of food products without altering their sensory and nutritional values. Finally, other
advantages are the HHP process reduces processing time, consumes less energy, and practically
has no effluents.
However, the main limitation of HHP is the high costs, and because water is required for
destroying microorganisms, HHP cannot be used for dry foods such as spices; or for foods which
contain enmeshed air, such as strawberries, Fellows (2009) explained. Moreover, HHP treatment
24
in milk was not as effective as in other food systems. Fat and protein content in milk seems to
protect the microorganisms against pressure, whereas the low pH of fruit juices can be an
additional inhibitory factor enhancing the effectiveness of HHP technology (Fan & Sampedro,
2010, p.38). In addition, foods characterized by a large number of voids or air spaces, such as plant
foods, may undergo a permanent deformation due to gas displacement and liquid infiltration or
compression and subsequent expansion of gas during pressurization and pressure release
respectively (Chen & Neetoo, 2012).
In conclusion, HHP has minimum impacts on the nutritional properties of food compared to
other food processing methods. There are few studies regarding the effects of HHP on the
nutritional properties of pressure-processed low acid juices; consequently, more research is needed
before conclusive statements can be made.
1.3.2.2 Ultraviolet light treatment
Ultraviolet light treatment is non-thermal or operates at temperatures below conventional heat
treatments. Ultraviolet (UV) is considered an energy domain of the electromagnetic spectrum lying
between the x-ray and visible domain (between 100-400 nanometers) (Koutchma, 2014b; Forney,
Koutchma and Moraru, 2009; Rupasinghe & Juan, 2012). UV can come from the sun or artificial
sources, with the risk to humans increasing as the wavelength gets shorter. In general, Churey et
al. (2014) mention that wavelengths range from 100 to 400 nm can be used for UV light
processing, and several types of lamps capable of producing UV light, such as low and medium
pressure mercury, arc lamps are used. UV is classified into three general areas by wavelengths:
UV-A, UV-B and UV-C as in Figure 1.5. UV-A has the longest wavelength range (320-400
nanometers) and is the type of radiation responsible for sunburns and is linked to skin cancer. UV-
25
B, at 280-320 nm, also plays a role in skin tanning and burning, but it is a less severe than UV-A.
UV-C, at 100-280 nm, has the shortest wavelength range of the three and is the type applied to
food and beverages (Forney et al., 2009, p.2; Bintsis, Robinson & Tzanetaki, 2000).
US FDA named UV-C at 200-280 nm the “germicidal range” because it “effectively inactivates
bacteria and viruses and pasteurize the liquid food products in appropriate designed reactors.” FDA
explains that the “germicidal properties of UV irradiation are mainly due to DNA mutations
induced through absorption of UV light by DNA molecules.” Today UV-C is commonly used on
juices and apple cider, grains, cheeses, baked goods, frozen products, fresh produce (except lettuce,
which wilts), liquid egg products and other foods and beverages (Siegner, 2014).
Forney, Koutchma, and Moraru (2009) have mentioned that using UV light in food treatment is
still limited even though its use is well approved in order to decontaminate surfaces, treat water
and disinfect air. In 2000, the FDA recognized UV light processing as an alternative safe
processing for juice pasteurization when the low-pressure mercury lamps giving 90% of the
emission at a wavelength of 253.7 nm were used for treatment (Churey et al., 2014; Koutchma,
2008). For example, in the USA, the Cider Sure 3500 is one of the widely used commercial non-
thermal processing machines for apple cider (Churey et al., 2014). In addition, US FDA and Health
Canada approved UV-light as an alternative treatment to thermal pasteurization of fresh juice
products such as apple juice (Koutchma, 2012; Forney, Koutchma & Moraru, 2009). Nevertheless,
Forney, Koutchma and Moraru stated that “the European Union EU considers UV light as an
irradiation. Regulations for the use of the irradiation process in the EU are not harmonized” (2009,
p. 27). Therefore, more research studies are now investigating the uses of UV light in the food
industry and its effects on the food products.
26
Figure 1.5- The different wavelengths of light and UV kinds (source: http://www.aquafineuv.com/UV-Science)
Application and Sources of UV light
There are several UV sources that are applied for surface decontamination. Continuous UV low-
pressure, medium-pressure mercury lamps (higher emission intensity in UV-C range), pulsed- UV and
excimer lamp (can be applied to foods). UV technology success relies on the correct matching of
parameters of UV sources (lamps) to specific UV applications such as water treatment or foods
processing. However, the effective characteristics of common UV light sources are used today for
water treatment, but they have not been approved for food treatment (Koutchma, 2008; Forney,
Koutchma & Ye, 2011 and Forney, Koutchma & Moraru, 2009, p. 33).
Liquid foods like fresh juice products transmit a relatively small amount of UV light because of the
presence of color compounds, organic solutes and suspended matter. As a result, this low transmission
reduces the UV pasteurization processes’ efficiency (Forney, Koutchma & Moraru, 2009). Therefore,
physical properties such as (pH, soluble solid contents), liquid density, viscosity and turbidity are key
challenges to pasteurizing juices using UV (Forney, Koutchma & Moraru, 2009).
27
Collimated Beam
In water treatment, laboratory dose-response data from collimated beam tests are commonly used
as a basis for determining the necessary delivered UV dose for full scale UV systems. In general, there
are several components that should be considered crucially in the design and construction of a bench
scale UV testing device. These include:
1- Shutter: To regulate the time of exposure factor in the fluence ~UV dose calculation.
2- Window: The lamp enclosure should be thermally stable, since the output of many UV lamps
is quite temperature sensitive. It is often useful to employ a quartz window to assure that no
change in air drafts occur when a shutter is used.
3- Power supply: It is very important to maintain a constant emission from the UV lamp over
exposures.
4- Collimating tube: The inner surface of the collimating tube should be ‘‘roughened’’ and
painted with a ‘‘flat black’’ paint to prevent reflection from the sidewalls of the collimating
tube.
5- Platform: Is where the Petri dish and stirring motor is placed for UV exposure. It should be
thermally and physically stable and easily raised or lowered.
6- Stirring: In order to assure equal fluence UV dose for all microorganisms in the suspension, it
is important to maintain adequate stirring during the UV exposure.
7- Lamps: May be either low pressure mercury vapor (monochromatic at 253.7 nm) or medium
pressure mercury vapor (polychromatic UV light). Lamps should be properly ventilated to keep
the temperature stable through the irradiation as shown in Figure 1.6 (Bolton & Linden, 2003).
28
Figure 1.6-Example of bench scale devices for conducting UV experiments (Bolton & Linden, 2003)
UV Reactor designs
Various continuous flow UV reactor designs are being used in fresh juice pasteurization. The first
design approach uses a thin film UV reactor to reduce the path length and decrease problems related
to loss of penetration. Thin film reactors are characterized by laminar flow with a symbolic velocity
profile. The highest liquid velocity that is twice as fast as the average of liquid velocity is observed in
the center. The two laminar flow designs illustrated in Figure 1.7 are a Taylor-Couette flow UV
reactor (a) and the thin film Cider Sure reactor (b). Each thin film annular reactor has a UV lamp,
quartz sleeve and remote power supply (Koutchma & Stewart, 2005; Koutchma et al., 2004).
A second design approach raises the turbulence within a UV reactor to deliver all materials very
close to the UV light during the treatment. For example, The UV module (Salcor Inc, CA) shown in
29
Figure 1.8 contains a coiled Teflon tube with 24 ultraviolet lamps and reflectors. The coiled tube
causes additional turbulence and a secondary eddy flow effect. It is also known as a Dean effect, and
results in more residence time distribution and uniform velocity. The lamps and reflectors both are
located inside and outside the coiled tube to increase not only UV irradiance of the flowing liquid, but
also its uniformity (Koutchma & Stewart, 2005; Koutchma et al., 2004).
Figure 1.7-Schematics of (a) a laminar Taylor - Couette UV reactor and (b) a laminar thin film reactor (Cider Sure)
(Koutchma & Stewart, 2005)
Figure 1.8-Schematics of turbulent channel reactor (a) and Dean flow reactor (b) (Koutchma & Stewart, 2005)
a b
30
1.3.2.2.1 Effects of UV-C light treatment on food
Effects on microorganisms in liquid foods
UV-C light technology has been proven to be an effective method of eliminating and reducing
microbial contamination for a wide range of liquid foods and beverages (Forney, Koutchma and
Moraru, 2009). Churey et al. (2014) and Forney, Koutchma and Moraru (2009) report that the main
principle of UV-C decontamination is based on the formation of photoproducts in the DNA of
microorganisms, which prevents DNA replication as illustrated in Figure 1.9. Low -pressure mercury
lamps with the highest emission at 253.7 nm is the most effective wavelength to inactivate
microorganisms (Koutchma, 2014). For instance, Cider Sure 3500 gives more than 5-log reductions
of Cryptosporidium parvum and Escherichia coli O157:H7 (Churey et al., 2014). In addition,
Alzamora et al. (2010) compared the effects of UV-C light and High-Intensity Ultrasound (USc)
treatments on E. coli and yeasts in fruit juices (orange and/or apple juice). Their results were that UV
light reduced E. coli in fruit juices about 4.5-log cycle reduction higher than USc, and E. coli cocktail
was more sensitive to UV-C light than S. cerevisiae yeasts cocktail. Therefore, UV light technology
could achieve the 5-log reduction that is recommended by the HACCP juice regulation.
31
Figure 1.9-The effectiveness of UVC on the DNA structure in the microorganisms (source:
https://thatscienceguy.wordpress.com/category/cancer/skin/)
Effects on nutritional quality and enzymes of juices
Few studies have investigated the impacts of UV-C light on the physical, chemical and sensory
characteristics of low acid juices. For example, Corrales et al. (2012) show that in terms of soluble
solids content and pH of tiger nut milk, UV-C with different doses did not cause any significant
changes. In addition, in terms of chemical features, a study by Beltrán, González and Velascoa
(2014) on coconut milk reported that UV-C light reduced phenolic compounds, but antioxidant
activity hardly changed. Finally, in terms of sensory quality, Butz et al. (2010) study the influences
of UV-C light on color, browning degree, and dynamic viscosity of watermelon juice. The color
a* of juice decreased when UV dose increased, but the browning degree increased when time and
dose of UV-C increased. The same study by Beltrán, González and Velascoa (2014) demonstrates
that UV-C light affected color parameters of coconut milk when they used a double tube type UV-
32
C light system at different flow rates 0–30.33 mL/s and times 0–30 min. Some changes in
enzymatic activity are produced by the impact of UV-C light on low acid juices. For example, in
watermelon juice, UV-C light inactivated the "polygalacturonase, cellulase, xylanase, b-D-
galactosidase, and protease" which are responsible for juice viscosity (Butz et al., 2010).
Therefore, further research studies are required on the effects of UV light on products’ quality.
1.3.2.2.2 Advantages and disadvantages of UV-C treatment
Even though the UV-C radiation is an alternative and cheap method to decreasing
microorganisms on fresh fruit and vegetable surfaces, it has some limitations. The first limitation
is that liquid foods and beverages have a large range of the physical and optical characteristics,
various chemical compositions that impact the transmittance of UV light (UVT), distribution of
dose, transferring of impetus, and inactivation of microorganisms, as a result. In addition, other
factors that include the design of UV reactors and fluid dynamics criteria affect the UV treatment
efficiency of low UV transmittance liquids (Forney, Koutchma &Ye, 2011).
Therefore, in liquids, UV absorption and scattering occurs because of solutes and particles and
these are the most crucial limiting factors that determine the UV-C infiltration depth (Koutchma,
2004). For instance, UV-C has lower efficiency disinfection in orange juice, because of colored
compounds and pulp particles which cause poor UV-C light transmission (Alzamora et al., 2010).
This limitation can be compensated by technologies using centrifugal forces or by effective
mixing.
In summary, it has been seen that thermal treatment and non-thermal treatment (HHP and UV-
C light) have various effects on low acid foods in terms of microorganisms’ inactivation and
products’ qualities. However, there is no scientific data available on the treatment of wheatgrass
33
juice by thermal or other novel treatments. Wheatgrass juice requires alternative technologies to
increase its shelf life and keep its nutrients. Therefore, it is important to study and explore the
effects of the previously mentioned technologies’ treatment on the quality of wheatgrass juice.
34
Chapter 2
2. Materials and Methods
2.1.Chemicals
Pectinase (P2611-50mL) from Aspergillus Aculeatus with activity ≥ 3.800 units/mL was
purchased from Sigma Aldrich (Oakville, Canada Co). Folin-Ciocalteu reagent (2N, Cat. # F9252-
1L), Gallic Acid standard (Mw.188.1, G-8647). Phenolphthalein, sodium hydroxide, 2,6
dichlorophenolindo-phenol (2,6 DPIP) reagent, oxalic acid, acetone, sodium carbonate Na2CO3,
Sodium nitrite NaNO2, sodium hydroxide NaOH, sodium phosphate buffer, phosphate buffer (75
mM, pH 7.4), aluminum chloride, p-phenylenediamine, 2,2-diphenyl-1-picrylhydrazyl (DPPH),
hydrogen peroxide, catechol and Lowry solution is: solution A, (NaOH and Na2CO3); solution B,
(Copper (II) sulfate pentahydrate CuSO4. 5 H2O) and solution C (sodium tartrate, dihydrate Na2
Tartrate. 2H2O) were all purchased from Sigma Aldrich (Oakville, Canada Co).
DPPH 3.5 mM stock (MW 394.32), DPPH 350 mM working solution, Trolox (Mw 250.29) stock
solution (20 mM), methanol MeOH, fluorescein working solution (8.68×10-5mM) and 2,2’- azobis
(2-amidopropane) di hydrochloride (AAPH) reagent were used.
2.2. Wheatgrass Juice Extraction
2.2.1. Experimental materials
Frozen raw wheatgrass was obtained from Evergreen plant company in Don Mills ON, Canada.
The grass was harvested in May 2015 by harvesting machine, after 6 weeks when it reached 6 to
8 inches. The grass was washed by water and then was put in plastic bags and stored in freezer
until used.
35
2.2.2. Juice extraction
The processing steps of wheatgrass juice are explained below as used by De, Karmakarb, Nsoa
and Sagua for banana juice (2014). Raw wheatgrass was washed by distilled water to remove
surface filth and soil. Then the wheatgrass was cut to small pieces (≈ 5 cm) by scissor and 50 g of
wheatgrass was weighed and put into a beaker. Based on preliminary experiments, wheatgrass to
water ratio of 1:4 (weight/volume) was used to maximize extraction. Therefore, 200 mL of distilled
water for each 50 g of grass were used in the experiment at temperature 23 ± 2 °C. Pectinase 2%
was added to the grass in water. For example, enzyme dose of 2% (v/w) indicated that 1 ml of
enzyme was added to 50 g of wheatgrass. The control sample was pressed right away without
adding pectinase enzyme; however, the samples with pectinase were kept for different incubation
times (20, 60 and 120 minutes). The wheatgrass was pressed by the Juice Fountain Compact juicer
(Model BJE200XL Issue-D12, Breville, Canada) as in Figure (2.1). The extracted juice was
collected and stored at -20 °C.
Table 2.1-Processing parameters for the wheatgrass juice extraction from the raw material
independent variables *Control
Without pectinase
Samples with pectinase
enzyme
Incubation temperature (°C) 23 ± 2 23 ± 2 23 ± 2 23 ± 2
Incubation time (min) _ 20 60 120
Enzyme Concentration _ 2% 2% 2%
36
Figure 2.1-The juice fountain compact juicer used for wheatgrass juice extraction
2.3.Physical and chemical analysis of untreated wheatgrass juice
Prepare juice for experiments. Frozen wheatgrass juice, which was brought from Evergreen
plant company in Don Mills ON, Canada, was used in all experiments as in Figure 2.2. The frozen
juice at -20 °C was thawed in cold water (10 °C) before all experiments to measure the nutrients
content.
Figure 2.2-Wheatgrass juice used in all experiments
37
Determination of pH. The pH value of the sample was measured using an Oakton
pH/conductivity/TDS/°C/°F meter (pH/CON S10 series; serial number 531157, Eutech
instruments, Singapore)
Determination of Total Soluble Solid (°Brix). The Total Soluble Solid of wheatgrass juice was
determined as Brix using an optical Refractometer (Fisher Scientific, Canada) (0o to 18o Brix scale)
at 20 ± 2°C
Titratable Acidity Determination (TA %). The TA was determined according to Awonorin and
Udeozor (2014) method. A 20 mL of the wheatgrass juice was measured into a conical flask and
2 drops of 1 % phenolphthalein indicator was added to the mixture. The sample was titrated with
0.1N NaOH against a white background. The result was recorded as soon as the first appearance
of a dark red color. Titration continued until the color persisted, and the results obtained were
calculated as follows in Equation (2.1):
T.A% = Number of mL of NaOH used / sample taken (mL) × 100 = (2.1)
Determination of vitamin C. Vitamin C was carried out according to the procedure of Rastogi’s
method (2005). A 30-40 mL of thawed wheatgrass juice was taken and added 0.2 g of oxalic acid.
