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EXTRACTION OPTIMIZATION AND IN-MATRIX STABILITY TESTING OF

MEMBRANE-CONCENTRATED COLORANT FROM PRICKLY

PEAR ( INDICA)

A Thesis

Presented to the

Faculty of

California State Polytechnic University, Pomona

In Partial Fulfillment

Of the Requirements for the Degree

Master of Science

In

Human & Science

By

Shannen N. Hilse

2018

SIGNATURE PAGE

THESIS: EXTRACTION OPTIMIZATION AND IN-MATRIX STABILITY TESTING OF MEMBRANE-CONCENTRATED BETALAIN COLORANT FROM PRICKLY (OPUNTIA FICUS INDICA)

AUTHOR: Shannen N. Hilse

DATE SUBMITTED: Summer 2018

College of Agriculture

Dr. Harmit Singh Thesis Committee Chair & Food Science

Dr. Jeremy T. Claisse Biological Sciences

Stephen J. Lauro CEO; colorMaker, Inc. Anaheim, CA

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ACKNOWLEDGEMENTS

First and foremost, I cannot express my gratitude and appreciation for my thesis advisor, Dr. Harmit Singh, Professor & Chair of the Human Nutrition & Food Science

Department. Beyond his command of food chemistry and creativity in its applications, his support, compassion, and overall optimism during this project kept me going and I cannot thank him enough.

To my committee members: Dr. Jeremy Claisse, who offered his statistical expertise in both the experimental design and data analysis of this project and Stephen

Lauro, whose industrial input, support, and encouragement was pivotal to its completion.

Thank you both for your willingness to be a member of my committee and for the time you have dedicated.

Additionally, I would like to give thanks to Dr. Gabriel Davidov-Pardo, Associate

Professor of the HNFS Department. Beyond his insight, enthusiasm, and genuine interest in this project, he served as a mentor for me during my time at Cal Poly Pomona and had a big hand in making the past two years such an invaluable experience.

I would be remiss to not acknowledge those within the food science family that offered support in their own distinct ways. To my labmates Shirin Mal Ganji and Carol

Pow Sang, and to my fellow graduate students Benjamin Steiner, Yuguang Zheng, Huiying

Hu, and Franz Fernandez: your advice was always welcome, your experience was always helpful, and your friendship made the long days in lab just a little more entertaining. Also to my undergraduate researcher, Sarah Caballero, who offered a unique perspective, helped with data collection, and never once complained when asked to wash glassware.

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To my parents, Steve and Debra, who made me the person I am today and whose love and support never faltered. To my sister Taryn, who has offered wisdom and advice not just during this project, but throughout my entire adult life. To John, my beginning, and

Ellie-Mae, my rock. To Christian, who has helped me in so many ways. And to my entire network of friends and family: your years of belief in me and my education got me here, and I am so grateful for all of you.

And finally, to all the students, staff, and faculty at California State Polytechnic

University, Pomona and those within the Human Nutrition and Food Science department

– thank you for the most difficult, stressful, challenging, memorable, and rewarding years of my life.

Research reported in this publication was supported by the MENTORES

(Mentoring, Educating, Networking, and Thematic Opportunities for Research in

Engineering and Science) project, funded by a Title V grant, Promoting Post-baccalaureate

Opportunities for Hispanic Americans (PPOHA) | U.S. Department of Education,

Washington, D.C. PR/Award Number: P031M140025. The content is solely the responsibility of the authors and does not necessarily represent the official views of the

Department of Education.

We would also like to thank Stephen Lauro at ColorMaker (Anaheim, CA) for funding and supporting this research.

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ABSTRACT

Although they offer more stability and lower production costs, consumers perceive synthetic colorants as undesirable. Accordingly, companies are looking to natural color sources for their products. Prickly are that are rich in pigment molecules known as . These water-soluble colorants are traditionally extracted from red table beets, but beet extract comes with complications – it imparts an undesirable taste and is high in nitrites, precursors to carcinogenic nitrosamines. In addition, beets (as opposed to red table beets) are genetically modified to improve their yield of sugar. This further complicates the product development challenges facing companies looking to avoid artificial colors. Consequently, a commercial betalain source other than red table beets is desirable to industry.

The purpose of this research was to increase the feasibility of a marketable betalain food colorant sourced from Opuntia ficus indica. This was achieved through three main objectives: (1) increased colorant yield with use of pectinase, cellulase, and hemicellulase enzymes to break down cell wall structures of the solids, (2) the purification and concentration of the colorant with athermal crossflow membrane filtration, and (3) testing the resulting betalain colorant against comparable natural and artificial colorants on the market when used in a common food matrix.

Interestingly, enzymatic treatments led to a decrease in betacyanin content the more crude enzyme was added. This could be explained by potential glucosidase activity of hydrolytic enzymes, leading to betalain degradation. Clarification with centrifugation and microfiltration (MF) caused transmittance to increase from the initial 4.1% to 14.33% post- centrifugation and an average 70.51% post-MF. Nanofiltration led to a 234.36% increase

v in betacyanins. When used to color a common food matrix ( dessert), both prickly pear and red beet saw rapid degradation at room temperature but showed significant stability under refrigerated conditions. This project resulted in a membrane-concentrated, natural betalain colorant sourced from prickly pear that can be used in refrigerated food items.

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TABLE OF CONTENTS

Signature Page ...... ii Acknowledgements ...... iii Abstract ...... v List of Tables ...... ix List of Figures ...... xi Chapter 1: Introduction ...... 1 Relevance of the Topic ...... 4 Specific Scope ...... 9 Chapter 2: Literature Review ...... 11 Natural Colorants, Betalains ...... 11 Phenolic Compounds ...... 12 Red & Alternatives ...... 13 Enzyme Use for Pigment & Phenolic Extraction ...... 15 Membrane Filtration ...... 18 Monitoring Physico-Chemical Properties of Prickly Pear ...... 25 Stability & Model Food Matrices ...... 28 Chapter 3: Materials & Methods ...... 31 Objectives and Hypotheses ...... 31 Flow Diagram ...... 32 Sample Preparation and Chemical Reagents ...... 32 Methodology ...... 33 Physico-Chemical Parameters ...... 33 Enzymatic Pretreatments ...... 36 Membrane Purification and Concentration via Micro- and Nano-filtration ...... 39 Stability of Prickly Pear Concentrate in a Model Food Matrix ...... 42 Chapter 4: Results & Discussion ...... 45 Enzymatic Pretreatment ...... 45 Varying Concentrations of Crude Enzymes...... 45

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Active Enzyme, Deactivated Enzyme, and a Blank Solution ...... 49 Membrane Purification and Concentration via Micro- and Nano-filtration ...... 55 Stability of Prickly Pear Concentrate in a Model Food Matrix ...... 63 L*, a*, b* Values ...... 63 Chroma and Hue Angle ...... 70 Chapter 5: Conclusions ...... 75 Objective 1 ...... 75 Enzyme Use to Increase Color Yield ...... 75 Membrane Purification and Concentration ...... 75 Objective 2 ...... 76 Maintaining Beneficial Properties of Prickly Pear ...... 76 Objective 3 ...... 76 Stability of Prickly Pear Concentrate in a Model Food Matrix ...... 76 Chapter 6: Project Limitations and Future Studies ...... 77 Project Limitations ...... 77 Samples ...... 77 Enzyme Treatments to Increase Yield ...... 77 Membrane Purification & Concentration ...... 78 Stability Testing in a Model Food Matrix ...... 78 Future Studies ...... 79 Enzyme Treatments to Increase Yield ...... 79 Membrane Purification and Concentration ...... 80 Stability Testing in a Model Food Matrix ...... 80 References ...... 82 Appendix A: Industrial Enzymes Specification Sheets ...... 99 Appendix B: Enzyme Pretreatment Results ...... 100 Varying Volumes of Crude Enzymes ...... 100 Active Enzyme, Deactivated Enzyme, and a Blank Solution ...... 103 Appendix C: Membrane Purification and Concentration Results ...... 105 Appendix D: Stability Test in a Model Food Matrix Results ...... 108

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LIST OF TABLES

Table 1. Formulation for ‘Cold’ and ‘Hot’ Mixtures Used in Gelatin Dessert Preparation ...... 42 Table 2. Initial and Final Concentration of Betacyanins in Prickly Pear Treated with Various Additives ...... 48 Table 3. Initial and Final Concentration of Betaxanthins in Prickly Pear Treated with Various Additives ...... 49 Table 4. Initial and Final Concentration of Betacyanins in Prickly Pear in Follow-Up Enzyme Treatment ...... 50 Table 5. Initial and Final Concentration of Betaxanthins in Prickly Pear in Follow-Up Enzyme Treatment ...... 51 Table 6. Membrane Flux Pre- and Post-Sample for Both Rounds of Microfiltration Performed ...... 57 Table 7. Membrane Flux Pre- and Post-Sample for Both Rounds of Nanofiltration Performed ...... 57 Table A. 1. Confirmed composition of Rapidase® enzyme (DSM 2015)...... 99 Table B. 1. Betacyanin Concentration in Prickly Pear Juice During Different Enzymatic Treatments Over 4 Hours ...... 100 Table B. 2. Betaxanthin Concentration in Prickly Pear Juice During Different Enzymatic Treatment Over 4 Hours ...... 101 Table B. 3. Total Soluble Solids (⁰Brix) in Prickly Pear Juice During Different Enzymatic Treatment Over 4 Hours ...... 102 Table B. 4. Betacyanin Concentration in Prickly Pear Juice During Follow-Up Enzyme Treatment Over 1 Hour ...... 103 Table B. 5. Betaxanthin Concentration in Prickly Pear Juice During Follow-Up Enzyme Treatment Over 1 Hour ...... 103 Table B. 6. Total Soluble Solids (⁰Brix) in Prickly Pear Juice During Follow-Up Enzyme Treatment Over 1 Hour ...... 103 Table B. 7. Initial and Final Phenolic Content in Prickly Pear Juice During Follow-Up Enzyme Treatment of 1 Hour ...... 104 Table B. 8. Initial and Final Capacity in Prickly Pear Juice During Follow- Up Enzyme Treatment of 1 Hour...... 104

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Table C. 1. Transmittance (%) of Prickly Pear Sample at Various Stages of Both Rounds of Centrifugation, Microfiltration, and Nanofiltration ...... 105 Table C. 2. Betalain Content of Prickly Pear Sample at Various Stages of Both Rounds of Centrifugation, Microfiltration, and Nanofiltration ...... 105 Table C. 3. Total Phenolic Content and Antioxidant Capacity of Prickly Pear Sample at Various Stages of Both Rounds of Centrifugation, Microfiltration, and Nanofiltration 106 Table D. 1. Measured L* Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored in Refrigerated Temperatures (4⁰C) Over the Course of 8 Weeks ...... 108 Table D. 2. Measured L* Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored at Room Temperature (25⁰C) Over the Course of 4 Weeks ...... 108 Table D. 3. Measured a* Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored in Refrigerated Temperatures (4⁰C) Over the Course of 8 Weeks ...... 109 Table D. 4. Measured a* Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored at Room Temperature (25⁰C) Over the Course of 4 Weeks ...... 109 Table D. 5. Measured b* Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored in Refrigerated Temperatures (4⁰C) Over the Course of 8 Weeks ...... 110 Table D. 6. Measured b* Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored at Room Temperature (25⁰C) Over the Course of 4 Weeks ...... 110 Table D. 7. Calculated Chroma Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored in Refrigerated Temperatures (4⁰C) Over the Course of 8 Weeks ...... 111 Table D. 8. Calculated Chroma Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored at Room Temperature (25⁰C) Over the Course of 4 Weeks ...... 111 Table D. 9. Calculated Hue Angles of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored in Refrigerated Temperatures (4⁰C) Over the Course of 8 Weeks ...... 112 Table D. 10. Calculated Hue Angles of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored at Room Temperature (25⁰C) Over the Course of 4 Weeks ...... 112

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LIST OF FIGURES

Figure 1. Flow diagram describing the methodology used in this project...... 32 Figure 2. Change in betacyanin content of prickly pear during various Rapidase® Fiber enzyme treatments over 4 hours...... 45 Figure 3. Change in betacyanin content of prickly pear during various ExtractSEB RLBE enzyme treatments over 4 hours...... 45 Figure 4. Change in betacyanin content of prickly pear during various DI water treatments over 4 hours...... 46 Figure 5. Change in betaxanthin content of prickly pear during various Rapidase® Fiber enzyme treatments over 4 hours...... 46 Figure 6. Change in betaxanthin content of prickly pear during various ExtractSEB RLBE enzyme treatments over 4 hours...... 46 Figure 7. Change in betaxanthin content of prickly pear during various DI water treatments over 4 hours...... 47 Figure 8. Change in betacyanin and betaxanthin content of prickly pear during 10% v/w treatments of various additives (deionized water, Rapidase® Fiber, deactivated Rapidase®, and ‘ blank’) versus control treatment over 1 hour...... 49

Figure 9. degradation pathway...... 54 Figure 10. Transmittance at different points of the clarification/concentration process of prickly pear juice for separate rounds...... 56 Figure 11. Concentration of betacyanins at different points of the clarification/concentration process of prickly pear juice for separate rounds...... 59 Figure 12. Concentration of betaxanthins at different points of the clarification/concentration process of prickly pear juice for separate rounds...... 59 Figure 13. Phenolic content (expressed as equivalent [GAE]) at different points of the clarification/concentration process of prickly pear juice for separate rounds...... 61 Figure 14. Antioxidant capacity (expressed as ascorbic acid equivalent antioxidant capacity [AAEAC]) at different points of the clarification/concentration process of prickly pear juice for separate rounds...... 62 Figure 15. Measured L* values of refrigerated (4⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 8 weeks...... 65

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Figure 16. Measured L* values of room temperature (25⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 4 weeks...... 65 Figure 17. Measured a* values of refrigerated (4⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 8 weeks...... 67 Figure 18. Measured a* values of room temperature (25⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 4 weeks...... 67 Figure 19. Measured b* values of refrigerated (4⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 8 weeks...... 69 Figure 20. Measured b* values of room temperature (25⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 4 weeks...... 69 Figure 21. Calculated chroma values of refrigerated (4⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 8 weeks...... 71 Figure 22. Calculated chroma values of room temperature (25⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 4 weeks...... 71 Figure 23. Calculated hue angle values of refrigerated (4⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 8 weeks...... 73 Figure 24. Calculated hue angle values of room temperature (25⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 4 weeks...... 73

Figure A. 1. Activity specification sheet for Rapidase® Fiber enzyme (DSM 2015). .... 99 Figure A. 2. Activity specification sheet for ExtractSEB RLBE enzyme (Enzyme Innovation 2018)...... 99

Figure C. 1. Standard curve of gallic acid used for phenolic content determination. .... 106 Figure C. 2. Standard curve of ascorbic acid used for antioxidant capacity determination...... 107

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CHAPTER 1

INTRODUCTION

Food colorants are used to accomplish a wide variety of goals from improving the product’s perceived freshness to increasing the product’s appeal to a certain demographic.

The earliest recorded history of the addition of colorants, archaeologists believe, dates back to around 1500 BC and Homer's Iliad when the use of was mentioned as a colorant for robes. Romans would use aloe, saffron, and elderberries as direct sources of colorants, and also added spices and floral agents that would add their own pigment to a finished drink.

