A COMPARISON AMONG DIFFERENT ALTERN ATIVES OF PROCESSED GREEN JUICE BLEND S: EVALUATION OF ENZYME ACTIVITY, BIOACTIVE COMPONENTS, AND MICROFLORA By GALO WENCESLAO CHUCHUCA MOR N A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2017
2017 Galo Wenceslao Chuchuca Mor n
To my parents who have worked so hard to provi de me the best education the most valuable gift that I have been given. To Evee who inspire d me to never give up chas i ng a dream.
4 ACKNOWLEDGMENTS First and foremost I would like to thank the Ecuadorian government for granting me the opportunity to purs u e ab roa d. It has been a n invaluable experience. I would like to express my gratitude to my major advisor, Dr. Ren e Goodrich Schneider for her guidance throughout this experience and her invaluable advice about build ing my career I also thank Dr. Keith Schneider for his tremendous help during my research project and his advice. I am also thankful to D r. Amy Harder for inspiring me to use education as a powerful means to help people. I would like to thank all of my professors and staff in the Food Science and Human Nutrition Department who ever taught and assisted me during my education I would like to acknowledge m y lab mates Jessica Lepper and Brittany Xu for the ir advi ce and encouragement Finally, I would especially like to thank my parents and sisters as well as my friends for always giv ing me unconditional support to achieve my goals.
5 TABLE OF CONTENTS page ACKNOWLEDGMENTS ................................ ................................ ................................ ...... 4 LIST OF TABLES ................................ ................................ ................................ ................ 7 ABSTRACT ................................ ................................ ................................ .......................... 8 CHAPTER 1 INTRODUCTI ON ................................ ................................ ................................ ........ 10 2 LITERATURE REVIEW ................................ ................................ .............................. 12 Green Juice Blends ................................ ................................ ................................ ..... 12 Green Juic e Blends Market ................................ ................................ ........................ 12 Juice Processing ................................ ................................ ................................ ......... 14 Consumer Perception ................................ ................................ ................................ 16 Food Safety Aspects of Juice ................................ ................................ ..................... 18 Regulatory Aspects of Juice Production ................................ ................................ .... 19 Quality Parameters of Green Juice Blends ................................ ................................ 21 Enzyme Activity ................................ ................................ ................................ .... 21 Chlorophyll and Total Carotenoids ................................ ................................ ...... 23 Antioxidant Capacity ................................ ................................ ............................ 26 Microbio logical Quality Aspects ................................ ................................ ........... 28 3 MATERIALS AND METHODS ................................ ................................ .................... 30 Retail Screening of Commercial Juices ................................ ................................ ..... 30 Sample Preparation of Model Juice ................................ ................................ ........... 31 Thermal Processing Treatments ................................ ................................ ................ 32 Enzyme Activity of Polyphenol Oxidase and Peroxidase ................................ .......... 33 Measurement of Chlorophylls and Total Carotenoids ................................ ............... 34 Measurement of the Total Antioxidant Capacity ................................ ........................ 35 Microbiological Analysis ................................ ................................ .............................. 35 Statistical Design and Analysis ................................ ................................ .................. 36 4 RESULTS AND DISCUSSION ................................ ................................ ................... 39 Product Quality Parameters in Commercial Green Juice Blends .............................. 39 Enzyme Activity of PPO and POD in Untreated and Thermally Treated Formulated Green Juice Blends ................................ ................................ ............. 43 Levels of Chlorophylls and Total Carotenoids in Untreated and Thermally Treated Formulated Green Juice Blends ................................ ................................ 47 Chlorophylls ................................ ................................ ................................ .......... 47 Total Carotenoids ................................ ................................ ................................ 49
6 Total Antioxidant Capacity in Untreated and Thermally Treated Formulated Green Juice Blends ................................ ................................ ................................ 51 Natural Microflora in Untreated and Thermally Treated Formulated Green Juice Blends ................................ ................................ ................................ ...................... 54 Comparison a mong the Thermally Treated Formulated Green Juice Blends and the Commercial Alternatives ................................ ................................ ................... 57 5 CONCLUSIONS AND SUGGESTIONS FOR FUTURE WORK ............................... 64 APPENDIX A SSOP OF THE JUICING MACHINE ................................ ................................ .......... 66 B SUMMARY OF QUALITY PARAMETERS OF FORMULATED GREEN JUICE BLENDS AFTER FOUR INDEPENDENT TRIALS ................................ .................... 67 Summary of the Enzyme Activity (PPO and POD) in Untreated and Thermally Treated Formulated Green Juice Blends ................................ ................................ 67 Summary of the Levels of Chlorophylls and Total Carotenoids in Untreated and Thermally Treated Formulated Green Juice Blends ................................ ............... 68 Summary of the Total Antioxidant Capacity in in Untreated and Thermally Treated Formulated Green Juice Blends ................................ ................................ 69 LIST OF REFERENCES ................................ ................................ ................................ ... 70 BIOGRAPHICAL SKETCH ................................ ................................ ................................ 83
7 LIST OF TABLES Table page 3 1 Label information of commercial green juice blends found at the retail market and evaluated in this study. ................................ ................................ .................... 38 4 1 Summary of product quality parameters in some commercial green juice blend s found at the retail market and evaluated in this study. .............................. 59 4 2 Overall results of the enzyme activity, chlorophylls & total carotenoids levels, and total antioxidant capacity among treatments. ................................ ................. 6 0 4 3 Overall normalized results of the enzyme activity, chl orophylls & total carotenoids levels, and total antioxidant capacity among treatments. ................. 61 4 4 Summary of natural microflora in unt reated green juice blends and thermally treated green juice blends. ................................ ................................ ..................... 62 4 5 Overall log reductions of APC in thermally treated gre en juice blends. ............... 63
8 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Master of Science A COMPARISON AMONG DIFFERENT ALTERN ATIVES OF PROCESSED GREEN JUICE BLEND S: EVALUATION OF ENZYME ACTIVITY, BIOACTIVE COMPONENTS, AND MICROFLORA By Galo Wenceslao Chuchuca M or n December 2017 Chair: Ren e Goodrich Schneider Major: Food Science and Human Nutrition Health conscious consumers associate drinking green juice blends with being healthy and lowering their sugar intake. The two primary objectives of this study were to examine commercial green juice blends alternatives at retail market, and to evaluate sever al product quality parameters of unprocessed and thermal pasteurized green juice blends. All products were evaluated for enzyme activities of polyphenol oxidase (PPO) and peroxidase (POD), levels of chlorophyll a and b total carotenoids, total antioxidant capacity, total coliforms, E. coli and total aerobic plate counts (APC). For the first objective, three high pressure processed (HPP) juices and one thermally processed juice were examined. Enzyme activities were higher in the HPP juices compared to the thermal ly processed juice. Chlorophyll levels appeared formula dependent. Levels of total carotenoids were similar among juices. Average levels of total coliforms were 2.56 0.07 log CFU/ml for one HPP juice and lower than the detection limit for the rema ining juices Levels of E. coli were lower than the detection limit in all juices APC, as a general quality indicator, varied widely among all juices.
9 For the second objective, a representative green juice blend was formulated, and then thermally treated in four independent trials. Thermal treatments applied to the juices were at 72 C for 15 s, and at 90 C for 30 s. In the control, average PPO and POD activities were 12.17 and 38.33 U/ml, respectively. After thermal processing, enzyme activities decrease d and were significantly different among treatments ( P < 0.05). PPO was more thermoresistant than POD. In the control, average levels of chlorophyll a chlorophyll b and total carotenoids were 56.04 3.04, 25.36 1.19, and 13.24 0.80 g/ml, respective ly. After thermal processing, the levels of chlorophylls decreased significantly and differed among treatments ( P < 0.05). Chlorophyll a was more affected than chlorophyll b during the mild thermal treatment. No significant differences existed between cont rol and the milder treatment for the levels of total carotenoids ( P > 0.05), but the levels decreased after the higher treatment. In the control, average antioxidant capacity was 0.67 0.07 mol of TE/ml, which increased after thermal treatments. No signi ficant differences existed between treatments ( P > 0.05). In the control, average levels of total coliforms and APC were 2.32 0.62 and 5.44 0.42 log CFU/ml, respectively. The level of E. coli was 1.00 log CFU/3 ml in only one of the four trials. After thermal processing, the levels of total coliforms and E. coli were lower than the detection limit Average levels of APC after mild and higher treatments were 2.79 0.30 and 2.09 0.08 CFU/ml, respectively. Overall, results showed that thermal pasteuriza tion reduced the enzyme activity of PPO and POD, levels of chlorophyll a and b total coliforms, E. coli and APC, especially at higher temperature. Furthermore, a milder thermal treatment increased the total antioxidant capacity and bioavailability of tot al carotenoids in green juice blends.
10 CHAPTER 1 INT R ODUCTION Health and wellness is a valuable market in the U nited S tates (US ) In 2016, its division of food and beverages was expected to approximately account for $66 milli ons of industry revenue (IBISWorld 2016) Furthermore, h ealth and wellness market also drives new food trends among consumers. A growing consumer segment is the h ealth conscious consumers who value food products with natural ingredients as well as those designated a s sugar free, low salt, or made from fruit and vegetables (Nielsen 2015) Drinking j uices and beverages is a convenient mean s to consume fruit and vegetables. Juice production had a domestic demand of $12.8 billions and an ind ustry revenue of $12.0 billions in 2016 (IBISWorld 2017) The fruit juice industry, however, has faced som e concerns about the relation between obesity and juice consumption (PBH 2015) By contrast, vegetable juice s are attract ive to consumers because of their low sugar content (Mintel 2017) Drinking vegetable juice is considered among the acceptable mean s for achieving a healthy intake of vegetable s according to t he 2015 2020 Dietary Guidelines for Americans (HH S and USDA 2015) Dark green leafy vegetables become popular among consumers because they are low calorie and great sources of nutrition These vegetables contain high levels of vitamins (mainly vitamin A, C, E, and K ) and minerals (iron, magnesium, potassium and calcium) as well as an abundance of carotenoids and antioxidants (Yan 2016) Therefore, g reen juice blends might be an attractive food product for the beverage industry and for consumers seeking to increa se their vegetable intake.
11 T he p r oduction o f green juice blends however, has challenges in meeting consumer expectations. One of the largest need s is to conserve the health benefits, both real and perceive, from vegetables and fruits, by minimizing the processing effects but without neglecting the product food safety. Since unpasteurized juice has limited shelf life and serious food safety implications thermal pasteurization is the most reliable processing alternative for the juice industry (Siv apalasingam and others 2004; Vojdani and others 2008) Furthermore, n on thermal technologies become the popular processing alternat ive among consumers that are looking for minimally processed food (Huang and others 2017) Nevertheless, there is l imited data reported about the effects of juice processing in green juice blends. The overall goal of this study is to provide technical information about green juice blends that could potentially guide both processors and consumers in their choices. Specifically, this study aims to first examine a range of commercial green juice blends at retail market, and to then evaluate several product quality parameters of a representative treated green juice blend. All products were evaluated for enzyme activities of polyphenol oxidase (PPO) and peroxidase (POD), levels of chlorophyll a and chloro phyll b total carotenoids, total antioxidant capacity, total coliforms, generic E scherichia coli and total mesophilic aerobic plate counts (APC).