Using a 10 mL aliquot of prepared wheatgrass juice and titrated with 2,6 dichlorophenoindo-
phenol (2,6 DPIP) reagent. The endpoint was marked by the appearance of the first permanent
dark purplish red color. The vitamin C was calculated by using the following formula (2.2):
Mass of vitamin C mg/L= molecular weight of vitamin C × C- 2,6 DPIP × V- 2,6 DPIP (2.2)
Where C-2,6 DPIP is the concentration of 2,6 dichlorophenolindophenol, and
V- 2,6 DPIP is the volume of 2,6 dichlorophenolindophenol in L used in experiment
38
Determination of Chlorophyll. The values of chlorophyll a and b were measured using the
method by Dong et al. (2012) with some modification. A 3 mL acetone (80%v/v) was added to 3
mL of the thawed wheatgrass juice in a 20 mL tube at room temperature. The liquid was then
filtered three times by a 114 Whatman laboratory Division filter paper (wet strengthened circles
125 mm Ø 100 circles; Springfield Mill, Maid stone, Kent; England). The absorbance values at A
647 nm and A 664 nm and A 750 nm were measured at room temperature using the Smart-Spec
Plus Spectrophotometer (Bio-Rad Laboratories, Hercules, Canada). The total chlorophyll was
calculated by measuring chlorophyll a and b values which were calculated using the following
Equations (2.3), (2.4) and (2.5):
Chlorophyll a=11.85 × A664−1.54 × A647 (2.3)
Chlorophyll b= 21.03 × A664−5.43× A647 (2.4)
Total chlorophyll = chlorophyll a + chlorophyll b (2.5)
Determination of Total Protein. Total protein was assayed according to the method used in
proteins protocol (Lowry protocol, n.d). Wheatgrass juice was diluted by distilled water (1mL
sample in 19 mL distilled water, Dilution Factor = 20). A 0.5 mL of diluted wheatgrass juice was
added to 10 mL glass tube, and then added 0.7 mL of Lowry solution. Sample was vortexed and
incubated for 20 min in dark place at room temperature. The sample was taken out after 20 min,
and 0.1 mL of diluted Folin Reagent was added, the sample was vortexed and incubated once more
for 30 min in darkness at room temperature. The protein was measured the absorbance using the
Smart-Spec Plus Spectrophotometer (Bio-Rad Laboratories, Hercules, Canada) at wavelength
750nm at room temperature by Equations (2.6) and (2.7):
Protein was calculated by using the slope of the standard curve equation
39
Y= 0.0056 X + 1.8625 (2.6)
For the sample protein µg/ml (X) = Y/M multiply by dilution factor (2.7)
Where Y is the absorbance value for the sample at 750nm, and
M is the slope from the protein standard curve
Determination of total phenols content TPC.
A modified Folin-Ciocalteu method used for determining the TPC of wheatgrass juice. First, the
serial dilutions of Gallic Acid were prepared from 0.5 mg/mL to 0.25, 0.125, 0.0625, 0.03125,
0.015625 mg/mL. Then about 25 μL different G.A. standards aliquots were transferred to the
appropriate wells in triplicates and a 25 µL H2O was used as blank and point zero of standard
curve. A 25 µL of samples with different dilutions were transferred to the appropriate wells in
triplicates. Then about 14 mL of 1/10 diluted FCR was poured into a plastic solution basin and
pipet 125 μL of diluted FCR using a multichannel pipettor into each of the wells. The 96-well
microplate was swirled gently and let stand for 8 to 10 min. About 14 mL of 7.5 % Na2CO3 was
poured into a plastic solution basin and pipet 125 μL into each of the wells using a multichannel
pipettor and was stand for 30 min or longer. The absorbance at 765 nm was read using the
Microplate Reader, and the linear regression was made to obtain the standard curve and calculate
the total phenolic contents in the samples (Singleton, Orthofer & Lamuela-Raventós, 1999).
Determination of Antioxidant activity
2, 2-Diphenyl-1-Picrylhydrazyl (DPPH) Assay.
The total antioxidant method was described by Herald, Gadgil and Tilley (2012). A 225 mL of
MeOH was added to blank wells only, do not add DPPH to blank wells. In addition, a 25 mL
40
MeOH or acetone (same solvent used for samples) and 200 mL DPPH were added to Control wells.
A 25 mL was added to sample/standard wells (properly diluted) to the appropriate wells in
triplicates, then add 200 mL DPPH (350 mM). Gently swirl and tap to mix, seal with plate sealing
tape and allow to react at room temperature for 6 hours in the dark. Tape was removed carefully
and sample was read at 517 nm. The percentage of DPPH quenched was determined as Equation
(2.8): %DPPH quenched = [1-(A sample-A blank)/(A control-A blank) x 100 (2.8)
The percentage of DPPH quenched was plot against the concentrations of Trolox, and the
antioxidant capacity of samples is calculated from the linear equation.
Oxygen Radical Absorbance Capacity (ORAC) Assay.
A 25 μL of phosphate buffer (75 mM, pH 7.4) was put in blank wells, and a 25 μL of Trolox
dilutions (6.25, 12.5, 25.0, 50.0, 100μM) were received in standard wells. Then, sample wells
received 25 μL of appropriately diluted samples. A 150μl of 8.68×10-5mM fluorescein working
solution was added into all experimental wells. The plate was allowed to equilibrate by incubating
for a minimum of 30 min in the Synergy HT Multi-Detection Microplate Reader at 37°C.
Reactions were initiated by the addition of 25 μL of 2,2’- azobis(2-amidopropane) dihydrochloride
AAPH reagent followed by shaking at maximum intensity for 10 seconds. The fluorescence was
then monitored kinetically with data taken every minute for up to 2 hours by Fluorescence
microplate reader (FLx800, Bio-Tek Instruments Inc., Winooski, VT, USA) (Prior et al., 2003).
Determination of POD and PPO enzymatic activity.
Peroxidase POD was assayed according to methods described by Ancos, Cano and Hernandez
(1997) with some modification. First, sample was centrifuged by centrifuge (Avanti J-20 XPI
centrifuge; Beckman Coulter, California, USA) at 5000 × g for 5 min in 50-mL centrifuged tubes.
41
Then, centrifuged wheatgrass juice was diluted by distilled water, so 1mL of centrifuged juice was
diluted by 99 mL of distilled water (1 mL: 99 mL / Dilution Factor=100). Peroxidase activity was
measured by using the Smart-Spec Plus Spectrophotometer (Bio-Rad Laboratories, Hercules, CA).
A 1 mL of diluted wheatgrass juice was used and 0.32 mL of 0.1 M potassium phosphate buffer
(pH 6) and 5% pyrogallol (0.16 mL, w/v) were added. The reaction was initiated by addition 0.16
mL of hydrogen peroxide (0.5% w/w). Peroxidase was measuring using UV spectrophotometer at
A420 nm at 25 °C. The enzyme activities were determined by measuring the absorbance for 5 min
each 30 seconds.
Polyphenol oxidase PPO was assayed according to methods described by Ancos, Cano and
Hernandez (1997) with minor modification. First, sample was centrifuged by centrifuge (Avanti
J-20 XPI centrifuge; Beckman Coulter, California, USA) at 5000 × g for 5 min in 50-mL
centrifuged tubes. Then, centrifuged wheatgrass juice was diluted by distilled water, so 1mL of
centrifuged sample was diluted by 1 mL of distilled water (1mL: 1mL / Dilution Factor=2).
Enzyme activity was measured by using 1 mL of diluted wheatgrass juice and 1 mL of catechol
(0.07M) in sodium phosphate buffer (pH 6.5, 0.05M) was added. The activity was measuring using
the SmartSpec Plus Spectrophotometer (Bio-Rad Laboratories, Hercules, CA) at 420 nm at 25 °C.
The enzyme activities were determined by measuring the absorbance for 10 min each 2 min.
The slope of the very first linear part of the reaction curve was taken as the POD/PPO specific
activity (Abs/min).
Color measurement. The color was detected according to method of Hu et al. (2012). Color
assessment was conducted at room temperature using Spectrophotometer CM-3500d (Konica
Minolta Sensing, Inc. made in Japan). Hunter L* (lightness), a* (greenness), and b* (yellowness)
42
values of wheatgrass juice were measured and the total color difference (ΔE) was calculated using
equation (2.9).
ΔE= [(L*−L0*) 2 + (a*−a0)
2 + (b*−b0)2]1/2 (2.9)
Where L* is lightness of treated juice,
L*0 is lightness of the control
a* is greenness of treated juice
a0* is greenness of the control
b* is yellowness of treated juice
b0* is yellowness of control
2.4.Thermal and Non-thermal treatments of wheatgrass juice
2.4.1. Thermal treatment (HTST)
Wheatgrass juice was pasteurized in a covered water bath (Serological Water bath 148007
Series, Boekel industries, INC Canada). Juice samples were pasteurized with the following
condition: 75°C at 15s which based on literature findings (Chen and Neetoo, 2012).
The thermal treatment was carried out at an atmospheric pressure. The small frozen wheatgrass
juice bags from Evergreen plant company (0.6 oz each) were thawed in cold water. Aliquots of 10
mL of the wheatgrass juice were transferred into sterile test tubes and subsequently spiked with
the appropriate concentration of target microbes (0.1 mL of 109 log of three different types of
bacteria (E. coli P36, Salmonella typhimurium WG 49 and Listeria innocua ATCC 51742). The
initial temperature of water bath was 75°C and treatment time was measured after the samples
reached the target temperature (75°C). All fresh wheatgrass juices were pasteurized at same
condition and were then placed in an ice bath and stored at 4 ± 2 °C until analysis. The time needed
43
for samples to reach 75°C was 6 minutes. The digital thermometer (S/N 80583726; Traceable
Fisher Scientific, Canada) was used to measure the temperature during the treatment.
2.4.2. Non-thermal treatment
2.4.2.1.High Hydrostatic Pressure
The HHP treatment was conducted on HHP unit (55 L; Grid path Solutions Inc., Stoney Creek,
ON, Canada) that is a Hyperbaric 55 L capacity chamber that could apply pressures up to 600
MPa. Hiperbaric 55 is an industrial production equipment includes:
A 55 L volume vessel (200mm diameter).
22 m2 surface requirement.
Automatic loading/ unloading system.
Ergonomics and speed (source: http://www.hiperbaric.com/en/hiperbaric55)
In the HHP treatment for microbial inactivation, the primary HHP processing conditions were
based on literature findings and recommendations (Chen and Neetoo, 2012). Three process
pressures of 400, 500, 600 MPa and times at 60, 90, and 180 seconds have been used to confirm if
they achieve the 5-log reduction of pathogenic bacteria in wheatgrass juice or not. In addition,
600 MPa for 3 min are typically used by industry to treat juice products.
Sample preparation and transportation for HHP treatment
The small frozen wheatgrass juice bags (0.6 oz each) were thawed in cold water. The juice was
dispensed in 10 mL aliquots into plastic pouches (their size 23 × 15.5 cm) (Winpak, MB, Canada)
and subsequently spiked with the appropriate concentration of target microbes (0.1 mL of 109 log
of three different types of bacteria. The pouches were vacuum sealed to exclude air and the
vacuumed bags were kept in -20 °C freezer over night until the HHP treatment was done. The
44
plastic bags were transported in a cold box to the HHP processing facility. Vacuum packed juices
10 mL were pressurized in a HHP unit (55 L; Grid path Solutions Inc., Stoney Creek, ON, Canada).
All treatments were undertaken at 11 °C unless otherwise stated.
2.4.2.2.UV-C parameters for wheatgrass juice treatment
1. Collimated beam and experiment set up
Inoculated wheatgrass juice samples were treated with UV-C irradiation using a collimated
beam apparatus as shown in Figure (2.3). Collimated beam is a bench scale apparatus is used to
determine UV response. Collimated beam has output of a UV lamp is directed onto a horizontal
surface, either down a long ‘‘collimator,’’ consisting of a cylindrical tube or by successive
apertures. Since there remains some dispersion in the beam, the beam is never collimated. The cell
suspension to be irradiated is placed on the horizontal surface below the bottom of the collimator
(Bolton & Linden, 2003). The system consisted of a 30 W low-pressure mercury vapor UV lamp
emitting at 254 nm (Trojan Technologies Inc., London, Canada).
Sample preparation for collimated beam
A 5mL of juice sample was added to the Petri dish (50 x 35mm; Kimax, Kimble Chase, Vineland,
NJ, United State) to obtain a sample depth of 0.5 cm. The UV intensity at the surface of the sample
(incident intensity (Io) (0.107 mW/cm2) was measured using a radiometer with UVX-25 sensor
(UVX, UVP Inc., CA, USA). The radiometer was placed at the same distance as the liquid
interface. A magnetic stirrer placed on the petri dish to have an adequate mixing during treatment.
The UV lamp was switched on for 45 min to reach the optimal working conditions before
irradiating the wheatgrass juice samples. Samples were directly inoculated with the three bacteria
(L. innocua ATCC 51742, E. coli P36 and Salmonella typhimurium WG49) to provide a final
inoculum of 107 CFU/mL. Samples were exposed to direct UV light and the degree of inactivation
45
of microorganisms occurs by UV radiation is directly related to the UV dose. The UV dosage can
be calculated as:
Eave was calculated using Equation (2.10):
Eave = E0 x PF x WF x DF x RE x t (2.10)
Where E0 is the measured intensity,
PF is the petri dish factor, WF is the water quality factor,
DF is the divergence factor, RE is the Reflection factor and t is time in seconds as illustrated in
Table (2.2) (Bolton & Linden, 2003).
Table 2.2-Parameters of the collimated beam during wheatgrass juice treatment
Factors Values
E0, mW/cm2 0.107
Reflection Factor 0.975
Absorption coefficient, cm-1 43.33
L (distance from the UV lamp to the surface
of cell suspension, cm
32
l = vertical path length, cm 0.5
t= Time, second 3600, 5400 and
7200
This Reflection Factor basically represents the part of the incident beam that enters the water. A
change of refractive index occurs whenever a beam of light passes from one medium to another.
This change of refractive index causes a small fraction of that light beam to be reflected off the
surface between the media. The average refractive indices are 1.000 and 1.372 for an area of 200-
300 nm. Therefore, after calculation, R=0.025 for these two media, the Reflection Factor is (1-R)
= 0.975.
The Petri Dish Factor is essentially the incident fluency ratio over the surface area of the test
Petri dish. The ratio is calculated by the average of the radiance incident over the whole area of
46
the Petri dish to the irradiance specifically at the center of the dish. Methodically, the Petri Factor
is determined by scanning the area of the Petri dish with a radiometer detector (every 5 mm) and
averaging the irradiance over the area of the Petri dish.
The Water Factor is defined as; Water Factor = 1-10-al/al ln (10)
Where, a = absorption coefficient (43.33 cm-1) or absorbance for a 1 cm path length, and l = vertical
path length (0.5 cm) of water in the Petri Dish
The absorption coefficient of the sample juice was determined from the slope of the linear plot
of absorbance vs. path length (cm) using spectrophotometer
The Divergence Factor is the average of the above function over the path length l of the cell
suspension. The divergence factor and the absorbance effects of water are considered together.
Divergence Factor = L/ (L + l)
L is the distance from the UV lamp to the surface of cell suspension (32 cm).
UV dose delivered was determined for the bioassay trails by calculating the average intensity with
a fixed time of UV exposure [Actual dose = Eave x time (s) = J/cm2] (Bolton & Linden, 2003).
Figure 2.3-Schematic graph of Collimated beam instrument (source: https://www.researchgate.net /figure/
44679202_ fig1_Fig-1-e-Collimated-beam-apparatus-employed-for-UVH-2-O-2-treatments)
47
2- Dean Flow Reactor and experiment set up
Because of a smaller particle size and a lower dissolved compounds concentration in juices, the
radiation depth penetration is almost small and most radiation is absorbed within a few millimeters.
As a result, the effectiveness of a radiation treatment system is significantly dependent on effective
UV reactor design. A helically cavity Quartz-coil is used to cause a secondary eddy flow. This
type of liquid flow results in secondary vortices (Dean Vortices) (Müller, Stahl, Graef, Franz, &
Huch, 2011).
