COMPARATIVE STABILITY OF PHYTONUTRIENTS

IN FUNCTIONAL BEVERAGES STORED UNDER DIFFERENT ENVIRONMENTS

THESIS

Presented in Partial Fulfillment of the Requirements for the Degree Master of Science

in the Graduate School of The Ohio State University

By

Shirley Yunita Witarsa, B.S.

Graduate Program in Food Science and Technology

The Ohio State University 2011

Thesis Committee:

M. Monica Giusti, Advisor

John Litchfield

Michael Mangino

Copyright by

Shirley Yunita Witarsa

2011

Abstract

The increasing consumer demand for foods that can enhance health, coupled to desire for lower sugar and calories intake has made functional beverages very popular.

One major challenge in functional beverages development is that most nutrients and vitamins added are sensitive to the processing and storage conditions. A new system for delivery of phytonutrients into a beverage was evaluated which uses an enclosed chamber attached to the bottle (caps) to keep the ingredients in the dry form until released just prior to consumption. This experiment investigated the ability of these caps to deliver and preserve the chemical integrity of nutrients used in the beverage industry including ascorbic acid, hydroxycitric acid, riboflavin, pyridoxine, pyridoxal phosphate, niacinamide, and caffeine.

Formulations containing known amounts of each nutrient were stored under four different conditions: in a cap attached to a bottle(1) or stored in a bag (2), hot-filled

(3) and dissolved in water (4). Three different storage environments were applied: at

37˚C incubator without light exposure, at room temperature and exposed to light, at room temperature without light exposure. Samples were analyzed by HPLC-PDA-MS.

Light exposure significantly reduced the stability of ascorbic acid, riboflavin, pyridoxine, and caffeine in commonly processed beverages. However, when ascorbic

ii acid, riboflavin, pyridoxine, and caffeine stored in dry environment (inside a cap), significantly higher stability was obtained even under light exposure.

Higher temperature storage significantly decreased the stability of ascorbic acid, hydroxycitric acid, and pyridoxal phosphate in commonly processed beverages.

However, dry storage of those compounds resulted in higher stability under the same storage condition. No significant difference in niacinamide degradation rate among treatments in all storage conditions. There was also no significant difference of phytochemicals stability observed when stored in a cap off or on a bottled water.

Light significantly affected the stability of betalains and anthocyanins when dissolved in water, resulting in clear or faded color solutions. Stability of these natural pigments increased when stored inside a cap.

Our results indicate that the delivery of functional ingredients in the dry form inside a cap would improve stability, lower the manufacturing cost due to reduced losses, and provide functional beverages with more consistent nutritional value for customers.

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Acknowledgements

The completion of this research reflects the supports and help of many people. I would like to thank Dr. M. Monica Giusti for the opportunity to join her lab, as well as for her direction, assistance, and guidance throughout the project. I would also like to acknowledge Liquid Health Labs, Inc. for their trust in allowing me to do this project and financial supports. I would like to thanks Dr. John Litchfield and Dr. Mike Mangino for their support and time in serving as my committee member. Additionally, I would like to thank my colleagues and friends: Scott Kottman, Kom Kamonpantana, Neda Ahmadiani,

Steven Simmons, Allison Atnip, Abby Synder, Andrew Barry, Kumala Marthina, and

Pauline Ie for their suggestions, help, and cooperation during our work in the lab.

Lastly, I would like to thank my father Burhan Witarsa, mother Nieni Prajogo, and brother Darwin Witarsa, who trust me and support me in pursuing my Master’s

Degree at The Ohio State University.

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VITA

June 27, 1986 …………… Born in Semarang, Indonesia

2005-2009 ………………. B.S. in Food Science, Iowa State University, Ames, IA 2009-2011……………….. Graduate Research and Teaching Assistant The Ohio State University, Columbus, OH

Field of Study

Major Field: Food Science and Technology

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Table of Contents

Abstract ...... ii

Acknowledgements ...... iv

VITA ...... v

List of Tables ...... ix

List of Figures ...... x

CHAPTER 1: INTRODUCTION ...... 1

CHAPTER 2: LITERATURE REVIEW ...... 3

2.1 Functional Food ...... 3

2.2 Functional Beverages ...... 4

2.2.1 Energy drinks ...... 5 2.2.2 Enhanced water ...... 6 2.3 U.S functional beverages market ...... 7

2.3.1 Market of functional beverages ...... 7 2.3.2 Market for enhanced water and energy drink ...... 8 2.4 Functionality of enhanced water and energy drink phytochemicals...... 9

2.4.1 Vitamins ...... 9 2.4.2 Energy Enhancing Ingredients ...... 10 2.4.3 Weight loss compounds ...... 12 2.4.4 Natural food colorant ...... 13 2.5 Stability of phytonutrients during Processing and Storage ...... 14

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2.5.1 Overview of phytonutrients stability ...... 14 2.5.2 Processing of functional beverage ...... 18 2.5.3 Packaging of Functional Beverage ...... 19 2.5.4 Nutrient Degradation ...... 19 2.6 Optimizing the delivery of nutrients in functional beverages ...... 20

2.6.1 Vitamin Overage ...... 20 2.6.2 Active Packaging ...... 21 2.6.3 Dry storage ...... 22 CHAPTER 3: MATERIALS AND METHODOLOGY ...... 24

3.1 Materials...... 24

3.2 Sample Preparation ...... 24

3.3 Accelerated Storage Study (30 days) ...... 25

3.4 Long term storage study ...... 27

3.5 HPLC-MS analysis ...... 29

3.6 Color measurement ...... 32

3.7 Statistical analysis ...... 33

CHAPTER 4: RESULTS AND DISCUSSION...... 34

4.1 Compounds separation and identification ...... 34

4.2 Composition of different enhanced water formulations ...... 37

4.3 Ascorbic acid stability ...... 42

4.4 Hydroxycitric acid stability ...... 46

4.5 Riboflavin stability...... 48 vii

4.6 Pyridoxine and pyridoxal phosphate stability ...... 53

4.7 Niacinamide stability ...... 58

4.8 Caffeine stability ...... 60

4.9 Color stability ...... 61

CHAPTER 5: CONCLUSION ...... 67

REFERENCES ...... 68

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List of Tables

Table 1. FDMx sales and forecast of functional beverages, at current prices, 2004-2014 ...... 7

Table 2.FDMx sales of functional beverages, by segment, 2004-2009 ...... 8

Table 3. Vitamins and some commonly used synonyms ...... 10

Table 4. Summary of vitamin stability ...... 15

Table 5. Recommended overages for selected nutrients and vehicles based on losses during processing ...... 21

Table 6. Conditions for HPLC separation in different formulations ...... 31

Table 7. Compound composition reported and obtained in this study ...... 41

Table 8. Degradation rate of different compounds from formulations stored in 37˚C incubator for

30 days ...... 43

Table 9. Degradation rate of different compounds from VH formulations stored at different storage conditions for 25 weeks ...... 44

Table 10. Degradation rate of different compounds from LS formulations stored at different storage conditions for 15 weeks ...... 52

Table 11. CIE L*, a*, b* values of VH formulation when stored under light exposure ...... 63

*, b* values of VH formulation when stored at room temperature under light exposure ...... 63

Table 12. CIE L*, a*, b* values of LS formulation when stored under light exposure ...... 64

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List of Figures

Figure 1. Nutrient loss in beverage production ...... 20

Figure 2. Cap storing beverage ingredients ...... 22

Figure 3. Nutritional information of GR, LL, and BR formulations ...... 26

Figure 4. Nutritional information of VH and LS formulation ...... 28

Figure 5. Delivery of ingredients from the cap ...... 30

Figure 6. HPLC chromatogram and UV spectra (obtained with the SPD-M20 A photodiode array) of compounds ...... 36

Figure 7. Separation of compounds in different formulations ...... 39

Figure 8. Ascorbic acid degradation in GR formulation stored at 37˚C incubator without light exposure ...... 42