With the growth of civilization came advancement of technologies, including food pigments. In 1856, the first synthetic organic (mauveine) was discovered by William

Henry Perkin. Following his discovery, over 700 colorants were developed over the next

50 years. These original man-made were derived from the chemical by-products of coal processing and often included poisonous materials like lead, mercury, and arsenic that would then be ingested by consumers. This carelessness quickly got the attention of the US

Federal Government, and in 1906, Congress passed the Food and Drugs Act (U.S. Food &

Drug Administration 2014). The purpose of this act was to ban the use of harmful colors in any food product and protect public health. At that time Dr. Bernard Hesse, a German- born dye expert employed by USDA, was asked to study colorants available in . He concluded that only 16 of the 80 colorants were probably harmless and he recommended only seven for general use in foods. Thus, the hundreds of original colorants available were severely reduced and continued to fall over the course of the next century with the addition of the Federal Food, Drug and Cosmetic Act (FFDCA) in 1938 and the Color Additive

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Amendment in 1960. Now, from the initial 80 synthetic food colorants used, just seven are approved for use in food by the US Food and Drug Administration (FDA) (U.S. Food &

Drug Administration 2014). Interestingly, of the original seven FDA-approved food colorants recommended by Dr. Hesse, only indigotine (FD&C Blue #2) and erythrosine

(FD&C Red #3) remain in production for use as food colorants today. Both (then

FD&C Red #2) and #1 were found to be harmful when consumed, and the remaining three colors fell out of popularity with food producers.

In present day, the FDA enforces the safety and appropriate use of color additives.

For all food additives, the FDA has an exemption for the addition of a substance. If that additive is “generally recognized as safe” (GRAS), then there is no need for the food product manufacturer to get premarket approval for inclusion of that substance by the FDA.

For color additives, however, no such exemption exists. Instead, the FDA has established specific regulations for color additives listed in Title 21 of the Code of Federal Regulations

(CFR). Color additives in the CFR fall under one of two categories: subject to the FDA’s certification process or exempt from the FDA’s certification process. Colorants subject to certification are man-made pigments synthesized from raw materials obtained from petroleum (formerly coal). Certified colors are commonly known as “artificial colors.”

There are seven food colorants currently listed for certification by the FDA. The list of colorants exempt from certification, however, is much greater. The 37 exempt color additives mostly include pigments derived from , , or . Colors exempt from certification are known as “natural colors.” Products utilizing color additives that are either not listed, used improperly, or do not conform to the specifications of the CFR are considered adulterated and are not allowed for market sale.

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Food colorants are used in a wide variety of everyday products, from and fruit to pickles and chips. Even toothpaste, , and cough syrup all contain FDA-permitted colorants. Food colorants are also used to impart an exotic sense to what would otherwise be nondescript foods. In Japan, for example, both McDonald’s and Burger King introduced what were known as “kuro burgers” – hamburgers where most of the components of the sandwich were dyed black. Bamboo charcoal was used to color the buns and cheese black, while squid ink was used in the sauce. While a black burger may seem unappetizing to most, the burger was successful as an advertising vehicle for fast food in Japan due solely to its unique coloring.

Besides the man-made variety, colorant molecules also occur in nature. These pigment molecules come in many forms: , betalains, , , and to name the most common. These pigment molecules not only serve as pollination and ripeness indicators in plants, but also play a role in by absorbing light energy and protecting plants against damage from UV light.

Anthocyanins appear as red, purple, or blue depending on the pH of their environment. They are found in a wide variety of fruits and including , , , red , and Concord . Betalains consist of two separate categories. Betacyanins classify the red-violet pigmented betalains including amaranthine, betanin, isobetanin, and neobetanin. Betaxanthins classify the yellow pigmented betalains including , dopamine-betaxanthin, and miraxanthin.

Unlike anthocyanins, betalains are rare in the plant world. Only 10 plant families produce betalains, all within Caryopyllales (cacti, carnations, , beets, etc.) and some

Basidiomycota (mushrooms). Betalains and anthocyanins have never been found to occur

3 naturally together in the same plant species. Coming across both betalains and in the same plant is feasible, but finding betalains and anthocyanins together in the same plant has so far been nonexistent.

Relevance of the Topic

As a whole, the colorant market is a multi-million dollar business. On a global scale, the industry’s worth was estimated at approximately $1.55 billion in 2011. Of that $1.55 billion, natural colorants amounted to $600 million in sales, approximately 40% of the total market. This figure increased by almost 29% from natural colorants’ figures in 2007, amounting to an annual growth of more than 7% in those four years. Beyond this advancement, it has also been demonstrated that globally, new launches of food and drink products’ usage of natural colorants outweighs the choice of synthetic colorants by 2:1

(Institute of Food Technologists 2013).

For decades, synthetic colorants have been the choice of manufacturers, but studies on these pigments have linked them to , increased estrogen levels, and hyperactivity, sleep disorders, aggression, and attention issues in children (Kanarek 2011; Stevens and others 2011; Weiss 2011; Arnold and others 2012; Millichap and Yee 2012; Nigg and others 2012; Stevens and others 2013b). Despite this, the level of synthetic colorants in the acceptable daily intake increased from 12 mg/capita/day in 1950 to 68 mg/capita/day in

2012 (Stevens and others 2013a). Although they offer more stability and are less costly to produce, consumers are increasingly regarding synthetic colorants undesirable.

As a result of both the scarcity of synthetic colorants still allowed by the FDA and the health concerns linked to the manmade pigments, more and more companies are

4 looking to natural sources of color for their products, which follows consumers’ recent demand for more wholesome food with ‘clean labels’. Improvements in technology have also contributed to the expedition of the natural colorant market. Growth for naturally derived pigments is expected to increase at an annual rate of 5-10%, which surpasses synthetic colorants’ anticipated annual growth of 3-5% (Downham and Collins 2000).

Despite their anticipated demand, the transition from artificial to natural colorants has been impeded by a number of factors. Natural colorants tend to lose their potency at a much faster rate than artificial colorants during storage. This is because the intensity of natural colorants is sensitive to temperature, light, and pH level. These environmental factors often cause certain colorants to degrade faster than others given the condition and storage of the product-in-question. A , for example, is an acidic product with an average pH range of 4.0 to 4.5. It is a perishable, dairy food item that is recommended to be held at a refrigerated temperature of 35-40°F and is sealed in a light blocking container.

Given these parameters, a natural colorant chosen for this product could be light- and heat- sensitive since both are not relevant to yogurt storage, but the pigments would have to be able to withstand an acidic environment. In some cases, colorants sensitive to environmental (light, pH) or production (heat) conditions can still be introduced into the food production cycle, but may require the installation of additional equipment or processing in the production line to mitigate color degradation. Understanding the behavior, structure, and sensitivity of these colorants is crucial to predict their feasibility in any food product.

Given the instability of natural colorants, it is reasonable to doubt their shift towards dominance of the coloring market in recent years, but they have a clear benefit over their

5 synthetic counterparts. These pigment molecules not only benefit the plants they are harvested from, but also offer antioxidant defense against free radical damage in the bodies of humans. Molecules like betalains and anthocyanins that protect plants during photosynthesis also act as chemopreventers in humans, fighting carcinogens and heart disease. Although natural colorants have lower stability, they have been proven to fight cancer while synthetic colorants, though highly immune to factors like light and temperature, have shown to cause cancer among other negative health effects. Consumers have become more aware of this contrast. As a result, the desire of pigments of a plant origin has never been higher.

While the use of natural food colorants is a noble and aspirational pursuit in the modern food industry, it is impossible to ignore the high costs associated with their production. Most natural colorants require separation and purification from the plant they are derived from. In some cases, such as with beta-, the colorant is abundant enough across a number of common sources that extraction and purification is a nearly- trivial cost, even if the ratio of bulk material to finished colorant is high. In other cases, such as with saffron, the rarity and thus cost of the base material (Crocus sativus) offsets the low cost and ease of harvesting the colorant. Finding a base plant or fruit that meets both criteria has been difficult to date.

In addition, while the impetus from consumers to move from artificial to natural colorants has pushed the food industry towards faster adoption of naturally produced pigments, the FDA has been more reluctant in its efforts to quicken the process. Only recently - in 2013 - did the FDA approve the limited use of Spirulina as a natural blue pigment in lieu of artificial blue dyes, and this was only after pressure from the M&M Mars

6 corporation. Part of this inertia, however, is for good cause as the health effects of some natural pigments are unknown or haven’t been studied long enough for the FDA to make a decision as to whether the pigment compound is GRAS or not. One major effect of this increased scrutiny is a rise in costs from FDA regulation, a hurdle that must be cleared by the natural colorant industry before a more widespread introduction into mainstream food industry use.

Looking past the FDA’s safety scrutiny, the increasing demand for natural colorants calls for ways to make the colorants more stable and to also find more natural sources of pigments to use in food. Red beet is the only betalain-based extract used on the market currently, due to its ability to produce more steadfast pigmentation in food products when compared to other readily-available natural sources of betalain. However, there are many negative consequences of its use, including an adverse earthy smell, high nitrate concentrations associated with the formation of cancerogenous nitrosamines, and a high risk of earth-bound contamination from heavy metals or chemical residues in the soil. Red beet color variability is also restricted due to betanin comprising most of its betalain content. Betalain sources are rare in the plant world, but recent studies have shown promise in cactus pears.

Prickly pears, or cactus pear fruits, have been gaining popularity with consumers due to their sweet taste and a growing concern amongst many consumers regarding the health benefits of the foods in their . Prickly pear is naturally high in minerals such as , , and , while also being high in and low in caloric content. Prickly pear extract has also been proven to have antioxidant properties, and has been shown to provide protection against ulcers, neurotoxins, and hepatotoxins

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(Galati and others 2003; Tesoriere and others 2004; Zou and others 2005; Kaur and others

2012). Prickly pear does not require much processing after ripening and harvest to be ready to eat, which - along with its health benefits - makes it a popular choice amongst consumers looking to lose weight and increase their fruit consumption.

The main hindrance to the growth and popularity of cactus fruits as a food staple, and for its use as a food colorant, is the narrow range of life cycle zones that allow for the harvest of cactus fruit. The prickly pear cactus grows well in a wide variety of areas and is tolerant of varied temperatures, soils, and moisture levels. Mexico is the primary source of commercial cactus fruit production, accounting for nearly half of all available cactus fruit for consumption, with Italy being the second main producer. The growth and harvesting periods for most cactus fruit occurs between the months of July and October; due to the short shelf life of harvested cactus fruit, this also coincides with an increase in consumption of cactus fruit. Prickly pear can also be found in parts of the Southwestern United States as well as other parts of Mediterranean Europe. However, in general, it is not grown as a item in these areas and thus has a very small commercial footprint in these regions.

The betalains abundant in prickly pear cactus fruit are water-soluble colorants that come in two forms: yellow-orange betaxanthin and red-violet betacyanin. Research on

Opuntia cacti has shown that the betacyanin/betaxanthin ratio varies widely in different prickly pear fruit (Moßhammer and others 2005a).

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Specific Scope

Increasing recognition of the prickly pear as both a potential natural colorant and antioxidant in foods has stimulated the need for chemical composition analysis of the fruit’s three elements: the skin, the pulp, and the . El Kossori and others (1998) determined the crude composition, fiber composition (, , , and ), composition, and composition of prickly pear. Results demonstrated significant concentrations of hemicellulose, cellulose, and pectin in all three elements of the fruit. Another study by Khatabi and others (2016) established that content in prickly pear is not only higher in the red variety than the yellow, but also that the per volume concentration of for both varieties is increased in the whole fruit as opposed to that of the juice alone. This implies the prickly pear pulp and seeds contain a quantity of polyphenols that is filtered out during the separation stage of juice processing.

These same principles can be applied to the use of enzymes with prickly pear, a practice common in fruit juice processing. juice production, for example, utilizes pectinase enzymes like pectin esterase and polygalacturonase to reduce the juice’s

(thickness) (Oszmiański and others 2009). Processors also use cellulase and hemicellulase enzymes to break down the cell wall, which leads to an overall 5-10% increase in juice production. Prior to any filtration or separation, the addition of enzymes like pectinase, hemicellulase, and cellulase will break down cell wall components found to be present in the pulp, skin, and seeds, thereby releasing the polyphenols found in the whole fruit rather than just the juice. The goal of this procedure would be to increase the concentration of polyphenols in the prickly pear extract above that which would be achieved by current means of processing.

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Once yield has been increased with an enzymatic pretreatment, the purification and concentration method of the juice is important because it determines how well the integrity of the pigment molecules will be maintained. Membrane filtration is a low-cost, athermal separation technique that will not influence the betalains and still yield a concentrate that is fresh and additive-free. Choice of membrane size will influence the permeate’s potential as either a juice or as a concentrated colorant.

Another important aspect in determining the feasibility of prickly pear concentrate as a natural colorant is its stability over a wide range of factors like temperature, time, pH, and light. By comparing these results against those of existing betalain colorants in the market as well as a synthetic counterpart, the value of this fruit’s natural pigments will become more evident. The potential success of these experiments will not only offer options and insight to food processors looking for alternative natural colorant sources, but also continue efforts in both ensuring the health of consumers and fulfilling their call for ‘clean label foods’.

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CHAPTER 2

LITERATURE REVIEW

Natural Colorants, Betalains

The color of a product is one of the most important attributes to food. It is the main determinant of consumer acceptability. Recent years have demonstrated a drastic shift in the worldwide colorant market as natural colorants exceeded synthetic colorant usage for the first time in 2011 (Institute of Food Technologists 2013). Consumers want wholesome, natural products as part of the ‘clean label’ movement, and it is the food industry’s responsibility to deliver.

One of the most common natural colorant molecules used in the market today are betalains. These water-soluble pigment compounds are not as examined as anthocyanins because of their relative scarcity in nature, restricted to just the plant order

(cacti, carnations, amaranths, beets, etc.) and some Basidiomycota (mushrooms) (Steglich and Strack 1990). Betalains come in two forms: the orange-yellow betaxanthin and the red- purple betacyanin. Both betaxanthins and betacyanins share a common moiety of betalamic acid [4-(2-oxoethylidene)-1,2,3,4-tetrahydropyridine-2,6-dicarboxylic acid]. Betacyanin and betaxanthin molecules have maximum absorptions at 540 and 480 nm, respectively

(Strack and others 2003).

Beyond the growth of the natural colorant market as a whole, the desirability of betalains has increased in recent years because of two main reasons: (1) they have been linked to numerous health advantages and (2) they are pH stable in a range of 3 to 7

(Stintzing and Carle 2004). These factors make them the choice for providing red-purple natural pigment to low acid foods due to anthocyanins’ instability at pH values greater than

11

3 (Stintzing and Carle 2004). Betalains have been proven to exhibit antioxidant activities, acting as radical scavengers in the body (Pedreño and Escribano 2000; Kanner and others

2001). Because of their prevention of oxidative processes, betalains can contribute to the prevention of several degenerative diseases in humans including , , immune disorders, neurodegeneration, , and others (Galati and others

2003; Tesoriere and others 2004; Zou and others 2005; Kaur and others 2012).

Phenolic Compounds

Aside from betalains, the most important biocompounds found in prickly pear fruit are phenolic compounds, which also exhibit antioxidant properties (Gentile and others

2004). Phenolic compounds consist of an aromatic ring with one or more hydroxyl groups.

Research by Kuti (2000) established that cactaceae plants and fruits are not only rich in flavonones, , glycosilated flavonols, and dihyrdroflavonols but also that these were major contributors to the plant’s overall antioxidant activity. Studies of other foods rich in flavonols have shown their role in degenerative diseases prevention including cancer, diabetes, and diseases of the gastric and cardiovascular variety (Galati and others 2003; Jacob and others 2008; Lampila and others 2009).

Phenolics include many various classifications including flavonoids, , , , and phenolic acids, among others. They serve many purposes in the plant kingdom, from pollination and ripeness indicators to protecting the plant against UV damage. Quantitative determination of polyphenols has been proven to be difficult due to their structural complexity and wide assortment. However, there are multiple methods

12 employed to determine polyphenols present in plant extracts (Møller and others 2009;

Pieroni and others 2011).