In the laboratory, a Dean Flow reactor was used for the UV-C treatment of wheatgrass juice. The
system consisted of a 30 W low-pressure mercury vapor UV lamp emitting at 254 nm (Trojan
Technologies Inc., London, Canada). The main component is a module which consists of a quartz
coil with a helically wound cavity as shown in Figure 2.4. The Dean flow UV unit characteristic
dimensions are given in Table 2.2. The experimental set up is shown in Figure 2.5. A 20 mL
wheatgrass juice was inoculated with three tested bacteria in the inlet tube and then the juice was
pumped through the coil by a Masterflex L/S pump (HV-77202-60, assembled in USA) into outlet
tube. The UV-C source was mounted in the center of the quartz rotor Figure 2.5. As fluid was
passing through the unit, it was exposed to the UV light at flow rate 2.6 cm3/s and at the
corresponding UVC intensity (1.5 mW/cm2) as illustrated in Table 2.3. The UV intensity (incident
intensity (Io) at the surface of the quartz rotor was measured using the digital UVX radiometer
(UVP LLC, Canada). The radiometer was placed at the same distance of the coil tube from the
lamp. The initial temperature of juice was maintained at 10 °C in all experiments. Microbial,
chemical, and physical analyses followed.
48
Table 2.3-Technical characteristic of the Dean Flow reactor
Characteristics Values
Flow rate, cm3/s 2.6
Coil tube volume, cm3 6.3
Hydraulic diameter of the quartz tube, cm 0.195
Inner diameter of coil, cm 2.93
Number of coil rotors 23
Coil Length, cm 211.6
Absorption Co-efficient of WGJ, cm-1 43.33 cm-1
Light irradiance (I0), mW/cm2 1.5
Velocity, m/s 0.892
Dynamic viscosity, Pa – s 1.66 ± 0.073
Density, kg/m3 980
Figure 2.4-The quartz coil of UV-C reactor used for treatment of wheatgrass juice
UV-C Lamp Sample OUT Sample IN
Quartz Coil
49
Figure 2.5. Dean Flow reactor experiment set up
Flow dynamics
First, flow dynamics in the UV reactor was evaluated for wheatgrass juice. Velocity (v) was
calculated by v= Q/A (cm/s)
Q is a volumetric flow rate (mL/s),
and A is a cross section area of the coiled tube, cm2
Dean vortices calculation
Different cycles with the same flow rate were used to investigate the effect of Dean vortices on
the UV-C inactivation of microorganisms and effects on nutrients. Reynolds number (Re) is the
ratio of inertial forces to viscous forces and is expressed in Equation 2.11. The viscosity of
wheatgrass juice was measured by viscometer (Small sample adapter (SC418 13R), Spindle #18)
Master-flex
L/S pump UV light
source Radiometer
50
to calculate the Reynolds number. The Dean number (De) is calculated by the Reynolds number
and the geometric data (dh and D) of the helical cavity tube (Müller et al., 2011).
Re= u × dh / v = u × dh × p / ɳ (2.11)
Where dh is the hydraulic diameter of the tube,
D is the diameter of the coil, u is the velocity (m/s),
ɳ is the dynamic viscosity (Pa _ s) and p is the density.
The Dean number was calculated as follows Equation (2.12):
De= Re √dh/D (2.12)
UV dose calculation in Dean Flow reactor
The light irradiance (I0) was measured at the surface of quartz rotor using the digital UVX
radiometer (UVP LLC, Canada) and its average value is given in Table 2.2
The UVC dosage was inversely proportional to the juice flow rate and was determined based on
the energy delivered per volume of juice. The dose, expressed as joules per milliliter, was
calculated theoretically using Equation (2.13).
UV-C Applied dose J/mL = Total UVC output power W / Flow rate mL/s (2.13)
30 x 0.3 x 0.7 / 2.6 =2.42 J/ml = 2420 J/l
30% efficiency of the UVC lamp need to be considered along with 70 % of UV Transmission of
quartz coil) (Müller et al., 2011).
UV absorbed dose per cycle = 0.7 × Intensity (I0) × Residence Time (s) (2.14)
Residence Time (s) = Volume of the coil / Flow rate (2.15)
6.3 ml / 2.6 ml/s = 2.42 s
UV dose = 0.7 × 1.5 mW/cm2 × 2.42 s = 2.54 mJ/cm2
51
Table 2.4- Overview of the different UV-C treatments at a flow rate of 2.6 cm3/s.
Cycle Re Dean
number
UV-C Applied dose
J/L
UV absorbed dose
mJ/cm2
1 2420 2.54
2 4840 5.08
3 7260 7.62
4 9680 10.16
5 1027 265 12100 12.7
6 14520 15.24
7 16940 17.78
8 19360 20.32
9 21780 22.86
10 24200 25.4
2.5.Microbes and cultivation methods
Bacteria used in this study were Escherichia coli P36 originally isolated from spinach (Warriner
et al., 2003). Salmonella typhimurium WG49 and Listeria innocua ATCC 51742 obtained from
the American Type Culture Collection (ATCC).
2.5.1. Escherichia coli P36 cultivation and enumeration
E. coli P36 suspensions were prepared from overnight culture grown aerobically at 37°C in
Tryptone Soy Broth (TSB; OXOID Ltd, Basingstoke, Hampshire, United Kingdom). The cells
were harvested by centrifugation (Avanti J-20 XPI centrifuge; Beckman Coulter, California, USA)
at 5000 × g for 10 min in 50-mL centrifuged tubes. Finally, the cell pellet was suspended in 0.9%
w/v sterile saline to give a cell density of 109 CFU/mL at 600 nm. For enumeration, a 0.1 mL of
wheatgrass juice was mixed thoroughly, added to 0.9 mL of sterile saline (0.9% w/v), and then
serially diluted to 10-5 (serial dilutions of the bacteria, treated and untreated samples are 0, -1, -2,
-3, -4 and -5). A 0.1 mL aliquot of each dilution was plated on Tryptone Soy Agar (Casein soya
bean digest agar) (TSA; OXOID Ltd, Basingstoke, Hants, United Kingdom) supplemented with
52
30 μg/mL sterilized kanamycin Sulfate. The plates were incubated at 37 °C for 24 h and the
colonies were counted and reported as CFU/mL using Equation (2.16).
CFU/mL = 1/ [# of colonies * aliquot plated (0.1mL) * dilution factor] (2.16)
2.5.2. Salmonella Typhimurium WG49 cultivation and enumeration
Suspension of Salmonella WG49 was prepared from overnight culture grown aerobically at 37°C
in Tryptone Soy Broth (TSB; OXOID Ltd, Basingstoke, Hampshire, England). The cells were
harvested by centrifugation (5000 rpm for 10 min at room temperature). Finally, the cell pellet was
suspended in 0.9 % w/v sterile saline to give a final optical density of 109 CFU/mL at 600 nm then
stored at 4 °C until required. For enumeration, serial dilutions of the bacterium, treated and
untreated samples until 10-5 were prepared in sterile saline and plated on Xylose lysine
deoxycholate agar XLD (Oxoid Ltd, Basingstoke, United Kingdom). The plates were incubated at
37 °C for 24 h, and the colonies were counted and reported as CFU/mL using Equation (2.16).
2.5.3. Listeria innocua ATCC 51742 cultivation and enumeration
Listeria innocua was cultivated in Tryptone Soy Broth (TSB; OXOID Ltd, Basingstoke,
Hampshire, England) incubated at 30 °C for 24 h. The cells were harvested by centrifugation (5000
rpm for 10 min at room temperature) and suspended in sterile saline to give a final optical density
of 109 CFU/mL at 600nm. The cell suspension was transferred to 4 °C until required. For
enumeration, serial dilutions of the bacterium and untreated samples and treated samples until 105
were prepared in sterile saline (0.9 % w/v) and plated onto Listeria Selective Agar Base (Oxford
Formulation; LSA; Oxoid Ltd, Basingstoke, United Kingdom) with Listeria Selective Supplement
(SR 0234E). The plates were incubated at 30 °C for 48 h and typical colonies were counted and
reported as CFU/mL using Equation (2.16).
53
2.6.Preparation of wheatgrass juice sample for different treatments to analyze the nutrients
2.6.1. HTST Pasteurization
In the thermal treatment, the wheatgrass juice samples from Evergreen juices company were
treated at condition 75 °C for 15s, which achieved the 5-log reduction for the tested bacteria. The
preparation of the sample for nutrient analysis was same as the microbial samples preparation, but
without microbial inoculation. After the samples were thermally treated, all nutritional analyses
were done as in section (2.3).
2.6.2. Non-thermal treatment
2.6.2.1. High Hydrostatic Pressure
Processing pressures of 500 MPa and 600 MPa for 60, 90 and 180 seconds for each pressure were
used to treat the wheatgrass juice for quality and nutrients analysis because the 5-log reduction for
three types of tested bacteria was achieved at these conditions. After the samples were treated by
HHP, all nutritional analyses were done as in section (2.3).
2.6.2.2.UV-C treatment
In UV-C Light treatment, the different cycles were evaluated for microbial reduction first. After
ten cycles through the system the 5-log CFU reduction of three tested bacteria was achieved and
the total UV absorbed dose was 25.4 mJ/cm2 for 10 cycles. Therefore, the sample was treated for
10 cycles for nutrients evaluation. As a result, after the samples were treated by Dean flow reactor,
all nutritional analyses were done as in section (2.3).
Storage conditions after treatment. The treated samples were stored at -20 °C in darkness until
physicochemical analyses were conducted. For all kinds of treatment, three different batches (n=3)
were considered and analyzed separately.
54
2.7.Experimental Design and Statistics
2.7.1. Microbial counts
The plates of E. coli P36 and Salmonella typhimurium WG49 were incubated at 37 °C for 24 h,
but the plates of Listeria innocua ATCC 51742 were incubated at 30 °C for 48 h. After the
incubation, the colonies were counted. The logarithmic count reduction (LCR) was calculated
using the following Equation 2.17:
LCR = Log10 Ni – Log10 No (2.17)
Where the: Ni = the initial microbial loading
No = the surviving numbers (post-treatment count). All trials were performed at least three times
using three replicates sample in each treatment.
2.7.2. Statistics of physiochemical analysis of wheatgrass juice nutrients
All experiments of nutrients analysis were done in triplicate for each treatment, and all data were
expressed as mean and standard deviation of triplicate observations. All the data were analyzed
using the (IBM SPSS Statistics 23) version software. Analysis of variance were performed by
(one-way ANOVA) and Tukey’s test. P ≤ 0.05 was considered statistically significant.
55
Chapter 3
3. Results
3.1.Wheatgrass Juice Extraction
The first objective of this study is to increase the extraction of juice from raw wheatgrass by
using pectinase enzyme. As shown in Figure 3.1, there was no significant (P ˃ 0.05) increase in
the juice extraction by using pectinase enzyme (≥ 3.800 units/mL) at concentration 2%.
Figure 3.1-Wheatgrass juice extraction from raw materials with and without pectinase enzyme at
different incubation times
3.2.Physical and chemical properties of raw wheatgrass juice from the evergreen plant
company
Different experiments were performed to assess the physical and chemical properties of raw
wheatgrass juice which are reported in Table 3.1. In term of physical properties, wheatgrass can
be considering a low acid juice with low solids content with variable vitamin C and chlorophyll
content Table 3.1.
5
10
15
20
25
30
Control Pectinase+20min Pectinase+60min Pectinase+120min
Am
ou
nt
in m
L/5
0 g
Extract of wheatgrass Juice
56
The use of DPPH and ORAC assays are reliable methods for determining the antioxidant ability
of wheatgrass juice. As shown in the Table 3.1, the juice has high antioxidants activity by using
two different assays DPPH and ORAC. The total phenolic content (TPC) of the wheatgrass juice
amount is also summarized in Table 3.1. The TPC was found to be high in the wheatgrass juice,
and the value of TPC in wheatgrass juice is 341.76 mg equivalent of Gallic acid/100 mL ± 15.46.
In term of quality parameters, the enzyme activity of POD and PPO enzymes were measured. It is
found that the wheatgrass juice had high peroxidase activity, but the polyphenol oxidase activity
was little low. Finally, color is also shown in Table 3.1 in terms of color values a* (greenness),
b*(yellowness) and L*(lightness).
Table 3.1-Physical and chemical properties of wheatgrass juice from Evergreen company (Don Mills
ON, Canada)
Properties *Values Properties *Values
pH 5.7 ± 0.0057 TPC a 341.76 ± 15.46
TSS (Brix) 3.16 ± 0.28 DPPH b 1511.6 ± 46.38
TA % 27.29 ± 0.241 ORAC c 5.30 ± 1.98
Vitamin C mg/100mL 9.21 ± 0.162 Peroxidase units/mL 0.810 ± 0.085
Chlorophyll mg/100mL 1.45 ± 0.042 Polyphenol oxidase units/mL 0.015 ± 0.0007
Protein mg/100mL 511.86 ± 32.87 Color L*
a*
b*
19.81 ± 0.237
-6.63 ± 0.383
19.60 ± 0.366
*Values are the Mean ± SD of three replicated a TPC expressed as mg equivalents of Gallic acid/100mL
b DPPH expressed as Trolox Eq. uM c ORAC expressed as Trolox Equivalent Mm
57
3.3.Effect of thermal and non-thermal treatment on microbial survival
3.3.1. Thermal treatment (HTST)
The microbial inactivation in wheatgrass juice treated with thermal processing (75 °C/15 s) was
investigated. Temperatures obtained during treatments were monitored using data-logger
(time/temperature profile) as in Figure 3.2, and the time needed for samples to reach 75°C was 6
minutes. The impacts of HTST treatment on L. innocua ATCC 51742, Salmonella WG49 and E.
coli P36 counts in the wheatgrass juice are illustrated in Table 3.2. The initial count of tested
bacteria in the juice prior to treatment was around 7-log10 CFU/mL. Thermal treatment at 75 °C
for 15s decreased the three tested bacteria counts to ˂ 2-log10 CFU/mL. D values of three different
bacteria were included in Table 3.2. Therefore, thermal pasteurization at 75 °C for 15s resulted in
sufficient reduction of Listeria innocua ATCC 51742, Salmonella WG49 and E. coli P36 in
wheatgrass juice to 5-log reduction and more.
Figure 3.2- Time/Temperature monitoring of wheatgrass juice during thermal treatment
0
10
20
30
40
50
60
70
80
0 0.5 1 1.5 2 2.5 3 3.5 4 4.5 5 5.5 6
Tem
pra
ture
°C
Time/min
58
Table 3.2-Microbial inactivation for inoculated wheatgrass juice with different bacteria after thermal
treatment (75 °C/15s)
*Values are the Mean ± SD of three replicated LCR Log Count Reduction CFU Colony-Forming Unit
Name of bacteria D values
(s)
*Initial Log10 (CFU/mL)
Ni
*Log10 (CFU/mL)
N0
*Log Reductions (Ni-N0)
LCR
Listeria innocua
2.8
7.15 ± 0.05
1.84 ± 0.05
5.31 ± 0.06
Salmonella WG49 2.4 7.36 ± 0.08 1.20 ± 0.14 6.16 ± 0.17
E. coli P36 2.8 7.12 ± 0.10 1.77 ± 0.32 5.35 ± 0.4
59
3.3.2. Non-thermal treatment
3.3.2.1.Effects of High Hydrostatic Pressure on bacterial inactivation
The initial L. innocua ATCC 51742 counts in the wheatgrass juice prior to pressure treatment was
7.11 ± 0.02 -log10 CFU/mL. Bacterial counts were 2.51 ± 0.52, 2.53 ± 0.41 and 2.28 ± 0.39-log10
CFU/mL after pressurization at 400 MPa for 60, 90 and 180 seconds, respectively Figure 3.3. In
addition, treatment at 500 MPa for 60, 90 and 180 seconds resulted in a reduction of 1.77 ± 0.17,
1.46 ± 0.12 and 1.4 ± 0.14-log10 CFU/mL, respectively. After treatment at 600 MPa for 60, 90 and
180 seconds, the Listeria innocua bacteria counts were reduced to 1.54 ± 0.24, 1.83 ± 0.25 and
1.42 ± 0.08-log10 CFU/mL, respectively. Therefore, the pressure treatments at 600 MPa time did
not resulted in higher inactivation compare with treatment at 500 MPa, but the inactivation was
higher than 400 MPa.
Figure 3.3-Microbial inactivation curve for inoculated wheatgrass juice with Listeria innocua after
HHP Treatment
0
1
2
3
4
5
6
7
8
9
10
0 30 60 90 120 150 180 210
Lo
g C
FU
/mL
(N0)
Time (s)
Listeria Innocua after HHP Treatment
600 MPa
400 MPa
500 MPa
60
Wheatgrass juice inoculated with Salmonella WG 49 was exposed to different pressure-time
combinations. Salmonella WG 49 was completely inactivated by the treatments in this study, and
their levels were below the detection limit of 1.0 log cycle when it exposure to 500 and 600 MPa
as shown in Figure 3.4. However, treatment at 400 MPa for 60s (4.33 ± 0.58) and 90s (4.69 ±
0.50) did not give rise to significant reductions in the bacterial population except for 400MPa at
180 s the LCR was 5.27. No significant differences in HHP resistance were observed for
Salmonella WG 49 strain at the pressure-time combinations assayed for 500MPa and 600MPa.