12 CHAPTER 2 LITERATURE REVIEW Green Juice Blend s Vegetable juice is defined as the unfermented liquid (but fermentable product) obtained from the edible part of one or more vegetables and preserved exclusively by physical means; it may be clear, turbid or pulpy (Bates and others 2001) Straight v egetable juice product s, such as carrot, beet, and celery juices, generally have pH values higher than 6 .0 (Danyluk and others 2012b) Wu and Shen (2011) categorize vegetables juices and beverages into four categories: 1) vegetable juice products from normal ly acidic produce, such as tomato juice; 2) acidic vegetable juice products that are mainly prepared with a low acid produce, but acidifying agent adjust product pH; 3) acidic vegetable juice products from fermented vegetables, such as sauerkraut juice; an d 4) non acidic vegetable juice products, such as carrot juice and asparagus juice. Green juice blends are generally acidic vegetable juice products (pH < 4.6) characterized by a formulation containing different green vegetables and leafy greens, such as c ucumber, celery lettuce, spinach, kale, parsley, and wheatgrass. S ome fruits mainly apple and citrus, may be added to enhance sweetness and flavors as well as to adjust product pH. Green Juice Blends Market In 201 6 v egetable juice accounted for 1.2% of the US juice production which had a total domestic demand of $12.8 billions and an industry revenue of $12.0 billions (IBISWorld 2017) Tomato, carrot, and red beet juices and beverages have been the most popular alternatives among vegetable juice products (Bates and others 2001; Grassin and Coutel 2010) Recently, h ealth and wellness market drives growth of
13 vegetabl e juice products with green juices lead ing the new trends during the last five year s (del Buono 2017) C o nsumers associate drinking green juice blend s with being healthy and they are attracted to the low sugar content of those drinks (Mintel 2017) This perception and interest contrast with the reality about fruit juice consumption I t has steadily decreased during the last years (from 2004 to 2014) because of some concerns about the relation between o besity and juice consumption (PBH 2015) There are many green juice blends on the market, which suggests consumer demand, but there is l imited information av ailable about the green juice blends market. Some US demographic findings regarding vegetable consumption suggest that adu lts over the age of 55 with annually incomes over $60 thousands consume the highest amounts of fruit and vegetables and adults under the age of 40 are consuming more fruit and vegetables than their counterparts 10 years ago (PBH 2015) Nevertheless, the consumption of vegetable is still lower among mainstream consumers. In the US less than 14% of adults in each state consumed the recommended amount of vegetables which is (Moore and Thompson 2015) Vegetable juice therefore might result attracted fo r a specific population segment and market such as health conscious consumers. Furthermore, consumers under 34 ye ars old are most willing to pay a premium for health attributes and th ose consumers also consider health attributes very important in food that they are purchasing (Nielsen 2015) Actually, the millennial generation is most i nterested in novel juice products that are both nutritious and delicious (IBISWorld 2017) Green juice ble nds market might be interested in those population segments.
14 Juice Processing Pasteurization in juice processing traditionally refers to an application of heat sufficient for the destruction of pathogens or the reduction of spoilage organisms (Parish 1997) Pasteurization includes heating and cooling phases (come up and come down time ), respectively, as well as a holding phase where food product is held at specific time and temperature to reduce its processing indicator, e.g., pathogen, enzyme, or spoilage organisms (Fellows 2009) Refrigerated juices are usually processed by high temperatur e short time (HTST) treatments with the intent to improve the retention of nutrients and fresh flavor profiles (McLellan and Padilla Zakour 2005) Mazzotta (2001) concluded that a thermal processing at 71.1 C for 3 s provide an effective treatment to achieve one log reduction of E. coli O157:H7, Listeria monocytogenes and Salmonella in acidic juice s. However, thermal pasteurization treatments targeting those vegetative cells are generally ineffective in the reduction of bacterial spores (Smelt and Brul 2014) The spores of Clostridium botulinum are a highly concern in public health but its risk could be reduced lowering the product p H lesser than 4.6 (Bell and Kyriakides 2000) When vegetable juice product s are low in acids (pH over 4.5), the juices require either pH reduction or severe thermal treatments at temperatures around 120 C (Bates and others 2001) For example, a thermal processing for salty asparagus juice filled into cans require s 116 C for 20 minu tes (Wu and Shen 2011) Nevertheless, t hermal processing designed to destroy vegetative pathogens may not completely inactivate endogenous enzymes or spoilage bacteria that may cause quality loss during storage and consequently shorten the product shelf life (Smelt and Brul 2014; Peng and others 2017) Juice processing treatments therefore may have
15 hi gher temperatures and longer holding times than processing parameters aiming to destroy the pertinent pathogen. For example, thermal processing treatments for orange juice are typically at 85 to 90 C for 10 25 s, and treatments for clarified apple juice a re at 85 C for 15 120 s and at 95 C for 10 30 s (Reyes De Corcuera and others 2014) In addition to thermal treatments, non thermal pasteurization technologies are promising alternatives to reduce the possible negative impact on thermal pasteurized food products such as losses of thermolabile nutrients. High pressure processing (HPP) is currently popular in juice industry. HPP juice s increased from 20 companies marketing such juices in 2010 to more than 100 companies in 2015 (Huang and others 2017) HPP t reatments for acid fruit juices are generally at 400 MPa for 10 min at 20 C to target pathogens such as E. coli O157:H7 as well a s yeasts and molds (Daryaei and Balasubramaniam 2012) For instance, HPP treatments at 500 MPa for 10 min at 25 C were used for processing of acidified broccoli apple juice blends to achieve 5 log reduction of E. coli as well as for processing of tomato juices to inactiv e polygalacturonase (Hsu 2008) Furthermore, higher pressure treatments and shorter holding times (615 MPa for 2 min at 15 C) achieved 6.4 log reduction of E. coli O157:H7 in carrot juice (Teo and others 2001) Although HPP treatments result a good alternative to minimize processing impact on sensory attributes, incomplete enzyme inactivation and following chemical reactions result in some drawbacks for this technology during the product shelf life (Oey and others 2008) For instance, HPP treatment at 500 MPa for 10 min at 25 C in tomato juice was limited to achieve the enzyme inactivation of pectin methylesterase (Hsu
16 2008) Furthermore feasible pressure treatments cannot inactive most of bacterial endospores at ambient temperatures; thus, high pressure treatment without thermal treatments is not an appropriate option for low acid vegetable juice processing (Tere fe and others 2014) A dditional non thermal technologies have also been evaluated for vegetable juice processing For example, ultrasound treatments for apple carrot blends (Gao and Rupasinghe 2012) p ulsed elec tric fields treatments for broccoli juice (Snchez Vega and others 2014) and orange carrot blends (Rivas and others 2006) h igh pressure carbon dioxid e processing for beetroot juice and gamma ir radiation for kale and carrot juice s (Song and others 2007) This denotes an especi al interest by researches and industry to provide non thermal treated juices. Nevertheless, t he juice matrix should be carefully evaluated as it significantly affects the efficacy of the potential processing treatment. For example, UV treatment of apple juice is feasible, while UV treatment of orange juice is not because of its cloudiness (Keklik and Demirci 2012) Finally, c ombination among thermal and non thermal treatments and hurdle approaches have also been put forth as plau sible alternatives for juice processing (Ross and others 2003) For instance, Evelyn and others (2017) found that 600 MPa at 70 or 75 C achieved higher bacterial and mold spore inactivation ( related to fruit spoilage ) compared to thermal processing treatments at the same temperatures. Consumer Perception Current c onsumers are looking for healthy options a nd embracing new trends in food products. In addition to the so (Huang and
17 others 2017) Likewise, f or the consumer segment who sacrifice costs and convenience for a high quality produc t, the top three attributes perceived as healthy include free from artificia l ingredients or additives high in healthy components and minimally processed (IFIC 2017b) N on thermal processing technologies therefore have gained popularity among co nsumers and food industry. For instance, consumers perceive that HPP technology provides food products with better quality and food safety (Frewer and others 2011) Nevertheless, it is imprecise whether consumer have a complete understanding of those perceived benefits or their thoughts are based on other consumer preferences (Asioli and others 2017) For instance, n early double of consumers see bagged baby carrot as processed food compare d to the same product, but organic ; d efining the term (IFIC 2017a) Perceived health benefits might not always correspond to reality. For exa mple, ready to drink juice and juice drink purchases in the US (from 2008 to 2012) with low sugar claims had the highest mean total sugar density compared to those juices with other claims or no claim (Taillie and others 2017) Finally, findings regarding the economic aspects show that the mainstrea m consumer may have a different attitude to the minimally processed technology. For instance, they are concerned that non thermal processing juice technologies could result in more expensi ve products and they generally are not willing to pay a premium for those technologies (Olsen and others 2010) Conversely, there is a growing consumer segment, the health conscious consumers, that are most willing to pay a premium for health attributes (Nielsen 2015)
18 Food Safety Aspects of Juice Several foodborne outbreaks have been associated with juice consumption. Many juices implicated in juice associated outbreaks were reported as unpasteurized (Sivapalasingam and others 2004) In general, f oodborne illness outbreaks due to bacterial pathogens and virus are the main concern because of their severe co nsequences at the public health level and the impact on the economy with loss of sales and trust (Sivapalasingam and others 2004; Van Boxstael and others 2013) Danylu k and others (2012a) reviewed outbreaks of foodborne disease associated with fruit and vegetable juices between 1922 to 2010. Among the most recent cases, Clostridium botulinum Salmonella Typhi and E. coli O157:H7 were found in pasteurized carrot juice, frozen orange pulp, and unpasteurized apple juice, respectively. This contamination occurred in juices sold at retail market in the US. In 2006, refrigerated thermal treated carrot juices were the source of an inter national outbreak of botulism that caused one deceased (at day 90 after illness onset ) and two ventilator dependents; the juices contained no chemical barriers to C. botulinum germination (Sheth and others 2008) Up to the present, no outbreak reports of microbial foodborne illness have been connected to the consumption of green juice blends However, numerous vegetables have been implicated in many foodborne illness outbreaks in the US For example, one of the most significant event occurred in October 2006 when a multistate outbreak of E. coli O157:H7 linked to fresh spinach caused 199 persons infected, 102 hospitalizatio ns, and 3 deaths (Sharapov and ot hers 2016) The risk of contamination by foodborne pathogens can originate from the primary production i.e. during growing and harvesting, through processing in the factory
19 In an epidemiologic study from 1973 to 2012, Herman and others (2015) identified norovirus as the leading cause of the outbreaks related to contaminated leafy greens, caused by poor food safety practices in the field and in restaurants. In addition Shiga toxin producing E. coli (STEC) and Salmonella were identified as t he common bacterial pathogens implicated. Foodborne illness due to STEC caused over 45% of hospitalizations and nearly half of the deaths. The number of confirmed outbreaks related to STEC contamination has increased in recent years. I rrigation water and s oil are considered risk factors for introduction of pathogens during the primary production where E. coli seems to be a suitable hygiene criterion for leafy greens (Castro Ibez and others 2015; Holvoet and others 2014) Even low levels of E. coli O157:H7 on produce during the field production could result an outbreak because of the likelihood of cross contamination during the post harvest handling (Danyluk and Schaffner 2011) Furthermore p ackinghouse practices might increase the risk of contamination with pathogens. In some instances, higher micr obial concentrations on produce had been found in th e final packing carton compared to the microbiological levels in the field (Ailes and others 2008) Schneider and others (2017) found similar results on tomatoes at different packinghouses, and they concluded that washing step at the packinghouse could result in a potential risk of cross contamination when poor food safety practices are implemented. Regulatory Aspects of Juice Production Since 2001, the US Food and Drug Administration (FDA) mandates that the pr ocessing technolo gy used in the juice production must be equivalent to a 5 log reduction of the pertinent microorganism and that the juice be produced under a HACCP plan. The pertinent microorganism refers to the most resistant microorganism
20 of public health significance that is likely to occur in the juice (FDA 2004) Hazard Analysis Critical Control Point (HACCP) is a food safety system to address any risk of contamination with biological, chemical, or physical hazards (FDA 1997) In a review focusing on the years between1995 and 2005, it appears the federal mandate of HACCP for juice industry was associated with a reduction in (but not total elimination of) juice associated outbreaks (Vojdani and others 2008) Refrigerated green juice blends, if they are 100% juices, are cover by the juice HACCP rule. As defined by t he federal law, juice refers to the aqueous liquid expressed or extracted from one or more fruits or vegetables (21 CFR Part 120). Therefore, the processing operations, i.e. thermal or non th ermal technology, of green juice blend s requires a HACCP plan. T he selection of the pertinent microorganism is of course critical for any processing regime for any green juice blends. Because most vegetable juice s have pH values higher than 4.6 they would be classified as low acid juices. The risk associated with spor es of non proteolytic and proteolytic strains of Clostridium botulinum must be considered as likely pertinent pathogens for low acid juice (FDA 2007) For acid vegetable juice (pH 3.7 to 4.6), e.g., green juice blends there may be ot her designated pertinent pathogens, such as Salmonella E. coli O157:H7, and Listeria monocytogenes (Mazzotta 2001) Unpasteurized green juice blends may still be offered at retail establishments or businesses that make and sell juice directly to consumers, e.g. the novel juice bars, because they are exempt from the juice HACCP regulation (FDA 2004) However, the FDA (2015) also warns consumers about the risk of serious illnesses from drinking untreated juice, especially for the high risk popu lation segment, such as children, older
21 adults, pregnant women, and people with weakened immune systems. The FDA recommends a warning label for untreated juice that stating the likely presence of harmful bacteria. Proper refrigeration is critical for untre ated juice as well as juices susceptible to potential C. botulinum contamination (FDA 2007) Qu ality Parameters of Green Juice Blend s Enzyme Activity Polyphenol oxidase (PPO) and peroxidase (POD) have been detected in most known fruit and vegetables, including dark green vegetables and leafy greens. The enzyme activity of PPO and POD are mainly related to enzymatic browning (Vmos Vigyz and Haard 1981) PPO catalyzes hydroxylation and oxidation reactions that involve phenolic compounds; browning pigments result from condensations of the enzyme reaction products, e.g. o quinones (Parkin 2008) Furthermore, POD utilizes hydrogen p eroxide as an electron acceptor and oxidize s donor compou nds; the reactions result in colored products (Hammer 1993) The enzyme activity of PPO and POD in green vegetables and leafy greens can fluctuate through the plant compone nts, e. g., roots, stalks, skin, pulp, seeds. For example PPO activity in cucumber fruit is present only in the skin, but POD activity may also be located in pericarp and carpel tissues (Miller and others 1990) Furthermore t he enzyme activity of PPO and POD can also be affected by vegetable va riety, maturity stage, pre harvest conditions and environm ental factors (Golbeck and Cammarata 1981; Vmos Vigyz and Haard 1981; del Amor and others 2008; Korus 2011; Boo and others 2011)
22 Enzyme inactivation by thermal treatments is mainly related to destabilization of covalent and noncovalent interactions. T he enzyme stability depends on matrix factors, such as pH, water content, and levels of salts and sugars (Ludikhuyze and others 2003) Therefore, such complexity makes difficult to extrapolate the thermal processing effects among different food systems. Furthermore, differences in enzyme systems in different plants (i.e. isozymes) mak e it difficult to pre cisely characterize enzymes. For example, overall thermal sensitivity of POD, among broccoli, asparagus and carrot, differed due to the relative proportions of the h eat labile and heat resistant fractions in each vegetable (Morales Blancas and others 2002) Traditionally, POD is identified as a more thermoresistant enzyme compared to PPO in leafy greens vegetables. Inactivation of PPO requires only short exposures to tempera tures between 70 to 90 C; conversely, inactivation of POD requires several minute exposures to temperatures between 80 to 100 C (Vmos Vigyz and Haard 1981; Parkin 2008) However, c ontrar y findings are also reported For example PPO appeared more thermoresistant than POD in blanched parsley pastes after blanching treatments at 80 C for 1 to 10 min (Kaiser and others 2012) Diverse results have been reported regarding the effects of non thermal treatments in green vegetab les and leafy greens. Terefe and others (2014) reviewed the effects of hi gh pressure processing in the enz yme activity of PPO and POD in some vegetables products. In general, PPO activity is extremely resistant to high pressure treatments and POD activity varies widely among products. But, s ome findings also suggest ed increase in the PPO and POD activities in products such as carrot, mushrooms, cloudy apple juice, tomato puree, and strawberry puree.