Red Beetroot & Alternatives

Beetroot extract or ‘beetroot red’ is the primary natural betalain colorant listed in

Title 21 of the Code of Federal Regulations (CFR). This crop contains two major pigment molecules: the red betanin and yellow vulgaxanthine I, among others like isobetanin, betanidin, and II (Gasztonyi and others 2001). Aside from the poor color variability exhibited in its betanin, beetroot as a pigment source has its drawbacks. The main fault is its high concentration of geosmin and pyrazines, which are attributed to an unpleasant peatiness that can result in negative sensorial impacts (Lu and others 2003).

Geosmin (trans-1,19-dimethyl-trans-(9)-decalol) is a bicyclic alcohol that is responsible for the earthy-aroma and flavor associated with red beets (Lu and others 2003). This taproot is also high in nitrates, which are precursors for carcinogenic nitrosamines. The Acceptable

Daily Intake (ADI) for nitrate is 3.7 mg/kg body weight per person per day according to the Scientific Committee for Food (SCF) (Opinion of the Scientific Panel on Contaminants

2008). Denitrification to yield a juice free of nitrates has been conducted by both ionic exchangers and Gram-negative bacteria Paracoccus denitrificans, which convert nitrate into gaseous (Grajek and Walkowiak-Tomczak 1997). However, these methods are expensive in operation and are not feasible to incorporate into industry.

Considering the narrow selection of betalain sources, attempts at finding alternatives to red beetroot extract is limited. However, there has been much promise in prickly pear considering its high concentration of both betalains and polyphenols as well

13 as its lack of adverse flavor impactors (Castellar and others 2003). Beyond its flavor, cactus pears (genus Opuntia) are a sensible crop due to their minimal soil and water requirements capable of growing in semi-arid to arid regions (Castellar and others 2006).

A major factor in the appeal of Cactaceae family over red beetroot is the broad color spectrum it offers. The Opuntia sp. (prickly pear), Hylocereus sp. (also known as or dragon fruit), Cereus sp., and Selenicereus sp. range from yellow-orange to red-violet, depending upon their concentration of betaxanthins and betacyanins (Stintzing and others

2002; Wybraneic and Mizrahi 2002; Herbach and others 2006).

Stintzing, Carle, Herbach, and Moßhammer have done a significant extent of research and collaboration to understand the betalains and antioxidant properties found in

Opuntia (Stintzing and others 2002; Herbach and others 2004; Stintzing and Carle 2004;

Moßhammer and others 2005a; Moßhammer and others 2005b; Stintzing and others 2005).

They have determined that the pulp of Opuntia sp. contains red-violet betacyanins as well as the yellow-orange betaxanthins. This contrasts against the pulp of the Hylocereus sp., which has been found to contain just betacyanins alone (Azeredo 2009). The of the

Hylocereus sp. is mostly red, but its pulp can also be colorless depending on the variety

(Kim and others 2011). For the Opuntia, both natural colorant molecules (betacyanin and betaxanthin) have been found to be abundant in both the peel and the pulp of the prickly pear (Vergara and others 2014). Because of these factors, the purple cactus pear (Opuntia ficus-indica) has been deemed by many as an appropriate alternative betanin source to replace the red beet (Delgado-Vargas and others 2000; Castellar and others 2003).

A recent study (Yeddes and others 2013) looked at the antioxidant capacity, phenolic content, flavonoids, and betacyanins present in both the peel and the pulp of

14 different varieties of prickly pear. Between O. ficus indica and O. stricta, O. stricta had a higher antioxidant activity despite O. ficus indica containing more total polyphenols. This is due to a larger concentration of betalains found in O. stricta. Concentration of certain molecules is not the only factor in determining antioxidant capacity. One must also consider factors such as reactivity towards the radical, the fate of the radical derived by the antioxidant, the relative localization or distribution, and the interaction between (Niki 2011). Yeddes and others (2013) also found that the pulp and peel of both Opuntia varieties also contained polyphenols and betalains. The peel of O. ficus indica exhibited a richness of betalain indicaxanthin, and O. stricta in the betalain betacyanin. For both varieties, however, the peel exhibited higher antioxidant activity than the pulp (1.66-

2.21 fold higher). These results agree with similar studies conducted by Diaz-Medina and others (2007) as well as Moussa-Ayoub and others (2011).

Enzyme Use for Pigment & Phenolic Extraction

Natural color sources typically contain less than 2% of pigment in the raw material.

This is in comparison to synthetic colorants, which typically yield more than 90% (Sensient

Food Colors 2017). This means that, despite being high in betalains, pigment extraction from prickly pear is far more costly than the synthetic alternative. In order to compete with man-made counterparts, increasing the efficacy of colorant extraction from natural sources is important. Several studies have shown that cell permeability is an important factor of extraction efficiency. Chamlermchat and others (2004) found that pulsed electric field treatment raised the betalain extracted from red beetroot, but this method does have many technological requirements.

15

The use of enzymes to increase yield in fruit juices is a simple technique widely used in the industry. For apple juice in particular, apple mash maceration by pectolytic enzymes has been proven to facilitate the pressing operation and increase phenolic content.

In 2004, Mihalev and others looked at the contribution that use of pectolytic enzymes have on the release of polyphenols in apple juice production. The results demonstrated the potential that optimization of enzyme composition can have on polyphenolic yield from the juice produced. To expand on the use of pectolytic enzymes, a study by Will and others

(2000) looked at the properties of apple pomace that had been liquefied with both pectolytic and cellulase enzymes. The phenolic content observed by the double-enzyme treated juice was significantly higher than the content observed in the “premium” juice treated conventionally by just pectolytic enzymes. Will and others (2000) displayed the efficiency of combining multiple enzymes in fruit juice extraction.

This concept of enzyme application can be utilized for prickly pear fruit, as shown by Makhdoom and others (2016). In this study, two portions of prickly pear juice were analyzed: a non-enzymatic treated sample and a sample that was treated with 2 mg of pectinase enzyme per liter of juice. The enzyme-treated sample exhibited high total content (3.28 mg/ml juice), with 2.97 mg/ml comprised of phenols and 0.31 mg/ml flavonoids, which increased over 11 percent from the original concentrations (2.95, 2.66, and 0.29 mg/ml juice, respectively). While the results of this study are promising for the use of enzymes in prickly pear, Makhdoom and others utilized only pectinase enzymes and monitored just phenolic content. A related study by Moßhammer and others (2005b) found that of the enzymes tested, Fructozym MA-XPress at a quantity of 80 ppm was most efficient at the degradation of prickly pear hydrocolloids. Further studies need to be done

16 with the use of enzymes in combination with pectinase similar to the study by Will and others (2000) on apple mash.

In 1998, El Kossori and others determined the crude composition (protein, non protein nitrogen, lipids, total , ash, ethanol-soluble , and ), fiber composition (hemicellulose, cellulose, pectin, and lignin), carbohydrate composition

(saccharose, , , , , , , and ), and mineral composition (Ca, Mg, Na, K, P, Fe, Cu, Zn, Mn, and Mb) of the different components of the prickly pear fruit. Results demonstrated significant concentrations of hemicellulose, cellulose, and pectin in the skin, pulp, and seeds. The skin fibers contained the largest amount of hemicellulose (20.8 ± 0.55 % of total fiber) while pectin was found to be most abundant in the pulp (70.3 ± 1.30 % of total fiber) and cellulose most abundant in the seeds (83.2 ± 0.25 % of total fiber). The high concentration of these and heteropolysaccharides that comprise the plant cell walls are of great interest considering the betalain and polyphenol molecules contained in the fruit components analyzed.

Another study by Khatabi and others (2016) established not only that red prickly pear has a higher polyphenol content than the yellow variety, but also that the rate of polyphenols for both varieties is superior in the whole fruit opposed to just the juice alone.

These results demonstrate that the prickly pear pulp, seeds, and skin contain a quantity of polyphenols that is filtered out during the separation stage of juice processing. This conclusion was in agreement with the project discussed earlier in which enzymatic

(pectinase) treatment was used to treat prickly pear juice to increase phenolic content

(Makhdoom and others 2016).

17

Aside from natural colorants, the recovery of highly valued compounds from food processing waste has been a pressing issue in recent years. The large quantity of these non- edible residues (fruit peels, seeds, and bagasse) cause environmental pollution, problems for company management, and a high amount of both food and profit loss (Ferrentino and others 2016). Repurposing this waste has been the focus of many projects, several concluding these residues are untapped sources of valued bioactive compounds including antioxidants, vitamins, and natural colorants that exhibit antioxidant properties. The largest obstacle in these efforts is finding the most appropriate extraction technique without compromising efficiency or impacting the environment. Stability of the extracted products also plays an important role as many compounds are sensitive to factors like light or heat.

The use of enzymes to increase the betalain and phenolic yield from fruit residues otherwise deemed as waste from fruit juice processing is of great economic and environmental interest.

Membrane Filtration

Because prickly pear fruit are low-acid fruits (pH > 4.5), juice processing requires heat treatment of at least 115.5°C for the management of microorganisms. However, even after thermal treatment, cactus pear is highly susceptible to microbial attack due not only to its high pH, but also the relatively low concentration of acids and high soluble solid content which can significantly shorten the shelf-life of the product (Joubert 1993).

Carrandi (1995) subjected prickly pear juice to several thermal treatments (100°C, 20 minutes each) and found that this process yields a product with good stability, but the color and flavor changed drastically, the juice acquiring an adverse taste. Efforts have been

18 made to stabilize the juice with mild thermal treatment and pH adjustment via juice, but that led to acetic of the product, meaning it could not be stored for long periods (Espinosa and others 1973).

Despite the complications that prickly pear juice has regarding its perceptivity to microbial contamination, there is still promise in its stability when it is concentrated.

Lowering the relative water activity acts as a protectant against microbial growth and helps not only extend the shelf life of the product, but also lowers packaging and storage costs significantly. Sáenz and others (1993) concentrated cactus pear juice to a total soluble solid level of 63-67 ⁰Brix via vacuum evaporator. This concentration method was efficient at creating a product with high microbial stability, but the sensory quality (color and aroma) was less than satisfactory. Another potential approach is freeze concentration. This method requires less energy than vacuum evaporation, but the equipment is expensive and successful concentration is not guaranteed (Köseoglu and others 1990).

Another possibility is the use of membrane operations. Clarification of fruit and plant juice extracts by means of membrane filtration is commonplace in industry and has been for some time. Unclarified juice is first centrifuged to remove larger insoluble particles that would otherwise foul the membrane filters before the remaining liquid fraction is fed to the filters for clarification. However, membrane fouling and process flux reduction over time remain major issues for membrane clarification and processing of fruit juices (Sarkar 2014), even after centrifugation. Historically, studies have focused on analyzing the most common fouling mechanisms of membranes of varied composition and pore size, and for a variety of fruit juices (Riedl and others 1998; de Oliveira and others

2012). More recent studies have focused on novel concepts regarding the reduction of

19 membrane fouling, such as de Oliveira’s study (2012) on enzymatic treatment of juice.

Membrane filtration is a simple method in theory, which makes it an appealing alternative. But besides its straightforwardness in operation, membrane technology also has several other benefits. The athermal processing method involves no phase change or chemical additives to accomplish concentration, which allows fruit juices to maintain their natural fresh taste while still remaining additive-free. The various membrane processes including microfiltration (MF), ultrafiltration (UF), nanofiltration (NF), and reverse osmosis (RO) have been successful in many juice processing procedures including purification, concentration, stabilization, and depectinization (Álvarez and others 1998;

Gökmen and others 1998). These processes yield a clarified product that is free of spoilage microorganisms while avoiding heat treatment.

Membrane filtration has been proven as a successful alternative method to obtain betalain extract while maintaining low temperatures. The evasion of high temperatures during the extraction process avoids the issue of betalain degradation during pigment purification and concentration. These processes have been effective both in the concentration of red beet juice (Bayindirli and others 1988) and the clarification of yellow, red, and purple cactus pear pulp (Cassano and others 2007; Cassano and others 2010;

Vergara and others 2014). The potential use of prickly pear extract as a natural colorant is heavily dependent on finding measures to concentrate pigment molecules since betalain stability has been found to have a positive correlation with relative betalain concentration

(Merin and others 1987).

20

Moßhammer and others have contributed significant research to understanding the stability and potential concentration methods of prickly pear. In a 2005 study, Moßhammer and others (2005b) found that crushing whole, fresh prickly pear fruits with a grinding mill and then separating the peels and seeds with a finisher prior to membrane filtration increased the yield from 37% to 47%. Moßhammer and others also recognized the potential problems the pre-clarified juice could present for the membrane in terms of its high viscosity level. In a follow-up study, enzyme treatment to the juice was deemed a crucial step in the concentration process (Moßhammer and others 2006). Rohapect B1L (AB

Enzymes, Darmstadt, Germany) was tested against Fructozym MA-X Press, two commercial-grade enzymes both recommended for fruit processing. The results showed

Rohapect B1L at a dosage of 350 ppm to be most efficient for hydrocolloid degradation

(Moßhammer and others 2006). Furthermore, the overall pigment retentions demonstrated using microfiltration (Moßhammer and others 2006) were greater than those of heat-treated juice (Moßhammer and others 2005b).

Data from a study by Vergara and others (2015) showed that betacyanins remaining in the filtered permeate composed 20.6% of the mass of the feed solution, and the total pigment in the permeate composed 20.4% of the mass of the feed solution. A study by

Cassano and colleagues (2007), however, showed that the pigment did not pass through the membrane, and instead was primarily found in the retentate. Vergara’s study surmised that the use of a series of membrane filters could be used to more successfully allow betalains to pass through filtration into the final permeate. The study also demonstrated the utility of microfiltration for clarifying prickly pear juice, as the membrane filter used was able to completely remove insoluble solids without using enzymatic pretreatment,

21 damaging betalains present in the finished permeate, or significantly degrading the performance of the membrane in terms of permeate flux. Finally, Vergara noted that ceramic membranes used in the study were more effective than polymeric membranes due to the ceramic membranes being more hydrophilic. However, ceramic membranes are more expensive than their polymeric counterparts, making polymeric membranes more feasible to incorporate into the large industrial scale.

While much has been written on the use of different types of filtration media, filter media pore sizes, and membrane fouling using both dead-end and cross-flow filtration methods, there is not much in the literature directly comparing dead-end and cross-flow filtration methods, especially in regards to juice clarification. This can be attributed to dead-end membrane setups being difficult to scale up from laboratory conditions, while cross-flow setups are common in large-scale, industrial conditions. (Shamsuddin and others 2015). One particular benefit that cross-flow membrane setups have over their dead- end membrane counterparts is that they inherently provide two output flows (permeate and retentate), each of which contain fractions of the original feed flow that may be useful for study individually whereas dead-end membrane setups only have a single output flow

(permeate).

Koltuniewicz and others (1995) performed a two-part study designed to determine filtration, fouling, and mass transfer performance of dead-end and cross-flow membranes using a simple oil-in-water emulsion as the feed material. In the first study, the researchers studied both dead-end and cross-flow filtration setups while varying flow rate, feed pressure, and feed temperature. The dead-end filtration setup also utilized a stirrer to inhibit the formation of cake-layer fouling on the membrane. Whilst the primary objective of this

22 portion of the study was to identify fouling mechanisms on membranes for both flow configurations, the data showed that the same membrane, subjected to cross-flow filtration techniques in lieu of dead-end filtration flow, was capable of sustaining a higher flu x rate over the course of a test run than a membrane subjected to dead-end flow. Additionally, the study determined that the mechanism and rate of fouling was consistent between the dead-end stirred cell and cross-flow feed flow regimes.