Figure 3.4-Microbial inactivation curve for inoculated wheatgrass juice with Salmonella WG 49
after HHP Treatment
Culture of Escherichia coli P36 in wheatgrass juice was exposed also to 400 MPa, 500MPa and
600 MPa at 60, 90, and 180 sec. for each pressure. The initial E. coli P36 counts in the wheatgrass
juice prior to pressure treatment was 7.11± 0.08-log CFU/mL as in Figure 3.5. E. coli P36 counts
were 2.51 ± 0.52, 2.53 ± 0.41 and 1.95 ± 0.07-log10 CFU/mL after treatment at 400 MPa for 60,
90 and 180 seconds, respectively. In addition, treatment at 500 MPa for 60 and 90 seconds resulted
0
1
2
3
4
5
6
7
8
9
10
0 30 60 90 120 150 180 210
Lo
g (
CF
U/m
L)
N0
Time (s)
Salmonella WG 49 after HHP Treatment
600MPa
500MPa
400MPa
61
in reduction of bacteria to 1.16 ± 0.22 and 1 ± 0.00-log10 CFU/mL, respectively. However,
treatments at 500MPa for 180s and at 600 MPa for 60, 90 and 180 seconds, gave the same
inactivation for E. coli (under the detection limit) Figure 3.5. Therefore, the pressure treatments
at 500 and 600 MPa time resulted in the reduction of Listeria innocua, Salmonella WG49 and E.
coli P36 in wheatgrass juice to 5-log reduction and more.
Figure 3.5-Microbial inactivation curve for inoculated wheatgrass juice with E. coli P36 after HHP
Treatment
D values calcuation after HHP: D values of the three different bacteria were determined at
pressure 400 MPa, 500 MPa and 600MPa for different times. D values of different bacteria are
showen in Table 3.3 and figures for calculation of D values are in the appendix. Figures 3.6, 3.7
and 3.8 were summarized the comparison of log reduction of different bacteria at different
pressures.
0
1
2
3
4
5
6
7
8
9
10
0 30 60 90 120 150 180 210
Lo
g (
CF
U/m
L)
N0
Time (s)
E.coli P36 after HHP Treatment
600MPa
500MPa
400MPa
62
Table 3.3-D values (s) achieved 1-log reduction for different microbes in wheatgrass juice after HHP
treatment at 400 MPa, 500 MPa and 600MPa for 60, 90 and 180s
Figure 3.6- Pressure and time response of E.coli P36, listeria innocua and salmonella WG49 in wheatgrass
juice after HHP treatment at 600MPa
0
1
2
3
4
5
6
7
8
0 30 60 90 120 150 180 210
Lo
g i
na
ctiv
ati
on
LC
R
Time (s)
Log inactivation for three bacteria at 600MPa
Salmonella WG49
E.coli P36
Listeria innocua
Microbes D values (s)
400MPa
D values (s)
500MPa
D values (s)
600MPa
E. coli P36 17 ± 0.2 12.9 ± 0.06 11 ± 0.06
Salmonella WG49 17.1 ± 0.05 11.5 ± 0.4 10.7 ± 0.33
Listeria Innocua 17 ± 0.6 14.1 ± 0.1 14.2 ± 0.8
63
Figure 3.7- Pressure and time response of E.coli P36, listeria innocua and salmonella WG49 in wheatgrass
juice after HHP treatment at 500MPa
Figure 3.8- Pressure and time response of E.coli P36, listeria innocua and salmonella WG49 in wheatgrass
juice after HHP treatment at 400MPa
0
1
2
3
4
5
6
7
8
0 30 60 90 120 150 180 210
Lo
g i
na
ctiv
ati
on
LC
R
Time (s)
Log inactivation for three bacteria at 500MPa
Salmonella
WG49E.coli P36
Listeria innocua
0
1
2
3
4
5
6
0 30 60 90 120 150 180 210
Lo
g i
na
ctiv
ati
on
LC
R
Time (s)
Log inactivation for three bacteria at 400MPa
Salmonella
WG49Listeria innocua
E.coli P36
64
3.3.2.2.Ultraviolet light treatment
1- Collimated beam
The following results of experiments were undertaken to determine the UV inactivation kinetics
of tested microbes in wheatgrass juice. The relative inactivation kinetics of the model bacteria was
determined in saline and wheatgrass was determined using collimated beam. The delivered dose
obtained for the various microbes in wheatgrass juice, and then D values (UV dose to support a 1-
log reduction) for the inactivation of microbes was calculated as in Figures 3.9, 3.10 and 3.11. As
shown in Tables 3.4 and 3.5. E coli P36 and Salmonella WG49 were found to be the most UV
resistant of the tested microbes and the Listeria innocua being the most sensitive one.
Table 3.4-D values (mJ/cm2) achieved 1 log reduction for different microbes in wheatgrass juice
Microbes D values (mJ/cm2) of
Saline
D values (mJ/cm2) of
Wheatgrass juice
E. coli P36 0.22 ± 0.01 2.1 ± 0.05
Listeria Innocua 1.31 ± 0.01 1.6 ± 0.3
Salmonella WG49 0.51 ± 0.02 2.1 ± 0.07
65
Table 3.5-Effect of UV dose on inactivation of different microbes in wheatgrass juice using
collimated beam
Microbes Time
(min)
Delivered dose
(mJ/cm2)
*Initial Log10
(CFU/mL) Ni
*Log10
(CFU/mL) N0
*Log Reductions
(Ni-N0)
E. coli P36 60 7.2 7.32 ± 0.07 3.5 ± 0.02 3.8 ± 0.02
90 10.8 7.32 ± 0.07 2.27 ± 0.19 5.05 ± 0.24
120 14.4 7.32 ± 0.07 2.26 ± 0.01 5.09 ± 0.1
Listeria
Innocua
60 7.2 7.32 ± 0.03 1.82 ± 0.3 5.5 ± 0.04
90 10.8 7.32 ± 0.03 1.10±0.14 6.22 ± 0.17
120 14.4 7.32 ± 0.03 1.10±0.18 6.22 ± 0.3
Salmonella
WG49
60 7.2 7.32 ± 0.4 4.31 ± 0.01 3.01 ± 0.02
90 10.8 7.32 ± 0.4 2.09 ±0.1 5.23 ± 0.12
120 14.4 7.32 ± 0.4 2.07 ± 0.02 5.25 ± 0.02
*Values are the Mean ± SD of three replicated LCR Log Count Reduction CFU Colony-Forming Unit
Figure 3.9-D value calculation for E.coli in wheatgrass juice with different UV doses using collimated beam
y = -0.4766x + 7.2229
R² = 0.9905
0
1
2
3
4
5
6
7
8
0 1 2 3 4 5 6 7 8 9 10 11 12
Lo
g N
0
UV Dose, mJ/cm2
E.coli P36 D=-1/K=-1/-0.476= 2.1 mJ/cm2
66
Figure 3.10-D value calculation for Listeria innocua in wheatgrass juice with different UV doses using collimated beam
Figure 3.11-D value calculation for Salmonella WG49 in wheatgrass juice with different UV doses using
collimated beam
The predicted UV doses in order to achieve a 5 log CFU reduction were verified by running
collimated beam studies using different times. From the results, applied dose 10.8 mJ/cm2 could
achieve 5 Log reduction in the verification study according to the predicted times for three tested
bacteria as illustrated in Figure 3.12.
y = -0.6028x + 7.03
R² = 0.9491
0
2
4
6
8
0 1 2 3 4 5 6 7 8 9 10 11 12
Lo
g N
0
UV Dose, mJ/cm2
Listeria Innocua
y = -0.475x + 7.42
R² = 0.9898
0
2
4
6
8
0 1 2 3 4 5 6 7 8 9 10 11 12
Lo
g N
0
UV Dose mJ/cm2
Salmonella WG49
D=-1/K=-1/-0.602= 1.6 mJ/cm2
D=-1/K=-1/-0.475= 2.1 mJ/cm2
67
Figure 3.12- UV-Dose response of E.coli P36, listeria innocua and salmonella WG49 in wheatgrass juice
using collimated beam
2- Dean flow UV reactor microbial results
Different tested bacteria inoculated in wheatgrass juice were exposed to different UV-Light doses
by cycles. The initial microbial counts in the juice prior to all UV treatments was around 7 log
CFU/mL. As illustrated in Figures 3.13/3.14/3.15, the UV doses were from 2.5 mJ/cm2 to 25.4
mJ/cm2 from one cycle to 10 cycles. E. coli and Salmonella WG 49 reduced about 0.5-log for each
cycle and reached the 5-log reduction at cycle 10 (Dose 25.4mJ/cm2); however, L. innocua
achieved the 5-log reduction at cycle 7 (Dose 17.78mJ/cm2). D values were calculated for different
tested bacteria after Dean flow UV treatment as shown in Table 3.6. Figure 3.16 is compared the
response of the three different bacteria to the UV treatment.
0
1
2
3
4
5
6
7
0 2 4 6 8 10 12 14 16
Lo
g i
na
ctiv
ati
on
of
test
ed
ba
cter
ia
UV Dose, mJ/cm2
E.coli
Listeria innocua
Salmonella
68
Table 3.6-D values (mJ/cm2) achieved 1 log reduction for different microbes in wheatgrass juice
after Dean flow UV treatment.
Figure 3.13-D value calculation for E. coli after Dean flow UV treatment (Quartz coli reactor)
Figure 3.14 -D value calculation for salmonella WG49after Dean flow UV treatment (Quartz coli
reactor)
y = -0.2039x + 7.03
R² = 0.9869
D=-1/K=-1/-0.203= 4.9 mJ/cm2
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Log
CF
U/m
L (
N0
)
UV dose (mJ/cm2)
D value of E.coli
y = -0.2141x + 7.07
R² = 0.8467
D=-1/K=-1/-0.214= 4.67 mJ/cm2
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Log
CF
U/m
L (
N0
)
UV dose (mJ/cm2)
D value of Salmonella WG 49
Microbes D values (mJ/cm2) of Wheatgrass
juice
E. coli P36 4.9 ± 0.11
Salmonella WG49 4.67 ± 0.5
Listeria Innocua 3.58 ± 0.06
69
Figure 3.15 -D value calculation for Listeria innocua after Dean flow UV treatment (Quartz coli
reactor)
Figure: 3.16-Effects of UV reactor upon inactivation of Listeria innocua, Salmonella WG 49 and E.
coli P36
y = -0.2796x + 7.22
R² = 0.9823
D=-1/K=-1/-0.279= 3.58 mJ/cm2
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Log
CF
U/m
L(N
0)
UV dose (mJ/cm2)
D value of Listeria Innocua
0
1
2
3
4
5
6
7
8
0 2 4 6 8 10 12 14 16 18 20 22 24 26 28
Lo
g C
FU
/mL
(N0)
Dose (mJ/cm2)
E.coli P36
Salmonella WG49
Listeria innocua
70
3.4. Effects of thermal and non-thermal technologies on nutrients content of wheatgrass
juice
3.4.1. Effects of thermal pasteurization on the nutritional quality of treated wheatgrass
juice
Thermal pasteurization of wheatgrass did not result in a significant (P ˃ 0.05) change in pH or TSS
(Table 3.5). The total acidity (TA) increased significantly after thermal processing (47.0 ± 3.00
%). The results regarding the effects of thermal treatment on vitamin C, chlorophyll and protein
content of wheatgrass juice are summarized also in Table 3.7. With thermal treated sample,
vitamin C was significantly lower (P < 0.05) than untreated samples. Moreover, the protein values
in wheatgrass juice were significantly decreased after thermal pasteurization. However, the total
chlorophyll of the pasteurized samples slightly increased, but it is not significantly different on the
chlorophyll of wheatgrass juice.
In addition, The TPC was significant decreased after thermal processing between treated samples
and untreated samples. The antioxidants value by DPPH and ORAC assays of wheatgrass juice
were 1511.6 ± 46.38 and 5.30 ± 1.98 respectively, after thermal treatment they were reduced
to1300 ± 104 and 3.4 ± 0.39 respectively. However, there were no significant influence in term of
antioxidants activity by DPPH, but ORAC significant decreased. The color of wheatgrass juice
after thermal treatment was investigated also. The L*, a* and b* values of the samples treated at
75 °C/15 s were significantly lower than those of the control sample (P ˂ 0.05) and the color
changed to browning green which is visually obvious.
The residual PPO and POD activities after the thermal treatments and in the control samples are
shown in Figure 3.17. Regarding the samples that were thermally treated in the water bath, there
71
was a significant decrease of residual PPO and POD activities. The residual content percentages
of PPO and POD activities were 61.8 and 25.9 %, respectively.
Table 3.7-Effects of pasteurization on the nutritional quality of wheatgrass juice
RC % Remaining Contents percentage *Values are the Mean ± SD of three replicates
a (TPC expressed as mg equivalents of Gallic acid/100mL b DPPH expressed as µmol L-1 Trolox equivalent (TE) mL-1 sample
c ORAC expressed as Trolox Equivalent mM
Different capital superscript letters in the same raw for each treatment correspond to significant differences (P < 0.05).
Properties *Untreated
sample
*Thermal treated
sample
RC
%
pH 5.7 ± 0.0057 AA 5.71 ± 0.0152 AA 100
TSS (Brix) 3.16 ± 0.28 AA 3.00 ± 0.0 AA 94.9
TA % 27.29 ± 0.241 AA 47.0 ± 3.00 AB 172.2
Vitamin C mg/100mL 9.21 ± 0.162 AA 6.69 ± 0.15 AB 72.6
Chlorophyll mg/100mL 1.45 ± 0.042 AA 1.74 ± 0.045AA 120
Protein mg/100mL 511.8 ± 32.87 AA 372.7 ± 3.20 AB 72.8
TPC (mg GAE/100 mL) a 341.76 ± 15.4 AA 218.9 ± 1.1 AB 64
DPPH b 1511.6 ± 46.38AA 1300 ± 1.04 AA
86
ORAC c 5.30 ± 1.98 AA
3.4 ± 0.39 AB
64.15
Color L* 19.81 ± 0.237 AA
17.65 ± 1.49 AB
89
a* -6.63 ± 0.383 AA -2.90 ± 0.8 AB
43.7
b* 19.60 ± 0.366 AA
13.27 ± 1.0 AB 67.7
∆E -5.54
72
Figure 3.17- Residual activity percentage of POD and PPO enzymes after thermal treatment
POD
PPO
0
20
40
60
80
100
Untreated sample
Treated sample
PO
D a
nd
PP
O R
esi
du
al
Acti
vit
y %
Thermal tretment (75 °C at 15s) POD PPO
73
3.4.2. Non-thermal technologies
3.4.2.1.High Hydrostatic Pressure
The pressures 500 MPa and 600MPa for 60, 90 and 180s were used to evaluate the impacts on
nutritional quality of wheatgrass juice based on achieving the 5-log reduction after microbial tests.
The effect of HHP treatment at 500 and 600MPa for 60, 90 and 180 s at pH, total soluble solids
content (TSS) and Titratable Acidity (TA) is summarised in Table 3.8. No significant changes in
pH and TSS (°Brix) were observed in the treated juice samples when compared with untreated
juice samples. However, TA significantly (P < 0.05) decreased after treatment at 500MPa for 60,
90 and 180 seconds (24.62 ± 1.17, 24.00 ± 0.8 and 24.44 ± 0.5 % respectively). Interestingly, at
600 MPa there was no significant (P ˃ 0.05) change in TA regardless of the treatment time applied
as in Table 3.8.
Table 3.8-Effects of HHP treatments on pH, TSS and TA of wheatgrass juice
*Values are the Mean ± SD of three replicates RC % Remaining Contents percentage
Different capital superscript letters in the same column for each treatment correspond to significant differences (P ≤ 0.05).
parameters Time (s) *pH RC% *TSS (°Brix) RC% *TA% RC%
Untreated - 5.7 ± 0.0057AA 100 3.16 ± 0.28 AA 100 27.29 ± 0.241 AA 100
500 MPa 60 5.70 ± 0.01 AA 100 3.16 ± 0.28 AA 100 24.62 ± 1.17 AB 90
500 MPa 90 5.70 ± 0.034 AA 100 3.16 ± 0.28 AA 100 24.00 ± 0.8 AB 88
500 MPa 180 5.73 ± 0.025 AA 99.47 3.16 ± 0.28 AA 100 24.44 ± 0.5 AB 89
600 MPa 60 5.67 ± 0.037 AA 99.47 2.77 ± 0.25 AA 87.47 28.95 ± 1.06 AA 106
600 MPa 90 5.68 ± 0.047 AA 99.64 2.88 ± 0.098 AA 91.15 29.10 ± 1.01 AA 106.6
600 MPa 180 5.68 ± 0.011 AA 99.64 2.89 ± 0.1 AA 91.47 29.00 ± 0.57 AA 106
74
The results regarding the effect of HHP treatment on vitamin C, chlorophyll and protein content
of wheatgrass juice are also listed in Table 3.9. No significant influences (p ˃ 0.05) in ascorbic
acid and protein were observed after treating juice samples at 500MPa and 600MPa for all different
times when compared with fresh untreated juice samples. However, after HHP treatment, the
chlorophyll increased to 50-70 % at 500 and 600MPa for different time. Therefore, there was
significant difference between untreated sample and pressurized treated samples, but there was no
significant difference between 500 MPa and 600 MPa treated groups.