23 Finally, the complexity of enzymes is also extended to their assays. Selection of substrate and reaction conditions impacts the enz yme activity evaluation of PPO and POD; optimized conditions may vary among vegetables, especially blends. For example (2005) found contrary thermal inactivation results of artichoke PPO after using different substrates in its activity assay T hermal inactivation treatments indicated effectiveness with increasing temperature and time to inactivate artichoke PPO using 4 methylcatechol and pyrogallol substrates, but artichoke PPO activity appeared unaffected using catechol substrate Nevertheless (2012) identified catechol as the most suitable substrate for parsley PPO after examining the enzyme kinetics and substrate specificity. Therefore, individual characterization of enzymes from green vegetables and leafy greens provides a basis for evaluation reactions and conditions of enzyme activity of the juice blends Chlorophyll and Total Carotenoids Chlorophylls and carotenoids are natural pigments found in vegetables. Chlorophylls mainly comprise two types: chlorophyll a and chlorophyll b Both comprise a macrocyclic tetrap yrrole complex (porph yr in ring) with an esterified phytol group a carbomethoxy group, a vinyl group, and an ethyl group ; the form a also includes a n ethyl group and the form b includes a formyl group (Schwartz and others 2008) Furthermore chlorophyll ring complexes carry magnesium as the central metal ion in the tetrapyrrole center that ensures absorption of light in the b lue to green red spectrum (Roca and ot hers 2016) Carotenoids comprise several different molecules and derivatives The basic structure include s a backbone of isoprene units where c yclic end groups might also be incorporated (Schwartz and others 2008) From the center of the molecule, carotenoids
24 are reversed resulting into two central side chains with lateral methyl gr oups ; this base structure can be modified by cyclization, hydrogenation and dehydrogenation, oxidation or any combination of them (Kaczor and others 2016) The numerous conjugated double bond structures serve a s the light absorbing chromophores that are responsible for the yellow to red color spectrum, a distinctive characteristic of carotenoids (Rodriguez Amaya 2001) Traditionally, carotenoids have been associated to health benefits because of their pro vitamin A activities carotene; eyes protection benefits e.g., lutein; and antioxidant capacity e.g., lycopene (Calvo 2005; Saini and others 2015) To a smaller extent chlorophylls have also been related to antioxidant capacity and potential health benefits such as cancer preventative activity (Ferruzzi and Blakeslee 2007; Szgen Nevertheless, as many health rela ted molecules, carotenoids could have different behaviors during the adsorption and metabolism. For instance, carotene have greater vitamin A value than their cis counterpart s; conversely cis forms of lycopene shows greater absorption than the tra ns configurations (Priyadarshani 2017) Green vegetables and leafy greens are an excellent source of chlorophylls and carotenoids. They contain significant levels of chlorophyll a and chlorophyll b violaxanthin, lutein, carotene isomers (Heino nen and others 1989; Burns and others 2003) These types of vegetables are usually associated to high chlorophyll content, but carotenoids should not be ignored. There is a clear association with higher carotenoid leve ls and darker green vegetables because carotenoids are located into the chloroplasts and leaves ha ve a high population of them (Britton and Khachik 2009)
25 Specific levels of carotenoids and chlorophylls might be a ffected by pre and post harvesting factors, such as cultivar genotype, ripening time, cultivation methods, farming practices, season, and climatic conditions (Kopsell and others 2004; Saini and others 2015; Walsh and others 2015) For instance, the levels of carotenoid and chlorophyll in kale differed through its maturity stages; the highest concentration of lutein occurre d in 1 2 week old leaves but chlorophylls ( a and b carotene reached their maximum concentrations at 2 3 weeks (Lefsrud and others 2007) These differences can be responsible for the range of values presented in the literature for these compounds. Furthermore, d uring thermal processing treatments of green vegetables and leafy greens pigments are also affected in differ ent grade. C hlorophylls are severely affected by heat Breakdown of chlorophylls results in the formation of pheophytins (displacement of magnesium ion) and pyropheophytins (displacement of carbomethoxy group) which occur in a lower extent for chlorophyll b because of its formyl group (Schwartz and others 2008) C onversely carotenoids might remain unaffected by th ermal treatments. Increases in apparent carotenoids levels could a lso occur because of carotenoids release from tissue cells (Walsh and others 2015) For example thermal sterilization treatments severely reduced the concentration of carotenoids and chlorophylls but non significant differences were found between high pressure treatments and cooking methods in the carotenoids levels among some xanthophyll rich foods, such as spinach, kale, parsley, and dill (Arnold and others 2014) In most of the schemes evaluated, thermal treatments reduced the levels of chlorophyll and consequently the color quality of the vegetables. Non thermal
26 treatments might result a good processing alternative. For instance, chlorophyll concentrations were unaffected at ext reme high pressure treatments (800 MPa) combined with thermal treatments not higher than 50 C in broccoli juice (Van Loey and others 1998) Furthermore, carotenoids are also pressure resistant molecules F or example, carotene and lycopene in pr es sure treated tomato juice at 600 MPa for 60 min (Butz and others 2002) Finally, HPP technology has a l imited effect on the natural pigments levels (chlorophyll and carotenoids) in fruit and vegetables products; however, color degradation might still occur related to incomplete inactivation of enzymes (Oey and others 2008) Antioxidant Capacity Although consuming fruits is the most common mean s to intake antioxidant from plants, some vegetables have importa nt levels of antioxidant compounds (Miller and others 2000; Chun and others 2005) A nti oxidative and bioactive plant compounds general ly include carotenoids, phenolics, alkaloids, nitrogen containing compounds, and organosulfur compounds (Liu 2004) A fter examining 56 vegetables, the main phenolic compounds with antioxidant activities were gallic acid, epicatechin, catechin, and quercetin (Deng and others 2013) V itamin C also had a relatively high contribution to the antioxidant activit ies in cucumber and red pepper (Chu and others 2002) Likewise the antioxidant capacity among Brassica vegetables varieties, such as broccoli, cauliflower, and kale, are mainly re lated to phenolic compounds and vitamin C, and in lesser e xtent to carotenoids and vitamin E Processing of green vegetables and leafy green have an impact on thei r antioxidant capacity. P hytochemicals in vegetables, such as phenolic acid and flavonoids, might degrade or modify their structures during thermal and non thermal
27 processing. New antioxidants compounds could be formed as well (Nayak and others 2015) Jimnez Monreal and others (2009) identified four possibilities about the increase in their the antioxidant capacity. These included (1) the liberation of anti oxidant components due to the cell wall disruptions; (2) production of stronger radical scavenging capacity by thermal chemical reactions; (3) reduction of oxidation stress because of the inactivation of oxidative enzymes; and (4) production of new antioxi dant components. However processing effects in the antioxidant activity of vegetables could result specific for each product and treatment. For example t he antioxidant activity capacity of spinach juices was significant reduced after thermal treatments ( at 75 and 100 C for 10 and 30 min each) but the antioxidant activity capacity of cabbage juices showed greater resistance to the same treatments (Roy and others 2007) Likewise significant reductions of the antioxidant activity were found in garlic and zucchini juices but higher increase s was found for tomat o and yellow bell pepper juices (Gazzani and others 1998) Differences among products might be related to the differences in the bound and the free fractions of antioxidants among vegetables For instance, free fraction of antioxidant in spinach was around 87%, but in cabbage was around 67% (Chu and others 2002) Finally, numerus assays are reported to m easure the antioxi dant capacity for beverage and food products Antioxidant assays might be an assay associated with lipid peroxida tions, e.g., thiobarbituric acid assay (TBA), malonaldehyde/ga s chromatography (MA/GC) assay, and carotene bleaching assay or an assay associated with electron or radical scavenging, e.g., the 2,2 diphenyl 1 picrylhydrazyl
28 azinobis(3 ethylben zothiazoline 6 sulfonic acid) (ABTS) assay, and ferric reducing/antioxidant power (FRAP) assay (Moon and Shibamoto 2009) The DPPH assay is popular because of its simplicity and highly sensitivity. Reduction by antioxidant compounds is measured by the decrease in DPPH abs orbance at 515 nm (Brand Williams and others 1995) Never theless, beverages and food products might contain different antioxidant molecules, and different reactions might occur among antioxidant assays (Stratil and others 2006; Zuluet a and others 2009) These differen ces can be responsible for the range of valu es presented in the literature. M icrobiological Quality Aspects In addition to pathogens, spoilage microorganisms are also studied in vegetables products because they are as sociated to loss of the product quality and to off flavors Those organisms include s everal b acteria, yeast, and molds The most common spoilage bacteria in vegetable product s are lacti c acid bacteria (LAB), Erwinia and Pseudomonas (Koo 2011) For instance, 46 isolated strains of pectolytic bacteria from three types of produce (green bell peppers, Romaine lettuce, and baby carrots) were identif ied as members of the genera of Pseu domonas Erwinia Bacillus Xanthomonas and Flavobacterium (Liao and Fett 20 01) LAB cause spoilage in fruits and vegetables products during storage at refrigerated temperatures; these bacteria lower pH and produce acetylmethylcarbinol and diacetyl, which provide off flavors to food products (Barth and others 2009) Nevertheless, its o ccurrenc e could differ among vegetables products. For example the average levels of LAB ranged 3.0 to 5.9 log CFU/g in fresh cut arugula, 4.3 to 7.6 log CFU/g in fresh cut carrot, 3.7 to 6.9 log CFU/g in fresh cut spinach, and 1.7 to 6.3 log CFU/g in fresh cut lettuce (Abadias and others 2008)
29 In general, yeast and molds are usually the main concerns after juice thermal processing. A large spectrum of fungi species is also related to loss of product quality in vegetables, e.g., Cladosporium Alternaria Aspergillus Colletotrichum Fusarium Penicillium Rhizopus Rhizoctonia and some mildews (Tournas 2005) Furthermore, some spore forming species related to spoilage are able to growth even at acidic conditions; traditionally, spores are not a main concern in acidic product. These thermal ly resisted organisms include Bacillus coagulans B macerans B. subtilis and Alicyclobacillus spp. (Worobo and Splittstoesser 2005) Finally, a plate count of aerobic mesophilic microorganisms (APC) has been used as quality indicator for fresh produce by several studies (Liao and Fett 2001; Johnston and others 2006; Abadias and others 2008; Ailes and others 2008) This indicator is for microorganisms that grow aer obically at mesophilic temperatures (typically between 20 and 45 C) on a food product but it does not di fferentiate types of bacteria (Swanson and others 1992) Although lower levels of APC could be associated with unspoiled products, higher co unts of APC do not always correlate with spoilage and they cannot be used only to predict the product shelf life (Barth and others 2009)