The use of spiral-wound membranes is a slight departure from the work of other studies that used tubular or flat-sheet membranes in their processes (Cassano and others

2007; 2010). While tubular and spiral-wound membranes are preferred in industry for their high ratio of membrane area to filter cartridge volume (Salgado and others 2015), flat-sheet membranes have been used in laboratory, pilot-scale settings as a representation of larger membranes used in industry-scale cross-flow filtration operations. In some cases, coupon samples of these larger membranes have been used in an attempt to determine their fouling and permeate flux properties. While a flat-sheet coupon of a spiral-wound membrane can be useful to characterize the properties of the filtration media, feed spacers between the filter layers of a completed spiral-wound membrane play an important role with respect to mass transfer and membrane fouling. Feed spacers allow for the separation of leaves of the membrane filtration media, creating multiple flow channels throughout the filter and flow instabilities within the filter. In turn, the unstable flow reduces membrane fouling, and the multiple feed channels allows the filter to achieve higher mass transfer rates using a laminar flow regime (Re less than 2000) and lower trans-membrane pressures (Koutsou and others

2009). Thus, it is important to characterize the efficacy of a complete spiral-wound membrane against that of a flat-sheet membrane coupon.

23

A study by Salgado and others (2015) was performed comparing flat-sheet and spiral-wound nanofiltration of must to determine the effects of process scale-up from laboratory to industry settings. Two membranes of similar properties and size – one flat- sheet and one spiral-wound – were selected from a membrane filter manufacturer for a direct comparison of the two processes. The study found that, while cake-layer fouling of the spiral-wound membrane was almost instantaneous after the introduction of the grape must feed due to the laminar flow regime within the filter, this layer of fouling remained relatively consistent in structure and thickness over the length of a test filtration run. This allowed for a consistent rejection of and mass transfer rate over the length of the filter run due to the formation of the cake-layer pseudomembrane. Conversely, the cake- layer fouling of the flat-sheet membrane became thicker as a function of time over the length of the filter run, despite increased shear stress caused by the flow regime over the membrane. This resulted in a less-constant rejection of sugars and a less-constant osmotic pressure on the membrane, which is undesirable in industry filtration.

Another study performed in 2012 by Patel and others sought to compare the permeate quality from spiral-wound nanofilters against that from flat-sheet membrane filtration of a reactive dye solution. Solute concentration was varied for three separate test runs on each filter type to determine the efficacy of each membrane in removing the dye and other dissolved solids from the initial feed solution. Trans-membrane pressure also varied for another three test runs on each filter to determine the effect of osmotic pressure on the mass transfer rate of both filters. Data from these test runs showed that, while the spiral-wound membranes were consistently more effective at removing dissolved solids and colorants from the feed solution, the flat-sheet membrane consistently had a higher

24 mass transfer rate across varied trans-membrane pressure inputs with the difference of mass transfer rate between the two worsening with increased osmotic pressure. This finding is in agreement with the study from Salgado and others (2015) noted above, wherein spiral- wound membranes performed better during laminar feed flow.

Monitoring Physico-Chemical Properties of Prickly Pear Juice

Before and after enzyme treatment and throughout membrane filtration, it is critical to monitor the physico-chemical properties of the prickly pear juice and resulting concentrate to determine the changes in betalain concentration, phenolic content, total soluble solids, and antioxidant capacity. Regarding antioxidant activity, Butera and others

(2002) found cactus fruit to contain double the antioxidants as the common , , pears, and tomatoes; therefore, it is important to maintain this positive trait associated with the prickly pear. There have been several assays developed for measuring total antioxidants in a variety of matrices including bodily fluids (Whitehead and others

1992), pure compounds (Arnao and others 1990), and various food extracts (-Evans and Miller 1995). No matter the matrix, each method relies on the generation of a synthetic colored free radical.

When considering the ABTS and DPPH assays, they are limited due to non- physiological radicals, but they are still convenient and widely recognized in the food science realm. The DPPH assay follows the reduction of the purple radical 2,2-Diphenyl-

1-Picrylhydrazyl (DPPH) to 1,1-diphenyl-2-picryl hydrazine, the colorless neutral. The

ABTS assay is similar in its operation, but it instead follows the reduction of the blue/green radical ABTS+.

25

A 2011 study conducted by Floegel and others looked at the 50 most popular antioxidant-rich foods in the US diet including fruits, vegetables, and beverages and determined their antioxidant capacity with the two most common assays utilized: DPPH and ABTS. The results showed that, when compared to the ORAC values of the same foods in the USDA database, ABTS was the superior assay in antioxidant assessment. This was the case in a variety of plant foods containing multiple types of antioxidant compounds including hydrophilic, pigmented, and lipophilic. Because of its broad range of antioxidant application, its superiority in accuracy compared to DPPH, and the refinement of its methodology over the years, ABTS is a respectable assay to determine antioxidant activity in food extracts. This method has been employed in a number of studies characterizing the antioxidant levels in Opuntia and its related species (Cassano and others 2007; Cassano and others 2010).

Several methods are available to determine the polyphenolic content in plant extracts including ferrous ammonium sulfate (FAS) indicator, the more complex Folin-

Ciocalteu reagent method, and high-performance liquid (HPLC) (Møller and others 2009; Pieroni and others 2011). Despite the assortment of methods available, colorimetric reactions using a UV/VIS spectrophotometer are often favored due to their simplicity, reliability, and low cost of operation (Pelozo and others 2008). However, a key component in this methodology is the use of a reference, or standard, which the phenolic content of the sample can then be compared to.

The Folin-Ciocalteu method is widely used to quantify the polyphenols in plant extracts. The reaction formed during this process can be indicative of the concentrations of several phenol groups, mostly via variations in color change due to unit mass (Glasl 1983)

26 and reaction kinetics (Folin and Ciocalteu 1927). In this method, a specific reagent

(the Folin-Ciocalteu reagent) reacts with polyphenols in the sample to form a blue chromophore comprised of a phosphotungstic-phosphomolybdenum complex (Schofield and others 2001). This reagent has been utilized to determine phenolic content across many fields from determination of phenols in plant methanol extracts (Cicco and others 2009;

Blianski and others 2013) to the revalorization of fruit waste as an antioxidant source

(Cardador-Martínez and others 2011).

Another critical aspect to understanding the treatment effects of plant extracts is being able to monitor individual colorant molecules and their relative concentrations in the sample. High-performance liquid chromatography (HPLC) was first applied in 1978 in the analysis of betalains in red beets () (Vincent and Scholz 1978). Over the next

40 years, the method improved to its current state, capable of identifying the 50 red betacyanins and 20 yellow betaxanthins that have been found in nature (Francis 1999).

Chemical characterization and separation of betalains has been accomplished extensively using HPLC (Vincent and Scholz 1978; Cai and others 2005; Cejudo-Bastante and others

2014). This is a useful tool in monitoring and quantifying both betacyanin and betaxanthin content in a plant-derived extract. However, the cost and time associated with HPLC does not make it ideal for use in most laboratories.

An alternative method to quantify betalains present within a sample is Beer-

Lambert Law. This method uses the concept that the absorbance of a material is directly proportional to the concentration of attenuating species within any given sample.

Compared to HPLC, Beer-Lambert Law has the disadvantage of only being able to quantify betalain molecules while HPLC can both quantify and identify. However, despite its

27 apparent drawbacks, this method is widely-recognized and has been employed by numerous studies to determine the betalain content within a sample (Herbach and others

2004; Cassano and others 2007; Cassano and others 2010; Castro-Muñoz and others 2014;

Cruz-Cansino and others 2015).

Stability & Model Food Matrices

Natural red colorants can be used in a variety of foods, but their stability relies heavily upon two factors: the food matrix they are introduced to and the storage conditions of that food (von Elbe and others 1974). Betalains have been shown to be affected by environmental factors such as pH, light exposure, oxygen, water activity, and enzymatic activities (Castellar and others 2003; Herbach and others 2004; Herbach and others 2006;

Azeredo 2009). In dairy products, fat content can have a major impact on the stability of natural pigment molecules, as demonstrated by studies (Giusti and Wrolstad

2002).

Aside from betalains of red beet or prickly pear, research by Cai and Corke (1999) compared the color properties and stability of extract in three model foods

(jelly, ice cream, and a model beverage) at different temperatures against red anthocyanins as well as a synthetic colorant. The results displayed great promise in the

Amaranthus betacyanins, which exhibited a more intense red color than the radish anthocyanins. At both 14°C and 25°C, red radish and Amaranthus demonstrated similar stability, but betalains’ sensitivity to heat showed at 37°C when radish stability surpassed that of the Amaranthaceae. Under most storage conditions, the synthetic colorant was more stable than both natural colorants.

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Caldas-Cueva and others (2016) observed the stability of betacyanins from

Ayrampo cactus (Opuntia soehrensii), a sister genus of the Opuntia known as “prickly pears”. Ayrampo extract was added to yogurt to test the stability of the betacyanins against those from red beet extract. While red is useful for a broad spectrum of food matrices including yogurt (Caldas-Cueva and others 2016), betacyanins – and other anthocyanins – can be lipophilic (Giusti and Wrolstad 2002) in addition to their sensitivity to pH. The study was performed with yogurt samples at two different levels of fat content

(0.1% and 3.0% by volume) to see how lipid content would influence the stability of the natural colorants compared to the synthetic. It was observed that fat content did not influence the color retention of the , but betacyanins from Ayrampo extract did show higher color retention (∼95%) after 4 weeks of storage compared to the red beet pigment (∼91%). The intensity of the Ayrampo extract was more comparable to artificial colorant FD&C Red #40, even when subjected to both extended storage time and heat treatment. This study also utilized betacyanins extracted from the seeds of the Ayrampo exclusively, supporting the notion of repurposing waste with observed success in pigment stability.

Another study by Hani and others (2015) studied the physico-chemical effects of producing gummy confections using red pitaya (Hylocereus polyrhizus) fruit extract.

While the primary outcome of the study was determining the effect that the red pitaya, or dragon fruit, pulp had on consistency and texture of the finished confectionary product, the researchers also analyzed the color intensity of the finished product based on colorimeter and sensory analysis. While the study found that the inclusion of red pitaya extract had a noticeable impact on the edible texture of the gummies and that the extract reduced the

29 hardness and rigidity of the final product, sensory panels concluded that these changes did not have an adverse effect on the desirability of the gummies. Additionally, the study found that the red pigmentation present in the gummies due to betacyanins present in the red pitaya was stable during an 8-week storage time, with gelatin-based gummy confections retaining more of their initial color than pectin-based gummies.

Betalains from related plant extracts have shown success in a variety of food matrices and at various storage conditions (Cai and Corke 1999; Castellar and others 2003;

Herbach and others 2006; Hani and others 2015; Caldas-Cueva and others 2016). Shelf- life studies on the extract alone have displayed prickly pears’ tolerance towards a variety of environmental factors, but there has yet to be a study monitoring the stability of betalain extract from Opuntia ficus indica in a model food matrix. Matrices to consider include low- acid foods that are not subjected to high temperatures and have a shorter shelf-life. In these conditions, betalains derived from prickly pear in their concentrated form could exhibit a great deal of success as a natural red colorant and offer an alternative to beetroot extract.

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CHAPTER 3

MATERIALS & METHODS

Objectives and Hypotheses

The specific objectives of this project, along with the corresponding aims and hypotheses, are as follows:

1. Create a concentrated colorant using prickly pear combining use of enzymes

and membrane filtration technology.

a. Use enzymes to increase the yield of betalains and polyphenols in

prickly pear mash.

H1A: The concentration of betalains and polyphenols will be greater

in enzyme-treated prickly pear mash when compared to the

control.

b. Use membrane filtration to concentrate and purify the prickly pear

colorant.

H1B: The transmittance (sample clarity) and concentration of

betalains will be higher after microfiltration and nanofiltration.

2. Maintain the beneficial properties associated with the natural colorant.

H2: The phenolic content and antioxidant capacity will be equal or

higher after microfiltration and nanofiltration.

3. Test the effectiveness of this colorant in a model food matrix.

H3: Prickly pear concentrate will demonstrate equal or less pigment

loss than red beet extract.

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Flow Diagram

This project was divided into four main stages which were used to meet the main objectives (Figure 1). These stages included: (1) enzyme pretreatment, (2) centrifugation and microfiltration, (3) nanofiltration, and (4) in-matrix stability testing.

Figure 1. Flow diagram describing the methodology used in this project.

Sample Preparation and Chemical Reagents

Prickly pear mash used in enzymatic pretreatments was made using ripe fruit sourced from the local supermarket. Prickly pears of the red variety were washed, crushed by hand, then blended with a Hamilton Beach B55 Single-Serve Blender (120V) for approximately 5 seconds (in order to break down larger pieces of skin/seeds). This blended sample was then used to test the effects of various amounts of crude enzyme mixtures.

Prickly pear puree used in the remainder of the project was purchased in a 441 lb. drum quantity from Stiebs (11767 Road 27 ½, Madera, CA 93637). Prickly

32 pear puree was made from the Opuntia ficus-indica species of red prickly pear cactus sourced from Peru and processed in Central California. The drum was ran through an industrial rocker and puree was divided into 5-gallon buckets and sealed for easier storage.

Sealed sample containers were stored at -24⁰C until required, then thawed overnight at 4⁰C.

Sucrose was purchased from IBI Scientific (Peosta, IA). Gelatin (250 Bloom) was purchased from Aspen Naturals (Denver, CO). Red dye 40 was purchased from Flavors &

Colors (, CA). Fumaric acid was purchased from TCI America (Portland, OR). Red beet concentrate and acidified carmine colorant were provided by colorMaker, Inc.

(Anaheim, CA). Petri dishes as well as all other chemical reagents were purchased from

VWR International (West Chester, PA).

Methodology

Physico-Chemical Parameters

Soluble solids content (SSC)/⁰Brix, pH, concentration of betalains, phenolic content, and antioxidant capacity of prickly pear were measured in triplicate. SSC was determined with an Atago PAL-ALPHA Digital Pocket Refractometer, 0.0 to 85.0% Brix.

The pH of samples was measured with a Fisher Scientific Accumet Basic AB15 pH Meter.

Data were presented as the mean ± standard deviation.

Determination of Betalain Content

Betacyanin and betaxanthin content was determined according to Castellar and others (2003) and Stintzing and others (2005) in mg equivalent betanin/L and mg equivalent indicaxanthin/L, respectively. Samples were diluted with McIlvaine buffer (pH

33

6.5, citrate-phosphate) to bring absorption values within optimum range. Betalain content was determined using the Beer-Lambert Law:

퐴 푥 퐷퐹 푥 푀푊 푥 1000 Betalain content [mg/L] = ɛ 푥 푙 where: A = absorbance, DF = dilution factor, MW = molecular weight (550 g/mol for betacyanins and 308 g/mol for betaxanthins), ɛ = extinction coefficient (60000 L/(mol cm) for betacyanins and 48000 L/(mol cm) for betaxanthins), and l = width of the spectrophotometer cell (1 cm).

Absorbance was observed at 535 nm for betacyanins and 484 nm for betaxanthins.

All readings were done on a Thermo Scientific Spectronic GENESYS 20 Visible

Spectrophotometer.

Determination of Phenols

Total phenolic content of each sample was determined with the modified Folin-

Ciocalteu colorimetric method as described by Dewanto and others (2002) and Singleton and others (1999). Prickly pear extract was diluted with deionized water to a known factor to bring the absorbance readings within the standard curve ranges of 0.0-600.0 µg of gallic acid/mL. In a cuvette, 83.33 µL of the diluted prickly pear extract was mixed with an additional 333.33 µL of deionized water followed by 83.33 µL of the Folin-Ciocalteu reagent (FCR). Cuvette was capped, inverted 3 times, then allowed to sit and react for six minutes. Following the six minutes, 833.33 µL of 7% carbonate (Na2CO3) was added before the final 666.67 µL addition of deionized water to bring the final volume of the cuvette to 2 mL. Samples were capped, inverted 3 times, then allowed to stand at room temperature for 90 minutes. After 90 minutes, the absorbance of each sample was measured at the wavelength of 760 nm. These absorbance values were then compared to the absorbance readings of the gallic acid standards with known concentrations to

34 determine the amount of phenolic compounds in each sample (See Appendix, Figure C. 1).