Figure 9. Ascorbic acid degradation in VH formulation stored at different storage environments for 25 weeks ...... 45

Figure 10.Hydroxycitric acid degradationin BR formulation stored at 37˚C without light exposure ...... 47

Figure 11. Riboflavin degradation in LL formulation stored at 37˚C incubator for 30 days

...... 48

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Figure 12. Riboflavin degradation in LS formulation stored at room temperature with and without light exposure for 15 weeks ...... 49

Figure 13. LS formulation chromatogram for sample stored at room temperature with light exposure ...... 51

Figure 14. Pyridoxine degradation in LS formulation stored at a) room temperature, dark; b) 37˚C incubator, dark; c) room temperature, light exposure ...... 54

Figure 15. Pyridoxine stability in VH formulation stored at 37˚C incubator, dark ...... 55

Figure 16. Pyridoxal degradation in VH formulation stored as a) hot-filled process sample, dark; b) premixed sample; c) cap sample ...... 57

Figure 17.Niacinamide degradation in LL formulation stored at 37˚C incubator for 30 days ...... 58

Figure 18. Niacinamide degradation in LS formulation stored in different environments for 15 weeks ...... 59

Figure 19. Caffeine degradation in GR formulation stored at 37˚C incubator for 30 days

...... 60

Figure 20. Caffeine degradation in LS formulation stored in different environments for 15 weeks...... 60

Figure 21. Color change in BR formulation after 21 days of storage at 37˚C incubator .. 62

Figure 22. Color change in VH formulation after 25 weeks of storage ...... 63

Figure 23. Color change in LS formulation after 15 weeks ...... 64

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CHAPTER 1: INTRODUCTION

Functional beverages can be defined as beverages enhanced with ingredients that can promote specific health benefits beyond general nutrition. A variety of functional beverages can be found easily in the market. Sales of functional beverages reached $9 billion in 2009 and are projected to increase to $9.7 billion by 2014. However, one of the technical challenges in beverage fortification is ingredient stability. Most nutrients and vitamins show limited stability to processing and storage conditions, such as heat, light, oxygen, and moisture.

A growing demand for solution to limit nutrient degradation in ready-to-drink beverages has resulted in numerous innovations. Dry storage is expected to reduce mobility and reactivity of the compounds and hence improve stability. Therefore, a new two-tiered dispensing bottle cap that stores ingredients in a dry enclosed chamber prior to consumption was developed. For consumer’s convenience, the cap is designed to fit water bottles and upon pushing the top section of the cap, stored ingredients can be released. Compounds solubilized in water after shaking, resulting on a drink ready to be consumed. This storage system is expected to be effective in different formulations of beverages.

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The objective of this study was to investigate the delivery and chemical integrity of nutrients used in the beverage industry including ascorbic acid, hydroxycitric acid, riboflavin, pyridoxine, pyridoxal phosphate, niacin, caffeine, and pigment color when stored in a cap as compared to traditional beverage processing methods.

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CHAPTER 2: LITERATURE REVIEW

2.1 Functional Food

Foods have long been known to have an important impact on health.

Inappropriate nutrient intake can lower human physical and cognitive performance,

increase risk of some diseases, and impact genetic potential (Milner, 2002). Around

2500 years ago, Hippocrates stated “Let food be thy medicine and medicine be thy

food”. However, due to the advance of drug technology in the 19th century, food as

one option of medications became less popular (Hasler, 2002). Then, healthcare cost

increased and medication may not increase quality of life. Therefore, during the 20th

century, development of functional foods to prevent chronic diseases of aging before

treatment became very important (Hasler, 2000). Scientists began to identify active

food components from plants (phytochemicals) and animals (zoochemicals) for this

purpose. These events, along with population becoming more health-conscious,

created the trend of functional food (Hasler, 2002).

There is no universal definition of functional foods. The first concept was

developed in Japan by the Ministry of Health and Welfare that introduced a

regulation to approve certain foods with documented health benefits(Arai, 1996). In

the United States, some organizations proposed definitions of functional foods.

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According to the National Academy of Sciences’ Food and Nutrition Board,

functional food is “any modified food or food ingredient that may provide a health

benefit beyond the traditional nutrients it contains” (Committee on Opportunities in

the Nutrition and Food Sciences, Food and Nutrition Board, 1994). The International

Life Science Institute defined it as “foods that, by virtue of the presence of

physiologically-active components, provide a health benefit beyond basic nutrition”

(International Life Sciences Institute, 1999). In 1990, the American Dietetic

Association defined them as “whole, fortified, enriched, or enhanced” food that

consumed as “… part of a varied diet on a regular basis, at effective levels”

(American Dietatic Association, 1999).

Guidelines for fortification of vitamins and other nutrients to food are based

on U.S. FDA 21 CFR Section 104.20 (g) which states that the nutrients added to food

should be stable during storage, distribution, and use; physiologically available from

the food; present at a level with assurance of no excessive intake; and suitable for its

intended purpose along with compliance to government safety regulation (Gregory

III, 2007).

2.2 Functional Beverages

The current trends of functional foods also affected the beverage industry.

Beverages have also been known as delivery systems of nutraceuticals (functional

food ingredients) and can provide other functions such as nourishment, enjoyment,

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relaxation, performance, and health (Gordon and Kubomura, 2003). A variety of functional beverages can be found easily in the market. Sales of carbonated soft drinks declined as the sales of fruit-based beverages, bottled water, and functional beverages increased significantly (ING Barings, 2001). Functional beverages can be defined as beverages enhanced with ingredients that can promote specific health benefits beyond general nutrition or modified through processes, such as fermentation of dairy products with probiotic bacteria. Functional beverages usually have an approved labeling claim from the FDA (Mintel, 2009).

There are some factors that drive the growth of the functional beverages market. The percentage of Americans who watch their diet increased from 28% in

2003-2004 to 52% in 2008-2009 due to their concerns with weight and cholesterol level. About two thirds of Americans are either obese or overweight. Therefore, the demand for low calorie products or functional products that aid in dieting increased.

Besides that, an increase in aging population between 2005 and 2015 is also expected to increase the demand of functional products that can help in maintaining good health and reducing risk of certain conditions, such as osteoporosis and cancer.

However, apart from these market drivers, price becomes a sensitive issue in deciding purchase of functional beverages, resulting in lower sales of certain beverage segment

(Mintel, 2010).

2.2.1 Energy drinks This segment includes beverages that contain calories and caffeine along with energy enhancer supplements, such as taurine, herbal extracts, or vitamin B

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(Heckman et al., 2010). Energy drinks were first introduced in Europe and Asia around 1960s due to consumers demand for dietary supplements that could enhance energy. However, it was not until 1997, that energy drinks became popular in the

United States market when Red Bull was first marketed (Reissig et al., 2009). The energy drink market is mainly targeted for teenagers and young adults, age 18-34 years old (Lal, 2007). Energy drinks are available for consumption as ready-to-drink beverages, powder mixes, and shots (Reissig et al., 2009). There have been some studies that show negative effect on physiological and cognitive performance after consuming energy drinks, due to the high amounts of sugar and caffeine present (IFT,

2007).

2.2.2 Enhanced water Enhanced water includes water fortified with vitamins or nutrients and contains claims other than hydration (Mintel, 2010). In 2000, Glaceau introduced one of the first enhanced water products, Energy Brands Vitamin Water. Enhanced water became popular since 2002 as the beverage industries competed to produce water fortified with vitamins, carbohydrates, electrolytes, and other supplements. Sales increased from $20 million in 2000 to $85 million in 2001 (Gamble, 2002).

Depending on the performance offered, different formulations of nutrients mix are available. Enhanced water may also contain energy-enhancer supplements with lower sugar content as an alternative to energy drinks.

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2.3 U.S functional beverages market

2.3.1 Market of functional beverages As seen in table 1, according to the marketing firm Mintel International

group, there was a strong sales growth of functional beverages (25%) between 2004

and 2008, reaching $9.2 billion in 2008. However, due to the economic recession,

overall sales declined by 2% in 2009. Sales remained stable in 2010 but it is

projected that the market will increase slowly to $9.7 billion by 2014 as a result of

improving economy and continued demand for healthy and energy-booster

beverages (Mintel, 2010).