Table 3.9-Effects of HHP treatments vitamin C, Chlorophyll and protein of wheatgrass juice
*Values are the Mean ± SD of three replicates RC % Remaining Contents percentage
Different capital superscript letters in the same column for each treatment correspond to significant differences (P ≤ 0.05)
The TPC is expressed as mg of Gallic Acid equivalents per 100 mL of wheatgrass juice. A
significant (P < 0.05) decrease was observed on the level of phenol compounds of treated HHP
samples. However, no significant effect between HHP treatment groups on total phenolic
compound content was detected. The residual contents of TPC after HHP was found to be in the
same range for all treatment conditions between (62.3-65.6%). In term of antioxidants, the HHP
significantly decreased both the total antioxidants activity as assessed by DPPH and ORAC assays
for the wheatgrass juice as shown in Table 3.10. The significant decrease was observed for all
Parameters Time(s) *Vitamin C
mg/100ml
RC% *Chlorophyll
mg/100ml
RC% *Protein mg/100ml RC%
Untreated - 9.21 ± 0.162 AA 100 1.45 ± 0.042 AA 100 511.86 ± 32.87 AA 100
500 MPa 60 6.77 ± 1.160 AA 73.5 2.18 ± 0.410AB 150 474.56 ± 51.34 AA 92.7
500 MPa 90 7.11 ± 1.287 AA 77.2 2.45 ± 0.431 AB 168.9 476.83 ± 65.97 AA 93.15
500 MPa 180 6.83 ± 1.527 AA 74.2 2.42 ± 0.184 AB 166.89 488.19 ± 47.97 AA 95.37
600 MPa 60 7.14 ± 1.707 AA 77.53 2.56 ± 0.135 AB 176.5 493.44 ± 64.16 AA 96.4
600 MPa 90 7.30 ± 1.567 AA 79.3 2.47 ± 0.460 AB 170.34 512.78 ± 80.06 AA 100.1
600 MPa 180 7.82 ± 1.108 AA 84.91 2.53 ± 0.554 AB 174.48 518.13 ± 30.67 AA 101.2
75
pressures and times, but within treated groups there were no significant differences. The residual
contents of antioxidants activity by DPPH and ORAC assays after HHP was found to be in the
same range for all treatment conditions between (50.7-55%).
Table 3.10-Effects of HHP treatments on the TPC and antioxidants activity of wheatgrass juice
* Values are the Mean ± SD of three replicates RC % Remaining Contents percentage
a (TPC expressed as mg equivalents of Gallic acid/100mL b DPPH expressed as µmol L-1 Trolox equivalent (TE) mL-1 sample
c ORAC expressed as Trolox Equivalent mM
Different capital superscript letters in the same column for each treatment correspond to significant differences (P < 0.05).
The effects of the different pressures on color values (L*, a* and b*) of the wheatgrass juice
are shown in Table 3.11. HHP did not have a significant impact on juice color when using
conditions required for 5-log inactivation of pathogenic microorganisms. No treatments cause a
significant color change because ∆E after each treatment was less than or equal to 3.0. The ranking
of ∆E was high-pressure treatment 600 MPa > high-pressure treatment at 500MPa. For instance,
the highest total color difference was observed in 600 MPa / 3 min treatment and the lowest total
color difference was observed in 500 MPa /1.5 min treatment. Therefore, total color difference
values increased from 0 to 3.1 did not indicate visual color differences after HHP treatment.
Parameters Time
(s)
*TPC a
(mg GAE/100 mL)
RC% *DPPH b RC% *ORAC c RC%
Control - 341.76 ± 15.46AA 100 1511.6 ± 46.38 AA 100 5.30±1.98 AA 100
500 MPa 60 216.43 ± 36.68AB 63.3 769 ± 19.03 AB 50.87 2.82±1.25 AB 53.2
500 MPa 90 224.34 ± 40.18 AB 65.6 760.14 ± 14.99 AB 50.28 2.82±1.40 AB 53.2
500 MPa 180 219.86 ± 37.48 AB 64.3 692.19 ± 20.98 AB 45.79 2.92±1.07 AB 55
600 MPa 60 213.03 ± 29.69 AB 62.3 711.08 ± 10.11 AB 47.04 2.69±1.25 AB 50.7
600 MPa 90 215.67±31.04 AB 63.1 748.69 ± 28.74 AB 49.5 2.83±1.42 AB 53.4
600 MPa 180 220.13±34.87 AB 64.4 684.27 ± 11.13 AB 45.26 2.86±1.57 AB 53.9
76
Table 3.11-Effects of HHP treatments on color of wheatgrass juice
* Values are the Mean ± SD of three replicates RC % Remaining Contents percentage
Different capital superscript letters in the same column for each treatment correspond to significant differences (P ≤ 0.05)
Enzyme activity PPO and POD
Finally, results regarding the effects of HHP treatments on enzymes activity of wheatgrass juice
are depicted in Figures 3.18 / 3.19. In term of polyphenol oxidase enzyme, even though the
wheatgrass juice does not have high PPO activity, there was slight increase in PPO in wheatgrass
juice after HHP treatment as shown in Figure 3.18. There was no significant difference between
untreated samples and treated samples at 500MPa and 600MPa for 60, 90 and 180s. The highest
increase was at 600 MPa for 90s and 180s (RC% was 126.6% and 124%, respectively).
In terms of the peroxidase enzyme, the POD decreased after HHP treatment, so there was
significant difference between untreated samples and treated samples at 500Mpa for 60s, 90s and
180s and 600MPa for 60s. However, there was no significant difference between untreated samples
and treated samples at 600MPa for 90s and 180s (RC% was 72.34% and 74.4%, respectively) as
shown in Figure 3.19.
Parameters Time(s) Color L* RC% Color a* RC % Color b* RC% ∆E
Control - 19.81±0.237AA 100 -6.63±0.383 AA 100 19.60±0.366 AA 100 0
500 MPa 60 20.08±0.327 AA 101.3 -6.49±0.453 AA 97.8 20.46±0.045 AA 104 0.91
500 MPa 90 20.05±0.315 AA 101.2 -6.57±0.130 AA 99 20.33±0.941 AA 103.7 0.77
500 MPa 180 19.23±0.489 AA 97 -6.55±0.079 AA 98.79 20.99±0.531 AA 107 1.26
600 MPa 60 19.01±0.298 AA 95.9 -5.76±0.060 AA 86.87 21.47±0.829 AA 109.5 1.9
600 MPa 90 18.80±0.306 AA 94.9 -6.77±0.077 AA 102.1 21.72±0.673 AA 110.8 2.3
600 MPa 180 19.28±0.269 AA 97.3 -6.17±0.142 AA 93 22.73±0.378 AA 115.96 3.1
77
3.3.4.2. UV-C treatment
The UV dose used to treat wheatgrass juice and to evaluate its impacts on nutritional quality was
based on doses necessary to achieve the 5-log reduction of three tested bacteria. UV dose
(25.4mJ/cm2) did not significantly influence on pH, total soluble solids and Titrable acidity as in
Table 3.12. In addition, the results regarding the effects of UV-C treatments on vitamin C,
chlorophyll and protein content of wheatgrass juice are summarized in Table 3.12. With UV
treated sample, vitamin C was not significantly different from untreated samples, but the total
chlorophyll of the pasteurized samples increased significantly by 102.7 % of untreated wheatgrass
juice. Protein values were not significantly affected by UV-C dose (25.4mJ/cm2), so the protein
content in wheatgrass juice following UV treatments decreased by 2.3 % only.
The Table 3.12 illustrates the total phenolic contents (TPC) and antioxidants of the wheatgrass
juice treated by UV-C. The UV treatment of wheatgrass juice did not affect the total phenolic
content. The antioxidants activity by assays DPPH and ORAC for the wheatgrass juice after UV
treatment was retained also as shown in Table 3.11. The residual contents of antioxidants activity
0
20
40
60
80
100
120
140
060
90180
PP
O R
esid
ua
l A
ctiv
ity
%
Time (s)
500 MPa 600 MPa
0
20
40
60
80
100
060
90180
PO
D R
esid
ua
l A
ctiv
ity
%
Time (s)
500 MPa 600 MPa
Figure 3.18-Residual activity percentage of PPO
enzyme in wheatgrass juice after HHP treatment
Figure 3.19-Residual activity percentage of POD enzyme
in wheatgrass juice after HHP treatment
78
of wheatgrass juice by DPPH and ORAC assay after UV treatment were found (78% and 92.4%),
respectively. Color of wheatgrass juice after UV processing was significantly increased in term of
L* and b* values, but a* value was not significantly affected from untreated sample after UV
treatment.
Table 3.12-Effects of UV-C treatment nutritional quality of wheatgrass juice
*Values are the Mean ± SD of three replicates RC % Remaining Contents percentage
a (TPC expressed as mg equivalents of Gallic acid/100mL b DPPH expressed as µmol L-1 Trolox equivalent (TE) mL-1 sample
c ORAC expressed as Trolox Equivalent Mm
Different capital superscript letters in the same row for each treatment correspond to significant differences (P < 0.05).
Properties Untreated sample UV-C treated sample RC %
Absorbed dose
(25.4mJ/cm2)
Re=
1027
De= 265
pH 5.7 ± 0.0057AA 5.76 ± 0.03 AA 101
TSS (Brix) 3.16 ± 0.28 AA 3.00 ± 00 AA 95
Titratable Acidity % 27.29 ± 0.241 AA 26.0 ± 1.37 AA 95.2
Vitamin C mg/100Ml 9.21 ± 0.162 AA 8.4 ± 0.72 AA 91
Chlorophyll mg/100Ml 1.45 ± 0.042 AA 2.94 ± 0.05AB 202.7
Protein mg/100Ml 511.86 ± 32.87 AA 500.3 ± 24.1 AA 97.7
TPC (mg GAE/100 mL) a 341.76 ± 15.46 AA 306.9 ± 8.3 AA 90
Antioxidants activity
DPPH assay b
1511.6 ± 46.38 AA
1179 ± 8.6 AA
78
ORAC assay c 5.30 ± 1.98 AA
4.90 ± 0.25 AA 92.4
Color L*
a*
b*
∆E
19.81 ± 0.237AA
-6.63 ± 0.383 AA
19.60 ± 0.366 AA
0
22.39 ± 1.06AB
-6.15 ± 0.07 AA
22.03 ± 0.5 AB
3.57
113
92.7
112.3
79
Enzyme activity POD and PPO
In term of polyphenol oxidase enzyme, there was slight decrease in PPO in wheatgrass juice after
UV treatment, but no significant difference was between untreated samples and treated samples at
dose 25.4mJ/cm2 as shown in Figure 3.20. The peroxidase enzyme was decreased after HHP
treatment, so there was significant influence after UV processing as shown in Figure 3.20.
Figure 3.20-Residual activity percentage of POD and PPO enzymes in wheatgrass juice after UV
treatment
POD
PPO
0
20
40
60
80
100
Untreated sample Dose 25.4mJ/cm2
PO
D a
nd
PP
O R
esid
ua
l A
ctiv
ity
%
UV-C Treatment
80
Chapter 4
4. Discussion
The overall objective of the study was to compare the effects of the two non-thermal treatments
HHP and UV light at 254nm on the fate of nutrients, enzymes and bioactive constituents of
wheatgrass juice treated under processing conditions that allowed achieving the 5-log reduction of
the pertinent pathogens. The experiments were conducted to measure the effects of the thermal
and HHP and UV treatments on inactivation of three bacteria (E. coli P36, Listeria innocua and
Salmonella WG 49) in wheatgrass juice to optimize conditions of equivalent processing and then
to measure the effects of heat, HHP and UV treatments on nutritional quality of wheatgrass juice.
4.1. Juice Extraction
This study assessed that how process variables of pectinase enzyme extraction of wheatgrass juice,
particularly incubation temperature (constant), incubation times (20, 60, 120 min), and pectinase
concentration (2 %) did not significantly affect the yield of the wheatgrass juice. The enzyme
extraction process is industrially applicable to improve the yield and quality of the juice especially
for apple and banana juice (De, Karmakarb, Nsoa & Sagua, 2014). The enzymatic hydrolysis of
wheatgrass did not improve the juice yield significantly, although there was a slight increase in
juice yield after different incubation times. The present study’s result was in contrast with result
reported by Chang et al. (1995) that the yield of plum juice was significantly increased with
different pectinase enzymes concentration from 0.05 % to 0.6%. In addition, the enzymatic
hydrolysis is assumed to help in producing carrot juice (Qin et al., 2005). As a result, it was found
that yield of the wheatgrass juice extracted was able to increase only by 13% by using the enzyme
pectinase from Aspergillus aculeatus at incubation time 120 min.
81
4.2. Physical and chemical properties of defrosted wheatgrass juice
Similar to other fruit and vegetables juices such as carrot and watermelon juice, the pH of
wheatgrass juice was (5.7 ± 0.0057) confirming the beverage can be classified as a low acid juice.
The main parameters tested in the untreated wheatgrass juice were vitamin C (9.21 ± 0.162
mg/100mL), chlorophyll (1.45 ± 0.042 mg/100mL), TPC (341.76 ± 15.4 mg EGA/100mL) and
antioxidants by assays (DPPH) and ORA (1511.6 ± 46.38 (TE) mL-1 and 5.30 ± 1.98 TE mM,
respectively). It is well known that phenolic compounds including flavonoids of plant origin are
mostly responsible for radical scavenging. They possess different antioxidant properties that can
be attributed to their therapeutic uses in different diseases. The present results showed that the
values of antioxidants are similar to or higher than those for some fruits and vegetables. The values
of antioxidants in wheatgrass juice were in the range for different extracts of turmeric, garlic,
spinach, onion, plum and carrot (Acharya et al., 2006).
4.3. Effects of tested treatments on microbial counts in wheatgrass juice
The FDA juice HACCP guidelines state that a 5-log10 CFU/mL reduction must be achieved for
processing technologies to be approved (US FDA, 2004). The effects of the thermal, high pressure
and UV-C treatments on microbial contamination of the wheatgrass juice are compared based on
their standardization in achieving the 5-log10 reduction of target microbes.
Heat treatment
First, thermal pasteurization treatments (75 °C for 15s), achieved the 5-log reduction of E. coli
P36, Listeria innocua ATCC 51742 and Salmonella typhimurium WG 49 and the heat resistance
of tested bacteria was not significantly different. The microbial counts determined in wheatgrass
juice were similar to results reported for other fruit juices, such as watermelon juice. In the
82
Tarazona-Díaz, and Aguayo’s (2013) work, E. coli, Salmonella spp. and L. monocytogenes were
analyzed in watermelon juice. Pasteurization at 87.7 °C for 20 s reduced the microbiological counts
of watermelon juice and no Salmonella spp. and L. monocytogenes were detected after the
treatment.
HHP treatment
Pressure treatments of 500 MPa and 600 MPa for 60, 90 and 180s achieved 5-log10 CFU/mL
reductions of the pathogen surrogates. Based on the results of present study, Listeria Innocua was
the most resistant bacteria, but E. coli and Salmonella were under the detection limit (more
sensitive). The results of HHP are similar to present study are, Fan and Sampedro (2010) reported
that treatment of carrot juice by HHP (615 MPa for 1-2 min at 15°C) reduced Salmonella
Enteritidis, S. Typhimurium, S. Hartford and E. coli cocktail by 6.67-log, 5.05-log, 5.31-log, > 7
log and 6.4 log, respectively. They also studied HHP treatment 600 MPa for 5 min at room
temperature for Listeria species (L. monocytogenes and L. innocua) in fruit juices which achieved
the 5-log reduction of L. monocytogenes in fruit juices. In Coconut water, HHP treatment has
effective results for elimination of E. coli O157:H7, Salmonella Typhimurium, and Listeria
monocytogenes. HHP processing at 500 and 600 MPa for 120 seconds supported more than 5-
log10 CFU/ml reduction of bacteria (Boyer et al, 2013). The same result for E. coli reduction (5-
log reduction) was achieved on low acid juice (melon juice) using 500 MPa pressure at room
temperature for 8 min (Chen and Neetoo, 2012). Therefore, even though the previous results from
different studies used different pressures, they could achieve 5-log reduction for different
microorganisms.