Final values were expressed as the mean (µg of gallic acid equivalents [GAE]/mL) ± standard deviation.

Determination of Antioxidant Capacity

Antioxidant capacity of each sample was determined using the improved ABTS radical cation decolorization assay according to Cassano and others (2007) and Rice-Evans and Miller (1994). ABTS was dissolved in deionized water to a concentration of 7 mM.

Oxidation of ABTS to ABTS+ radical cation was completed with 2.45 mM potassium persulfate (final concentration) in a volumetric ratio of 1:0.5 (ABTS:potassium persulfate)

(Re and others 1999). This mixture was allowed to react in the dark at room temperature for 12-16 hours. After the 12-16 hour period, the ABTS+ solution was diluted with ethanol to an absorbance of 0.70 ± 0.02 and equilibrated at 30⁰C. Prickly pear extract was diluted with deionized water to a known factor to bring the absorbance readings within the standard curve ranges of 0.0-300.0 µg of ascorbic acid/mL. For each sample, 20 µL of diluted sample was added to 2000 µL of diluted ABTS+ radical. Cuvettes were capped, mixed thoroughly, and left to sit for up to six minutes to react. After six minutes, the absorbance reading of each sample was recorded at 734 nm. The decolorization of the blue/green

ABTS+ chromophore was measured as the percentage of inhibition calculated relative to ascorbic acid equivalents under the same conditions (See Appendix, Figure C. 2). Ascorbic acid stock standards were diluted so that, after adding 20 µL of the different levels of ascorbic acid dilutions into 2.0 mL of the ABTS+ assay, there was a 20-80% inhibition of the blank absorbance. Final values were expressed as the mean compared to Ascorbic Acid

Equivalents Antioxidant Capacity (AAEAC) ± standard deviation.

35

Enzymatic Pretreatments

Varying Volumes of Crude Enzymes

Two crude enzyme mixtures were tested on their efficiency in increasing betalain concentration in raw prickly pear mash. The two enzymes used were Rapidase® Fiber

(DSM Food Specialties USA, Inc. | South Bend, IN) and ExtractSEB RLBE (Enzyme

Innovation | Chino, CA). Rapidase® Fiber is a liquid pectinase enzyme with arabinolytic activities from Aspergillus niger as well as cellulolytic activities from Trichoderma longibrachiatum. This crude enzyme is advertised in fruit and applications to solubilize pectin and cell wall structure, decrease mash viscosity, and improve juice and color extraction (DSM Food Specialties 2015). ExtractSEB RLBE is also a liquid pectinase enzyme that has high pectolytic activities of both polygalacturonase (endo-PG) and pectinlyase (endo-PL) along with hemicellulase activities of endo-arabanases and arabinogalactanases. This enzyme was developed specifically for the extraction and depectinization of red fruits during pressing, leading to clarification, an increase in color extraction, and an increase in juice yields (Enzyme Innovation 2018). Both enzyme mixtures have an optimum operating pH and temperature of 4.0 and 45-50⁰C (See

Appendix, Figure A. 1-2). However, due to the extreme heat sensitivity of betalains, all treatments were done at room temperature. No pH-adjustment was needed since prickly pear mash consistently measured within a pH range of 4.0-5.0, which was optimum for chosen enzyme mixtures.

Treatments began with 200 mL of prickly pear mash loaded in a beaker. Separate treatments were done for each additive (deionized (DI) water [to observe dilution effects],

Rapidase® Fiber, and ExtractSEB RLBE) at different concentrations (10, 7.5, 5, 2.5, and

36

1% v/w) as well as one control run. Immediately following treatment addition, mash was mixed for approximately 5 seconds followed by an ‘Initial’ sampling taken and stored at

-24⁰C for later analysis. Treatment then began with sample mixed with a Panasonic MX-

SS1 Hand-Held Immersion Blender set on the lowest setting and equipped with the wire whisk attachment. Mixer was also connected to a Staco® Powerstat Variable

Autotransformer Adjust-A-Volt® 3PN500B (AV6) that was set to 48 percentage of maximum output voltage, which ensured the slowest mixing speed possible (approximately

60 rpm) with available equipment. Treatment was carried out at room temperature (25⁰C) for a total of 4 hours with samplings taken at 15, 30, and 45 minutes and 1, 1 ½, 2, and 4 hours. Samples were frozen immediately after sampling and stored at -24⁰C until time of analysis. The betalain content and ⁰Brix of each sample at each time point was then determined in triplicate (See Appendix, Table B. 1-3).

To determine if treatment (enzyme v. deionized water v. control) and amount of additive (10, 7.5, 5, 2.5, and 1% v/w) had a significant effect on the dependent variables

(betacyanin and betaxanthin content), a One-Way ANOVA analysis with a Tukey Post Hoc

Test was ran on each sample at the first (initial) and final (4 hours) collection points using

IBM SPSS Statistics Software. Separate analyses were ran on each dependent variable.

Active Enzyme, Deactivated Enzyme, and a Blank Solution

A follow-up enzyme treatment was conducted to observe the effect of active enzymes on the sample. A 200 mL portion of prickly pear mash was divided into 5 separate beakers. Each beaker was subjected to a different treatment – 10% v/w deionized water,

10% v/w Rapidase® Fiber, 10% v/w deactivated Rapidase® Fiber, 10% v/w glycerol blank, and a control. Pectolytic and cellulolytic enzymes within Rapidase® Fiber were

37 deactivated by heating in a 90⁰C water bath for 5 minutes, then cooling to room temperature prior to addition to prickly pear sample (Naderi and others 2010). To see the effects of the major crude enzyme mixture composites, a ‘glycerol blank’ was also made following the specification sheet given by DSM Food Specialties (See Appendix, Table A. 1). This

‘glycerol blank’ consisted of 45% glycerol, 5% KCl, 5% NaCl, 5% , and 40% deionized water in the place of enzyme solutions, producing a clear solution. Following treatment addition, mash was mixed for approximately 5 seconds followed by an ‘Initial’ sampling taken and stored at -24⁰C for later analysis. Treatment then began with samples mixed on a Talboys Advanced Multi-Position Stirrer 5 (120V) at 600 rpm for 1 hour.

Samplings were taken at 15, 30, 45, and 60 minutes and frozen (-24⁰C) immediately until time of analysis. The betalain content and ⁰Brix of each sample at each time point were then determined in triplicate (See Appendix, Table B. 4-6). The initial and final phenolic content and antioxidant capacity were also determined for each treatment in triplicate (See

Appendix, Table B. 7-8).

To determine if treatment had a significant effect on the dependent variables

(betacyanin and betaxanthin content) over the course of time, a One-Way ANOVA analysis with a Tukey Post Hoc test was ran on each sample at initial and final time points using

IBM SPSS Statistics Software. Separate analyses were ran on each dependent variable.

38

Membrane Purification and Concentration via Micro- and Nano-filtration

Centrifugation via Basket Centrifuge

Prior to membrane filtration, prickly pear puree was clarified with a Rousselet

Robatel RA12Vx Laboratory/Bench Top Centrifuge equipped with a polypropylene back screen and a 1-3 micron polypropylene filter bag. Centrifuge was ran at 3000 rpm with prickly pear puree loaded into the feed tube until 4000 mL of filtrate was collected. This filtrate was then further clarified via microfiltration.

Purification via Microfiltration

Microfiltration (MF) of prickly pear puree was carried out with the FMA BT1812

Membrane Test Unit (Filtration Energy Solutions, San Diego, CA) equipped with a 5-7 liter stainless feed tank with drain valve, a positive displacement pump (1.1 GPM at 1000 psi capacity), and digital pressure monitoring systems for the permeate, concentrate, feed, and flow. The microfiltration membrane of choice was a spiral-wound polyethersulfone

(PES) TriSep TurboClean® 1812-MF01-31 filter with a molecular weight cut off (MWCO) of 0.1 microns (Microdyn Nadir, Goleta, CA).

Membrane was pre-soaked with deionized water for 30 minutes with open pressure prior to sample addition. The flux (rate of filtrate) of the membrane pre-sample filtration was measured in triplicate and determined with the following:

푄 퐽 = 푝 퐴푚 2 where: J = flux (gallons/ft /day), Qp = filtrate flow rate through membrane (gallons/day), 2 and Am = surface area of membrane (ft ).

After the 30-minute pre-soak treatment, water was drained from the membrane and

2000 mL of centrifuged sample was added to the feed tank. Temperature was monitored

39 via thermometer in the feed tank and kept under 30⁰C with the addition of ice packs on the feed lines when needed. Pressure was maintained at approximately 37 psi and permeate was collected in 200 mL increments. When sample in the feed tank ran low, 1000 mL of additional centrifuged prickly pear puree was added; this was repeated for the last 1000 mL of centrifuged sample. Once all permeate had been collected, retentate was drained from the feed tank and concentrate line.

Once all sample (permeate and retentate) had been removed from the membrane, the microfilter was rinsed with deionized water, drained, then repeated until feed was clear.

This clear deionized water was then ran through the membrane for 30 minutes. Following the water rinse, membrane unit was drained, then reloaded with a sodium hydroxide solution (pH 10.0) which was ran for 30 minutes followed by another round of deionized water (30 minutes). The flux post-sample filtration was determined and compared to that of the flux pre-treatment. Membrane rejection coefficients of different components of filtered juice were calculated with the following equation:

[퐶표푚푝표푛푒푛푡]푝 푀푒푚푏푟푎푛푒 푟푒푗푒푐푡푖표푛 푐표푒푓푓푖푐푖푒푛푡 = (1 − ) [퐶표푚푝표푛푒푛푡]푓

where: [Component]p = Component concentration in permeate and [Component]f = Component concentration in feed.

Concentration via Nanofiltration

Following microfiltration, the resulting permeate was concentrated via nanofiltration (NF). The nanofiltration membrane of choice was a spiral-wound cellulose- acetate (CA) TriSep TurboClean® 1812-SBNF-31 filter with a MWCO of 2000 Daltons

(Microdyn Nadir, Goleta, CA).

40

Membrane was pre-soaked with deionized water for 30 minutes with a constant pressure of 57 psi prior to sample addition. The initial flux was determined in triplicate before water was drained from feed tank and 2000 mL of MF permeate was added. Ice packs were used for temperature regulation, pressure was maintained at approximately 190 psi, and permeate was collected in 100 mL increments. When sample in the feed tank ran low, the remainder of the MF permeate was added. Once all permeate had been collected, retentate was drained from the feed tank and concentrate line and NF filter was cleaned in the same manner as the MF membrane. Post-treatment flux was determined in triplicate along with rejection coefficients for various components.

To determine if there was any significant difference in the dependent variables

(transmittance, [betalains], phenolic content, and antioxidant capacity) over the course of the treatment (initial, post-centrifugation, MF permeate/retentate, and NF permeate/retentate), a One-Way ANOVA with a Tukey Post Hoc Test was ran with IBM

SPSS Statistics Software. Separate analysis was done for each dependent variable at each filtration/concentration stage for each round of treatment.

Both MF and NF membranes were stored in a 0.5% sulfite solution until next use.

Entire filtration process (centrifugation, microfiltration, and nanofiltration) was performed in duplicate.

Further Concentration via Dehydrator

To best mimic comparable natural food colorants currently on the market, prickly pear concentrate resulting from nanofiltration was further concentrated via dehydration. A

25-mL portion of NF retentate was added to a petri dish, then ran in a Nesco® American

Harvest® Food Dehydrator & Jerky Maker at 35⁰C for 24 hours. This dehydrated sample

41 was then resuspended in glycerin in a w/w ratio of 4:1 (dried prickly pear concentrate:glycerin).

Stability of Prickly Pear Concentrate in a Model Food Matrix

Food Matrix Preparation

To test the stability of prickly pear concentrate as a food colorant, a proper food matrix needed to be developed; the matrix chosen with industrial suggestion was gelatin dessert. Gelatin dessert formula was created according to the Gelatin Manufacturers

Institute of America (GMIA) Gelatin Handbook (2012). Total resulting formulation consisted of 86.5% , 9.0% Gelatin (250 Bloom), 2.5% Fumaric Acid, and 1.2%

Sodium Citrate. Preparation began by first weighing out two separate powder mixtures –

¼ of the total formulation weights for the ‘cold’ mixture and ¾ of the weights for the ‘hot’ mixture. Total weights measured for each mixture are shown below (Table 1):

Table 1. Formulation for ‘Cold’ and ‘Hot’ Mixtures Used in Gelatin Dessert Preparation

Following the dry mixture preparations, 7.0 grams of gelatin powder (250 bloom) was also weighed and set aside. In a beaker, 180 mL of deionized water was brought to a gentle boil. While water was boiling, ‘cold mixture’ was added to 60 mL deionized water

(room temperature) and stirred until mostly dissolved. Gelatin powder (7.0 g) was then added to the cold mixture and let stand for approximately 1 minute. While gelatin was pre-

42 soaking, the ‘hot mixture’ powder was added to the boiling water and stirred with a stir bar until dissolved. Hot mixture was then gradually added to the cold mixture, swirling gently on occasion, until both mixtures were combined. Resulting mixture was then stirred continuously for 5 minutes, then cooled to room temperature. Procedure was repeated 4 times to prepare matrix for all 4 colorants tested as well as the control.

Colored Gelatin Dessert Sample Preparation

Once appropriate mixtures were developed, colorants were added in various concentrations to achieve relative intensity to one another. Total amounts of each colorant added to their respective mixture were: prickly pear concentrate (1.5 g), red beet concentrate (0.5 g), acid proof carmine (0.5 g), and FD&C Red 40 (0.05 g).

With the colorants added, six petri dishes containing 25 mL of the liquid mixture were made for each colorant type (24 total) and the control (6 total), then left for 12 hours to chill at 4⁰C until set. Samples of each colorant type/control were then divided and subjected to different storage temperatures – half at refrigeration temperature (4⁰C) and half at room temperature (25⁰C) – for 1-2 months in the dark (Cai and Corke 1999). Each pigment and temperature combination was ran in triplicate. Stability tests were ran in duplicate.

Color Analysis

Color measurements were recorded once a week according to the method adapted by Caldas-Cueva and others (2016) with a CR-400 Chroma Meter (Konica Minolta, Osaka,

Japan). Readings were taken at the center of the inverted petri dish for each sample with a white backdrop. Color measurement results were expressed as L*, a*, b* values (CIELab

System), chroma [C* = (a*2 + b*2)½], and hue angle (h = tan-1(b*/a*)).

43

To determine the stability of each colorant over the course of time, a One-Way

ANOVA analysis was ran for each dependent variable (L*, a*, b*, chroma, and hue angle) using IBM SPSS Statistics Software. A separate analysis was done for each storage condition (temperature) and colorant additive (prickly pear, red beet concentrate, carmine,

Red 40, and control). Statistical analysis values were expressed as the mean (n=6) ± standard deviation for triplicate samples of duplicate rounds.

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CHAPTER 4

RESULTS & DISCUSSION

Enzymatic Pretreatment

Varying Concentrations of Crude Enzymes

Various concentrations of crude enzyme mixtures Rapidase® Fiber and

ExtractSEB were used to check the effect on betacyanin content, betaxanthin content, and

⁰Brix; the observed effects were similar. Larger amounts of enzyme (% v/w) led to a greater decline in betalain content (Figure 2-3, 5-6). A similar trend was also observed with increasing additions of DI water, but the decline in betalains was not as drastic (Figure 4,7).