Table 1.FDMx sales and forecast of functional beverages, at current prices, 2004- 2014 Year Sales at current % change prices ($ million) 2004 7,396 - 2005 7,883 6.6 2006 8,489 7.7 2007 9,047 6.6 2008 9,264 2.4 2009 9,064 -2.2 2010 (fore) 9,208 1.6 2011 (fore) 9,315 1.2 2012 (fore) 9,456 1.5 2013 (fore) 9,595 1.5 2014 (fore) 9,733 1.4 Extracted from: Mintel, based on Information Resources, Inc. InfoScan Reviews Information

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2.3.2 Market for enhanced water and energy drink

Table 2.FDMx sales of functional beverages, by segment, 2004-2009 Year Functional Energy Enhanced Functional Functional Functional fruit juice& drinks water soy, rice,& tea yogurt juice drinks almond- drinks/ based smoothies drinks $ million 2004 5,534 276 207 485 401 493 2005 5,500 458 334 523 511 557 2006 5,542 667 513 561 601 605 2007 5,531 863 754 618 676 604 2008 5,548 965 825 679 654 591 2009 5,374 1,026 772 691 659 542 2010 5,410 1,073 808 736 645 536 (fore) 2011 5,309 1,242 840 773 636 516 (fore) 2012 5,174 1,467 884 809 627 496 (fore) 2013 5,041 1,689 927 846 617 476 (fore) 2014 4,909 1,908 969 883 607 456 (fore) Source: Mintel, based on Information Resources, Inc. InfoScan Reviews Information

Focusing on energy drinks and enhanced water segments (table 2), sales of energy drinks grew more than other categories and kept increasing by 6.3% in 2009, while growth of enhanced water sales in previous years stopped in 2008 and declined by 6.9% in 2009 due to competition from powdered drink mixes. However, growth prediction of energy drinks in later years is not as high as observed in 2005 and 2007, which may be due to negative impact of energy drinks in their high amount of sugar 8

and caffeine contents. Financial issues were the major cause in decreasing sales of

enhanced water. Line extensions in powdered mixes with lower selling prices may

improve sales in this category (Mintel, 2010).

2.4 Functionality of enhanced water and energy drink phytochemicals

2.4.1 Vitamins Micronutrients are added to beverages for restoration of vitamin loss during

processing or for fortification. Vitamins are mainly divided into two groups (table 3),

water and fat soluble vitamin (Ottaway, 2009). The Recommended Dietary

Allowance (RDA) is developed as a nutritional reference standard intake of vitamins

(Gregory III, 2007).

There are various functions offered by different vitamins. Ascorbic acid,

some carotenoids, and vitamin E have been known for their antioxidant property.

Some vitamins, including niacin, thiamin, riboflavin, biotin, pantothenic acid,

vitamin B6, vitamin B12, and folate, function as coenzymes or their precursors.

Vitamin A and D involves in genetic regulation. Vitamin A is also good for vision.

Certain vitamin has specific function in different reactions, such as ascorbate in

hydroxylation reactions and vitamin K in carboxylation reactions (Gregory III,

2007).

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Table 3. Vitamins and some commonly used synonyms Vitamin Synonyms Fat-soluble Vitamin A Retinol Vitamin D2 Ergocalciferol Vitamin D3 Cholecalciferol Vitamin E α-, β-, γ-Tocopherols and α-tocotrienol Vitamin K1 Phylloquinone, phytomenadione Vitamin K2 Menaquinone, farnoquinone Vitamin K3 Menadione

Water Soluble Vitamin B1 Thiamin Vitamin B2 Riboflavin Vitamin B6 Pyridoxal, pyridoxine, pyridoxamine Vitamin B12 Cobalamins, cyanobalamin, hydroxocobalamin Niacin Nicotinic acid (Vitamin PP) Niacinamide Nicotinamide (vitamin PP) Pantothenic acid - Folic acid/ folates Folacin (Vitamin M) Biotin Vitamin H Vitamin C Ascorbic acid Source: Ottaway, 2009

Several diseases have also been associated with inadequate intake of certain vitamins. Lack of vitamin D can cause rickets which is characterized by skeletal malformation in growing children. Scurvy is a disease due to vitamin C deficiency. A paralyzing disease, called beriberi, is caused by lack of vitamin B1 consumption. Folate, vitamin B6, and vitamin B12 can prevent certain types of anemia (Williams, 2001).

2.4.2 Energy Enhancing Ingredients There are various energy drinks available in the market, specifically targeted for young males (Heckman et al., 2010). Caffeine is commonly used in combination

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with other compounds such as taurine, vitamin B, L-carnitine, and glucuronolactone.

Sugar is sometimes added as a source of rapid energy. Some factors that affect selection of energy blends are overall taste, amount of energy, duration of active energy, and health properties (Ottaway, 2009). Consumption of less than 400 mg caffeine daily is not associated with adverse effects. Most energy drinks contain 72-

150 mg of caffeine per serving, while brewed coffee, tea, and cola beverages can range from 134-240 mg/serving (Nawrotet al., 2003).

Caffeine (1,3,7- trimethylxanthine) has been shown as an active ingredient that stimulates the central nervous system. Caffeine acts as an adenosine receptor blocker in the brain due to its similarity in chemical structure with adenosine. This blockage causes the sleep promoting effects of adenosine to stop and neurons speeding up, allowing consumers to stay awake (Ferre, 2008). Other studies found positive effects of caffeine consumption on energy utilization, exercise performance, processing information, and awareness (Cysneiroset al., 2007).

Another supplement ingredient, such as taurine (2 aminoethanesulfonic acid), has been associated with its role in variety physiological functions (Brosnan and Brosnan, 2006), performance endurance, and reduction of lactic acid formation after exercise (Imagawaet al., 2009). L-carnitine is believed in improving exercise performance by enhancing muscle fatty acid oxidation, altering glucose homeostasis, enhancing acylcarnitine production, modifying training responses, and improving muscle fatigue resistance (Brass, 2000).

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2.4.3 Weight loss compounds Normal body weight based on body mass index (BMI, in kg/m2) ranges from 18.5 to 24.9. In the United States, 34% adult population have BMI index above

25 which is classified as overweight and additional 31% is obese with BMI over 30

(Pittler and Ernst, 2004). Low physical activity and energy expenditure is one factor that causes high percentage of overweight and obese population (Heini and Weinsier,

1997). However, compliance in conventional weight-management program is poor, resulting in the development of safe and effective dietary supplements as an additional way in reducing body weight (Blancket al., 2001). These supplements have become very popular because they are less demanding as compared to a special diet or increasing physical activity, no prescription is needed, and they are perceived as natural compounds (Saperet al., 2004).

Different mechanisms have been proposed in targeting to body weight loss.

Chromium and ginseng are thought to modulate carbohydrate metabolism, which influencing weight and body composition (Anderson, 1998 and Sotaniemin et al.,

1995). Glucomannan, psyllium, and guar gum are expected to increase satiety (Saper et al., 2004). Glucomannan is a dietary fiber that acts as a bulking agent by increasing the moisture content of food during digestion (Walsh et al., 1984). Guar gum increases the viscosity of bowel contents and fullness, resulting in lower appetite and food intake (Blackburn et al., 1984). A study conducted with chitosan suggested its role in preventing dietary fat absorption (Ernst and Pittler, 1998).

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Hydroxycitric acid (HCA) from the extract of certain Garcinia sp. fruit has been known for its weight loss properties in reducing fat synthesis. Previous research showed that hydroxycitric acid can inhibit the citrate cleavage enzyme, which is responsible for the synthesis of a de novo fatty acid. It also increased the rates of hepatic glycogen synthesis, resulting in suppressed food intake and body weight loss

(Heymsfieldet al., 1998).The fruits itself are not palatable due to their acidic flavor, therefore people are looking for an extract of hydroxycitric acid as a dietary supplement (Jena et al., 2002).