UV treatment
83
Finally, UV-C doses from 2.54 to 22.86 mJ/cm2 (from cycle 1 to 9) did not achieve the 5-log10
CFU/mL for E. coli and Listeria innocua. However, at an optical density 254 nm, UV-C treatments
at dose 25.4 mJ/cm2 (cycle 10) achieved 5-log10 CFU/mL for all tested bacteria. By comparing
results of different bacteria in the same UV-light experimental conditions with different UV doses,
it can be concluded that the most UV-resistant bacteria were E. coli and Salmonella. However,
Listeria innocua was more sensitive to UV light than E. coli and Salmonella. Those results were
in agreement with Yaun et al. (2004) who reported that UV resistance of Salmonella and E. coli
O157:H7 did not differ, but in contrast with Gabriel and Nakano (2009) who demonstrated
that Salmonella Typhimurium was less resistant than E. coli O157:H7 and Listeria
monocytogenes. Disagreements in the literature may be due to the wide variation of UV resistance
between strains. It is well known that the physicochemical characteristics of the treatment media
(wheatgrass juice) may change the bactericidal efficacy of most food processing technologies. It
is demonstrated that the difficulty of UV light treatment in achieving a 5 log10 reduction due to the
low penetration capacity of UV photons on liquid foods has prompted several authors to develop
hurdle strategies combining UV light with other novel processing techniques or milder
conventional preservation methods (Gayan et al., 2012).
84
Figure 4.1-Microbial inactivation of three tested bacteria after heat, HHP and UV treatments
4.4. Effects of heat, HHP and UV on physical/chemical properties of the wheatgrass juice
4.4.1. pH, Total soluble solids and Titratable Acidity
Nutrient content of wheatgrass juice is another focus of this study to be emphasized after
thermal, HHP and UV-light treatments. pH is a simple measurement which gives useful
information on the potential chemical changes that take place in food during processing and
storage. pH and total soluble solid did not change after different treatments, but the Titratable
Acidity of treated wheatgrass juice exhibited an increasing and decreasing trend after different
treatments.
Heat
Thermal pasteurized treatment at 75°C for 15s did not have significant effects on the pH values
and TSS of wheatgrass juice. However, thermal pasteurization increased the titratable acidity of
wheatgrass juice significantly. The previous study by Tarazona-Díaz, and Aguayo (2013) showed
the same results in their study for watermelon juice treated by thermal processing (87.7 ◦C for 20
0
1
2
3
4
5
6
7
8
Untreeated sample Thermal (75°C/15s) HHP
(600MPa/180s)
UV (25.4mJ/cm2)
Lo
g c
fu/m
L (
N0
)
Treatments
Microbial counts
E.coli P36
Salmonella WG49
Listeria Innouca
85
s). It is illustrated that pH and TSS of watermelon juice did not change, but the TA increased after
treatment. Moreover, a study on cucumber juice showed that thermal pasteurization did not have
an effect on the pH of juice when it treated at 850C for 15 seconds (Dong et al., 2012).
HHP treatment
In the current study, it was found that none of the HHP treated samples had changes in the pH
values and total soluble solid of wheatgrass juice (p > 0.05). However, TA significantly lower after
treatments at 500MPa for 60, 90 and 180 seconds, but at 600 MPa there was no significant changes
in TA. Similar effects result with the current study have been found in previous research studies.
Hu et al. (2012) reported that in watermelon juice treated by HHP, total soluble solid and pH of
juice did not change. In addition, the pH values and TSS of cucumber juice (Dong et al, 2012) and
pH values of carrot juice (Patterson, McKay, Connolly, & Linton, 2012) treated with HHP were
the same as those of untreated samples. However, different results have also been observed in term
of titratable acidity. Hu et al. (2012) reported that HHP treatment, titratable acidity of watermelon
juice did not change. However, other study shows that the TA of cucumber juice drinks during
storage exhibited an increasing trend.
UV treatment
In the present study, UV-C treated samples (dose 25.4mJ/cm2) had no significant changes in the
pH values, TSS and TA of wheatgrass juice. Similar results from this study have been found in
previous study on watermelon juice. UV-C (dose 37.5 J/mL) treatment in watermelon juice using
helix Teflon®-coil resulted in no-significant effects on pH and °Brix (Feng et al., 2013).
4.4.2. Vitamin C
Ascorbic acid is an indicator of nutritional quality in fruit juices. Wheatgrass juice also contains
vitamin C which is an essential nutrient for humans. Vitamin C aids in the synthesis of collagen
86
in addition to protecting against oxidative damage. Vitamin C consumption has been shown to
protect against stomach, oral, and lung cancers, improve cholesterol, and prevent scurvy. Vitamin
C is very sensitive to heat and degrades very quickly during pasteurization (Bodla & Mujoriya,
2011).
Heat treatment
The thermal treatment decreased the vitamin C of wheatgrass juice significantly. To discuss the
effects of thermal treatment on wheatgrass juice was compared with other low acid juices as melon
juice. Same results have been found in previous research in term of thermal pasteurization. The
HTST treatment caused a considerable loss of vitamin C content of melon juice (51%) (Hu et al.,
2009). Moreover, Chen et al. (2009) reported that HTST pasteurization produced significant
decreases in ascorbic acid in the melon juice which agrees with the present study on wheatgrass
juice.
HHP treatment
However, high pressure did not induce significant loss of vitamin C content of wheatgrass juice.
In addition, there was no significant difference between different times at same pressure. Hence,
the high pressures treatment at 500 and 600 MPa did not affect the vitamin C of the wheatgrass
juice, being similar to the results about the effect of the high pressure treatment on the fruit and
vegetable matrices when they applied 600–800 MPa (Butz et al., 2003). Being similar, the vitamin
C of carrot juice did not change significantly after the high pressure treatment at 250 MPa (25 C
for 5 or 15 min) (Dede, Alpas, and Bayındırlı, 2007). As a result, the change in ascorbic acid
content of juices by different HHP (500MPa/600MPa) combinations was not statistically
significant.
87
UV treatment
After UV-C treatment at 25.4 mJ/cm2, vitamin C in wheatgrass juice did not affect significantly
(loss 3% only). Most studies reported the effects of UV treatments on vitamin C in high acid juices
such as apple and orange juice. Previous reviews showed that the average residual vitamin C
content was 83.7 ± 11.9%. Orlowska and others (2013) reported that UV treatment of apple juice
at 10 mJ/cm2 reduced the vitamin C content by only 1.3%. In addition, UV-treated samples of
pineapple juice at 53.4 mJ/cm2 retained a higher residual content during storage period than
thermal treated samples (Chia et al., 2012). Therefore, ascorbic acid content following the
pasteurization, HHP and UV-C methods varied and UV-C treatment was effective to retain the
vitamin C of the treated wheatgrass juice than HHP treatments as in Figure 4.2.
4.4.3. Chlorophyll
Total chlorophyll, the pigments responsible for the characteristic green color of fruits and
vegetables, is highly susceptible to degradation during processing, resulting in color changes in
food (Burdurlu, Karadeniz & Koca, 2006).
Heat treatment
Thermal treatment did not affect the total chlorophyll of wheatgrass juice. This result is in contrast
with Van Loey et al. (1998), who found that thermal treatment at 100 °C/37 min resulted in a 90%
decrease in the total chlorophyll content of broccoli juice. In addition, Dong et al. (2012) in the
same study on cucumber juice shows that chlorophyll a and b of cucumber juice increased after
thermal processing. Therefore, Klein and Lurie (1991) believed that thermal pasteurization of
fruits and vegetables could enhance the chlorophyll degradation during storage for two reasons.
Firstly, the stability of the chlorophyll molecule decreased after thermal pasteurization, and non-
88
enzymatic browning increased. Secondly, thermal pasteurization induced the high-temperature
catalysis of the chlorophyll degradation mechanism.
HHP treatment
However, in the non-thermal treatment (HHP), the significant increase of chlorophyll in treated
wheatgrass juice in this study was evaluated. The present result was different on the previous
studies on low acid juices as cucumber juice. Dong et al. (2012) showed that chlorophyll a and b
of cucumber juice had not significantly changed after HHP treatments, but they were significantly
higher than those of the thermally pasteurized (85 °C/15s) samples. Regarding chlorophylls, high
pressure treatment caused no degradation or slight increases, while HPHT processes degraded both
chlorophylls. Wang et al. (2012) also found no significant differences in both chlorophylls between
raw and pressure treated (600 MPa, 5 min) samples of spinach. The increase of chlorophyll content
described in this work might be caused by the cell disruption occurred during HP treatment, which
results in the release of chlorophyll, yielding a more intense bright green color on the vegetable
surface (Sánchez, Baranda, & Marañón, 2014).
The increase of chlorophylls content described in previous work by Krebbers et al. (2002) might
be caused by the cell disruption occurred during HP treatment, which results in the release of
chlorophyll, yielding a more intense bright green color on the vegetable surface. This effect was
seen in green beans after HP treatment of 500 MPa for 1 min (Sánchez, Baranda, & Marañón,
2014). The reason why total chlorophyll of wheatgrass juice increased after HHP needs to be
further studied.
89
UV treatment
The other non-thermal processing is UV-C treatment which increased the total chlorophyll of
wheatgrass juice significantly. There is limited data on the effects of UV on the chlorophyll of low
acid juices. However, previous results indicated that the chlorophyll contents were affected by UV
radiation. The chlorophyll a, b, and total contents of desert plants were decreased compared with
the control values and reduced with the enhanced UV radiation (Salama, Watban & Al-Fughom,
2011).
Protein
The thermal treatment of wheatgrass juice decreased the protein significantly (loss 27%).
However, the non-thermal treatments (HHP and UV-C) did not decrease the protein content of
wheatgrass juice significantly. The treatments comparison in terms of vitamin C, chlorophyll and
protein was summarized in Figure 4.2.
Figure 4.2-Comparision of the residual contents of vitamin C, chlorophyll and protein in wheatgrass juice after
different treatments
0
50
100
150
200
250
Untreeated sample Thermal (75C/15s) HHP (600MPa/180s) UV (25.4mJ/cm2)
Res
idu
al
con
ten
t %
wheatgrass juice samples
Vitamin C
Chlorophyll
Protien
90
Total phenolic compounds and antioxidants
To compare the influence of the HTST, HHP and UV-C processes, the total phenolic acid with
antioxidant properties in wheatgrass juice were quantified. Phenolic compounds are secondary
metabolites in plants that are known to be essential for giving health benefits and for developing
the color and flavor of fruit juices. Phenolic compounds degrade, oxidize, or polymerize quickly
during processing and storage. Therefore, total phenolic content is an important indicator of the
quality of fruit juice (Ghafoor & Choi, 2012).
Heat treatment
The thermal pasteurization significantly affected TPC of wheatgrass juice. The loss of TPC was
about 36%. There is limited data are available on the effects of thermal processing on TPC and
antioxidants of wheatgrass juice. One study reported that the total phenols in carrot juice were
significantly decreased after the HTST process (Zhang et al., 2016). In terms of antioxidants, the
thermal treatment significantly decreased antioxidants activity of wheatgrass juice by DPPH and
ORAC assays. The present result is similar to those of a previous study on carrot juice. The
antioxidant capacity of carrot juices using the DPPH and FRAP assays showed a significant
decrease after HTST treatments (Zhang et al., 2016).
HHP treatment
In present study of the HHP processing, the total phenols of wheatgrass juice were decreased.
However, the total phenols after HHP treatment was found to vary in previous studies. Zhang et
al. (2016) mentioned that the total phenols in carrot juice were better preserved after the HHP
process. In addition, Barba, Esteve, and Frigola (2010) found that the total phenols in vegetable
beverages showed no significant difference after HHP treatment (100–400MPa/9min). The change
in total phenols could be attributed to plant cell disruption caused by HHP treatment, leading to a
91
higher extractability of these types of compounds. In this study, the decrease in total phenols in
carrot juice could be attributed to a balance between a higher extraction rate and non-enzymatic
oxidation degradation of total phenols (Zhang et al., 2016). Then, the HHP processing significantly
reduced antioxidants activity of wheatgrass juice by DPPH and ORAC assays. It is similar to a
study by Zhang et al. (2016). The antioxidant capacity of carrot juices using the DPPH and FRAP
assays showed a significant decrease after HHP.
UV treatment
The total phenols of the UV-C treated wheatgrass juice decreased by 10% which is probably due
to the oxidation degradation of phenolic compounds and the polymerization of phenolic
compounds with proteins. Few reports concerning the effects of the UV dosage on the phenolic
content of low acid juices are available. A study by Feng et al. (2013) the treatment of watermelon
juice using helix Teflon®-coil resulted in no significant effects on total phenols.
The UV treatment showed slight decrease on antioxidants activity in the DPPH assays, but it was
not significant. The residual contents in antioxidants of wheatgrass juice by both DPPH and ORAC
assays were 78 and 92.4 %, respectively. Similarly, a study by Corrales et al. (2012) reported that
Tiger-nut milk antioxidants by DPPH assay did not change by UV dose 4.3J/cm2. The treatments
comparison in terms of TPC and antioxidants is summarized in Figure 4.3.
92
Figure 4.3-Comparision of the residual contents of TPC and antioxidants in wheatgrass juice after different
treatments
Color
Color is one of the important quality characteristics of fruits and vegetables and major factors
affecting sensory perception and consumer acceptance of foods. Total color variation (ΔE) is the
more interpretable factor when examining color attributes (Dong et al., 2012).
Heat treatment
Thermal treatment caused a significant decrease in the color of wheatgrass juice because ∆E after
treatment was lower than 0 (ΔE = -5.5). The L*, b* and a* values of the wheatgrass juice treated
at 75 °C/15s were significantly lower than those of the control. This is probably due to thermal
pasteurization resulting in enzymatic browning which was visually clear after treatment. Some
new compounds could be produced in HTST-pasteurized wheatgrass juice, which were associated
with a cooked-off odor. This result was similar to those of previous studies. Dong et al. (2012)
found that the L* value of the cucumber samples treated at 85 °C/15 s was significantly decreased.
0
20
40
60
80
100
120
Untreated sample Thermal (75°C/15s) HHP (600MPa/180s) UV (25.4mJ/cm²)
Res
idu
al
con
ten
ts %
Wheatgrass juice samples
TPC
Antioxidants
DPPH
Antioxidants
ORAC
93
HHP treatment
However, HHP did not affect the L*, b* and a* values of the wheatgrass juice. This result was
different to those of previous studies. Two different results were reported that the color of
cucumber drinks (Dong et al., 2012) and watermelon juice (Hu et al., 2012) are changed after high
pressure treatments. Dong et al. (2012) reported that L* values of the cucumber drinks treated at
400 MPa/4 min and 500 MPa/2 min were significantly higher than those of the control which
indicated that the HHP-treated samples were brighter than the control. In addition, a study by Hu
et al. (2012) showed that color of watermelon juice after HHP treatments (600 MPa/15min) was
changed. L* value increased, but b* and a* had no significant difference compared to control.
UV treatment
UV treatment (25.4 mJ/cm2) did not effect on the a* values of the wheatgrass juice, but L* and
b* values increased significantly after treatment. This difference may result from the texture and
microstructure varieties of wheatgrass juice, because these varieties cause changes in the nature
and the extent of internally scattered light and the distribution of surface reflectance. Wheatgrass
juice is a highly pigmented product due to its high chlorophyll content. In literature, it was reported
that highly pigmented juices are less affected by the processing and storage. High color pigments
concentrations provide a better masking effect on color differences. These type of juices have more
acceptable color after the processing (Lee & Coates, 1999).
This result was different to those of previous studies. Feng et al. (2013) reported that L*, b* and
a* values of the watermelon treated at 37.5J/mL did not affect which indicated that the UV-treated
samples were brighter than the control. Nevertheless, a study by Butz et al. (2011) showed that
color of watermelon juice after UV treatments (2421J/L) was changed. L* and b* values decreased,
94
but a* value increased compared to control. The treatments comparison was summarized in
Figure 4.4.
Figure 4.4-Comparision of the residual contents of color values (L*, a* and b*) in wheatgrass juice after
different treatments
Enzyme activity
Enzymes such as polyphenol oxidase and peroxidase can be involved in the deterioration of
food products that cause changes in their sensory qualities, such as undesirable color and flavor or
nutritional changes. Therefore, one of the main purposes of fruit juices treatment via pasteurization
is to inactivate the enzymes to increase the shelf life of fruit juices (Fellows, 2000).
Heat treatment
Thermal treatment significantly decreased the PPO and POD activities of wheatgrass juice which
are the key enzymes in enzymatic browning of fruits and vegetables. However, there have been
few studies about enzyme activity in low acid juices following thermal treatment. Enzymes, such
as pectin methyl esterase and polygalacturonase, are inactivated when they are treated at 650C,
770C and 800C for 30 min, 1 min and 10-60 s, respectively.