30 CHAPTER 3 MATERIALS AND METHODS Retail Screening of Commercial Juices The first section of this study examined commercial green juice blend s found at the retail market. Generally, options of green juice blends include different green vegetables and leafy greens, such as cucumber, celery lettuce, spinach, kale, parsley, and wheatgrass. Refrigerated green juice blends offered at ret ail are usually high pressure treated or thermally treated. For this work, three high pressure processed (HPP) juices and one thermally processed juice were comprehensively evaluated. The HPP juices included Suja ber Greens TM (Suja Life, LLC, San Diego, C A), Naked Pressed TM Bright Greens TM (Naked Juice Co., Monrovia, CA), and Evolution Fresh TM Essential Greens with Lime (Evolution Fresh, Inc., Rancho Cucamonga, CA). The thermally processed juice was Naked Kale Blazer (Naked Juice Co., Monrovia, CA). The label information of these commercial juices is summarized in Table 3 1. All products were evaluated for enzyme activities of polyphenol oxi dase (PPO) and peroxidase (POD) as well as the levels of chlorophyll a chlorophyll b total carotenoids, total anti oxidant capacity, total coliforms, generic E scherichia coli and total aerobic plate counts (APC). The pH and Brix were also measured. For this purpose, three bottles of each juice brand were pooled to complete one liter of juice. Bottles of juice s were purchased in a local supermarket and kept unopened in refrigeration prior to analysis. The shelf life of the products was unknown, however the remaining shelf life, i.e. days to the enjoy by or best by date of the commercial juices ranged from 12 to 43 da ys when the assays were performed
31 Sample Preparation of Model Juice The second section of this study examined a formulated, representative green blended fresh juice and two thermal treatments of that same juice. This study mainly focused on the quality at tributes of vegetable juices; thus, fruit juices (with the exception of lemon juice) were excluded from the juice formulation. A representative green juice blend was formulated and prepared according to the ingredients stated in a commercial juice availabl e at the retail market. Evolution Fresh TM Green Devotion is a green juice blend that fulfilled the requirements of readily available ingredients that were observed as ingredients in other commercial green juice blend s Three different formulas were prepare d and tasted in comparison to the commercial alternative as a reference. The final formula included 55% ( v/v ) celery juice 20% ( v/v ) cucumber juice 8% ( v/v ) spinach juice 5% ( v/v ) kale juice 5% ( v/v ) Romaine lettuce juice 4% ( v/v ) lemon juice and 3% ( v/v ) flat leaf parsley juice The model product was a medium acid juice with an average pH value of 4.3 and 4.0 Brix Ingredients, fresh and unpacked produce, were purchased in a local supermarket before 24 h to complete experiments and kept in refriger ation prior to use Each ingredient was individually cl eaned under running tap water and t he excess water was drained off. A masticating juice maker ( Omega J8006 Omega Products, Inc., Harrisburg, PA) individually extract ed juices from minor to major ingre dients. Then juices were immediately cooled in ice water bath to preve nt variations of enzyme activity. A freshly batch of one liter was prepared pooling each juice according to the formula and the pH and Brix were measured. The juice maker was cleaned f lowing a standard cleaning procedure detailed in Appendix A Finally the final blend wa s subdivided in three batches: untreated juice (Control), Treatment 1, and Treatment 2
32 Thermal Processing Treatments Pasteurization treatments for juices are often specific for each juice product and process, especially in validation studies associated with a 5 log reduction of the pertinent microorganism (Peng and others 2017) In this document, l iterature review s were focused on thermal processing treatments of acidic green juice blend s. Selection of the thermal processing treatments were based on typical commercial condi tions for juice pasteurization Treatment 1 represented a mild thermal treatment (representative of a label terminology of lightly pasteurized or gourmet pasteurized) at 72 C for 15 s This standard treatment could accomplish a 5 log reduction of E. coli O157:H7, Listeria monocytogenes and Salmonella in acidic juices (Mazzotta 2001) Treatment 2 r epresented a typical thermal treatment of many juices at 90 C for 30 s. These parameters are commonly used in industry for enzyme inactivation and spoilage microorganism control (Reyes De Corcuera and other s 2014) The t hermal process es were performed using a water bath at three degrees higher than the processing temperatures, i.e. 75 and 93 1 C to minimize come up times. Eight 15 m l glass tubes were filled with 10 ml of juice closed with plastic cap s and submerged into a water bath ( Precision GP 05, Thermo Fisher Scientific, Waltham, MA ). The water level completely exceeded the level of the juice aliquots. An additional glass tube containing the same volume of juice was used to record the temperatur e. After reaching the processing temperatures, tubes were kept submerged into the water bath until the holding time for each treatment was reached. Then, tubes were removed from the water bath and immediately cooled until 35 to 45 C in an ice water bath i n order to avoid cooling lags
33 Enzyme Activi ty of Polyphenol Oxidase and Peroxidase Enzyme extractions and assay of the enzyme activities were completed as described by Terefe and others (2010) with some modifications. The activi ti es of PPO and POD were analyzed in triplicate for all samples To extract the enzymes, 4.5 ml of juice were mixed with 4.5 ml of extraction solution and agitated using a vortex mixer for 30 s The extraction solution in 0.2 M sodium phosphate buffer (pH 6.5) included 4% (w/v) poly(vinylpolypyrrolidone) (PVPP), 1% (v/v) 4 (1,1,3,3 Tetramethylbutyl) phenyl polyethylene glycol (Triton TM X 100), and 1 M NaCl. M ixtures were centrifuged for 30 min at 4 500 rpm (5250 x g) and 4 C ( Allegra X 15R, Beckman Coulter, Inc., Brea, CA), with t he supernatants comprising the enzyme extracts. For measurement of the P PO activity, 200 L of enzyme extract were mixed with 3 ml of 0.07 M catechol. Catechol solution was prepared before assays in 0.05 M sodium phosphate buffer (pH 6.5). For blank readings, 0.1 M sodium phosphate buffer (pH 6.5) solution were used instead of enzyme extract at the same volume. Enzyme reactions were measured in kinetic/time mode at 420 nm and ambient temperature for 5 min using an UV visible spectrophotometer (DU 730, Beckman Coulter, Inc., Brea, CA). One PPO activity unit (U) was expressed as an increase of 0.1 unit in absorbance per min. Results were expressed as U/ml of juice. For measurement of the POD activity, enzyme extracts were mixed with 1.5 ml of 0.05 M sodium phosphate buffer (pH 6.5), 0.2 ml of p phenylenediamine diluted at 1% in 0.05 M sodium phosphate buffer (pH 6.5), and 0.2 ml of 1.5% hydrogen peroxide. It was used different amounts of enzyme extract to obtain accurate absorbance readings. It was used 7.5, 75 and 200 L of enzyme extract for the assays of Cont rol, Treatment 1, and Treatment 2, respectively. For the high pressure processed commercial juices, it
34 was used from 5 to 10 L of enzyme extract. For the thermal ly pasteurized commercial juice, it was used 200 L of enzyme extract. Enzyme reactions were m easured in kinetic/time mode at 485 nm and ambient temperature for 10 min using an UV visible spectrophotometer (DU 730, Beckman Coulter, Inc., Brea, CA). One POD activity unit (U) was expressed as an increase of 0.1 unit in absorbance per min. Results wer e expressed as U/ml of juice Measurement of Chlorophylls and Total Carotenoids Extraction procedures were completed as described by Lee and Castle (2001) with some modifications. Analysis was completed in triplicated for each treatment. A stock extraction solution was prepared containing 50% (v/v) hexane, 25% (v/v) acetone, and 25% (v/v) ethanol. An aliquot of 2.5 ml of juice was mixed with 5 ml of extraction solution and agitated using a vortex mixer for 30 s. Then, mixtures were centrifuged at 4500 rpm (3260 x G) for 5 min (Sorval ST 8, Thermo Fisher Scientific, Waltham, MA) One ml of the supernatants was collected and diluted with acetone until absorbance readings were accurate Absorbance was measure at 662, 645 and 470 nm using an UV visible spectrophotometer (DU 730, Beckman Coulter, Inc., Brea, CA). Levels of chlorophyll a c hlor o phyll b and total carotenoids were estimated using mathematical models established by Lichtenthaler (1987) Results were expressed as g per ml of juice (g/ml). Model are described below: Chlorophyll a (Ch a ) = 11.24 A 662 2.04 A 645 (3 1) Chlorophyll b (Ch b ) = 20.13 A 645 4.19 A 662 (3 2) Total Carotenoids = (1000 A 470 1.90 Ch a 63.14 Ch b )/214 (3 3)
35 Measurement of the Total Antioxidant Capacity Total antioxidant capacity of juice treatments were analyzed by DPPH ( 2,2 diphenyl 1 picrylhydrazyl ) assay as described by Martnez Flores and others (2015) with some modi fications. Analysis was completed in triplicated for each treatment. One milliliter of juice was mixed with 5 ml of methanol. Mixtures were agitated using a vortex mixer for 30 s and kept in darkness for 90 min at ambient temperature. Then, mixtures were centrifuged at 4500 rpm (3260 x g) for 10 min (Sorval ST 8, Thermo Fisher Scientific, Waltham, MA). The supernatants were used to quantify the antioxidant capacity of samples. A stock solution of 1 mM DPPH was prepared with methanol and kept at room temperature and darkness for no longer than one month The working solution of 0. 2 mM DPPH was prepared only before assays. In addition, a standard curve assay was needed for the assay. Thus, 6 hydroxy 2,5,7,8 tetra methylchroman 2 carboxylic acid (Trolox) solutions from 0.05 mM to 0.80 mM in methanol were prepared prior assays. Two hundred microliters of each sample extract, Trolox solutions, and methanol (acting as a blank) were mixed with 3.8 ml of 0.2 mM DPPH solution. All mixtures were kept at room temp erature in darkness for 45 min when the reaction reached a constant level. The decrease in absorbance was measured at 515 nm using an UV visible spectropho tometer (DU 730, Beckman Coulter, Inc., Brea, CA). Results were expressed as moles of Trolox equival ent (TE) per ml of juice (moles of TE/ml). Microbiological A nalysis Ten milliliters were sampled by triplicate from each treatment and commercial juice. Samples were kept in refrigeration prior to analysis. One milliliter of each sample was diluted with 9 ml of sterile buffered peptone water (Oxoid, Thermo Fisher Scientific,
36 Waltham, MA). Following serial dilutions were prepared with 1 to 9 ml of sterile buffered peptone water. To determine total mesophilic aerobic plate counts (APC), one hundred microlite rs of each dilution were spread plated in duplicate onto standard methods agar (Remel, Thermo Fisher Scientific, Waltham, MA ). All of the dishes were incubated for 48 48 h at 35 C. To determine total coliforms, one ml of each dilution was plated by duplic ate onto center o f bottom 3M Petrifilm TM E. coli / Coliform Count (EC) ( 3M Company, Maplewood, MN). All petrifilms were incubated for 24 h at 35 C. Results of generic E. coli required additional incubation of 24 hours at 35 C. When sample counts resulted below the assay limit of detection (10 CFU/ml) for total coliforms and APC, the data point was assigned 5 CFU/ml (0.70 log CFU/ml); halfway value between 0 and the detection limit (Ailes and others 2008) Because of the low loads of E. coli that results were reported as log CFU per 3 ml of juice (the total amount of juice analyzed per sample) Statistical Design and Analysis Nine quality attributes were evaluated among the commercial green juice blends at retail market as well as for the representative, formulated treated green juice blend The product quality attributes included enzyme activity of PPO and POD as well as the levels of chlorophyll a chlorophyll b total carotenoids, total antioxidant cap acity, total coliforms, generic E. coli and APC All of the assays were performed in triplicate starting with the enzyme activity assay and ending with the microbiological analysis. The assays were completed in less than 12 h to minimize the storage effec ts in the samples For each quality attribute, mean and standard deviation were calculated and reported.