2.4.4 Natural food colorant Color has been known as an important factor in food industries for its aesthetic value, flavor and taste perception, and appetite stimulation (Bayarriet al.,

2001). Due to current awareness regarding toxicity of artificial food colors, people gain more interest in natural pigments as color additives (Bridle and Timberlake,

1997). Acceptance of nature-derived color alternatives is supported by their healthy properties and high quality (Stinzing and Carle, 2004). There are many sources of pigments that have been known for their potentials as natural food colorants, including carotenoids, betalains, and anthocyanins (Bridle and Timberlake, 1997).

Carotenoids are oil-soluble pigments that give yellow to red color

(Timberlake and Henry, 1986). The carotenoids group also functions as vitamin A precursors and antioxidant (Cognis Corporation, 2002). Commonly used water- soluble pigments are derived from betalains and anthocyanins. Red beets, which are part of betalains group, are commercially used as source of red-purple color 13

(Mortensen, 2006). Betalains have been known to provide protection againsts stress-

related disorders (Kanneret al., 2001), exhibitanti inflammatory effects (Gentil et al.,

2004), and to have antioxidant activities (Butera et al., 2002 and Stinzing et al.,

2005). Anthocyanins give wide variety of colors from red to blue (Timberlake and

Henry, 1986). Anthocyanins are also known for their antioxidant activity (Abuja et

al., 1998; Wang and Jiao, 2000) and its role in preventing stomach, colon or rectal

cancer (Gee and Johnson, 2001; Kanner and Lapidot, 2001). Commonly fruit and

vegetable extracts that contain anthocyanins are derived from red cabbage, red radish,

purple sweet potato, black carrot, cherry, elderberry, and blackberry (Stinzing and

Carle, 2003).

2.5 Stability of phytonutrients during Processing and Storage

2.5.1 Overview of phytonutrients stability A technology challenge occurs when incorporating fat-soluble vitamins in a

beverage and with different stability of vitamins during processing or storage. A

summary of vitamin sensitivity toward different physical and chemical factors can be

seen in table 4.

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Table 4. Summary of vitamin stability Nutrient Humidity Light Heat Max. cooking loss (%) Vitamin A S U U 40 Ascorbic acid (C) U U U 100 Biotin (B7) S S U 60 Vitamin B12 U U S 10 Vitamin D S U U 40 Folate S U U 100 Vitamin K S U S 5 Niacin (B3) S S S 75 Pantothenic acid (B5) U S U 50 Vitamin B6 S U U 40 Riboflavin (B2) S U U 75 Thiamin (B1) U S U 80 Tocopherols (E) S U U 55 Note: These conclusions are oversimplifications and may not accurately represent stability under all circumstances S= stable; U= unstable (Extracted from: Harris, 1971)

Vitamin C is one of the most commonly added micronutrients. However, L- ascorbic acid can be easily oxidized to form dehydroascorbic acid in aqueous solution, which further degraded rapidly to 2,3-diketogulonic acid. The formation of

2,3-diketogulonic acid is irreversible, resulting in no vitamin C activity (Nyyssonenet al., 2000). Various factors that can affect degradation of vitamins C include presence of oxygen, light exposure, metal ions (Cu2+, Ag+, Fe3+), alkaline pH, and high temperature (Lee and Kader, 2000).

One of the most unstable forms of vitamin B is thiamin (vitamin B1), which is greatly affected by moisture content (DSM Nutritional Product and USAID,

2011).Degradation of riboflavin (vitamin B2) is mainly affected by light. Direct sunlight gives more impact as compared to fluorescent light in affecting riboflavin 15

stability. Other factors that may influence stability of riboflavin are reducing agents and increasing pH (Ottaway, 2009). Niacin is considered as the most stable B vitamin. Degradation is limited in presence of oxygen, light, and heating in both aqueous and solid systems which make it stable in foods and drinks production

(Ottaway, 2009). Type of thermal processing, light, and water exposure affect the stability of pyridoxine. Folic acid degrades in the presence of light, oxidizing or reducing agent, and acidic or basic environments. Stability of pantothenic acid toward heat decreases when subjected to an alkaline environment. Biotin in unstable in both basic and acid conditions (DSM Nutritional Product and USAID, 2011).

Vitamin A rapidly degrades when exposed to light and oxygen. Retinol is more unstable during heating as compared to palmitate ester. Vitamin E is considered as a stable vitamin. However, the unesterified tocopherol is more sensitive due to free phenolic hydroxyl group. The most stable form is α-tocopheryl acetate, which is only hydrolyzed to free tocopherols in strong acid or basic environment. There are two types of vitamin D, which are vitamin D3 (cholecalciferol) from animal tissue and vitamin D2 (ergocalciferol) from plants. Both types are sensitive to light and acids. In the presence of oxygen, vitamin D3 is more stable than D2 due to a less double bond structure. The commonly added form of vitamin K in food is vitamin K1

(phytomenadione) derived from plants. This vitamin is unstable to sunlight, alkali, and slowly degraded in presence of oxygen (Ottaway, 2009)

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The molecular structure of caffeine makes it a stable compound even at high temperatures used during roasting or brewing (245˚C). Small amount of caffeine may be lost due to sublimation at 178˚C (Tarka and Hurst, 1998). Caffeine degradation is affected by pH of the medium. Complete degradation was observed at pH 6, while less than 15% degradation was attained at pH 4 (Gokulakrishnan, 2006).

Hydroxycitric acid (HCA) exists as free acid or lactone. The lactone form is more stable but inactive. The free acid is the active form of HCA that can inhibit citrate lyase but unstable and can be converted to the lactone form easily. Therefore, potassium or calcium salts are used to stabilized the free acid form and retain the activity (MDidea, 2011). Super Citrimax® (HCA_SX) is a calcium/potassium salt of hydroxycitric acid. This salt form is more soluble and more bioavailable. It contains of 95% calcium/potassium-HCA and provides 60% hydroxycitric acid. HCA_SX is stable at temperature of 30˚C with 65% relative humidity for over three years (Soni, et al., 2004).

Unlike synthetic dyes, natural food color extracts are more unstable. Color stability of anthocyanins and betalains is affected by chemical structures, concentration, pH, solvents, temperature, oxygen, light, enzymes, and other substances presence (Rein, 2005). In anthocyanins, intense red color is produced in pH below 3. At pH 4 to 5, anthocyanin solutions become colorless. Further increases of pH alter the color to purple and blue. Upon heat treatment and storage, color changes from blue to yellow (Jackmanet al., 2007). Betalains exhibits a wider

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stability range of pH from 3 to 7 (Stinzing and Carle, 2004). Outside of this range, betalains become more reactive and induce degradation mechanisms (Herbach et al.,

2006).

2.5.2 Processing of functional beverage One of the most important steps in functional beverage production is processing to ensure that the beverage meets its shelf life requirement. Producers need to establish the pH of product to determine processing parameters. For enhanced water which has low pH (<4.4), hot filling and chemical preservation can be used as a preservation method (Eckert and Riker, 2007).

Potassium sorbate, benzoic acid, and sulfur dioxide are commonly used as preservatives (Ecker and Riker, 2007). However, minimized or no chemical preservation is more desirable in functional beverages. A hot filling process is generally employed for beverages that do not contain preservatives. In this process, the product is heated through a heat exchanger to a minimum of 180˚F, filled at that temperature, then cooled down to ambient temperature. Beverages expand slightly when heated and shrink as they cool down; generating vacuum that allows the cap to create a seal (Ecker and Riker, 2007; Rankenet al., 1997; Papaspyrides and

Vouyiouka, 2009).The advantage of hot filling is that the hot liquid can eliminate microbial contaminants on the inner surface of the bottle and closure (Ranken et al.,

1997).