0
20
40
60
80
100
120
140
Untreeated sample Thermal (75°C/15s) HHP (600MPa/180s) UV (25.4mJ/cm2)
Res
idu
al
con
ten
ts %
Treatments
L*
a*
b*
95
HHP treatment
In contrast, HHP treatment, phenol oxidase (PPO) levels in the wheatgrass juice did not
significantly change after any of the treatments with the high pressure. Normally, this would be an
undesired effect because the PPO enzyme causes browning within the wheatgrass juice, as well as
many other fruits. Most juice processing is used to reduce the activity of these enzymes and prevent
them from causing juice browning changes (Murasaki-Aliberti et al., 2009). The untreated
wheatgrass juice did not contain much of this enzyme to begin with. This could be because of
wheatgrass maturity or higher level of total solids.
HHP treatment can change the enzyme activity by changing the conformation of enzyme protein.
In similarity, for instance, in watermelon juice treated by HHP, polyphenol oxidase and peroxidase
decreased when juice treated at 200, 400 and 600MPa (Hu et al., 2012). Nevertheless, Boyer et al.
(2013) demonstrate that coconut water treated by HHP (500 and 600 MPa for 120 sec.) had no
changes on polyphenol oxidase. Thus, two different results have been shown from different
studies, so more research need to be done for enzyme activities in the low acid juices after the
HHP treatment.
UV treatment
In the UV treatment, phenol oxidase (PPO) levels in the wheatgrass juice did not significantly
change; however, POD enzyme reduced significantly (RC%= 19.3) after the UV treatment
(25.4mJ/cm2). Likewise, Peroxidase activity of tiger nut milk decreased at UV-C dose
(4.23mJ/cm2), and only 14% residual activity was recovered after a treatment at the highest UV-C
doses (Corrales et al., 2012).
96
The treatments comparison in terms of enzymes activity of wheatgrass juice was summarized in
Figure 4.5. Finally, Table 4.1 is summarized the values of different nutrients in wheatgrass juice
after HTST (75°C/15s), HHP (600MPa/180s) and UV (25.4mJ/cm2).
Figure 4.5-Comparision of the residual contents of POD and PPO enzymes in wheatgrass juice after different
treatments
POD
PPO
0
20
40
60
80
100
120
140
Untreated sample Thermal (75 °C/15s) HHP(600MPa/180s) UV (25.4mJ/cm2)
PO
D a
nd
PP
O R
esi
du
al
Acti
vit
y %
Treatments
97
Table 4.1-Summary of physical and chemical properties values of wheatgrass juice after heat, HHP
and UV treatments
*Values are the Mean ± SD of three replicated ** Values are in mg/100mL
a TPC expressed as mg equivalents of Gallic acid/100mL b DPPH expressed as Trolox Eq. uM
c ORAC expressed as Trolox Equivalent Mm
Properties *Untreated
Wheatgrass
juice
*HTST
(75C/15s)
*HHP
(600MPa/180s)
*UV (Dose
25mJ/cm2)
Most resistant
bacteria
----
Similar Listeria innocua E. coli and
Salmonella WG49
pH 5.7 ± 0.0057 5.71 ± 0.0152 5.68 ± 0.011 5.76 ± 0.03
TSS (Brix) 3.16 ± 0.28 3.00 ± 0.0 2.89 ± 0.1 3.00 ± 00
TA % 27.29 ± 0.241 47.0 ± 3.00 29.0 ± 0.57 26.0 ± 1.37
Vitamin C** 9.21 ± 0.162 6.69 ± 0.15 7.82 ± 1.108 8.4 ± 0.72
Chlorophyll** 1.45 ± 0.042 1.74 ± 0.045 2.53 ± 0.55 2.94 ± 0.05
Protein** 511.8 ± 32.87 372.7 ± 3.20 518.13 ± 30.67 500.3 ± 24.1
TPC a
341.76 ± 15.4 218.9 ± 1.1 220.13 ± 34.87 306.9 ± 8.3
Antioxidants:
DPPH b 1511.6 ± 46.38 1300 ± 1.04 684.27 ±11.13 1179 ± 8.6
ORAC c 5.30 ± 1.98
3.4 ± 0.39
2.86 ± 1.57
4.90 ± 0.25
Color L* 19.81 ± 0.237
17.65 ± 1.49
19.28 ± 0.26
22.39 ± 1.06
a* -6.63 ± 0.383
-2.90 ± 0.8
-6.17 ± 0.142
-6.15 ± 0.07
b* 19.60 ± 0.366
13.27 ± 1.0 22.73 ± 0.378 22.03 ± 0.5
Enzymes Activity:
POD
0.81 ± 0.085
0.2 ± 0.04
0.6 ± 0.05
0.15 ± 0.08
PPO 0.015 ± 0.0007 0.009 ± 0.0003 0.018 ± 0.0006 0.012 ± 0.0007
98
Chapter 5
5. Conclusion
The primary objective of the study was to compare the performance of high pressure processing
and UV treatment on microbial inactivation, nutrient levels, along with sensory characteristics of
wheatgrass juice. By using the 5-log10 CFU reduction of pathogen surrogates as a metric for
successful treatments differences between the non-thermal pasteurization techniques were
observed.
1- Extraction
An extraction process based on low temperature and high concentration of commercial enzyme
with different incubation times was outlined in the present study. The present study revealed
that enzymatic treatment did not cause significant increase in the yield of wheatgrass juice.
2- A variation of pathogens response to thermal, High Hydrostatic Pressure and UV light
susceptibility was observed among the three tested bacteria such E. coli, Listeria Innocua and
Salmonella WG49. Heat sensitivity of three bacteria was similar. However, in HHP, it has been
demonstrated that listeria innocua was much more resistant to pressure than E. coli and
Salmonella WG49. The E. coli and Salmonella WG49 were the more resistant to UV-C than
listeria innocua which was more sensitive to UV-C processing.
3- Equivalent processing conditions to achieve the 5-log10 reduction of the most resistant
pathogens of concern have been established for each treatment: heat (75°C/15s), UV treatment
(dose 25.4mJ/cm2) and HHP at 600MPa for 180s, and those parameters were used to further
compare their effects on nutritional quality of wheatgrass juice.
4- No processing effects on pH and TSS content as compared to control samples were observed
after all treatments. This means pH and TSS of wheatgrass juice have the same sensitivity to
those treatments; however, the TA increased significantly after HTST treatment.
99
5- The residual content of vitamin C following UV light (91%) and HPP treatment (84.9%)
showed only minor reduction compare to raw untreated juice product, but HTST decreased
vitamin C significantly.
6- The increase in chlorophyll content after UV was higher than after HHP and HTST treatments.
7- The HTST and HPP treatments resulted in similar retention of TPC and antioxidant capacity
of wheatgrass juice, but the UV treatment preserved the TPC and antioxidants.
8- The effects on color of wheatgrass juices ranked using ∆E value was UV-C treatment > high
pressure treatment > thermal treatment. Furthermore, each treatment had a different influence
on the color of the wheatgrass juice. Being different to the thermal and HHP treatments, UV-
C treatment increased the ∆E. Therefore, the high pressure treatment at 600 MPa/180 and UV-
C treatments were effective to keep the color of the treated wheatgrass juice as the control
compared to the thermal treatment.
9- The residual activity of juice enzyme POD was decreased the most following UV-C and HTST
treatments (19.3% and 25.9%) respectively, as opposed to HHP (74.4%) treatment. However,
residual activity of juice enzyme PPO was decreased more following HTST and UV-C (61.8%
and 84.4%), respectively, as opposed to HHP (124%) treatment.
10- From the results obtained, it can be recommended that HHP treatment would be the preferred
pasteurization treatment for wheatgrass juice.
100
Future Work
Lower process pressures have to be tested (425, 450, and 475 MPa) if they can achieve the desired
5-log reduction in wheatgrass juice to save time and money. More time selections have to be tested
with changing the pressures. In addition, different flow rates should be tested with the UV Dean
flow reactors. There needs to be more studies about these varying pressures/UV doses and times
along with some added antimicrobials, slight heat, or changes in pH. More tests have to be done
with some other families of bacteria with HHP and UV. More tests have to be done to know reasons
for increasing or decreasing of some nutrients such as chlorophyll, antioxidants and TPC after non-
thermal treatments (HHP and UV-C). Finally, further research is required to determine the effects
of UV light and HHP on other fruits and vegetables enzymes such as pectin methyl esterase PME,
and lipoxygenase LOX.
101
References
Acharya, R. Devasagayam, T. P. A Kulkarni1, S.D. Rajurkar1, N.S. Reddy, A. V. R. & Tilak, J.C. (2006).
Evaluation of the antioxidant activity of wheatgrass (Triticum aestivum L.) as a function of growth
under different conditions. Phytotherapy Research. Res. 20, 218–227.
Acharya, R. Kulkarni, S.D. Nair, A.G.C. Rajurkar, N.S & Reddy, A.V.R. (2005). Determination of
elemental concentration profiles in tender wheatgrass (Triticum Aestivum L.) using instrumental
neutron activation analysis. Food Chemistry Journal. (95) 699–707.
Agriculture and Agri-Food Canada. (2016). Canada’s fruit and vegetable preserving and specialty food
industry. Received from: http://www.agr.gc.ca/eng/industry-markets-and-trade/statistics-and-
market-information/by-product-sector/processed-food-and-beverages/canadas-fruit-andvegetable
- preserving-and-specialty-food-industry/?id=1375206764774
Agriculture and Agri-Food Canada. (2012). Fruit Juices in the United States. International Markets Bureau
(Agriculture and Agri-Food Canada, International Markets Bureau. American Eating Trends Report,
2012). Received from: http://www.agr.gc.ca/eng/industry-markets-and-trade/statistics-and-market-
information/agriculture-and-food-market-information-by-region/united-states-and-mexico/market-
intelligence/fruit-juices/?id=1410083148489
Ahmed, J. and Shivhare, U.S. (2012). Thermal processing of vegetables; Thermal Food Processing: New
Technologies and Quality Issues. Second edition. Sun, D (Ed). Contemporary Food Engineering
Science. Taylor & Francis Group: CRC press. 384-406.
Alzamora, S.M. Char, C.D. Guerrero, S.N. &Mitilinaki, E. (2010). Use of High-Intensity Ultrasound and
UV-C Light to inactivate some microorganisms in fruit juices. Food Bioprocess Technology;
(2010) 3:797–803
102
Ancos, B.DE, Cano, M.P, Hernandez, A. (1997). High pressure and temperature effects on enzyme
inactivation in strawberry and orange products. Journal of Food Science. 62, No.1,1997- 85.
Awonorin, S.O and Udeozor, L.O. (2014). Chemical properties of tiger nut-soy milk extract. IOSR
Journal of Environmental Science, Toxicology and Food Technology (IOSR-JESTFT) e-ISSN:
2319-2402, p- ISSN: 2319-2399.Volume 8, Issue 3 Ver. 1 (April 2014), PP 87-98
Balasubramaniam, V., Martínez-Monteagudo, S. I., & Gupta, R. (2015). Principles and application of high
pressure–based technologies in the food industry. Annual Review of Food Science and Technology Annu.
Rev. Food Sci. Technol., 6(1), 435-462. doi:10.1146/annurev-food-022814-015539
Barba, F. J., Esteve, M. J., & Frigola, A. (2010). Ascorbic acid is the only bioactive that is better preserved by
high hydrostatic pressure than by thermal treatment of a vegetable beverage. Journal of Agricultural and
Food Chemistry, 58(18), 10070–10075.
Bar-Sela, G. Goldberg, H. Fried, G. and Tsalic, M. (2007). Wheat grass juice may improve hematological
toxicity related to chemotherapy in breast cancer patients: a pilot study. Nutrition and Cancer
journal, 58(1), 43–48.
Benincasa. P, Galieni, A. Guiducci, M. Manetta, A.C. Pace, R.Pisanteb, M and Stagnari, F. (2014).Phenolic
compounds in grains, sprouts and wheatgrass of hulled and non-hulled wheat species. Journal of Science
Food Agriculture. 2015; 95: 1795–1803
Bintsis, T. Robinson, R.K. & Tzanetaki, E.L. (2000). Existing and potential applications of ultraviolet
light in the food industry – a critical review. Journal of the Science of Food and Agriculture;
80:637-645
103
Bodla, R.B & Mujoriya, R. (2011). A study on wheat grass and its Nutritional value. Food Science and
Quality Management journal, ISSN 2224-6088 (Paper), Vol 2, 2011.
Bolton, J. R., & Linden, K. G. (2003). Standardization of methods for fluence (UV Dose) determination in bench-
scale uv experiments. J. Environ. Eng. Journal of Environmental Engineering, 129(3), 209-215.
doi:10.1061/(asce)0733-9372(2003)129:3(209)
Bonfili, L., Amici, M., Cecarini, V., Cuccioloni, M., Tacconi, R., Angeletti, M., . . . Eleuteri, A. M. (2009). Wheat
sprout extract-induced apoptosis in human cancer cells by proteasomes modulation. Biochimie, 91(9),
1131-1144. doi: 10.1016/j.biochi.2009.06.001
Boyer, R., O'keefe, S. & Williams, R. (2013). Use of High Pressure Processing to reduce food borne
pathogens in coconut water. Faculty of Virginia Polytechnic Institute. Blacksburg, Virginia. pp 62.
Burdurlu HS, Karadeniz F and Koca N. (2006). Effect of pH on chlorophyll degradation and color loss in blanched
green peas. Food Chemistry 100: 609–615.
Butz, P. Lia, W. Ma, Y. Rüfer, C.E. Trierweiler, B. Xu, Y. Zhang, C. & Zhao. X. (2010). Comparison
of Thermal, Ultraviolet-C, and High pressure treatments on quality parameters of watermelon
juice. Food Chemistry Journal; 126 (2011) 254-260.
Celik, M.P. (2012). High Hydrostatic Pressure (HHP) inactivation of foodborne pathogens in low-acid
juices. International Journal of Food Science and Technology; 48, 673–677
Chakraborty, R. Das, A. & Raychaudhuri, U. (2012). Effect of freeze drying and oven drying on
antioxidant properties of fresh wheatgrass. International Journal of Food Sciences and Nutrition,
63(6): 718–721
104
Chang, C. Lai, L. Wang, C. Wang, D. (2006). Catalytic characteristics of peroxidase from wheat grass. Journal
of Agriculture and Food Chemistry, 54, 8611−8616-8611
Chang, T.S., Siddiq, M., Sinha, N.K. and Cash, J.N. (1995). Commercial Pectinases and the yield and quality of
Stanley plum juice. Journal of Food Processing and Preservation 19: 89-101.
Chen, J. Feng, Z. Hu, X. Song, L. Wu, J. & Zhang, J. (2009). Influence of thermal and dense-phase carbon
dioxide pasteurization on physicochemical properties and flavor compounds in Hami melon juice.
Journal of Agriculture and Food Chemistry; 57, 5805–5808 DOI: 10.1021/jf900752b
Chen, H. and Neetoo, H. (2012). Application of High Hydrostatic Pressure technology for processing and
preservation of foods. Progress in Food Preservation (First Edition). Edited by Rajeev Bhat, Abd
Karim Alias and Gopinadhan Paliyath. Published 2012 by John Wiley & Sons, Ltd.
Chia S, Rosnah S, Noranizan MA, Wan Ramli W. (2012). The effect of storage on the quality attributes
of Ultraviolet-irradiated and thermally pasteurized pineapple juices. Intl Food Res J 19(3):1001–
10.
Churey, J.J. Usaga, J. Worobo, R. W &Zakour, O.I.P. (2014). Determination of the validation frequency
for commercial UV juice processing units. Journal of Food Protection, Vol. 77, No. 12, 2014,
2076–2080
Corrales, b. M. Fernández, R. Souza, P.M.D. Mario R. Stahl, M.R. (2012). Effects of the decontamination
of a fresh tiger nuts' milk beverage (horchata) with short wave ultraviolet treatments (UV-C) on
quality attributes. Innovative Food Science and Emerging Technologies 13 (2012) 163–168
Danyluk, M.D. Goodrich-Schneider, R.M. Schneider, K.R. Harris, L.J. and Worobo, R.W. (2012).
Outbreaks of foodborne disease associated with fruit and vegetable juices, 1922–2010. Received
105
from: https://www.researchgate.net/publication/274510377_ Outbreaks_ of_ foodborne _
disease_ associated_with_fruit_and_vegetable_juices_1922-2010
De, S. Karmakarb, S. Nso, E.J. and Sagua, S.T. (2014). Optimization of low temperature extraction of
banana juice using commercial pectinase. Food Chemistry 151(2014)182–190
Dede S, Alpas H, Bayındırlı A. (2007). High Hydrostatic Pressure treatment and storage of carrot and
tomato juices: antioxidant activity and microbial safety. J Sci Food Agric 87(5):773–82.