Figure 2. Change in betacyanin content of prickly pear during various Rapidase® Fiber enzyme treatments over 4 hours.

Figure 3. Change in betacyanin content of prickly pear during various ExtractSEB RLBE enzyme treatments over 4 hours.

45

Figure 4. Change in betacyanin content of prickly pear during various DI water treatments over 4 hours.

Figure 5. Change in betaxanthin content of prickly pear during various Rapidase® Fiber enzyme treatments over 4 hours.

Figure 6. Change in betaxanthin content of prickly pear during various ExtractSEB RLBE enzyme treatments over 4 hours.

46

Figure 7. Change in betaxanthin content of prickly pear during various DI water treatments over 4 hours.

Since a decline in betalain content was observed in treatments of DI water,

Rapidase® Fiber, and ExtractSEB RLBE, statistical analyses were conducted to see if there was a significant difference for each treatment at initial and final treatment times. Upon initial 10% v/w addition of the enzyme treatments, betacyanin content of the prickly pear mash had an immediate decrease of 45.06, 64.74, and 72.45% for deionized water,

Rapidase® Fiber, and ExtractSEB respectively relative to the control. After 4 hours, those same treatments were 44.50, 78.27, and 74.44% lower in betacyanins than the control

(Table 2). At initial treatment time, 5% and 7.5% Rapidase® treatments were significantly lower than all other additives. This was true for the 4-hour collection as well with 7.5%

Rapidase® seeming to cause the steepest decline in betacyanin content at both intervals.

47

Table 2. Initial and Final Concentration of Betacyanins in Prickly Pear Treated with Various Additives

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3). Separate One-Way ANOVA (Initial, p<0.001; 4 Hours, p<0.001) and Tukey’s HSD tests were done for each time point. a-m Mean values in the same column are significantly different at p < 0.05 using Tukey’s HSD test.

The negative correlation observed between enzyme dosage and betacyanin content agreed with the results of a similar study attempting to use enzymes to increase betacyanins in Hylocereus polyrhizus (Naderi and others 2010). They found that increasing the dosage of their chosen pectolytic enzyme, Pectinex Ultra-SPL isolated from Aspergillus aculeatus, resulted in steeper decline in the main betacyanins present. The treatment with the highest enzyme concentration (2% w/v) led to a 59.11, 77.57, and 44.88% decline in betanin, phyllocactin, and hylocerenin relative to the control.

When looking at betaxanthin concentrations, the 10% v/w treatments led to a

42.37% (DI water), 39.48% (Rapidase® Fiber), and 48.22% (ExtractSEB RLBE) decrease after 4 hours when compared to the control (Table 3). Active enzymes appeared to have less of a negative effect on betaxanthins than betacyanins in prickly pear samples.

48

Table 3. Initial and Final Concentration of Betaxanthins in Prickly Pear Treated with Various Additives

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3). Separate One-Way ANOVA (Initial, p<0.001; 4 Hours, p<0.001) and Tukey’s HSD tests were done for each time point. a-i Mean values in the same column are significantly different at p < 0.05 using Tukey’s HSD test.

Active Enzyme, Deactivated Enzyme, and a Blank Solution

Due to the negative effects that the crude enzymes had on betalains in the previous treatment, a follow-up treatment was designed to best determine what component of the enzyme mixtures was causing the decline in both betacyanins and betaxanthins.

Figure 8. Change in betacyanin and betaxanthin content of prickly pear during 10% v/w treatments of various additives (deionized water, Rapidase® Fiber, deactivated Rapidase®, and ‘glycerol blank’) versus control treatment over 1 hour.

49

Addition of DI water, deactivated Rapidase®, and glycerol blank did cause both betacyanins and betaxanthins to decrease, but the decline caused by active Rapidase® was more significant, especially in betacyanin content (Figure 8). To see the significance of the decrease in betalain content for each treatment, statistical analyses were conducted at initial and final treatment times.

Table 4. Initial and Final Concentration of Betacyanins in Prickly Pear in Follow-Up Enzyme Treatment

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3). Separate One-Way ANOVA (Initial, p<0.001; 1 Hour, p<0.001) and Tukey’s HSD tests were done for each time point. a-e Mean values in the same column are significantly different at p < 0.05 using Tukey’s HSD test.

All treatments resulted in a significant decrease in both betacyanins and betaxanthins when compared to the control; however, the decline observed in Rapidase®

Fiber samples was significantly larger than all other treatments. After the initial addition of each additive, treatments of DI water, Rapidase® Fiber, deactivated Rapidase®, and the glycerol blank caused 8.00, 42.59, 18.10, and 11.81% drops in betacyanins, respectively.

Betacyanin levels remained stable over the course of the 1-hour treatment for all additives except Rapidase® Fiber. The 42.59% decrease in betacyanin content initially observed with this treatment increased to a total 63.45% loss of betacyanin after 1 hour of Rapidase®

Fiber addition (Table 4). Treatments had similar effects on betaxanthin content of prickly pear mash. Additives DI water, Rapidase® Fiber, deactivated Rapidase®, and glycerol blank caused 8.20, 20.19, 10.91, and 9.74% drops in initial betaxanthins (Table 5).

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Table 5. Initial and Final Concentration of Betaxanthins in Prickly Pear in Follow-Up Enzyme Treatment

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3). Separate One-Way ANOVA (Initial, p<0.001; 1 Hour, p<0.001) and Tukey’s HSD tests were done for each time point. a-d Mean values in the same column are significantly different at p < 0.05 using Tukey’s HSD test.

These percent decreases were relatively similar to those seen in betacyanins, except for Rapidase® Fiber, which had less than half of the influence on initial betaxanthin levels

(20.19% decrease) as its impact on initial betacyanin levels (45.29% decrease). However, just as seen with betacyanins, the betaxanthin content for each treatment stayed the same for all additives aside from Rapidase® Fiber; the crude enzyme’s impact on betaxanthins’ decline grew from 20.19 to 33.61% loss over the course of 1 hour relative to the control.

From the results of this follow-up enzymatic treatment, it was concluded that the active enzymes in Rapidase® Fiber were responsible for significant loss of betalains observed in the previous treatment. The Rapidase® Fiber that had been deactivated by heat treatment prior to addition into prickly pear had nearly the same initial betacyanin levels

(94.563 mg/L) as the glycerol blank (101.823 mg/L) and DI water (106.223 mg/L) treatments (Table 4). The level of betacyanins within the deactivated Rapidase® treatment did lower slightly over 1 hour, but only by approximately 5 mg/L as opposed to active

Rapidase® enzyme mixture that caused roughly a 24 mg/L loss in betacyanin content over

1 hour of treatment beyond its significant initial 50 mg/L decrease (Table 4).

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A possible explanation for the negative impact on betacyanins displayed by both

Rapidase® Fiber and ExtractSEB RLBE is the potentiality of glucosidase activity by these two enzymes. The primary betacyanin in prickly pear, betanin, is subject to transformation to its respective aglycone, betanidin, through β-glucosidase activity. This reaction results in a bathochromic shift of about 4 nm, which can be beneficial if used to obtain a deep violet red (Herbach and others 2006). However, the aglycone has significantly less stability than its corresponding and is more susceptible to oxidation (Stintzing and Carle

2004).

As suggested by Zakharova and Petrova (2000), deglycosylation is just the first step in rendering these compounds accessible to oxidizing enzymes. Enzymes like β- glucosidase, peroxidases, and polyphenoloxidases responsible for betalain degradation and turnover are usually bound within the membrane (Shih and Wiley 1981; Wasserman and

Guilfoy 1983; Zakharova and Petrova 2000). As shown in a 1984 study on red beet root, partial cell wall digestion by enzymes like pectinase and cellulysin (a crude cellulase) were fair conditions for the release of decolorizing enzymes (Wasserman and Guilfoy 1984).

Due to the enzyme combinations used in this study (pectinase, hemicellulase and cellulase), cell wall components were likely broken down, thereby releasing cell contents (pigment molecules) as well as freeing peroxidase and polyphenoloxidase enzymes to oxidize betanin and betanidin, respectively.

A similar study on betacyanins in Beta vulgaris (red beet) found that oxidation of betanidin formed betanidin while oxidation of betanin formed betalamic acid as well as several oxidized cyclo-DOPA-5-O-α-D-Glucoside (Martínez-Parra and Muñoz

2001). These resulting products were like those of acidic, alkaline, or thermal degradation

52 of betanin (Figure 9). Oxidation of betacyanins yields products (cyclo-DOPA and cyclo-

DOPA-5-O-α-D-Glucoside) that are derivatives of 5,6-dihydroxindole and related structures, which are the key intermediates in melanogenesis (Wybraniec and Michalowski

2011). This all ultimately leads to the loss of red color and subsequent browning, which masks any remining bright red hue (Stintzing and others 2000).

Addressing the speed at which the effects of enzyme addition was seen in betalain concentrations of this study, similar studies have addressed the efficiency of peroxidase in the catalysis of betalains. In a 2001 study, betanidin and betanin concentrations decreased

76% and 18% just 15 seconds following reaction start (Martínez-Parra and Muñoz 2001).

This difference seen in catalysis efficiency of betanidin and betanin by peroxidase also suggested the involvement of both glucosidases and peroxidases in the catabolism of betalains, with β-glucosidase used to hydrolyze betanin (Martínez-Parra and Muñoz 2001).

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Figure 9. Betanin degradation pathway. (Source: Stintzing and Carle 2004)

54

Membrane Purification and Concentration via Micro- and Nano-filtration

Due to the negative impact both crude enzymes had on the betalain content within the sample, prickly pear juice was not subjected to any pretreatment prior to clarification steps, though this meant no decrease in viscosity of the sample nor throughput capacity and efficiency of the membrane (Lee and others 1982). The effectiveness of centrifugation via basket centrifuge as well as clarification and concentration via microfiltration (MF) and nanofiltration (NF) were analyzed. Phenolic content as well as antioxidant capacity at separate stages were also monitored due to the increased interest in functional foods and bioactive compounds in recent decades.

To determine the efficiency of sample clarification, transmittance was the main attribute considered. The sample used in the first round had a higher initial transmittance than the sample in round 2; this was most likely due to settling of larger particles within the sample pail during storage. Despite the slight difference in initial clarity, centrifugation did have similar effects both rounds, increasing initial transmittance about 3.5-fold for each treatment (See Appendix, Table C. 1). Figure 10 shows sample transmittance (%) at different stages for both rounds of treatment.

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Figure 10. Transmittance at different points of the clarification/concentration process of prickly pear juice for separate rounds. a-f Mean values within each round are significantly different at p < 0.05 using Tukey’s HSD test. Error bars represent SD.

There was a noticeable difference between performance of the microfilter between round 1 and round 2 with the permeate of the latter reaching significantly higher transmittance after filtration (100%) while the first only increased to just above 40%

(Figure 10). This contrast was because of a filter change; the filter used in the second round was unused while the filter used in round 1 had been utilized in preliminary trials. These results show the influence membrane fouling can have on filtration efficiency. As the pores of the filter become plugged, the membrane will experience irreversible fouling, ultimately affecting the long-term operation (Bessiere and others 2005).

Membranous flux during both MF and NF was monitored before and after sample filtration for this reason – as an indication of spoilage as well as expected filtration performance. For round 1 of MF, the pre-sample flux was nearly identical to that of round

2; however, after running filtration of the prickly pear juice, the flux of the MF filter used in round 1 decreased by 58.42% compared to the second filter that experienced an 85.99%

56 decline (Table 6). The steeper decline in flux rate seen in the second round of microfiltration implies more efficient clarification of the sample. Lower membrane flux suggests more solids were trapped in the pores of the filter due to the membrane itself being tighter and not allowing as many particles to pass through. However, regardless of MF performance, a juice with less solids resulted in both cases, as shown in the subsequent nanofiltration which had a consistent permeate flux for both rounds (Table 7).

Table 6. Membrane Flux Pre- and Post-Sample for Both Rounds of Microfiltration Performed

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3).

Table 7. Membrane Flux Pre- and Post-Sample for Both Rounds of Nanofiltration Performed

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3).

Despite the variations in membrane flux, both rounds of filtration yielded a clear nanofiltered permeate (NP) of 100% transmittance due to the NF filter’s high rejection rate.

However, in the main sample of interest (nanofiltered retentate [NR, NF Retent]/prickly pear concentrate) the clarity was significantly lower in round 1 (25.67%) than round 2

(96.73%) (See Appendix, Table C. 1). This was because the NR of round 1 garnered large

57 particles that were not rejected by the looser MF filter, leading to a permeate and NF starting point with lower clarity.

Unlike transmittance, pigment levels during both rounds of filtration behaved similarly at each stage. Values of betacyanins and betaxanthins (mg/L) in the prickly pear juice following centrifugation averaged 57.383 and 41.080. These levels were slightly lower than those reported by Stintzing and others (2003), but higher than those observed by Cassano and others (2010). However, levels of these pigments vary widely depending upon origin of the juice itself, so variability was to be expected (Fernández-López and

Almela 2001; Butera and others 2002; Stintzing and others 2003; Khatabi and others 2016).

Despite the prickly pear in the second round having a higher concentration in both betacyanins and betaxanthins following centrifugation, MF permeate had similar levels of betalains after microfiltration (Figure 11-12).

The concentration of betalain pigments via nanofiltration also had nearly identical results in duplication with the first round’s NR containing 111.28 and 69.07 mg betacyanins and betaxanthins/L and the second round’s containing 110.70 and 66.37 mg betacyanins and betaxanthins/L of sample, respectively. Results of post hoc analyses showed these pigment levels to be significantly greater than those seen at all previous steps within the filtration process for each individual round except for betaxanthins in MF retentate of round 2 (See Appendix, Table C. 2). When considering the levels of betalains in the feed, which in this case was the resulting permeate of microfiltration, nanofiltration led to an average increase of 237.02% of betacyanins and 217.89% of betaxanthins from the initial values (See Appendix, Table C. 2).

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Figure 11. Concentration of betacyanins at different points of the clarification/concentration process of prickly pear juice for separate rounds. a-e Mean values within each round are significantly different at p < 0.05 using Tukey’s HSD test. Error bars represent SD.

Figure 12. Concentration of betaxanthins at different points of the clarification/concentration process of prickly pear juice for separate rounds. a-e Mean values within each round are significantly different at p < 0.05 using Tukey’s HSD test. Error bars represent SD.

To determine the effectiveness of the chosen nanofilter in the concentration of pigments, the rejection coefficient was calculated for each betalain molecule classification.

For betacyanins, nanofiltration yielded an average rejection coefficient of 0.819 while filtration of betaxanthins resulted in a coefficient of 0.749. This meant that the chosen

59 nanofilter of cellulose acetate with a molecular weight cut off (MWCO) of 2000 Daltons

(Da) allowed for approximately 78.4% retention of betalain molecules in the retentate; the remaining 21.6% was lost to the permeate.

The use of a nanofilter with a lower MWCO could have resulted in more efficient concentration with less pigment molecules lost to the permeate. It has been shown that using membranes composed of the same material but lowering the MWCO from 5000 Da to 1000 Da results in a shift of 20% to 83% rejection rate of betanin by the filters (Bayindirli and others 1988). This was also seen in another study that observed when filtering red beetroot, lowering the size of the membrane from a 5000 Da MWCO to 1000 and 500 Da resulted in a 95 and 98% decrease in amount of betanin lost to the permeate (Mereddy and others 2017).