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2.5.3 Packaging of Functional Beverage A polyethylene terephthalate (PET) based plastic bottle is commonly used as a container in the enhanced water beverage industry. This plastic is considered as low-cost packaging materials. However, many factors need to be considered in choosing packaging, such as container shaping, size flexibility, barrier properties, and transparency (Ashurst, 1994). Due to the high temperature applied in hot-filling process followed by fast cooling, packaging materials used need to be modified by increasing the glass-transition of PET to maintain bottle shape stability. Addition of comonomer or second polymer improves the performance of bottle during filling process. However, it leads to a higher cost and heavier container (Papaspyrides and

Vouyiouka, 2009).

2.5.4 Nutrient Degradation As seen in figure 1, ingredients start to degrade even before processing and keep degrading during storage of the finished product. Most losses of vitamins and minerals occur during thermal processing (DSM Nutritional Product, 2011).Vitamin

B1, panthothenic acid, and vitamin C are considered quite labile in heat treatment

(Chaudhari, 2005; Rysstad and Johnstone, 2009)

Packaging also affects beverage quality during storage by influencing the interaction between products and environments. Some important changes that may occur include aroma loss, migration of monomers and additives from packaging materials, and entering of oxygen and light (Rysstad and Johnstone, 2009). Certain compounds, such as riboflavin and vitamin C, are sensitive to light and oxygen

19

exposure. Light protection can be achieved by aluminum-foil lined cartons, however

producers prefer plastic bottle to attract consumers attention (Rysstadet al., 2003)

Premix storage loss

Processing loss Finished product storage loss

Source: Chaudari, Fortitech Strategic Nutrition, 2008

Figure 1. Nutrient loss in beverage production

2.6 Optimizing the delivery of nutrients in functional beverages

A growing demand for solution to limit nutrient degradation in ready-to-drink

beverage results in numerous innovations.

2.6.1 Vitamin Overage Overage refers to the additional amount of fortificant added to cover loss of

nutrients so the products still deliver the targeted level of nutrients at the time of

consumption. Overage is calculated based on the estimation of loss and expresses as a

20

percentage based on the level claimed on the label ((DSM Nutritional Product and

USAID, 2011; Ottaway, 2009). Since each compound degrades at different rate, technologists need to determine the amount of overage required prior to production.

The amount added has to be the minimum necessary and still within the safety level of consumption for each vitamin (Ottaway, 2009). Table 5 shows recommended overages for certain nutrients in different food applications ((DSM Nutritional

Product and USAID, 2011). Overages can be determined by conducting shelf life studies with different temperatures during storage (Ottaway, 2009)

Table 5. Recommended overages for selected nutrients and vehicles based on losses during processing Food Vitamins A D E B1 B2 Niacin B5 B12 C Milk Pasteurized 20 20 10 25 15 15 30 15 30 UHT 30 30 30 50 40 20 30 30 100 Dry milk 40 40 20 20 20 20 20-30 40 50 Milk 20 20 10 25 15 15 30 20 30 dessert Drinks Juices 30-40 40 15-25 40-50 30-40 20-50 30-50 70 20-80 Drink 15 15 10 10 10 10 10 10 mixes Extracted from: DSM Nutritional Product and USAID

2.6.2 Active Packaging Active packaging is a packaging that offer functions other than a mere barrier to the outside environment (Rooney, 1995a). This system predicts environmental changes and responds by changing properties. Its main purpose is to

21

maintain product quality prior to consumption (Brody, 2001a). In beverages, oxidation and microbial growth are the main cause of deterioration. Oxidation can alter color, flavor, and nutritional value, while microbial growth affects the safety

(Dawson, 2003). Different properties of active packaging include oxygen scavengers, moisture-regulating materials, antimicrobial polymers, flexible barrier materials, and light protection packaging (Fraunhover Ivy).

2.6.3 Dry storage One of the latest solutions is modified caps and closures to store dry ingredients inside a cap. When the cap is pushed, ingredients will be released.

Shaking the bottle will allow ingredients to dissolve in water and the beverage will be ready to drink (Scott, 2010).

Figure 2. Cap storing beverage ingredients

22

In dry storage, mobility and reactivity of compounds will be limited, resulting in more stable nutrients and increasing beverage shelf life. Nutrient overage can be limited and a hot-filling process can be substituted with a cold-filling process, which requires less energy and less plastic for the bottle used. For convenience, the cap is designed to fit major water bottles available by offering a variety of sizes from

26.7-mm, 28-mm, 38-mm, and 43-mm (Fuhrman, 2009 and Scott, 2010).

Reported limitations of this new cap category include ability to store only small amount of powder, preventing some companies from using bulky natural sweeteners. Carbonation is also not applicable yet for this product. It will also be difficult to lower the selling price; however it is expected that the cap-equipped beverages will cost between $1.99 and$2.49 each, which is comparable to Glaceau’s

Vitamin Water by Coca-Cola (Casey, 2008).

23

CHAPTER 3: MATERIALS AND METHODOLOGY

3.1 Materials

The nutrient containing caps (PowerCap® ) were provided by Liquid Health

Labs, Inc. (Deerfield, NH). Ascorbic acid, riboflavin, niacinamide, and caffeine

standards were provided by Flavor Systems International, Inc. (Cincinnati, OH).

Pyridoxal-5phosphate standard were purchased from MP Biomedical, LLC (Solon,

OH). Optima® LC/MS grade water, 99.9% methanol, 99.9% acetonitrile, Certified

ACS Plus 96.1% sulfuric acid, and Certified ACS 88% formic acid were purchased

from Fisher Scienctific (Fair Lawn, NJ). Bottled water (500 ml, Dasani) was

purchased from local grocery store (Columbus, OH).

3.2 Sample Preparation

Five different formulations were analyzed and the drink codes were GR, LL,

BR, VH, and LS. Four different treatments were applied to each formulation prior

starting the storage study:

a. Cap on bottle: A cap containing ingredients (1200 mg) was attached to a bottle of

water. This treatment was done to observe any possibility of water migration to

the inner part of cap that might affect the stability of phytonutrients

24

b. Cap in a bag: Ingredients (1200 mg) were stored in a cap. Caps of the same

formulation were then stored in a plastic bag, without attachment to a bottle.

Stability of compounds was monitored in this completely dry environment.

c. Hot fill: Ingredients in a cap were dissolved in 500-ml water, distributed into a

pre-sterilized container, and loosely capped. The beverage mix was then sterilized

using heat treatment at 180˚F for 30 minutes and cooled to ambient temperature.

This treatment was done to observe the stability of compounds in solution and the

effect of heat treatment.

d. Premixed: Ingredients in a cap were dissolved in 500-ml water and distributed to

pre-sterilized container. This treatment was done to observe the stability of

compounds in solution and monitor any microbial growth that might affect the

safety for consumption.

3.3 Accelerated Storage Study (30 days)

Three different formulations in the caps (GP, LL, and BR formulation) were

used as samples in this study. The composition of each formulation can be seen in

figure 3. These formulation samples were evaluated under the following conditions:

a. Cap on bottle

b. Cap in a bag

c. Hot-fill

d. Premixed

25

For treatment c and d, 2 caps were used to generate 2 true replicates. Each cap was dissolved in a 500-ml water (d) and hot-filled (c), then transferred into 20 pre-sterilized 4-ml vials for the 30-days storage trial.

GR FORMULATION BR FORMULATION

LL FORMULATION

Figure 3. Nutritional information of GR, LL, and BR formulations

Source: Liquid Health Labs, Inc., 2009

26

All samples were stored in the dark in an incubator oven set at 37⁰C. During

the 30-day storage, six time points (day 0, 3, 7, 14, 21, 30) were chosen to measure

the concentration of each selected nutrient. In GR formulation, the concentrations of

ascorbic acid and caffeine were monitored. For LL, the concentrations of ascorbic

acid, niacin, and riboflavin were quantified. The concentration of hydroxycitric acid

(Super Citrimax®) in BR formulation was also observed over time. For every

treatment, measurements were done in duplicate, taking a new individual vial, bottle

or cap as a replication. Each sample was then injected to the HPLC. The

concentration was calculated based on a calibration curve prepared with the pure

standards, and then averaged.