Dhamija, M. Drabu, S. Gupta, A. Padalia, S. and Raheja, I. (2010, April-June). Multitude potential of
wheatgrass juice (Green Blood): an overview. Chronicles of Young Scientists. 1.2: p23
Dong, P. Huang, W. Zhao,L. Liao. X. Liu, F. Wang, S. &Xiong, L. (2012). Comparing the effects of
High Hydrostatic Pressure and thermal pasteurization combined with nisin on the quality of
cucumber juice drinks. Innovative Food Science and Emerging Technologies: 17, 27-36.
Fan, X. & Sampedro, F. (2010). High Hydrostatic Pressure processing of fruit juices and smoothies:
research and commercial application. Case Studies in Novel Food Processing Technologies -
Innovations in Processing, Packaging and Predictive Modelling; first published 2010 Woodhead
publishing limited, Edited by Christopher J. Kustin, D.K and Feeherry, F.E. Elsevier Ltd 2010. pp.
34-72.
Fellows, P. (2009). Food processing technology: Principles and Practice (Third Edition). Woodhead
Publishing Limited. CRC Press LLC. P 241-249
Feng, M., Ghafoor, K., Seo, B., Yang, K., & Park, J. (2013). Effects of Ultraviolet-C treatment in Teflon®-
coil on microbial populations and physico-chemical characteristics of watermelon juice.
Innovative Food Science & Emerging Technologies, 19, 133-139. doi:10.1016/j.ifset.2013.05.005
106
Food Quality News.com. (2016). E. coli in organic wheatgrass. (Food outbreaks). Received from:
http://www.foodqualitynews.com/Food-Outbreaks/IKEA-Hanoju-DEEN-and-Lidl-in-
recalls/(page)/4).
Food Safety News. (2013). More details emerge in recall of sprouts for listeria risk. Received from:
http://www.foodsafetynews.com/2013/02/more-details-emerge-in-recall-of-sprouts-for-listeria-
risk/#.V_2ok48rLIU
Forney, L.J. Koutchma, T. & Moraru, C.I. (2009). Sources of UV light. sun, D. (ED), Ultraviolet Light in
Food Technology: Principles and Applications. Contemporary Food Engineering Science. Taylor
& Francis Group: CRC press. 33-50
Forney, L. Koutchma, T. & Ye, Z. (2011). Characterization and simulation of Ultraviolet Processing of
liquid foods using computational fluid dynamics. Innovative Food Processing Technologies:
Advances in Multiphysics Simulation, (First Edition). Edited by Kai Knoerzer, Pablo Juliano, Peter
Roupas, and Cornelis Versteeg. 2011 by John Wiley & Sons, Ltd. 303-322.
Fruit and Vegetable Juices: U.S. Market Trends. (2013, May 9). PR Newswire, Academic OneFile.
Retrieved from: http://www.reportlinker.com/p01172039/Fruit-and-Vegetable-Juices-US-
Market-Trends.html#utm_source=prnewswire&utm_medium=pr&utm_campaign=Juice
Gabriel AA, Nakano H. (2009). Inactivation of Salmonella, E. coli and Listeria monocytogenes in
phosphate-buffered saline and apple juice by ultraviolet and heat treatments. Food Control 20:443–
446.
Gandhi, V. Kamboj, J.K. & Rana, S. (2011). Living life, the natural way – wheatgrass and health.
Functional Foods in Health and Disease Journal, 1(11):444-456
107
Gayan, E., Serrano, M. J., Raso, J., Alvarez, I., & Condon, S. (2012). Inactivation of Salmonella enteric
by UV-C Light alone and in combination with mild temperatures. Applied and Environmental
Microbiology, 78(23), 8353-8361. doi:10.1128/aem.02010-12
Ghafoor, K., and Choi, Y. (2012). Polyphenol oxidase deactivation in juice from “Campbell Early” grapes
by heating under vacuum pressure. Journal of Food Process Engineering, 35, 391–402.
Guerzoni, M.E. Kamdem, S.L.S. Lanciotti, R. Patrignani, F. & Vannini, L. (2010). Potentialities of High-
Pressure homogenization to inactivate Zygosaccharomyces bailii in fruit juices. Journal of Food
Science; Vol. 75, Nr. 2, 2010
Health Canada. (2007). Unpasteurized fruit juice and cider. Received from: http://www.hc-sc.gc.ca/fn-
an/securit/facts-faits/unpast_fruit_juices-jus_fruits_cidre_nonpast-eng.php
Herald, T. J., Gadgil, P., & Tilley, M. (2012). High-throughput micro plate assays for screening flavonoid
content and DPPH-scavenging activity in sorghum bran and flour. Journal of the Science of Food
and Agriculture J. Sci. Food Agric., 92(11), 2326-2331. doi:10.1002/jsfa.5633
Hu, X. Liu, Y. Zhao, X. & Zou, L. (2012). Effect of High Hydrostatic Pressure on overall quality
parameters of watermelon juice. Food Science and Technology International Journal; 19(3) 197–
207. DOI: 10.1177/1082013212442194.
Klein, J. D., & Lurie, S. (1991). Postharvest heat treatment and fruit quality. Postharvest News
Information, 2, 15–19.
Koutchma, T. (2012). Conventional processing systems of producing safe foods: pasteurization and
sterilization. Handbook of Food Safety Engineering, First Edition. Edited by Da-Wen Sun. © 2012
Blackwell Publishing Ltd. Published 2012 by Blackwell Publishing Ltd. pp 355-370.
108
Koutchma, T. (2014a). Fundamentals of HPP Technology/ In-Container HPP Principle; Adapting High
Hydrostatic Pressure (HHP) for Food Processing Operations; pp.5. Guelph, ON, Canada:
Agriculture and Agri-Food Canada, Guelph Food Research Center.
Koutchma, T. (2014b). Preservation and shelf life extension; UV applications for fluid foods. Guelph,
ON, Canada: Agriculture and Agri-Food Canada, Guelph Food Research Center. First edition
Koutchma, T. (2008). UV Light for processing foods. National Center for Food Safety and Technology,
Illinois Institute of Technology, IUVA News; Vol. 10 No. 4, pp 24-29
Koutchma, T., Keller, S., Parisi, B., and Chirtel, S. (2004). Ultraviolet disinfection of juice products in laminar
and turbulent flow reactors. Innovate. Food Science. and Emergent Technology., 5, 179-189
Koutchma, T. and Stewart, C. (2005). Applications and efficacy of UV Light for foods. The national center
for food safety and technology, Illinois institute of technology, 6502 S. Archer Road, Summit-
Argo, IL 60501.
Koutchma, T., Popović, V., Ros-Polski, V., & Popielarz, A. (2016). Effects of Ultraviolet Light and High-
Pressure Processing on quality and health-related constituents of fresh juice products.
Comprehensive Reviews in Food Science and Food Safety Comprehensive Reviews in Food
Science and Food Safety, 15(5), 844-867. doi:10.1111/1541-4337.12214
Krebbers B, Matser AM, Koets M, Bartels P and Van den Berg R. (2002). Quality and storage-stability of
high-pressure preserved green beans. Journal of Food Engineering 54: 27–33.
Lee, H.S and Coates, G.A. (1999). Thermal pasteurization effects on color of red grapefruit juices. Journal
of Food Science. 64(4):663–666
109
Lowry protocol. (n.d). Proteins (Lowry) protocol. Received from: http://web.itu.edu.tr/~dulekgurgen
/Proteins.pdf
Luo, S. Wang, W. & Zhang, Y. (2013). Stability and sensory quality of fresh fruit and vegetable juices:
evaluated with chromatic aberration. Journal of Food Processing and Preservation, ISSN 1745-
4549, 38 (2014) 1259–1268.
Meyerowitz, S. (1999). Wheatgrass nature’s finest medicine. The complete guide to using grasses to revitalize
your health. Sixth Edition. pp. 49-50
Müller, A., Stahl, M. R., Graef, V., Franz, C. M., & Huch, M. (2011). UV-C treatment of juices to
inactivate microorganisms using Dean vortex technology. Journal of Food Engineering, 107(2),
268-275. doi:10.1016/j.jfoodeng.2011.05.026
Murasaki-Aliberti, N. C., Silva, R. M. S., Gut, J. A. W., & Tadini, C. C. (2009). Thermal inactivation of
polyphenoloxidase and peroxidase in green coconut (Cocos nucifera) water. International Journal
of Food Science and Technology, 44, 2662-2668.
Osaili, T.M. (2012). Developments in the thermal processing of food. progress in food preservation, First
Edition. Edited by Rajeev Bhat, Abd Karim Alias and Gopinadhan Paliyath. Published 2012 by
John Wiley & Sons, Ltd. pp 211-230
Patterson, M. F., McKay, A. M., Connolly, M., & Linton, M. (2012). The effect of high hydrostatic
pressure on the microbiological quality and safety of carrot juice during refrigerated storage. Food
Microbiology, 30(1), 205–212.
Prior, R.L., Hoang, H., Gu, L., Wu, X., Bacchiocca, M., Howard, L., Hampsch-Woodill, M., Huang, D.,
Ou, B., Jacob, R., 2003. Assays for hydrophilic and lipophilic antioxidant capacity (oxygen radical
110
absorbance capacity (ORACFL)) of plasma and other biological and food samples. Journal of
Agricultural and Food Chemistry 51, 3273–3279.
Qin, L., Xu, S.Y. and Zhang, W.B. (2005). Effect of enzymatic hydrolysis on the yield of cloudy carrot
juice and the effects of hydrocolloids on color and cloud stability during ambient storage. Journal
of Science Food Agriculture 85: 505-512.
Rastogi, S.C. (2005). Chemical constituents, digestion and absorption of food. Experimental Physiology
(Second Edition). New Age International (P) Limited Publishers. P.117
Ray, B. and Bhunia, A. (2008). Fundamental food microbiology. Fourth Edition. Taylor & Francis Group,
LLC. Boca Raton, FL.
Rupasinghe, H. P., and Juan, L. (2012). Emerging preservation methods for fruit juices and beverages. Food
Additive. doi:10.5772/32148
Salama, H. M., Watban, A. A., & Al-Fughom, A. T. (2011). Effect of ultraviolet radiation on chlorophyll,
carotenoid, protein and proline contents of some annual desert plants. Saudi Journal of Biological
Sciences, 18(1), 79-86. doi:10.1016/j.sjbs.2010.10.002
Sánchez, C., Baranda, A. B., & Marañón, I. M. (2014). The effect of High Pressure and High Temperature
processing on carotenoids and chlorophylls content in some vegetables. Food Chemistry, 163, 37-45.
doi:10.1016/j.foodchem.2014.04.041
Siegner, C. (January 27, 2014). Does Ultraviolet mean ultra-safe. Food Safety News. Received from:
http://www.foodsafetynews.com/2014/01/pasteurization-does-ultraviolet-mean-ultrasafe/#.V7p1-6Je_qT
111
Singleton, V. L., Orthofer, R., & Lamuela-Raventós, R. M. (1999). [14] Analysis of total phenols and other
oxidation substrates and antioxidants by means of folin-ciocalteu reagent. Oxidants and Antioxidants Part
A Methods in Enzymology, 152-178. doi:10.1016/s0076-6879(99)99017-1
Tarazona-Díaz, M. P., & Aguayo, E. (2013). Influence of acidification, pasteurization, centrifugation and storage
time and temperature on watermelon juice quality. Journal of the Science of Food and Agriculture J. Sci.
Food Agric., 93(15), 3863-3869. doi:10.1002/jsfa.6332
U.S. Food and Drug Administration. (2015). Analysis and evaluation of preventive control measures for the
control and reduction/elimination of microbial hazards on fresh and fresh-cut produce: Chapter IV.
Outbreaks associated with fresh and fresh-cut produce. incidence, growth, and survival of pathogens in
fresh and fresh-cut produce. Received from: http://www.fda.gov/Food/FoodScience Research/ Safe
Practices for Food Processes /ucm091265.htm
U.S. Food and Drug Administration. (2004). Guidance for Industry: Juice HACCP hazards and controls guidance
first edition; Final Guidance. Retrieved August 11, 2016, Received from: http://www. fda. gov/
food/guidanceregulation/guidancedocumentsregulatoryinformation/ucm072557.htm#ii .
U.S. Food and Drug Administration. (2014). Kinetics of Microbial Inactivation for Alternative Food Processing
Technologies: High Pressure Processing. Retrieved August 13, 2016, from http://www.fda.gov/ Food/
FoodScienceResearch/SafePracticesforFoodProcesses/ucm101456.htm
Van Loey, A., Ooms, V., Weemaes, C., Van den Broeck, I., Ludikhuyze, L., Indrawati, et al. (1998). Thermal
and pressure–temperature degradation of chlorophyll in broccoli (Brassica oleracea L. italica) juice: A
kinetic study. Journal of Agricultural and Food Chemistry, 46(12), 5289–5294.
112
World Health Organization WHO. (December 2015). Healthy diet. Received from: http://www.who. int/
mediacentre/ factsheets/fs394/en/
Yaun BR, Sumner SS, Eifert JD, Marcy JE. 2004. Inhibition of pathogens on fresh produce by ultraviolet energy.
Int. J. Food Microbial. 90:1– 8.
Zhang, Y., Liu, X., Wang, Y., Zhao, F., Sun, Z., & Liao, X. (2016). Quality comparison of carrot juices processed
by high-pressure processing and high-temperature short-time processing. Innovative Food Science &
Emerging Technologies, 33, 135-144. doi:10.1016/j.ifset.2015.10.012
113
Appendix
Figure 1 -D value calculation for E. coli P36 after HHP treatment at 600MPa for different time
Figure 2 -D value calculation for Salmonella WG49 after HHP treatment at 600MPa for different
time
y = -0.0912x + 7.11
R² = 0.8846
D=-1/K=-1/-0.0912= 11
0
1
2
3
4
5
6
7
8
0 30 60 90 120
Lo
g C
FU
/mL
N0
Time (s)
E.coli / 600MPa
y = -0.0926x + 7.22
R² = 0.8846
D=-1/K=-1/-0.0926=10.7
0
1
2
3
4
5
6
7
8
0 30 60 90 120
Lo
g C
FU
/mL
N0
Time (s)
Salmonella WG49 / 600MPa
114
Figure 3 -D value calculation for Listeria innocua after HHP treatment at 600MPa for different time
Figure 4 -D value calculation for E. coli P36 after HHP treatment at 500MPa for different time
y = -0.0708x + 7.11
R² = 0.924
D=-1/k=-1/0.07=14.2
0
1
2
3
4
5
6
7
8
0 30 60 90 120
Lo
g C
FU
/mL
N0
Time (s)
Listeria /600MPa
y = -0.0775x + 7.11
R² = 0.8995
D=-1/K=-1/-0.0775= 12.9
0
1
2
3
4
5
6
7
8
0 30 60 90 120
Lo
g C
FU
/mL
N0
Time (s)
E.coli / 500MPa
115
Figure 5 -D value calculation for Salmonella WG49 after HHP treatment at 500MPa for different
time
Figure 6 -D value calculation for Listeria innocua after HHP treatment at 500MPa for different time
y = -0.0869x + 7.22
R² = 0.9609
D=-1/K=-1/-0.0869=11.5
-1
0
1
2
3
4
5
6
7
8
0 30 60 90 120
Lo
g C
FU
/mL
N0
Time (s)
Salmonella / 500MPa
y = -0.0708x + 7.11
R² = 0.9151
D=-1/K=-1/-0.0708=14.1
0
1
2
3
4
5
6
7
8
0 10 20 30 40 50 60 70 80 90 100
Lo
g C
FU
/mL
N0
Time (s)
Listeria / 500MPa
116
Figure 7-D value calculation for E. coli P36 after HHP treatment at 400MPa for different time
Figure 8-D value calculation for Salmonella WG49 after HHP treatment at 400MPa for different
time
y = -0.0588x + 7.11
R² = 0.8821
D=-1/K=-1/0.0588=17
0
1
2
3
4
5
6
7
8
0 10 20 30 40 50 60 70 80 90 100
Lo
g C
FU
/mL
N0
Time (s)
E.coli /400MPa
y = -0.0583x + 7.22
R² = 0.9264
D=-1/K=-1/0.0583=17.1
0
1
2
3
4
5
6
7
8
0 10 20 30 40 50 60 70 80 90 100
Lo
g C
FU
/mL
N0
Time (s)
Salmonella / 400MPa
117
Figure 9-D value calculation for Listeria innocua after HHP treatment at 400MPa for different time
y = -0.0588x + 7.11
R² = 0.8821
D=-1/K=-1/-0.0588=17
0
1
2
3
4
5
6
7
8
0 10 20 30 40 50 60 70 80 90 100
Lo
g C
FU
/mL
N0
Time (s)
Listeria / 400 MPa