37 For the second part of the study, it was analyzed an unprocessed juice (Control) and two thermal treatments. Treatment 1 represented a standard mild pa steurization at 72 C for 15 s. Treatment 2 represented a higher thermal treatment at 90 C for 30 s. The experiment was completed in four independent occasions during August 2017. Each occasion was reported as Trial 1, Trial 2, Trial 3, and Trial 4. The s ame treatments were performed in each trial. Results were individually analyzed per trial. To analyze overall results between treatments, mean and standard deviation among the four trials were also calculated and reported. To compare thermal treatments, r esults were normalized to relative values by dividing the results in treated juices over the results in untreated jui ces for each quality attribute. Furthermore, m icrobial reductions, i.e., the initial count minus the final count were estimated to examine significant differences between treatments. Data analysis was performed using RStudio (RStudio, Inc., Boston, MA). The rejection criteria of null hypothesis in favor of the alternative hypothesis was p value less than 0 .05 ( P < 0.05). Analysis of variance was performed to compare treatments for each trial set as well as to compare the overall normalized results between treatments. When differences existed among treatments, m ultiple comparisons were performed by Tu key Kra mer method (Bower 2009) Finally t student test was performed to compare microbial reductions.
38 Table 3 1. Label information of c ommercial green juice blend s found at the r etail market and evaluated in this study Size Processing Treatments Ingredients a Shelf Life Remaining b Suja ber Greens TM 12 fl. oz. (354 ml) High Pressure Processed Cucumber Juice, Celery Juice, Grapefruit Juice, Green Chard Juice, Green Leaf Lettuce Juice, Lemon Juice, Kale Juice, Spinach Juice, Parsley Juice, Tea (Water, Peppermint Tea Leaf, Spearmint Leaf) 31 days Naked Pressed TM Bright Greens TM 12 fl. oz. (354 ml) Cold Pressed c Apple Juice, Cucumber Juice, Celery Juice, Romaine Lettuce Juice, Lemon Juice, Wheatgrass Juice, Lemon Oil 12 days Evolution Fresh TM Essential Greens with Lime 15.2 fl. oz. (450 ml) High Pressure Processed Celery Juice, Cucumber Juice, Spinach Juice, Romaine Lettuce Juice, Kale Juice, Lime Juice, Parsley Juice 16 days Naked Kale Blazer 15.2 fl. oz. (450 ml) Pasteurized d Orange Juice, Kale Puree, Apple Juice, Cucumber Juice, Spinach Juice, Celery Puree, Ginger Juice, Lemon Juice, Natural Flavors 43 days a The bold ingredients are green vegetables or leafy greens. b The remaining days to the enjoy by or best by date labeled on the bottles. c Cold pressed usually refers to a high pressure processing treatment, however that was not stated on the label. does indicate that all juices are pasteurize d d Pasteurized usually refers to a thermal processing treatment.
39 CHAPTER 4 RESULTS AND DISCUSSION Product Quality Parameters in Commercial Green Juice Blend s The results of nine pr oduct quality parameters examined in representative commercial green juice blen ds are summarized in Table 4 1. The examined juices included three high pressure treated (HPP) juices (Suja ber Greens TM Naked Pressed TM Bright Greens TM and Evolution Fresh TM Essential Greens with L ime) and one thermally treated, i.e. pasteurized, juice (Naked Kale Blazer ) The product quality parameters included the enzyme activity of polyphenol oxidase ( PPO ) a nd peroxidase ( POD ) as well as the levels of chlorophyll a chlo rophyll b total carotenoids, total antioxidant capacity, total coliforms, generic E scherichia coli and total mesophilic aerobic plate counts (APC). The pH values and Brix were reported as well. All juices were of their content of d ark vegetables and leafy greens even though some products included fruit j uices as their first ingredient. The vegetables used for green blend juices often included leafy green options, such as kale, spinach, chard, lettuce, wheatgrass, and parsley. Gree n vegetables, such as cucumber and celery, were commonly used as well. The average Brix values for Naked Kale Blazer and Naked Pressed TM Bright were higher (9.4 0.0 and 8.0 0.0) than Suja ber Greens TM and Evolution Fresh TM Essential Greens with Lime (4.2 0.0 and 3.8 0.0). Higher values could be related to the apple juices used for those products. Furthermore, all juices were also acidic vegetable juices with pH values from 3.76 0.03 to 4.29 0.02. T he use of citrus ( mainly lemon) juices likely contrib uted acidifying the blends.
40 T he enzyme acitivity of PPO and POD was generally higher in the HPP juices compared to the thermal treated juice. Findings suggested that HPP might be less effective to reduce the enzyme activi ty of POD compared to the thermal ly treated juice. The commercial HPP juices had POD activ ities ranged from 139.61 9.89 to 63.28 0.39 U/ml; conversely, the POD was inactive (0.05 0.01 U/ml) in the thermal treated juice Naked Kale Blazer The enzyme activity of PPO ranged from 7.91 to 9.89 U/ml among the commercial juices. The PPO activity was quite similar among the commercial juices despite the different formulations, but the thermally treated juice had lower PPO activity in general. In general, PP O and POD are resistant to HPP treatments. Seyderhelm and others (1996) concluded that POD and PPO had the highest barostability in Tris buffer at 600 and 700 MPa compared to other enzymes ( catalase, phosphatase, lipase, pectin esterase, lactoperoxidase and lipoxygenase ) Furthermore, Hernndez and Cano (1998) found that HPP treatments in tomato puree from 50 to 500 MPa did not show any significant effect in PPO activity and a small reduction in POD activity Likewise, findings by Park and others (2002) showed that PPO activity inactivation in pressure treated carrot juices was around 2 0 % and 4 0% after HPP treatments for 10 min at 2 00 and 400 MPa, respectively Keenan and others ( 2012) examined HPP treatments for a fruit beverage that mainly contained strawberry and apple. T reatments at 450 MPa for 5 min achieved small reduction (35%) of PPO activity, but severe treatments at 600 MPa for 10 min did achieved 83% reduction of PPO activity The levels of chlorophylls ( a and b ) among juices appeared formula dependent. For instance, t he average level of chlorophyll a in the therma l l y treated Naked Kale
41 Blazer was 18.55 9.89 g/ml but t he average levels of chlorophyll a in the high pressure processed Suja ber Greens TM and Evolution Fresh TM Essential Greens were 7.57 0.70 and 35.22 0.77, respectively. There was not a trend among the average of chlorophyll a in the juices evaluated. Nevertheless, t he levels of chlorop hyll a were higher than the levels of chlorophyll b in all of the commercial juices examined The proce ssing effect in the chlorophyll levels might be marginal among the pressure treated commercial juices. For example Zhao and others (2013) found non significant differences in the levels of chlorophyll a and b among pressure treated cucumber drinks ( 400 MPa for 4 min and 500 MPa for 2 min ) and control Similarly, Wang and others (2012) observed non significant differences in the levels of chlorophyll a and b in spinach puree after HPP treatments at different pressures (200, 400, and 600 MPa) and processing time (5, 1 5, and 25 min). Van Loey and others (1998) described a high stability of chlorophyll in broccoli juice after severe HPP treatments at 800 MPa with temperatures not exceeding 50 C. Conversely, differences existed amon g HPP and thermal treatments. Butz and others (2002) compared the pressure effect at 600 MPa in the chlorophylls levels of minced broccoli at 25 and 75 C during 10, 20 and 40 min. Significant reductions were observed for those treatments at 75 C. Contrary to the processing effects in chlorophylls the levels of total carotenoids and total antioxidant capacity might be superior in thermally treated juice s compared to pressure treated juices. Fo r instance, in the thermally treated Naked Kale Blazer the average level of total carotenoids and total antioxidant capacity were 7.35 0.16 g/ml and 1.80 0.10 mol of TE/ml respectively Conversely, among the HPP juices, the average total caroteno ids ranged 2.30 0.20 to 5.47 0.16 g/ml and the total
42 antioxidant capacity ranged 0.32 0.07 to 0.40 0.09 mol of TE/ml. The formula effect, however, should not be ignored. T hermal treatments might enhance the bioavailability of antioxidant molecule s such as carotenoids and phenolic compounds For example Chen and others (2015) found that the t otal antioxidant activity in asparagus juice was significant higher in the thermal ly treated samples (121 C for 3 min) compared to the control and the pressure treated samples at different pressures (200, 400, and 600 MPa) and treatment times ( 10 and 20 min) Conversely, additional findings suggested non significant differences among treatments. For a vegetable beverage that included celery and cucumber, Barba an d others (2010) found non significant differences in the total antiox idant capacity among the thermally treated samples ( 90 C for 15 s and 98 C for 21 s ) and the pressured treated samples at 400 MPa for 5 to 9 min S ignificant reductions however, were found for treatments from 100 to 300 MPa for 3 to 9 min. Finally, Andr s and others (2016) supported the previous findings after examining HPP treatments for a carrot beverage that contained some fruit juices. Their work showed non significant differences among the thermally treated samples (80 C for 3 min) and the pressure treated samples at different pressures (450 and 600 MPa) for 3 min C arotenoids molecules usually have high stability with respect to processing technologies Bu tz and others (2002) observed non significant differences in the levels carotene in tomato homogenates after pressure treatments at 600 MPa for 60 min as well as after thermal treatments at 95 C for 60 min. Furthermore, Barba and others (2010) also found non sig nificant differences in the total carotenoids for a thermally treated vegetable beverages (90 C for 15 s and 98 C for 21 s),
43 however significant reductions were observed among the pressure treated beverages (from 100 to 400 MPa for 2 to 9 min). F indings by Andrs and others (2016) also supported the insignificant effects by thermal treatments (80 C for 3 min) in a carrot beverage that contained some fruit juices but their work found higher antioxidant capacity after HPP treatments for 3 min at 450 MPa and 600 MPa. Finally, microbiological quality parameters showed that, a s was expected, the average levels of generic E. coli were lower than the detection limit in all of the commercial juices examined. The average levels of t otal coliforms also were lower than the detection limit except in the pressure treated Suja ber Greens TM ( 2.56 0.07 log CFU/ml ) Lastly the average levels of APC among the high pressure pro cessed juice range d from 2.93 to 4.20 log CFU/ml, but it was lower than the detection limit in the thermal ly treated juice. Findings by Zhao and others (2013) found that, after HPP treatment at 500 MPa for 2 min, the initial levels of APC in a cucumber drink was around 3.0 log CFU/ml and it increased less than 1 log after 50 days at 4 C ; however, HPP treatment at 400 MPa for 4 min and thermal treatment at 85 C for 15 s reached APC levels around 3.5 log CFU/ml and then increased around 1.5 log after storage at 4 C for 50 days. Nevertheless, all of the examined commercial juices had different shelf life periods as well as the elaborati on dates and processing parameters were unknown. Enzyme Activity of PPO and POD in Untreated and Thermal ly Treated Formulated Green Juice Blends The overall enzyme activities of PPO and POD in untreated and thermal ly treated green juice blends are summariz ed in Table 4 2. This model, formulated green juice blend (pH 4.27 0.03 and Brix 3.8 0.3) was prepared and then thermally treated at 72 C for 15 s (Treatment 1), and at 90 C for 30 s (Treatment 2). As was expected, the
44 activities of PPO and POD were higher in the untreated juices compared to the thermally treated juices. The average enzyme activity of PPO in untreated green juice blends was 12.17 0.30 U/ml among four independent trials. After the milder and the higher thermal treatments, the PPO ac tivity significantly decreased to 8.35 0.28 and 7.48 0.33 U/ml, respectively. Significant differences were found among treatments and the control ( P < 0.05). Furthermore, t he average enzyme activity of POD in untreated green juice blends was 38.33 7.05 U/ml among four independent trials. POD activity ranged from 28.37 to 43.77 U/ml (summarized in Appendix B). After the milder and the higher thermal treatments, the POD activity significantly decreased to 8.59 1.60 and 1.79 0.73 U/ml, respectively Significant differences were found between the treatments and the control ( P < 0.05), but not between the thermal treatments ( P > 0.05). The enzyme activity of POD had higher variation in untreated green juice blends. Differences in the enzyme activity o f vegetables could be related to maturity stage or growing conditions, such as weather and farming procedures (Vmos Vigyz and Haard 1981) For instance, Korus (2011) found significant differences in the POD activity among the maturity stages in three different cultivars of kale in two different ye ars POD activity in kale ranged a pproximately of fresh matter of sample; h igher levels generally occurred at the last maturity stage. Likewise, Boo and others (2011) found differences on the POD activities in lettuce at various combinations of day/night temperatures. The POD activity in samples was highest at 25/20 C, but the lowest activity was found for samples at lowe r temperatures ( 1 0/13 C). Nevertheless, the g rowing conditions f or the ingredients used in the green juice blends were unknown.