3.4 Long term storage study

VH and LS formulationsfrom PowerCap were stored at different conditions

for 25 weeks (VH) and 15 weeks (LS). Nutritional information of this formulation

can be seen in figure 4.

These 2 formulation samples were evaluated under the following conditions:

a. Cap on bottle

b. Cap in a bag

c. Hot fill

d. Premixed

27

For treatment c and d, 3 caps were used to generate 3 true replicates. Each cap was dissolved in a 500-ml water (treatment d) and hot-filled (treatment c), then transferred into 15-ml sterile centrifuge tubes for the storage trial.

VH FORMULATION LS FORMULATION

Figure 4. Nutritional information of VH and LS formulation Source: Liquid Health Labs, Inc

Samples were divided to three different storage conditions: (1) in a controlled temperature incubator set at 37⁰C in the dark; (2) at room temperature with light exposure; (3) at room temperature without light exposure. Light was controlled by fluorescent bulbs (120 VAC, 60 Hz, 23” L) mounted on top of the shelf. Dark environment was controlled by storing samples inside a closed cabinet. Six different

28

time points (week 0, 5, 10, 15, 20, 25) were chosen to monitor the stability of

nutrients in VH formulation and five different time points (week 0, 2, 5, 10, 15) for

LS formulation. Concentration of ascorbic acid, niacin, pyridoxine, and pyridoxal-5-

phosphate were monitored in the VH beverage. For the LS formulation,

concentrations of niacin, caffeine, riboflavin, and pyridoxine were observed. At each

time point, 3 replicate of treatment a (bottles) and b (caps) in each storage condition

were collected. For treatment c and d, 2 tubes from the same pre-mixed beverage

samples were taken from each storage condition. Each sample was then injected into

an HPLC system. The concentration was calculated based on a calibration curve

prepared with the pure standards, and then averaged.

3.5 HPLC-MS analysis

Prior to analysis, for treatment a and b, ingredient inside the caps were

completely mixed with 500 ml of bottled water. The cap is designed to fit major water

bottles available in the market and upon pushing the top section of the cap, stored

ingredients can be released. Compounds were solubilized in water by shaking,

resulted on finished product (figure 5).

29

Figure 5. Delivery of ingredients from the cap

The sample was then filtered through a Whatman 13mm diameter of polypropylene syringe filter device with 0.45 µm pore size (Fisher Scientific, Fair

Lawn, NJ). Solvents used were also filtered using a Millipore nylon membrane with

0.45 µm pore size (Fisher Scientific, Fair Lawn, NJ). All samples and standards except for BR formulation were analyzed using a high performance liquid chromatography (HPLC) (Shimadzu Scientific Instruments, Inc., Columbia, MD) system equipped with LC-20AD pumps and SIL-20AC auto sampler and coupled with SPD-M20A photodiode array (PDA) detector and single quadrupole electron spray ionization (ESI) LCMS-2010 EV mass spectrometer (MS) detector. Data was collected using LCMS Solutions Software (Version 3, Shimadzu, Columbia, MD). A reverse-phase Acclaim Polar Advantage II (PA2) C18 column with a 3.5 µm pore size and 4.6 x 150 mm long (Dionex Corporation, Sunnyvale, CA) attached to a 4.3 x

10 mm Acclaim Polar Advantage II 5 µm guard cartridge (Dionex Corporation,

Sunnyvale, CA) was used. This column provides enhanced hydrolytic stability from

30

pH 1.5-10 and allows resolution for single-run analysis of polar and non-polar

analytes. Compared to the conventional C18 reversed-phase columns, Acclaim PA2

are compatible with a 100% aqueous mobile phase (Dionex Corporation, 2010).

Compounds were eluted using a gradient of mobile phase A: 0.015% formic

acid in water and B: 13/87 methanol-acetonitrile mix as follows: 0 to 3 min, 0% B; 10

min, 0-100% B; 12 min, 100% B; 15 min, 100-0% B; 16 min, 0% B. Flow rate was

set to 0.8 ml/min. Temperature was set to 28˚C. Depending on compounds

absorbance and concentration, both wavelength and injection volume used were

adjusted for different formulations (Table 6).

Table 6. Conditions for HPLC separation in different formulations Formulation UV detection (nm) Volume of injection (µl) GR 254 10 LL 254 15 VH 254, 280 30 LS 254, 280 10, 100 BR 210 50

Total flow was split and 0.2 ml of the flow went to the mass spectrometer

detector. Positive ion mode was applied under following condition: 1.5 L/min of

nebulizing gas flow, +4.50 kV interface bias, 200˚C block temperature, -2.5 V focus

lens, -50 V entrance lens, -3.6 V pre-rod bias, -3.5 V main rod bias, 1.k kV detector

voltage, and 2000 amu/sec scam speed. Total Ion Count (TIC) was performed from

31

200-1500 m/z and Selective ion Monitoring (SIM) was applied to identify the

molecular ions of selected compound. .

The BR formulation and Super Citrimax® (hydroxycitric acid) standard were

analyzed using a high performance liquid chromatography (HPLC) (Shimadzu

Scientific Instruments, Inc., Columbia, MD) system equipped with LC-6AD pumps

and SIL-20AHTautosampler and coupled with a SPD-M20A photodiode array (PDA)

detector. Data was collected using LCMS Solutions Software (Version 3, Shimadzu,

Columbia, MD). A reverse-phase Symmetry C18 column with a 3.5 µm pore size and

4.6 x 150 mm long (Waters Corp., Milford, MA) attached to a 4.6 x 22 mm

Symmetry 2 µm guard column (Waters Corp., Milford, MA) was used. Compounds

were eluted using isocratic flow of 8 mM sulfuric acid for 20 minutes with flow rate

set 1.0 ml/min (Jayaprakasha and Sakariah, 2002).

Identification of peaks was achieved by comparing retention time, spectra,

and mass-to-charge ratio with standards and published literature. Quantification was

achieved by comparing peak area with a standard curve.

3.6 Color measurement

Beverage sample was poured into 1-cm path length plastic cells. Empty cell

was used as a blank standard. CIE L*, a*, b* values were measured using a

ColorQuest XE spectrophotometer (Hunter Lab, Hunter Associates Laboratories, Inc.,

Reston, VA). Measurements were done using total transmission mode, a D65 light

32

source, and 10˚ observer angle. Chroma and hue angle were also calculated. Values

obtained were analyzed using EasyMatch QC computer software.

3.7 Statistical analysis

Analysis was done using SPSS v 19 covariate analysis. Compound

concentration was set as dependent variable. Treatments, temperature, and presence

of light were treated as fixed factors and storage time (days or weeks) as covariate

variables. Alpha level of p<0.05 acceptance of the null hypothesis was used to

determine significant difference.

33

CHAPTER 4: RESULTS AND DISCUSSION

4.1 Compounds separation and identification

Compounds of interest were separated and identified by using an HPLC

(High Performance Liquid Chromatography) system. Figure 6 demonstrates

chromatograms and spectral characteristics of different compounds typically found in

enhanced water beverages. All compounds, except for hydroxycitric acid, were

analyzed using a single HPLC method and running conditions recommended by

Dionex Corporation (2009) for simultaneous detection of water and fat soluble

compounds. Hydroxycitric acid however, did not resolve well under those conditions,

so a separate reversed phase column and method were used. Identification of peaks

was achieved by comparing the retention time, mass to charge ratio (m/z) monitored

by the mass spectrophotometer, and spectral characteristic of each compound and

pure standards.