In the present study, the chosen nanofilter had a MWCO of 2000 Da but there was still a high rejection rate observed towards betalains, which have low molecular weights

(betacyanins: 550 Da; betaxanthins: 308 Da). Other studies using membranes with large pore sizes of 1000 Da also saw high betalain rejection rates of 83 and 92%, despite the low molecular weights of these pigment molecules (Bayindirli and others 1988; Mereddy and others 2017). This phenomenon can be explained assuming interactions of betalains with compounds otherwise-rejected by the membranes (Cassano and others 2010). Betalains are not only hydrophilic in nature, but because of the polarity of the O—H bond of its phenol group, betanin forms hydrogen bonds with other phenol molecules and different H-bonding systems (Slimen and others 2017). These complexes would be much larger than the pigment molecules on their own and could then be rejected by the nanofilter while those betalains that have not formed interactions are still capable of permeating through.

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The polarity of the membrane was also considered as a potential contributor to the observed betalain rejection of the nanofilter. However, the membrane of choice was made from cellulose-acetate. This synthetic fiber is hydrophilic in nature, capable of donating 8 and receiving 49 hydrogen bonds (NCBI 2018). As previously stated, betalains are also polar molecules, so any repulsion forces were not expected.

Figure 13. Phenolic content (expressed as gallic acid equivalent [GAE]) at different points of the clarification/concentration process of prickly pear juice for separate rounds. a-d Mean values within each round are significantly different at p < 0.05 using Tukey’s HSD test. Error bars represent SD.

Initial levels of phenols observed were greater than those of previous studies

(Stintzing and others 2005; Cassano and others 2010) but did not reach those seen by

Abdel-Hameed and others in 2014. Average initial antioxidant capacity of 504.235 µg

AAEAC/mL was also lower than that reported by the aforementioned study (Abdel-

Hameed and others 2014). However, MF and NF of prickly pear juice increased these levels an average of 148.80% for phenols and 124.03% for antioxidant capacity; in both cases, concentration in the NR was significantly higher than that observed in all previous stages (See Appendix, Table C. 3). When analyzing both rounds individually, the second round of filtration did consistently have higher levels of both polyphenols as well as 61 antioxidants at each stage (Figure 13-14). This was attributed to the higher initial concentration of betalains in the centrifuged sample of the second round.

Figure 14. Antioxidant capacity (expressed as ascorbic acid equivalent antioxidant capacity [AAEAC]) at different points of the clarification/concentration process of prickly pear juice for separate rounds. a-d Mean values within each round are significantly different at p < 0.05 using Tukey’s HSD test. Error bars represent SD.

Betalains like betanin are phenolic and are readily detected by assays monitoring phenolic content of the sample like the Folin Ciocalteu method. As such, an increase in betalain concentration would contribute to increased phenol detection in terms of GAE units. A similar effect would be expected in methods quantifying antioxidant capacity. Betanin has been shown to be 1.5-2.0-fold better at radical scavenging than -3-O-glucoside and cyanidin (Borkowski and others 2005), the latter of which exhibited one of the highest anti-radical activities as determined by the Trolox equivalent antioxidant capacity (TEAC) assay at pH 7.4 (Rice-Evans and others 1996). Betalains have even shown to have more efficient in vitro antioxidant activities than ascorbic acid, , and phenolic acids (Walle 2011; Del Rio and others 2013; Gandía-Herrero and others 2013; Khan 2015). Therefore, the significant increases in both antioxidant capacity

62 as well as phenolic content can be linked to the increase in betalain concentration observed in addition to any other compounds (polyphenols, etc.) that were also likely separated and concentrated to culminate in the finalized NF retentate.

Stability of Prickly Pear Concentrate in a Model Food Matrix

L*, a*, b* Values

Gelatin dessert was prepared using prickly pear concentrated colorant and other standard colors; the stability with time and temperature was evaluated using L*, a*, and b* values. The L*, a*, and b* values for gelatin dessert samples of various colorant types were not uniform at the start due to the variability in colorant intensity, as shown by previous studies (Fernández-López and others 2013). Therefore, looking at the trends of each variable relative to time and storage temperature for pigments independently was critical.

When studying colorants, L* is an important parameter because it is correlated with the color intensity of a sample (Cai and Corke 1999). If L*, or lightness, were to show an increase in a relatively clear food matrix such as gelatin, it would be indicative of color fading. In refrigerated conditions, prickly pear and red beet samples did show an increase in lightness while carmine and Red 40 showed high stability (Figure 15). When running post hoc analyses on L* values (recorded in triplicate for two rounds) over time, prickly pear samples showed no significant increase or decrease while red beet showed significant increase in lightness almost each week (See Appendix, Table D. 1). When each round is considered individually, a high amount of variability was seen in the measurements for prickly pear (Figure 15). This was attributed to different betalain concentrations of the starting colorant after running through the dehydrator and being resuspended in glycerin.

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At room temperature, Red 40 was the only colorant that did not undergo significant color change (See Appendix, Table D. 2). Samples colored with carmine had a slow, steady increase in lightness over the 4-week monitoring period which differed from the trends observed for both betalain colorants (Figure 16). L* values for both red beet and prickly pear colored gelatin dessert showed significant lightness increase after the first week (See

Appendix, Table D. 2). After the initial 12-point increase in its lightness, the L* values for red beet remained steady during the remainder of the trials, close to the values of the control gelatin dessert that contained no colorants. This immediate rise in lightness demonstrated the speed at which higher temperatures cause the degradation of betalain molecules, causing the red beet samples to jump to the same L* values as the unpigmented samples.

Rapid degradation by higher storage temperatures was also seen in prickly pear, which increased a significant 6.15 points in its L* values after the first week. Following this initial increase, however, there was a significant decline in lightness that went below the samples’ initial levels (See Appendix, Table D. 2). This was due to mold growth that, after week 2, continued to grow in all prickly pear samples kept at 25⁰C. The red beet, carmine, and Red 40 colored gelatin dessert also had mold growth, but not until week 3-4.

All samples experienced mold growth likely due to failure to properly sterilize all equipment before storage (Cai and Corke 1999). The increased mold susceptibility in prickly pear samples was attributed to possible contamination during the dehydration and glycerin resuspension stages of the prickly pear concentrate. Also, as mentioned previously, cactus pear juice has a relatively low concentration of acids and high soluble solid content, which makes it highly susceptible to microbial attack (Joubert 1993).

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Figure 15. Measured L* values of refrigerated (4⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 8 weeks. Results are shown for three triplicate samples prepared in duplicate rounds (n=6).

Figure 16. Measured L* values of room temperature (25⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 4 weeks. Results are shown for three triplicate samples prepared in duplicate rounds (n=6).

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In color determination, a* values correspond to the redness of the sample (Hani and others 2015). In both refrigerated and room temperature conditions, Red 40 samples experienced little-to-no degradation in redness while carmine showed more significant decrease in a* at room temperature, dropping a total of 9.14 points at 25⁰C as opposed to

2.79 points at 4⁰C (See Appendix, Table D. 3-4). Samples colored with carmine, however, did not display as significant a decline in a* values as those seen in the betalain pigments

(Figure 17-18). In refrigerated conditions, red beet samples experienced significant decline in a* each week over the 8-week monitoring period, values declining a total of 58.30% from initial levels (See Appendix, Table D. 3). Prickly pear samples showed an even steeper decline, a* values lowering nearly 82% from initial levels (See Appendix, Table

D. 3). Even with this larger drop, it is important to note that starting a* values for prickly pear gelatin dessert were nearly half of those for red beet. As previously mentioned, the concentration of betalains plays an important role in the relative stability of the pigment molecules themselves (Merin and others 1987). This could explain the why a* values of the prickly pear samples fell at a greater rate than the red beet, further justifying the need for a tighter nanofilter to inhibit loss of betalain molecules to the permeate and result in a more concentrated colorant.

The values for a* within the gelatin dessert samples stored at room temperature behaved similarly to L* values. For red beet and prickly pear colored gelatin dessert, decline was immediate with effects seen after one week. Following the 14.56 and 23.25- point drop of week 1, prickly pear and red beet colored samples remained relatively unchanged, with a* comparable to the control gelatin dessert.

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Figure 17. Measured a* values of refrigerated (4⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 8 weeks. Results are shown for three triplicate samples prepared in duplicate rounds (n=6).

Figure 18. Measured a* values of room temperature (25⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 4 weeks. Results are shown for three triplicate samples prepared in duplicate rounds (n=6).

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The b* values of samples (yellow-blue dimension) behaved similarly to both L* as well as a*. Red 40 as a colorant was stable and showed insignificant change in both storage conditions (See Appendix, Table D. 5-6). Carmine showed steady increase in b* under both conditions; however, the increase observed at 25⁰C was significantly greater than that seen at 4⁰C as evident by the larger slope of the trendline at room temperature (Figure 19-

20). When refrigerated, both prickly pear and red beet showed a steady decline in b* values over 8 weeks, and at room temperature, a significant increase of 4.89 and 13.25 points happened within the first week followed by a leveling of b* values for the remainder of storage (See Appendix, Table D. 6).

In a study that looked at color stability of yogurt samples containing betacyanins of ayrampo (Opuntia soehrensii) extract and red beet extract, a* and b* values had no significant change after 35 days of storage at 4⁰C (Caldas-Cueva and others 2016). For the present study, however, a* and b* values both showed significant change after the first week when stored at 4⁰C. This could have been because of the high concentration of sucrose within the gelatin dessert samples as research by Cai and Corke has shown that a food matrix with more than 13% sucrose causes betacyanin content to decrease (1999).

These findings suggest that betalain pigments might be best suited for a refrigerated food matrix that is lower in sugar content, such as a yogurt.

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Figure 19. Measured b* values of refrigerated (4⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 8 weeks. Results are shown for three triplicate samples prepared in duplicate rounds (n=6).

Figure 20. Measured b* values of room temperature (25⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 4 weeks. Results are shown for three triplicate samples prepared in duplicate rounds (n=6).

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Chroma and Hue Angle

Color purity/saturation, as indicated by calculated chroma (C), varied between samples depending upon both the colorant used as well as the temperature the samples were stored. For color monitoring, a higher chroma value corresponds to a more vivid sample

(Francis 1985); this was seen after initial preparation when samples colored with Red 40 were noticeably more vivid than the others, which was supported by the larger initial chroma values. At 4⁰C, the control and Red 40 gelatin dessert samples had no significant change over 8 weeks while C of carmine colored samples experienced a steady decline

(See Appendix, Table D. 7). The loss of saturation within the prickly pear samples was similar to the loss of carmine samples, which each had a trendline slope of -0.4519 and

-0.3711 over the 8 weeks. Red beet samples, with a trendline slope of -1.8464, experienced the greatest loss of color purity under refrigerated conditions (Figure 21).

At room temperature, carmine samples had a steeper decline than at refrigerated temperatures (Figure 22). However, this 26% loss in chroma of carmine samples did not compare to the 46 and 63% decline seen in prickly pear and red beet samples after just the first week, further displaying betalains’ sensitivity to higher temperatures in comparison to other natural pigments. As with L*, a*, and b* values, the chroma of prickly pear and red beet gelatin dessert samples remained relatively stable after this first week, implying all major observable effects that temperature had on color purity was rapid (See Appendix,

Table D. 8). These results confirmed other studies that found higher temperatures of 25⁰C or more greatly accelerated the degradation of betacyanins in jelly and dessert gels (Driver and Francis 1979; Cai and Corke 1999).

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Figure 21. Calculated chroma values of refrigerated (4⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 8 weeks. Results are shown for three triplicate samples prepared in duplicate rounds (n=6).

Figure 22. Calculated chroma values of room temperature (25⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 4 weeks. Results are shown for three triplicate samples prepared in duplicate rounds (n=6).

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When dealing with pigments, hue angle indicates sample color (0 or 360⁰ = red; 90⁰

= yellow; 180⁰ = green; 270⁰ = blue) (Francis 1985). A sample that is more violet-red has a lower hue angle closer to 0⁰ (Cai and Corke 1999). Initial values of the individual colorant types showed the similarity in red beet and carmine with more of a violet-red hue at angles

352 and 353, which differed from prickly pear with a brighter red at angle 12 and Red 40 with a bright orange-red hue at angle 35 (See Appendix, Table D. 9-10). Under storage at both 4 and 25⁰C, uncolored samples and samples colored with Red 40 had stable hue with no significant change. As shown in figures 23 and 24, carmine-colored samples experienced a change in hue nearly 10-times greater at the higher temperature when comparing the slopes of the trendlines. This change, however, was not as great as the shift in hue angle seen in the betalain pigments. Prickly pear and red beet increased by 67 and

35⁰ over 8 weeks of refrigerated storage, each sample growing closer to the yellow hue of the uncolored gelatin dessert at an angle of 100 (See Appendix, Table D. 9). As with previous variables, the effects of higher temperature (25⁰C) on the hue angle were significant after just one week of storage. The prickly pear colorant plateaued in its deterioration and its hue remained unchanged after week 1, but red beet continued to shift to a more yellow hue until the second week of monitoring. After week 2, both colorants’ samples had nearly reached the hue angle of the uncolored control gelatin dessert (See

Appendix, Table D. 10).

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Figure 23. Calculated hue angle values of refrigerated (4⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 8 weeks. Results are shown for three triplicate samples prepared in duplicate rounds (n=6).

Figure 24. Calculated hue angle values of room temperature (25⁰C) gelatin dessert samples colored with prickly pear, red beet, acidified carmine, and Red 40 monitored over 4 weeks. Results are shown for three triplicate samples prepared in duplicate rounds (n=6).

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For all variables, Red 40 and the control samples showed no significant change at both refrigerated and room temperature storage. Carmine consistently had a higher degradation rate at 25⁰C than at 4⁰C, implying sensitivity to heat. Effect of higher temperatures, however, was most drastically seen in gelatin dessert samples colored with betalain pigments, which was in agreement with findings by Fernandez-Lopez and others

(2013) looking at the thermal stability of various red food colorants. For all variables, prickly pear and red beet samples had significant degradation under refrigerated temperatures. At room temperature, the heat caused almost immediate degradation of pigment molecules with L*, a*, b*, chroma, and hue angle of samples plateauing in significant change after one week of storage in most cases. These results further demonstrate the importance of storage condition considerations when incorporating both prickly pear and red beet colorants into a food matrix given how much their degradation rate depends on temperature (Saguy and others 1978).

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CHAPTER 5

CONCLUSIONS

Objective 1

Enzyme Use to Increase Color Yield

The two chosen crude enzyme mixtures, Rapidase® Fiber and ExtractSEB RLBE, had negative effects on the raw prickly pear mash. Larger amounts of enzyme added (% v/w) led to greater declines in both betacyanins and betaxanthins, with 10% v/w additions of both Rapidase® Fiber and ExtractSEB lowering initial betacyanin levels 64.74 and

72.45% relative to the control. Follow-up treatment with active and deactivated enzymes along with controls not only supported the degradation effects of enzymes observed in the first round of treatments, but also showed that treatment with active crude enzymes did not yield a significant increase in bioactive compounds of interest. Therefore, the first hypothesis (H1a) was not supported and the first objective could not be fully achieved.

Membrane Purification and Concentration

Despite the negative results of the first aim of this objective, the second proved to be successful in the purification and concentration of prickly pear juice. Microfiltration removed up to 94.53% of turbidity and led to a stable flux rate with subsequent filtrations.

The cellulose acetate nanofilter had a 78.4% retention rate during the concentration of betacyanins and betaxanthins, increasing the levels of each colorant by 237.02 and

217.89%. With the significant increases in both transmittance and betalain content following membrane filtration, the hypothesis associated with this specific aim (H1b) was supported. This meant that half of the first objective’s goals were reached.