45 To evaluate the effects between treatments, a more suit able approach is to compare the decrease of enzyme activity on a relative basis, i.e. reporting final enzyme activity over initial enzyme activity Reporting absolute values can make comparisons between data problematic. The remaining fractions of enzyme activity after treatments are summarized in Table 4 3. Comparison s in terms of the relative enzyme activity suggested that PPO was more thermoresistant than POD in green juice blends. The remained factions of PPO activities were 0.69 0.02 and 0.61 0.02 after thermal treatments at 72 C for 15 s and at 90 C for 30 s respectively. Conversely, the remained fractions of POD activities were 0.23 0.04 and 0.05 0.01 after thermal treatments at 72 C for 15 s and 90 C for 30 s, respectively. Significant differences were found among treatments for both enzymes ( P < 0.0 5). These findings were in agreement with those reported by Kaiser and others (2012) PPO was more thermoresistant than POD in blanched parsley paste during treatments from 1 to 10 min at 80 C. Non significant differences were actually found in PPO activity between the control (unprocessed sample) and the treatment a t 80 C for 1 min. However, when the study treated an un blanched parsley paste at the same temperature and time, i.e. 80 C for 1 min, completely inactivation of PPO and POD were achieved. Likewise, Park and Fricker (1977) found that spinach POD extrac t at pH 6.0 was reduced 90% of its activity after treatments at 80 C for 40 s and at 90 C for were reduced. The same reduction at 80 C was completed in 20 s for the POD extract
46 at pH 4.0. The average pH in the green juice blends was 4.27 0.03. The milder treatment at 72 C for 15 s reduced almost 80% of POD activity. Among the thermal treatments evaluated for the representative green juice blend, it seemed that the hold ing times did not suffice to achieve the PPO inactivation. For instance, Heimdal and others (1994) found that isolated lettuce PPO was stable in treatments to 70 C for 5 min but steadily decreased at 80 and 90 C for 5 min. Likewise, after thermal treatments to isolated enzymes from cucumber, Miller and others (1990) found that the sensitivity of PPO and POD to thermal treatments similarly increased after 70 C for 10 min, completing the enzyme inactivation after thermal treatment s at 90 to 100 C for 10 min Moreover different substrates used during the assays of PPO activity could yield diverse results for thermal ly treated samples. For instance, significant inactivation of POD activity ( more than 50%) was reported at 75 C for 30 min using pyrogallol and 4 methylcatechol in the assays of artichoke PPO but it appeared unaffected when catechol was used in the assays Likewise, the normalized activity of PPO in celery roots after thermal treatment at 80 C was approximately 0.75 for L dopa substrate, but it was less than 0.10 for catechol and pyrogallol substrates (Yagar 2004) Nevertheless, catechol was the best sub strate for parsley PPO after comparing its enzyme kinetic parameters among 4 methylcatechol, pyrogallol, L dopa, dopamine, and catechin Limited evidence exists about the most suitable substrate to evaluate the enzyme activity of PPO and POD in green juice blends.
47 L evel s of Chlorophylls and Total Carotenoids in Untreated and Thermally Treated Formulated Green Juice Blends The levels of chlorophyll a chlorophyll b and total carotenoids were examined in untreated and thermal ly treated green juice blends Overall results are summarized in Table 4 2. This model, formulated green juice blend (pH 4 .27 0.03 and Brix 3.8 0.3) was prepared and then thermal ly treated at 72 C for 15 s (Treatment 1), and at 90 C for 30 s (Treatment 2). Furthermore, reporting absolute values can make comparisons between data problematic. To evaluate the effects between treatments, a more suit able approach is to compare the decrease of the levels of chlorophylls and total carotenoids on a relative base, i.e. final concentrations over initial concentrations The normalized results of the levels of chlorophyll s and total carotenoids after the thermal treatments are summarized in Table 4 3. Chlorophylls The average levels of chlorophyll a and chlorophyll b in un treated green juice blends were 56.04 3.04 and 25.36 1.19 g/ml, respectively After the mil der thermal treatments the average levels of chlorophyll a and chlorophyll b significantly decrease d to 38.17 2.63 and 20.49 1.29 g/ml respectively. Like wise, after the higher thermal treatments, the average levels of chlorophyll a and chlorophyll b significantly decrease d to 32.68 2.63 and 14 .66 1.03 g/ml, respectively. Significant differences were found in the absolute levels of chlorophyll a between the control and the treatments ( P < 0.05), but no n significant differences existed between the treatments ( P > 0.05). Conversely, significant differences were found in the absolute levels of chlorophyll b among the control and the treatments ( P < 0 .05).
48 The average level of chlorophyll a was twofold the level of chlorophyll b in untreated green juice blends The levels of chlorophyll a and b in the control ranged from 52.94 to 59.94 g/ml and from 23.85 to 26.47 g/ml respectively (summarized in Ap pendix B ) T he overall a : b ratio of chlorophylls in higher plants is usually 3 (Schwartz and others 2008) It was likely that s ome levels of chlorophylls were r etained in the pomace after juicing process Comparison in terms of the normalized levels of chlorophylls suggested that chlorophyll a was more thermolabile than chlorophyll b in green juice blend after a mild thermal treatment at 72 C for 15 s The relative levels of chlorophyll a and chlorophyll b after the milder treatment were 0.68 0.05 and 0.81 0.04 respectively. The higher thermal sensitivity of chlorophyll a compared to chlorophyll b has been reported as well in thermal ly treated bro ccoli juices (Van Loey and others 1998; Weemaes and others 1999) thermal ly treat ed parsley paste (Kaiser and others 2012) therma l l y treated cilantro puree (Rudra and others 2008) and thermal ly treated cucumber drink s (Zhao and others 2013) Schwartz and others (2008) explained that higher stability of chlorophyll b is associated with electron withdrawing effect of its formyl group. Nevertheless chlorophyll a and chlorophyll b re sulted affect at the same extent after the high thermal treatment at 90 C for 30 s in green juice blends T he relative levels of chlorophyll a and chlorophyll b after the higher treatment were 0.58 0.03 and 0.58 0.01, respectively. Significant differences were found among the control and the treatments for the relative levels of chlorophyll a and b ( P < 0.05). Kaiser and others (2012) evaluated the levels of chlorophyll a and chlorophyll b in thermal ly treated parsley paste s at 80 to 100 C for 1 to 10 min Although overall results indicated
49 si gnificant reductions of the chlorophyll levels at higher temperatures, some increases in the chlorophyll levels occurred at 5 and 7 min at 80 C compared to treatments at 1 and 10 min. Those variations could be result of cell disruptions and then release o f chlorophyll contents. Furthermore, a fter cooking kale leaves, Korus (2013) found reduction of 23% in the levels of chlorophyll b and 17% in the levels of chlorophyll a Those values were lower than those reported in this study. Reduction in the levels of chlorophyll a and chlorophyll b in green juice blends was 42% for both after the high thermal treatment at 90 C for 30 s. A higher reduction in the chlorophyll levels could be explained by the pH effect ; the average pH value in the representative green juice blend was 4.27 0.03. Acid ic conditions enhance the conversion of chlorophyll to pheophytin (Gunawan and Barringer 2000; Koca and others 2007) For instance, Rudra and others (2008) suggested that chlorophylls were less heat stable in cilantro purees at pH 5.5 than cilantro purees at pH 7.5 and pH 8.5. Finally, Zhao and others (2013) studied the t hermal treatments effects on the chlorophylls levels in a cucumber drink (pH 6.60 and 3.0 Brix) during 50 days at 4 C Although s ome peaks in the levels of both chlorophylls were reported in the middle of that period the levels of chlorophyll s were lower than the initial concentrations after the thermal treatment at 85 C for 15 s. Conversely, Castillejo and others (2016a) found that chlorophylls were unaffected in a cidic vegetable beverages (tomato, red pepper, broccoli, and spices) during 58 days at 5 C. Total Carotenoids T he average level of total carotenoids in untreated green juice blends was 13.24 0.80 g/ml among four ind ependent trials. The total carotenoids in untreated juices
50 ranged from 12.51 to 14.37 g/ml. After the mild thermal treatment at 72 C for 15 s a nd the high thermal treatment at 90 C for 30 s the level s of total carotenoids were 14.04 0.77 and 11.83 0.90 g/ml, respectively. Significant differences in the absolute level s of total carotenoids were found between the thermal treatments ( P < 0.05), but any treatment significantly differed with the control ( P > 0.05). carotene were the most representative in the untreated green juice blend s Heinonen and others (1989) carotene were the highest carotenoids found in spinach, lettuce, celery, parsley, and cucumber. Likewise, Arnold and others (2014) carotene as the main carotenoids for kale and parsley. Comparison s in terms of the relative levels of total carotenoids suggested that carotenoids were unaffected by the milder thermal tre atme nt. The relative level of total carotenoids was 1.06 0 .06. Non significant difference existed between the control and the milder treatment ( P > 0.05). Conversely, the hi gher thermal treatment significantly differed with the control ( P < 0.05). The relative level of total carotenoids was 0.89 0.05 after the higher treatment. In vegetable beverage s that included celery and cucumber Barba and others (2010) found that the levels of total carotenoids resulted unaffected after thermal treatme nts at 90 and 98 C for 15 to 21 min. However, those comparisons were in absolute values. Treated green juice blends also resulted unaffected compared to the control in terms of absolute values. Likewise, Korus (2013) found non significant carotene in kale after cooking procedures. Conversely, Lessin and other s (1997) found that the levels of carotene
51 significantly increased in boiled broccoli, canned collard, and canned spinach. Th e i ncrease s of carotenoids levels were related to increase of the disruptions of carotenoprotein complexes. In more sever e thermal treatments, Arnold and others (2014) found non significant differences carotene between untreated and thermal ly treated kale jars at 121 C for 5 to 20 min But significant carotene were found f or parsley jars at the same treatment s T he levels of l utein were also reduced in both products at the same treatment s Conversely Updike and Schwartz (2003) found that th e levels of lutein increased in canned kale and spinach after thermal treatments. However, those increments in the lutein levels were mainly in the cis configuration levels ; actually, th e levels of trans configuration decreased in both products after the thermal treatments carote ne was also reported by Lessin and others (1997) A significant increase in carotene was found in canned collard. The carotene (trans and cis) increased from 27 3.9 to 409.7 g/g of dry tissue but it was mainly related to an inc rease of the cis configuration. N evertheless, researchers still question whether cis isomers of carotenoids molecules could have the same health benefits as that proposed for their all trans counterparts. Findings could differ among carotenoid structures. For instance, carotene have better absorption than their cis counterparts; conversely cis lycopene shows greater absorption than the ir trans configurations (Priyadarshani 2017) Total Antioxidant Capacity in Untreated and Thermally Treated Formulated Green Juice Blends The overall total antioxidant capacity in untreated and thermal ly treated green juice blends are summarized in Table 4 2. This model, formulated green juice blend (pH
52 4.27 0.03 and Brix 3.8 0.3) was prepared and then therma l l y treated at 72 C for 15 s (Treatment 1), and at 90 C for 30 s (Treatment 2). The average total antioxidant capacity in untreated green juice blends was 0.67 0.07 mol of TE /ml After the milder thermal treatment, the average total antioxidant capacity was 0.82 0.10 mol of TE /ml There were no significant differences between that treatmen t and the control ( P > 0.05). Conversely, the average total antioxidant capacity was significantly increased after the higher treatment ( P < 0.05). The average total antioxidant capacity was 0.85 0.09 mol of TE /ml Non significant differences existed be tween the thermal treatments ( P > 0.05). Nevertheless reporting absolute values can make comparisons between data problematic. To evaluate the effects between treatments, a more suit able approach is to compare to compare the decrease of antioxidant capaci ty on a relative base, i.e. final total antioxidant capacity over their initial levels The normalized total antioxidant capacity after treatments are summarized in Table 4 3. Examining these results, the total antioxidant capacity resulted significantly higher in any thermal treatment compare d to the control ( P < 0.05). Non significant were found between the thermal treatments ( P > 0.05). After the milder and the higher treatments, the relative values of total antioxidant capacity were 1.23 0.05 and 1.2 7 0.03, respectively. In general, antioxidant losses during processing in fruit and vegetables are related to the high thermal sensitivity of v itamin C and in a lesser extent due to carotenoids degradation (Kalt 2005) In thermal ly treated green juice blends, the total antioxidant capacity significantly increased after the highes t thermal treatment. However, total carotenoids decreased after the highest thermal treatment as it was