Due to the different UV absorbance profiles of compounds, simultaneous

detection at wavelengths of 210, 254, and 280 nm were chosen. Based on previously

reported information (Jayaprakasha and Sakariah, 2002), a wavelength of 210 nm was

used to monitor hydroxycitric acid. For simplification, a wavelength of 254 nm was

chosen to monitor a variety of compounds such as ascorbic acid, caffeine, thiamin,

34

riboflavin, and niacinamide simultaneously. Detection of vitamin B6 (pyridoxine and

pyridoxal phosphate) was more sensitive at 280 nm. Both pantothenic acid (vitamin

B5) and cyanocobalamine (vitamin B12) were not successfully detected. It may have

happened because pantothenic acid has a small structure of unconjugated organic

acids which make it less UV active, while cyanocobalamine (vitamin B12), a very

complex compound, exhibits higher absorptivity at different wavelength (Dionex

Corporation, 2009). No fat-soluble compounds were evaluated in this study.

.

35

Figure 6. HPLC chromatogram and UV spectra (obtained with the SPD-M20 A photodiode array) of compounds 36

4.2 Composition of different enhanced water formulations

Five different enhanced water beverage formulations were evaluated for

phytonutrient stability. These formulations were obtained as dry mixes in a powder

form inside a cap for direct delivery into a water bottle. From each formulation we

chose different compounds to evaluate the delivery efficiency, concentration, and

stability under different storage conditions.

The GR formulation was claimed to have role in immunity, digestive health,

wellness, cellular energy, and mental acuity energy. Major compounds present in the

GR formulation were vitamin C, caffeine, and arabinogalactan as a prebiotic fiber,

zinc, and selenium. Vitamin C was present in the form of ascorbic acid, which

typically exists with its oxidation product dehydroascorbic acid (DHAA) when

dissolved in aqueous solution. Both compounds have vitamin C activity; however

DHAA has a low UV absorption. Therefore, only ascorbic acid concentration was

quantified as an indicator of vitamin C degradation under different treatments and

storage conditions. Caffeine and ascorbic acid were quantified in the GR formulation

(figure 7).

The LL formulation is a caffeine-free energy supplement that contains

vitamin C, vitamin B2 (riboflavin), vitamin B3 (as niacinamide), vitamin B1

(thiamin), vitamin B6 (pyridoxine HCl), folic acid, vitamin B12, and lycopene. From

those, ascorbic acid, riboflavin, and niacinamide (figure 7) were separated and

successfully quantified. Other compounds were present in low concentrations

37 in the drink and this might be the reason why no signal detected. Lycopene could not be detected in the drink, most likely due to lack of solubility in water and the solvent used for analyses.

The BR formulation was designed as a weight loss supplement that contains hydroxycitric acid and yerba mate. Hydroxictric acid, in the form of Super Citrimax with 70% concentration of active ingredients, had a distinct peak at 210 nm. Other peaks appeared were not identified. Peaks detected might come from citric acid or a salt form of hydroxycitric acid.

The VH formulation had a more complex matrix which was shown by some unidentified peaks. It contains niacin, panthothenic acid, vitamin B12

(cyanocobalamin), vitamin B6 (pyridoxal phosphate), vitamin C, vitamin D, zinc, and electrolyte blend for rehydration, endurance, and immunity. Ascorbic acid, niacin, pyridoxal phosphate, as well as pyridoxine were identified in this formulation.

The LS formulation contains ingredients that are claimed to provide hangover protection, energy enhancement, hydration, and alcohol detoxification such as vitamin B2, B3, B6, B12, B5, caffeine, glucaronolactone, and electrolyte blend.

Degradation of vitamin B2(riboflavin), B3(niacinamide), B6(pyridoxine), and caffeine were monitored over time.

38

Figure 7. Separation of compounds in different formulations

39

In general, the caps containing ingredients delivered the targeted amount of each compound as claimed on the label (table 7). Measured values were the average of compounds weight obtained at the starting time point (day 0) when the ingredients from the cap were dissolved in 500-ml bottled water. Higher concentration obtained above the targeted level was probably due to the overage added. As for ascorbic acid, small amount of overage might be added because ascorbic acid may slowly degrade even in powder form (Kennedy et al., 1989). However, the amount of overage was lower (10-27%) than the overage commonly added to commercially available juices, which ranged from 20-80% (DSM Nutritional Product and USAID).

Quantity variability during filling the caps might be the cause of the lower amounts detected as compared to the amounts claimed. For example, the amounts of riboflavin quantified in LL obtained at day 0 (1.3 mg) was less compared to the targeted value (1.7 mg). However, the average riboflavin weight obtained at a later time point, at day 7, was similar to the claim (1.95 mg). It has also been reported that caffeine increased water solubility of riboflavin (Guttman and Athalye, 1960) which may explain why the amount of riboflavin found in the LS formulation was higher than the targeted value.

40

Table 7. Compound composition reported and obtained in this study GR LL BR VH LS Ascorbic acid 500 120 - 60 - (mg) 635±10 133±13 66±8 Riboflavin - 1.7 - - 0.440 (mg) 1.3±0.4 0.775±0.055 Niacinamide - 20 - 5.3 (mg) 16±4 6.3±0.7 Pyridoxine - 2 - - 1.3 (mg) Not detected 1.5±0.9 Pyridoxal - - - 2 - (mg) 2.7±0.4 Caffeine 100 - - - 100

41 (mg) 122±27 114±2

Hydroxycitric - - 500 - - acid (mg) 478±47 Other Selenium, zinc, Thiamin, folic Calcium(as Pantothenic acid, Cyanocobalamin, pantothenic ingredients arabinogalactan, acid, hydroxycitrate), cyanocobalamin, acid, glucaronolactone, maltodextrin, cyanocobalamin, chromium vitamin D, zinc, magnesium chloride, citric acid, natural lycopene, polynicotinate, magnesium chloride, potassium chloride, flavor, stevia, maltodextrin, potassium (as potassium chloride, monopotassium phosphate, natural color, citric acid, stevia hydroxycitrate), monopotassium potassium glucarate, citric malic acid leaf extract, yerba mate, stevia phosphate, acid, potassium D-glucarate, natural lime and leaf extract, maltodextrin, citric maltodextrin, natural and lemon flavor, maltodextrin, citric acid, natural raspberry, artificial flavor, malic acid, natural turmeric, acid, natural berry peach,acai, acesulfame potassium, fruit beta-carotene, flavor, natural pomegranate, cranberry and vegetable juices (for lime color strawberry and flavor, stevia, malic color), sucralose, silicon purple carrot color acid, natural colors dioxide Note: : reported value; measured value

41

4.3 Ascorbic acid stability

Storing the formulation in dry environment (in the caps) significantly

maintained the stability of vitamin C (p-value < 0.01). However, as expected, when

the ingredients were mixed with water, the ascorbic acid became unstable and

degraded rapidly (table 8). Lee and Labuza (1975) found that as the water activity and

moisture content increased, the half life of ascorbic acid decreased due to the

increased mobility and lower viscosity in dilute aqueous phase. After one month,

ascorbic acid in GR premixed beverages was almost completely degraded, while

about only 50% of ascorbic acid concentration was retained in the GR hot-fill

processed beverages (figure 8). There was high variability observed in color and

ascorbic acid concentration among samples stored dissolved in water even if

subjected to the same conditions. These results strongly indicate that water and heat

accelerated the degradation of compounds

1.8

1.6

1.4 1.2 1 0.8 0.6

0.4 concentration (mg/ml) concentration 0.2 0 0 10 20 30 day Figure 8. Ascorbic acid degradation in GR formulation stored at 37˚C incubator without light exposure

42

Table 8. Degradation rate of different compounds from formulations stored in 37˚C incubator for 30 days Degradation rate2 Formulation Compounds Treatments1 (g/L per day) cap on bottlea -3 cap onlya -3 Ascorbic acid hot fillab 10.08 premixedb 34.2 GR cap on bottlea -3 cap onlya -3 Caffeine hot filla 0.686 premixeda 0.204 cap on bottlea 4.63 cap onlya 3.67 Ascorbic acid hot fillb 63.47 premixedc 88.88 cap on bottlea 0.58 cap onlya 0.42 LL Niacinamide hot filla 0.35 premixeda 0.4 cap on bottlea 0.051 cap onlya 0.055 Riboflavin hot filla 0.058 premixeda 0.064 cap on bottlea 1.51 Hydroxycitric cap onlya 1.51 BR acid hot fillab 6.43 premixedb 8.45 RT= room temperature 1 Values in each column with same letter are not significantly different (α=0.05) 2 Degradation rate was obtained from the negative slope value of linear regression analysis 3 Indicates positive slope