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Objective 2

Maintaining Beneficial Properties of Prickly Pear

Due to the increasing value and interest in bioactive ingredients, the phenols and antioxidants naturally present in prickly pear juice were a focal point throughout the purification and concentration process. Micro- and nanofiltration with the use of the crossflow membrane unit not only led to a successful increase in sample transmittance and pigment molecules, but also yielded a concentrate that, on average, was 148.80 and

124.03% higher in both phenols and antioxidant capacity relative to centrifuged prickly pear juice. This significant increase in bioactive compounds supported the associated hypothesis (H2) and the second objective of this project was reached.

Objective 3

Stability of Prickly Pear Concentrate in a Model Food Matrix

Data collected for samples stored at room temperature (25⁰C) showed that L*, a*, b*, chroma, and hue angle for prickly pear and red beet colorants followed very different patterns than carmine and Red 40. This was expected due to betalains’ sensitivity to heat.

At refrigerated temperatures, however, the difference between the two sets was not as drastic. Future studies that utilize a more uniform starting point for L*, a*, and b* values between the different colorants will be very insightful to the speed of degradation of these various pigment sources. However, results from this study support the feasibility of prickly pear as a replacement for red beet and potentially carmine when stored at refrigerated temperatures. Therefore, the hypothesis associated with the third objective (H3) was successfully supported.

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CHAPTER 6

PROJECT LIMITATIONS AND FUTURE STUDIES

Project Limitations

Samples

For the enzymatic pretreatment, the only prickly pears utilized were those available for purchase at a local market. However, prickly pears from various regions have been shown to contain differing amounts of betacyanins and betaxanthins as well as antioxidants. The use of the tested enzymes could prove to be beneficial for a project focused on phenols or antioxidants and less on the color of the product itself. Prickly pear cactus fruits lower in betanin, the betacyanin theorized to have the most sensitivity to degradation effects of the enzyme mixtures, could have also been used.

For the membrane purification and concentration trials, commercially available prickly pear juice was the only sample ran because of this project’s relation to industry.

While the results were positive and in favor of this method to yield a concentrated colorant, testing the effects on raw prickly pear mash could also be a feasible means of natural colorant production.

Enzyme Treatments to Increase Yield

Due to the large number of sample collections as well as the negative influence observed on betacyanin and betaxanthin content caused by enzyme addition, phenolic content and antioxidant capacity of prickly pear mash was not monitored over the course of all treatments. Though there was no significant difference in these parameters seen in the initial and final (1 hour) samples of the follow-up treatment, the increased interest in

77 bioactive ingredients to the food industry advocates for future studies focused on the effect of enzyme addition on these compounds.

Membrane Purification & Concentration

During the purification and concentration of prickly pear juice with the crossflow membrane unit, a clarified and concentrated product was achieved. However, as shown by the difference in MF permeate transmittance from round 1 compared to round 2, the use of a new microfilter is critical in the filtration process. During nanofiltration, there was also betalain content lost to the permeate feed due to the high MWCO of the membrane filter.

The use of a nanofilter with a lower MWCO could have resulted in a more suitable concentrate that eliminated the need for the use of the dehydrator prior to incorporation into the model food matrix. The purchase of new micro- and nanofilters was not feasible for this project. However, this study provides useful information in the efficiency of the chosen membranes and can be used to guide similar projects in the future.

Stability Testing in a Model Food Matrix

During the comparison of several colorants incorporated in gelatin dessert, the amount of colorant added to the gelatin dessert mixture prior to pouring into individual petri dishes for monitoring was determined by visual judgement. However, the larger preparations of each colorant proved to be deceptive; once divided into 25 mL aliquots and allowed to set within the petri dishes, it became apparent that the starting colors of the samples were quite distant from one another. The samples colored with prickly pear were lighter than the red beet and carmine counterparts. Further studies monitoring stability of prickly pear concentrate should utilize instrumentation to best achieve initial color levels

78 that are uniform across the colorants tested so a more accurate comparison of color degradation over time can be made.

Future Studies

Enzyme Treatments to Increase Yield

The crude enzymes tested during this study were limited to just 2 mixtures that were given by industry recommendation. Testing different crude enzyme mixtures with varying degree of pectolytic, hemicellulolytic, and cellulolytic activity would yield better insight and potential support for the betalain degradation effects observed during this project.

Monitoring the complete visible spectrum during enzyme treatment as opposed to just the wavelengths at maximum absorbance for betacyanins and betaxanthins would also be of interest to efficiently monitor wavelength shifts and so the shape of the spectral curve is not disregarded.

The effects the tested enzyme mixtures have on prickly pear mash can be further monitored by high performance liquid chromatography (HPLC). This method will not only quantify betalain content within the sample but can also be used to identify specific colorant molecules present as well as the products resulting from their degradation. This information can offer insight into the specific interactions between the enzymes and the pigment molecules within the prickly pear.

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Membrane Purification and Concentration

As mentioned in the project limitations, the use of nanofilters with lower MWCO will likely yield a more acceptable prickly pear concentrate and should be a topic of future studies. Spoilage monitoring of resulting permeate and retentate (microbial studies) could also be a potential topic for future studies since membrane processes have been shown to produce concentrated product that is a clarified fraction free of spoilage microorganisms

(Cassano and others 2007). This is in agreement with another study that found increasing the concentration of prickly pear in wool dyeing solution led to an increased inhibition zone of all tested microorganisms (Bacillus subtilus, E. coli, and Sphyl A.) for dyed wool

(Ali and El-Mohamedy 2011).

Sensory analysis of resulting products of micro- and nanofiltration in terms of consumer acceptability could also be of interest. This is due to the fact that previous studies have shown unsatisfactory acceptability rankings for concentrated juice produced by alternative methods (Alfa-laval centrifuge vacuum evaporator) on a 9-point Hedonic scale

(Cassano and others 2007). This was due to damage to the color and the herbaceous aroma after the concentration process (Sáenz and others 1993).

Stability Testing in a Model Food Matrix

This project further supported the well-documented sensitivity that betalains have towards higher temperatures. As such, betalains as a food colorant are most feasible for incorporation into refrigerated/frozen products with a shorter shelf life. This study exclusively measured colored food samples stored in refrigerated conditions and compared them to those kept at room temperature. However, monitoring food matrices colored with prickly pear and competing colorants at temperatures common to frozen products could be

80 beneficial since betacyanins have been shown to have high color retention (94.4%) after

18 weeks under these conditions (Cai and Corke 1999). The incorporation of additives like ascorbic acid and isoascorbic acid into the food matrix along with the prickly pear concentrate should also explored since supplementation with these antioxidants has been shown to prevent betalain pigment degradation during thermal treatment and could increase the colorant’s stability during storage (Bilyk and Howard 1982; Han and others 1998).

Stability tests of this study were limited to samples colored with only one amount/concentration of each colorant type. However, the use of natural prickly pear extracts has been found to be heavily dependent on the concentration of the pigment molecules themselves with betalain stability having a positive correlation with its relative concentration (Merin and others 1987). Testing the colorants at different concentrations would not only show the influence higher concentrations have on product stability, but would also be useful for further determining the feasibility of membrane-concentrated prickly pear colorant, given a side-by-side comparison of the amount of colorant needed to reach similar hue and chroma levels as the alternatives.

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APPENDIX A Industrial Enzymes Specification Sheets

Figure A. 1. Activity specification sheet for Rapidase® Fiber enzyme (DSM 2015).

Figure A. 2. Activity specification sheet for ExtractSEB RLBE enzyme (Enzyme Innovation 2018).

Table A. 1. Confirmed composition of Rapidase® Fiber enzyme (DSM 2015)

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APPENDIX B Enzyme Pretreatment Results

Varying Volumes of Crude Enzymes

Table B. 1. Betacyanin Concentration in Prickly Pear Juice During Different Enzymatic Treatments Over 4 Hours

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3).

100

Table B. 2. Betaxanthin Concentration in Prickly Pear Juice During Different Enzymatic Treatment Over 4 Hours

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3).

101

Table B. 3. Total Soluble Solids (⁰Brix) in Prickly Pear Juice During Different Enzymatic Treatment Over 4 Hours

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3).

102

Active Enzyme, Deactivated Enzyme, and a Blank Solution

Table B. 4. Betacyanin Concentration in Prickly Pear Juice During Follow-Up Enzyme Treatment Over 1 Hour

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3).

Table B. 5. Betaxanthin Concentration in Prickly Pear Juice During Follow-Up Enzyme Treatment Over 1 Hour

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3).

Table B. 6. Total Soluble Solids (⁰Brix) in Prickly Pear Juice During Follow-Up Enzyme Treatment Over 1 Hour

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3).

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Table B. 7. Initial and Final Phenolic Content in Prickly Pear Juice During Follow-Up Enzyme Treatment of 1 Hour

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3).

Table B. 8. Initial and Final Antioxidant Capacity in Prickly Pear Juice During Follow-Up Enzyme Treatment of 1 Hour

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3).

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APPENDIX C

Membrane Purification and Concentration Results

Table C. 1. Transmittance (%) of Prickly Pear Sample at Various Stages of Both Rounds of Centrifugation, Microfiltration, and Nanofiltration

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3). Separate One-Way ANOVA (Round 1, p<0.001; Round 2, p<0.001) and Tukey’s HSD tests were done for each round. a-f Mean values in the same column (round) are significantly different at p < 0.05 using Tukey’s HSD test.

Table C. 2. Betalain Content of Prickly Pear Sample at Various Stages of Both Rounds of Centrifugation, Microfiltration, and Nanofiltration

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3). Separate One-Way ANOVA (p<0.001 for all) and Tukey’s HSD tests were done for each variable during each round. a-e Mean values in the same column (betacyanins/betaxanthins) for the same round are significantly different at p < 0.05 using Tukey’s HSD test.

105

Table C. 3. Total Phenolic Content and Antioxidant Capacity of Prickly Pear Sample at Various Stages of Both Rounds of Centrifugation, Microfiltration, and Nanofiltration

Values are expressed as the mean of triplicate measurements ± standard deviation (n=3). Separate One-Way ANOVA (p<0.001 for all) and Tukey’s HSD tests were done for each variable during each round. a-d Mean values in the same column (phenols/antioxidant capacity) for the same round are significantly different at p < 0.05 using Tukey’s HSD test.

Figure C. 1. Standard curve of gallic acid used for phenolic content determination.

106

Figure C. 2. Standard curve of ascorbic acid used for antioxidant capacity determination.

107

APPENDIX D

Stability Test in a Model Food Matrix Results

Table D. 1. Measured L* Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored in Refrigerated Temperatures (4⁰C) Over the Course of 8 Weeks

Values are expressed as the mean of triplicate measurements ± standard deviation of duplicate trials (n=6). Separate One-Way ANOVA (Prickly Pear, p=0.506; Red Beet, p<0.001; Carmine, p=0.016; Red 40, p=0.950; Control, p=0.997) and Tukey’s HSD tests were done for each colorant type. a-f Mean values in the same column (colorant) are significantly different at p < 0.05 using Tukey’s HSD test.

Table D. 2. Measured L* Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored at Room Temperature (25⁰C) Over the Course of 4 Weeks

Values are expressed as the mean of triplicate measurements ± standard deviation of duplicate trials (n=6). Separate One-Way ANOVA (Prickly Pear, p<0.001; Red Beet, p<0.001; Carmine, p<0.001; Red 40, p=0.224; Control, p=0.129) and Tukey’s HSD tests were done for each colorant type. a-c Mean values in the same column (colorant) are significantly different at p < 0.05 using Tukey’s HSD test.

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Table D. 3. Measured a* Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored in Refrigerated Temperatures (4⁰C) Over the Course of 8 Weeks

Values are expressed as the mean of triplicate measurements ± standard deviation of duplicate trials (n=6). Separate One-Way ANOVA (Prickly Pear, p<0.001; Red Beet, p<0.001; Carmine, p=0.002; Red 40, p=0.005; Control, p=1.000) and Tukey’s HSD tests were done for each colorant type. a-i Mean values in the same column (colorant) are significantly different at p < 0.05 using Tukey’s HSD test.

Table D. 4. Measured a* Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored at Room Temperature (25⁰C) Over the Course of 4 Weeks

Values are expressed as the mean of triplicate measurements ± standard deviation of duplicate trials (n=6). Separate One-Way ANOVA (Prickly Pear, p<0.001; Red Beet, p<0.001; Carmine, p<0.001; Red 40, p=0.695; Control, p=0.556) and Tukey’s HSD tests were done for each colorant type. a-d Mean values in the same column (colorant) are significantly different at p < 0.05 using Tukey’s HSD test.

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Table D. 5. Measured b* Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored in Refrigerated Temperatures (4⁰C) Over the Course of 8 Weeks

Values are expressed as the mean of triplicate measurements ± standard deviation of duplicate trials (n=6). Separate One-Way ANOVA (Prickly Pear, p<0.001; Red Beet, p<0.001; Carmine, p<0.001; Red 40, p=0.182; Control, p=1.000) and Tukey’s HSD tests were done for each colorant type. a-i Mean values in the same column (colorant) are significantly different at p < 0.05 using Tukey’s HSD test.

Table D. 6. Measured b* Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored at Room Temperature (25⁰C) Over the Course of 4 Weeks

Values are expressed as the mean of triplicate measurements ± standard deviation of duplicate trials (n=6). Separate One-Way ANOVA (Prickly Pear, p<0.001; Red Beet, p<0.001; Carmine, p<0.001; Red 40, p=0.783; Control, p=0.835) and Tukey’s HSD tests were done for each colorant type. a-e Mean values in the same column (colorant) are significantly different at p < 0.05 using Tukey’s HSD test.

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Table D. 7. Calculated Chroma Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored in Refrigerated Temperatures (4⁰C) Over the Course of 8 Weeks

Values are expressed as the mean of triplicate measurements ± standard deviation of duplicate trials (n=6). Separate One-Way ANOVA (Prickly Pear, p=0.015; Red Beet, p<0.001; Carmine, p=0.001; Red 40, p=0.008; Control, p=1.000) and Tukey’s HSD tests were done for each colorant type. a-h Mean values in the same column (colorant) are significantly different at p < 0.05 using Tukey’s HSD test.

Table D. 8. Calculated Chroma Values of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored at Room Temperature (25⁰C) Over the Course of 4 Weeks

Values are expressed as the mean of triplicate measurements ± standard deviation of duplicate trials (n=6). Separate One-Way ANOVA (Prickly Pear, p<0.001; Red Beet, p<0.001; Carmine, p<0.001; Red 40, p=0.668; Control, p=0.815) and Tukey’s HSD tests were done for each colorant type. a-c Mean values in the same column (colorant) are significantly different at p < 0.05 using Tukey’s HSD test.

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Table D. 9. Calculated Hue Angles of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored in Refrigerated Temperatures (4⁰C) Over the Course of 8 Weeks

Values are expressed as the mean of triplicate measurements ± standard deviation of duplicate trials (n=6). Separate One-Way ANOVA (Prickly Pear, p<0.001; Red Beet, p<0.001; Carmine, p<0.001; Red 40, p=0.921; Control, p=0.999) and Tukey’s HSD tests were done for each colorant type. a-h Mean values in the same column (colorant) are significantly different at p < 0.05 using Tukey’s HSD test.

Table D. 10. Calculated Hue Angles of Gelatin Dessert Samples Colored with Various Natural and Synthetic Colorants Stored at Room Temperature (25⁰C) Over the Course of 4 Weeks

Values are expressed as the mean of triplicate measurements ± standard deviation of duplicate trials (n=6). Separate One-Way ANOVA (Prickly Pear, p<0.001; Red Beet, p<0.001; Carmine, p<0.001; Red 40, p=0.997; Control, p=0.846) and Tukey’s HSD tests were done for each colorant type. a-d Mean values in the same column (colorant) are significantly different at p < 0.05 using Tukey’s HSD test.

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