53 described in the last section. Furthermore, c hlorophyll a and b have also been related to antioxidant activity in vegetables 13) but they were severed affected after thermal treatments in green juice blends Therefore, it was likely that the main role in the antioxidant capacity in the treated green juice blend s was by the effect of phenolic and flavonoid contents. Bunea and others (2008) found that increase in the antioxidant capacity in boiled spinach occurred because simple phenolic compounds released after the breakdown of more complex structures containing them, i.e. cellulose networks. Chu and others (2002) compared the free and bound fractions of phenolic compounds in celery, cucumber, lettuce, and spinach. They all had bound fractions of phenolics between 12.5 to 28.5%. Although thermal treatments see m to enhance the release of phenolic compounds, longer treatments at high temperatures could produce a negative impact. Kaiser and others (2012) found that thermal ly treated parsley pastes at 90 C for 7 to 10 min decreased the phenolic contents and the antioxidant capacity. Results after treatments at 80 C did not d iffer with the untreated pastes. Additionally Sun and others (2007) suggested that flavonoids were among the mainly antioxidants related to the antioxidant capacity of asparagus and broccoli juices. These includ ed rutin, querc et in, and kaempferol sophor os ides. Flavonoid compounds could result exceptionally thermoresistant. Hostetler and others (2012) studied the flavonoid profiles in parsley and Chinese celery juices. Non significant differences existed in thermal ly treated parsle y and celery juices after thermal treatments at 100 to 121 C for up to 30 min. In addition, the pH effect was also evaluated in celery juices. Non significant differences existed at pH 3.0 to 5.0 after thermal processing at 121 C
54 for 15 to 60 min. Nevert heless, the study found that thermal treatments did effect the conversion of apiin to apigenin 7 O glucoside which could improve the absorption. Finally, differences in the assays to evaluate the total antioxidant capacity could yield mixed results. For instance, Barba and others (2010) compared the total antioxidant capacity in a vegetable beverage that included celery and cucumber after thermal treatments at 90 C for 15 to 21 min. Non significant differences existed among the control an d the thermal treatments by ABTS assay; conversely, significant differences existed among them by ORAC assay. Sun and others (2007) suggested the use of more than one method to evaluated the antioxidant activity. N atural Microflora in Untreated and Thermally Treated Formulated Green Juice Blends T he levels of total coliforms, generic E. coli and APC were examined in untreated and thermal ly treated green juice blends This model, formulated green juice blend (pH 4 .27 0.03 and Brix 3.8 0.3) was prepared and then thermal treated at 72 C for 15 s (Treatment 1), and at 90 C for 30 s (Treatment 2). Results are summariz ed in Table 4 4. The average level of total coliform s in untreated green juice blends was 2.32 0.62 log CFU/ml The levels ranged from 1.51 to 2.90 log CFU/ml in four independent trials. After the milder and the higher thermal treatment s the average levels of total coliform s were lower than the detection limit (1.00 log CFU/ml). Significant differ ences were found between the control and the thermal treatments ( P < 0.05), but non significant differences existed between the treatments ( P > 0.05). E. coli resulted unusual but still possible in untreated green juice blends. The level of E. coli was 1.00 log CFU/3 ml in onl y one of the four trials, which 1.00 log CFU /ml was found in one of three samples (1 ml) of juice analyzed Therefore, it
55 resulted more accurate to report the levels of E. coli by total volume of juice analyzed. Moreover, t he mi lder thermal treatment sufficed to reduce that level of E. coli in green juice blends. After the milder and the higher thermal treatments, the levels of E coli were lower than the detection limit (1.00 log CFU/ml). Furthermore, t he average level of APC in untreated green juice blends was 5.44 0.42 log CFU/ml. The levels ranged from 4.91 to 5.85 log CFU/ml in four independent trials. After the milder and the higher thermal treatments, the average levels of APC were 2.79 0.30 and 2.09 0.08 log CFU/ml, respectively. Significant differences were found among the control and the thermal treatments ( P < 0.05). To compare the effects of the thermal treatments on the APC levels, the log reductions are summarized in Table 4 5. Significant differences existed between thermal treatments ( P < 0.05). The milder thermal treatment at 72 C for 15 s achieved a log reduction of 2.65 0.30 log CFU/ml and the higher thermal treatment 90 C for 30 s achieved a log reduction of 3.35 0.37 log CFU/ml. Overall f indings in the untreated green juice blends were in agreement with the microflora found in fresh produce. For instance, Johnston and others (2006) evaluated fresh produce in final boxes for distribution. The levels of total coliforms, E. coli and APC were 2.54, 1.27, and 6.50 log CFU/g respectively Furthermore Valentin Bon and others (2008) found that the total bacteria l count i n bagged cut spinach and lettuce mix es ranged from 4.0 to 8.3 log CFU /g and from 4.5 to 7.9 log CFU/g respectively. Ailes and others (2008) examined the occurrence and levels of APC, total coliforms and E. coli on several leafy greens and vegetables including most of the ingredients used for the green juice blend. The total average of APC was 6.0 0.04 log CFU/g ; the
56 highest loads were found in cantaloupe, cilantro, mustard greens, and cilantro. The total average of total coliforms was 2.2 0.05 log CFU/g; the highest loads were found in arugula, cantaloupe, and parsley. Lastly the total av erage of E. coli was 1.0 0.03 log CFU/g; c antaloupe, cabbage, and cilantro had the highest percentages of occurrence (29 to 25%). Therefore, it was likely that parsley contributed with the most load of APC and total coliforms among the ingredients used f or the green juice blend. In addition, t he lower incidence of E. coli were also reported in several vegetables such as spinach (Ilic and others 2008) and lettuce (Holvoet and others 2014) Among 2,531 fresh produce commodities, Most Probable Number ( MPN) values of 1,000 E. coli cells/g were found in only six samples and MPN values < 10 E. coli cells/g (detection limit) were found in 2,442 samples (USDA 2011) Moreover similar levels of APC were also found in untreated juice and beverages. For instance, Song and others (2007) found the average levels of APC in untreated kale juice was 6. 80 log CFU/ml, but total coliforms were higher than those levels previously reported on fresh produce. The average levels of total coliforms in kale juice was 6.86 log CFU/ml. After storage at 10 C for three days, APC and total coliforms increased to levels around 10 log CFU/ml. Findings in untreated green juice blends were in agreement with those reported by Zhao an d others (2013) In a cucumber drink (3.0 Brix and 6.6 pH), the initial level of APC was 5.28 log CFU/ml Those levels decreased to approximately 3.50 log CFU/ml a fter a thermal treatment at 85 C for 15 s which resulted higher than th ose reported after the milder thermal treatment in green juice blends at 72 C for 15 s. After 50 days of storage at 4 C, the average levels of APC in cucumber drink increased by 1.60 log CFU/ml.
57 Finally, Castillejo and others (2016b) found lower levels of APC in an acidified vegetable drink (4.3 Brix and 4.5 pH) that contained cucumber, spinach, and broccoli. The level of APC was 4.4 log CFU/g in untreated samples. After thermal treatments, the levels of APC reduced to 1.7 to 1.8 log CFU/g. Non sign i ficant differences existed between the thermal treatment s at 80 C for 3 min and 90 C for 45 s Findings were c ontrary to the treatments used for the green juice blend s; h owever, Castillejo and others (2016b) used 75 mg/l NaClO to sanitize their ingredients prior to the drink preparation. Comparison among the Thermally Treated Formulated Green Juice Ble nds and the Commercial A lternatives Comparisons among the thermal ly treated formulated green juice blends and the commercial juice alternatives at retail market sowed that the enzyme activity of POD and PPO in the thermal ly treated juices were lower than the activities found in the commercial high pressure processed juices. T he PPO activity in the thermal ly treate d juices was similar with the activity found in the commercial thermal ly treated juice. Nevertheless, the commercial thermal treatment appeared to be a more sever e treatment than those used in this study (72 C for 15 s and 90 C for 30 s) because the enzy me activity of POD in the commercial thermal ly treate d juice was complet e ly inactive (0.05 0.01 U/ml). The levels of chlorophyll did not show any trend among the juice alternatives i.e. the formulated, representative juice and the commercial juices The levels of chlorophylls therefore appeared formula dependent. Conversely, the total carotenoids among the commercial high pressure processed juices showed similar figures but the levels of total carotenoids were superior in the thermal ly treated juice s. Likewise, the
58 antioxidant capacity was higher in the thermal ly treated juices compared to the commercial high pressure processed alternatives. The commercial thermal ly treated juice showed the highest total antioxidant capacity (1.80 mol of TE /ml ) amon g the juice alternatives. Although that could be explained bec ause of its fruit juice content, some of the HPP juices also included fruit in their ingredients and their antioxidant capacity were lower. T hermal treatment therefore appeared to increase the a ntioxidant capacity in green juice blends. Finally, m icrobiological quality parameters showed that the commercial green juice blends had lower lev els of total coliforms. Similarly, the evaluated thermal treatments for the formulated representative green j uice blend effectively reduce d the initial levels of total coliforms. Conversely, t he levels of APC differed among all juices The commercial thermal treatment appeared to be a more severe treatment than those used in this study T he shelf life differences among the products however, limited the comparison among the m Lastly, generic E. coli was not reported in any juice alternative as was expected Current commercially available juices all are apparently appropriately processed.
59 Table 4 1. Summary of pr oduct quality parameters in some commercial green juice blends found at the retail market and evaluated in this study. Suja a ,e Naked Pressed b ,e Evolution Fresh c ,e Naked d ,e pH 4.26 0.02 3.76 0.03 4.29 0.02 3.91 0.03 Brix 4.2 0.0 8.0 0.0 3.8 0. 0 9.4 0. 0 PPO (U/ml) f 9.89 0.42 9.67 0.07 10.22 0.90 7.91 1.13 POD (U/ml) f 139.61 9.89 34.58 1.47 63.29 0.39 0.05 0.01 Chlorophyll a (g/ml) 7.57 0.70 11.14 0.18 35.22 0.77 18.55 0.23 Chlorophyll b (g/ml) 5.45 0.56 6.00 0.11 18.71 0.43 2.69 0.06 Total Carotenoids (g/ml) 2.30 0.20 2.39 0.04 5.47 0.16 7.35 0.16 T. Antioxidant Capacity (mol of TE/ml) 0.32 0.07 0.40 0.09 0.40 0.03 1.80 0.10 Total Coliforms (log CFU/ml) 2.56 0.07 0.70 0.00 0.70 0.00 0.70 0.00 APC (log CFU/ml) 4.20 0.20 2.93 0.13 3.19 0.20 0.70 0.00 a Suja ber Greens TM ; 31 days to the enjoy by or best by date. b Naked Pressed TM Bright Greens TM ; 12 days to the enjoy by or best by date. c Evolution Fresh TM Essential Greens with Lime; 16 days to the enjoy by or best by date. d Naked Kale Blazer; 43 days to the enjoy by or best by date. e Values given are the means ( standard deviation) for one independent experiment. f
60 Table 4 2. Overall results of the enzyme activity, chlorophylls & total carotenoids levels, and total antioxidant capacity among treatments. PPO (U/ml) d,e,f POD (U/ml) d,e,f Chlorophyll a (g/ml) d,e Chlorophyll b (g/ml) d,e Total Carotenoids (g/ml) d,e Antioxidant Capacity (mol of TE/ml) d,e Control a 12.17 0.30 a 38.33 7.05 a 56.04 3.04 a 25.36 1.19 a 13.24 0.80 a,b 0.67 0.07 b Treatment 1 b 8.35 0.28 b 8.59 1.60 b 38.17 2.63 b 20.49 1.29 b 14.04 0.77 a 0.82 0.10 a,b Treatment 2 c 7.48 0.33 c 1.79 0.73 b 32.68 2.82 b 14.66 1.03 c 11.83 0.90 b 0.85 0.09 a a Control refers to untreated green juice blend s b Treatment 1 was completed at 72 C for 15 s. c Treatment 2 was completed at 90 C for 30 s. d Values given are the means ( standard deviation ) for four independent experiments e Significant differences ( P < 0.05) existed among treatments with different lett ers within the same column. f min
61 Table 4 3. Overall normalized results of the enzyme activity, chlorophylls & total carotenoids levels, and total antioxidant capacity among treatments PPO POD Chlorophyll a Chlorophyll b Total Caroten oids Antioxidant Capacity Control a 1.00 0.00 a 1.00 0.00 a 1.00 0.00 a 1.00 0.00 a 1.00 0.00 a 1.00 0.00 b Treatment 1 b 0.69 0.02 b 0.23 0.04 b 0.68 0.05 b 0.81 0.04 b 1.06 0.06 a 1.23 0.05 a Treatment 2 c 0.61 0.02 c 0.05 0.01 c 0.58 0.03 c 0.58 0.01 c 0.89 0.05 b 1.27 0.03 a a Control refers to untreated green juice blend s b Treatment 1 was completed at 72 C for 15 s. c Treatment 2 was completed at 90 C for 30 s. d Values given are the means ( standard deviation) for four independent experiments. e Significant differences ( P < 0.05) existed among treatments with different letters within the same column.
62 Table 4 4 Summary of natural microflora in untreated green juice blends and thermal ly tr eated green juice blends Treatments Total Coliforms (log CFU/ml) e,f E. coli (log CFU/3 ml) APC (log CFU/ml) e,f Trial 1 a Control b 2.68 0.15 a < 1.00 5.85 0.38 a T reatment 1 c 0.70 0.00 b < 1.00 3.07 0.09 b T reatment 2 d 0.70 0.00 b < 1.00 2.16 0.15 c Trial 2 Control 2.90 0.05 a 1.00 5.70 0.14 a T reatment 1 0.70 0.00 b < 1.00 2.71 0.20 b T reatment 2 0.70 0.00 b < 1.00 2.16 0.15 c Trial 3 Control 1.51 0.29 a < 1.00 5.31 0.28 a T reatment 1 0.70 0.00 b < 1.00 3.00 0.07 b T reatment 2 0.70 0.00 b < 1.00 2.00 0.30 c Trial 4 Control 2.17 0.21 a < 1.00 4.91 0.13 a T reatment 1 0.70 0.00 b < 1.00 2.40 0.17 b T reatment 2 0.70 0.00 b < 1.00 2.06 0.10 b Overall Control 2.32 0.62 a n/a 5.44 0.42 a Treatment 1 0.70 0.00 b n/a 2.79 0.30 b Treatment 2 0.70 0.00 b n/a 2.09 0.08 c a Trials were independent repetitions of the experiment completed during August 2016. b Control refers to untreated green juice blend s c Tre atment 1 was at 72 C for 15 s. d Treatment 2 was at 90 C for 30 s. e Values given are the mean s ( standard deviation ) per trial set f Significant differences ( P < 0.05) existed between treatments with different letters within each trial set and overall results
63 Table 4 5 Overall log reduction s of APC in thermal ly treated green juice blends Log Reduction of APC (log CFU/ml) c,d T reatment 1 a 2.65 0.30 b T reatment 2 b 3.35 0.37 a a Treatment 1 was completed at 72 C for 15 s b Treatment 2 was completed at 90 C for 30 s c Values given are the means ( standard deviation) for four independent experiments d Significant differences ( P < 0.05) existed between treatments wi th different letters within the same column
64 C HAPTER 5 CONC LUSIONS AND SUGGESTIONS FOR FUTURE WORK In general, results showed that the traditional thermal pasteurization used by the juice industry effectively reduced the enzyme activity of peroxidase ( POD ) in green juice blends, and to a lesser extent the activity of polyphenol oxidase ( PPO ) J uice processors could consider t he inactivation of PPO as suitable criteria for thermal processing efficacy for green juice blends C onsumers who are seeking minimally processed food may decide that thermal paste urization is not desirable due to this enzyme inactivation. Chlorophyll levels in green juice blends were negatively affected by thermal treatments. High pressure processing, however, seems to these effects ; thus HPP (or cold pressed) seems more attractiv e to consumers bec ause of the perceived benefits. Conversely juice processor could highlight that thermally treated green juice blends p rovide higher antioxidants and similar levels of carotenoids compared to fresh unprocessed juices. Health benefits fro m carotenoids are extensively well described in the literature. Gourmet pasteurization might be a good alternative for juice processor of green juice blends for some consumers Furthermore, c onsidering that thermal ly treated juices are low priced than the high pressure processed juices (due to presumably lower processing costs) thermal treatment could represent good alternative for consumer whose main concern is to obtain the health benefits related to vegetables at an intermediate price More studies, how ever, are needed to validate thermal treatment s in compliance with the J uice HACCP Regulation Shelf life studies would also provide more information to establish the suitable pasteurization treatment s that minimize the
65 processing effects without neglectin g product food safety and quality. In these types of studies Escherichia coli O157:H7 would reasonably be considered the pertinent microorganism for thermal processing designs of green juice blends. Potential contamination of E. coli in unpasteurized gree n juice blends might cause consumers (specially the vulnerable groups) who perceiv e unpasteurized juices healthier than pasteurized juices to reconsider their position Additional work could also include a comprehensive juice processing study examining th ermal and non thermal alternatives, for the same representative formula which would provide accurate results to conclude benefits and drawbacks from each technology for the green juice blend processing. Finally, f uture work could also be performed on the s ensory aspects of green juice blends such as color degradation. Chlorophyll is readily associated with the green color of the juice blends; however it can degrade by processing effects, the product pH or en vironmental conditions, e.g., light exposure Understanding the effect of color on juice blend acceptability is an interesting area of further study. From a flavor perspective juice processors might consider the use of fruit juices to enhance flavor and sweetness. Different formulas could be evaluated among potential consumers although health conscious consumers may be less likely to buy these blends due to the sugar content from fruits. In summary, there are several areas of inquiry that are possible for green juice blends that help more fully unders tand the risks, and potential benefits (both real and perceived) to the health conscious consumer.
66 APPENDIX A SSOP OF THE JUIC ING MACHINE A sanitation standard operating procedure to clean was elaborated for the masticating juice machine after its use. The procedure was developed according to manufacture recommendation. After concluding each trial, the procedure was followed as it is described below: 1. Unplug the equipment. 2. Remove the drum set from the housing part and place it in the sink 3. Take the removal parts off the drum set: the funnel, the auger, the juicing screen, and the end cap. 4. Remove chunk residues from the juicer parts under running tap water. 5. Soak items in hot tap water. 6. Rub each juicer part including sieve and collection bowls with scrubber sponges and dishwashing liquid soap. 7. Brush the juicing screen using a cleaning brush and dishwashing liquid soap. 8. Rinse items in running hot tap water until the suds are gone. 9. Place items upside down. Allow all surface to air dry. 10. Put t he unit back together. A validation of the cleaning procedure was completed during the preliminaries studies. The juicing screen, the most demanding part of the juice maker for cleaning was swabbed using a wet swab with sterile peptone water over 10 cm 2 APC was from 2 to 4 CFU/10 cm 2
67 APPENDIX B SUMMARY OF QUALITY PARAMETERS OF FORMULATED GREEN JUICE BLENDS AFTER FOUR INDEPENDENT TRIALS Summary of the Enzyme A ctivity (PPO and POD) in Untreated and Thermally T reated Formulated Green Juice B lends Trials a Treatments PPO ( U / ml ) e f ,g POD ( U / ml ) e f ,g 1 Control b 11.96 1.09 a 28.37 1.15 a T reatment 1 c 8.20 0.50 b 6.85 0.30 b T reatment 2 d 7.22 0.63 b 1.00 0.09 c 2 Control 12.44 1.66 a 38.32 2.39 a T reatment 1 8.22 0.76 b 10.68 1.05 b T reatment 2 7.31 0.40 b 1.38 0.18 c 3 Control 12.40 0.46 a 43.77 1.01 a T reatment 1 8.77 0.99 b 8.07 0.50 b T reatment 2 7.96 0.51 b 2.60 0.09 c 4 Control 11.87 1.04 a 42.85 0.50 a T reatment 1 8.22 0.80 b 8.75 0.18 b T reatment 2 7.42 0.78 b 2.17 0.03 c a Trials were independent repetitions of the experiment completed during August 2016. b Control refers to untreated green juice blends. c Treatment 1 was at 72 C for 15 s. d Treatment 2 was at 90 C for 30 s. e Values given are the means ( standard deviation) per trial set. f Significant differences ( P < 0.05) existed between treatments with different letters within each trial set. g
68 Summary o f the Levels of Chlorophylls and Total Carotenoids in Untreated and Thermally Treated Formulated Green Juice Blends Trials a Treatments Chlorophyll a (g/ml) e ,f Chlorophyll b (g/ml) e ,f Total Carotenoids (g/ml) e ,f 1 Control b 56.79 1.33 a 26.13 0.38 a 13.18 0.48 a,b T reatment 1 c 37.98 1.75 b 20.94 1.10 b 13.59 0.76 a T reatment 2 d 32.46 1.27 c 15.21 0.39 c 11.93 0.13 b 2 Control 52.94 2.53 a 23.85 0.89 a 12.51 0.63 a T reatment 1 34.64 0.54 b 18.70 0.41 b 13.18 0.17 a T reatment 2 28.96 1.70 c 13.26 0.63 c 10.52 0.43 b 3 Control 59.94 0.73 a 26.47 0.15 a 14.37 0.18 a T reatment 1 39.25 1.37 b 20.58 0.42 b 14.66 0.66 a T reatment 2 35.71 1.62 c 14.58 0.60 c 12.43 0.44 b 4 Control 54.51 0.37 a 25.01 0.21 a 12.89 0.64 b T reatment 1 40.82 0.29 b 21.74 0.11 b 14.71 0.33 a T reatment 2 33.59 0.98 c 15.60 0.17 c 12.43 0.47 b a Trials were independent repetitions of the experiment completed during August 2016. b Control refers to untreated green juice blends. c Treatment 1 was at 72 C for 15 s. d Treatment 2 was at 90 C for 30 s. e Values represent mean standard deviation per trial set f Significant differences ( P < 0.05) existed between treatments with di fferent letters within each trial set.
69 Summary of the Total Antioxidant C apacity in in Untreated and Thermally Treated Formulated Green Juice Blends Trials a Treatments Total Antioxidant Capacity (mol of TE/ml) e ,f 1 Control b 0.58 0.03 b T reatment 1 c 0.74 0.03 a T reatment 2 d 0.74 0.03 a 2 Control 0.68 0.09 a T reatment 1 0.72 0.06 a T reatment 2 0.90 0.12 a 3 Control 0.74 0.03 b T reatment 1 0.94 0.07 a T reatment 2 0.94 0.07 a 4 Control 0.66 0.06 b T reatment 1 0.86 0.03 a T reatment 2 0.80 0.03 a a Trials were independent repetitions of the experiment completed during August 2016. b Control refers to untreated green juice blends. c Treatment 1 was at 72 C for 15 s. d Treatment 2 was at 90 C for 30 s. e Values represent mean standard deviation per trial set f Signi ficant differences (P < 0.05) existed between treatments with different letters within each trial set.
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83 BIOGRAPHICAL SKETCH Galo W. Chuchuca Morn was born in Machala, Ecuador. Galo earned the degree of food e ngineering from the Escuela Superior Politnica del Litoral (ESPOL) in February 2013. He worked in the foo d industry for a few years and then decided to return to schoo l In August 2015, he moved to Gainesville, FL to work towards his degree of Master of Science in food s cience and human nutrition from the University of Florida. He graduated in the winter of 2017 and plans to pursue a career in academia in May 2018.