43

Table 9. Degradation rate of different compounds from VH formulations stored at different storage conditions for 25 weeks Treatments at Degradation rate2 Treatments at Degradation rate2 Treatments at Degradation rate2 Compounds 37C, dark1 (g/L per day) RT, light1 (g/L per day) RT, dark1 (g/L per day) cap on bottlea 0.96 cap on bottlea -3 cap on bottlea -3 a 3 a a 3 Ascorbic cap only - cap only 0.147 cap only - acid hot fillb 20.35 hot fillb 20.35 hot fillb 20.35 premixedc 26.59 premixedc 26.59 premixedc 26.59 Pyridoxine cap on bottlea 0.015 cap on bottlea 0.008 cap on bottleab 0.003 cap onlya -3 cap onlya 0.009 cap onlya -3 hot filla 0.044 hot fillb 0.049 hot fillb 0.045 44 b a c

premixed 0.006 premixed 0.014 premixed 0.009 cap on bottle not detected cap on bottle not detected cap on bottle not detected Pyridoxal cap only not detected cap only not detected cap only not detected Phosphate hot filla 0.707 hot filla -3 hot filla -3 premixeda 0.585 premixeda -3 premixeda -3 RT= room temperature 1 Values in each column with same letter are not significantly different (α=0.05) 2 Degradation rate was obtained from the negative slope value of linear regression analysis 3 Indicates positive slope

44

A similar trend was also observed in the LL formulation which was also

subjected to 37˚C storage for 1 month. Significantly higher degradation rate (p-

value<0.01) was obtained in pre-dissolved formulation (table 8). Since a lower

concentration of ascorbic acid (0.2 mg/ml) was present in the drink, complete

degradation was observed in premixed samples, while more than 80% of ascorbic

acid degraded in hot filling processed samples only after 1 week of storage.

0.25

0.2

cap on bottle-37C cap on bottle-RT,light 0.15 cap on bottle-RT,dark cap only-37C 0.1 cap only-RT,light cap only-RT,dark

concentration (mg/ml) concentration Hot-filled process 0.05 Pre-mixed in water

0 0 5 10 15 20 25 30 week Figure 9. Ascorbic acid degradation in VH formulation stored at different storage environments for 25 weeks

Ascorbic acid (VH formulation) remained stable when stored in a cap for 6

months under different storage conditions (figure 9). Complete degradation of

ascorbic acid was obtained at week 5 in all samples when mixed with water, resulting

45

in same degradation rate of ascorbic acid when stored at different conditions (table 9).

More time points prior to the 5-week measurement might show some differences on

the ascorbic acid degradation trend among treatments. Some studies (Kanneret al.,

1982; Vanderslice and Higgs, 1984; Maeda and Mussa, 1986) have been done to

study the stability of ascorbic acid in solution. In juices packagedin clear bottles, the

primary causes of vitamin C destruction were oxidation by residual oxygen in the

headspace (Gresswell, 1974), anaerobic decomposition (Keffordet al., 1959), and

light exposure (Goussaultet al., 1978). After 5 weeks of storage at room temperature,

0.26 mg/ml of ascorbic acid in bottled orange juice degraded (Maeda and Mussa,

1986), which was also shown in our study.

4.4 Hydroxycitric acid stability

A statistically significant difference between treatments was obtained in the

storage of hydroxycitric acid (table 8, p value< 0.01). Hydroxycitric acid showed

significantly higher stability when kept dry in a cap. Faster degradation, showed by a

higher slope (figure 10), was observed in premixed beverage samples, both with and

without heat treatment process. The half-life of hydroxycitric acid in water was two

months, as compared to over a year when kept dry.

46

1.2

1.1

1 PowerCap on a bottle

y = 0.0009x + 0.9381 0.9 powerCap only y = -0.001x + 0.9725 0.8

Hot-filled process 0.7

y = -0.0061x + 0.9094 concentration (mg/ml) concentration 0.6 Dissolved PowerCap in water 0.5 y = -0.0085x + 0.8971

0.4 0 5 10 15 20 25 30 day

Figure 10.Hydroxycitric acid degradation in BR formulation stored at 37˚C without light exposure

Many studies were done in understanding the effectiveness of hydroxycitric acid in body weight loss. However, limited information is available on the stability of hydroxycitric acid when it is added to food or beverages. The stability data of Super

Citrimax® (HCA-SX) suggests that this compound is stable for over three years when stored at temperature 30˚C and relative humidity of 65% (Soni et al., 2004). Another product specification states that hydroxycitric acid in solution (5% in water) was stable when stored at higher temperature (37˚ and 45˚C) for a minimum of 90 days

(MDidea). However, the concentration used was much higher compared to the

47

hydroxycitric acid present in the BR formulation (1 mg/ml). Therefore, small amount

of degradation might be unnoticeable.

4.5 Riboflavin stability

Riboflavin degradation was evaluated in both accelerated storage study (LL

formulation, table 8) for 30 days and long term storage study (LS formulation, table

10) for 15 weeks.

0.005 0.0045 cap on a bottle

0.004 y = -4E-05x + 0.0034 0.0035 cap in a bag 0.003 y = -4E-05x + 0.0028 0.0025 Hot-filled process 0.002 y = -8E-05x + 0.0046 0.0015 Premixed y = -6E-05x + 0.0042 concentration (mg/ml) concentration 0.001 0.0005 0 0 5 10 15 20 25 30 day

Figure 11. Riboflavin degradation in LL formulation stored at 37˚C incubator for 30 days

48

0.0025 cap on bottle, 25C light

0.002 cap on bottle, 25 C dark cap only, 25C light 0.0015 cap only, 25C dark

hot fill, 25C light 0.001

hot fill, 25C dark concentration (mg/ml) concentration 0.0005 premixed, 25C light

premixed, 25C dark 0 0 5 10 15 week Figure 12. Riboflavin degradation in LS formulation stored at room temperature with and without light exposure for 15 weeks

From the previous literature, which examined the stability of vitamins in

extrusion process which required thermal processing, riboflavin (vitamin B2) was

considered to, be heat stable (Riaz, et al., 2009). However, as seen in figure 11, the

concentration of riboflavin decreased significantly over time for all treatments (p-

value< 0.01). Therefore, different environments with longer storage times were

applied to further observe riboflavin degradation among treatments. Even though

there was a significant different among treatments stored in the same conditions, the

degradation rate of riboflavin when mixed with the water and stored under light

exposure was significantly higher as compared to other treatments and storage

conditions (figure 12, table 10, p-value<0.01). In the LS formulation, 65-73% of the

49

riboflavin content was lost when quantified after 2 weeks of storage. There was also a formation of another peak in the HPLC chromatogram which was only appeared in premixed sample in water exposed to light. This compound was still unidentified, however it was assumed as a degradation product of riboflavin. A similar trend of riboflavin degradation was obtained when the compound was stored in a 37˚C incubator and at room temperature without light exposure.

Light exposure had a greater impact on riboflavin stability as compared to the temperature storage. In milk, only 12% of its riboflavin was lost after boiling for

30 minutes while up to 30% loss was occurred after being exposed to sunlight

(Wishner, 1964). Another study performed on macaroni resulted in more than 50% riboflavin degradation within 1 day (Woodcock et al., 1982). Different components, such as hydroxyl radical, superoxide anion, singlet oxygen, riboflavin cationic and anionic radicals, may be involved in the destruction of riboflavin in foods (Choe et al., 2005). Due to the exposure to light, fragmentation of riboflavin will result in the formation of lumichrome in neutral or acid solution and lumiflavin in alkaline solution (Pan et al., 2001). An unidentified peak appeared when the compound was dissolved in water and stored under light exposure (figure 13). It might be represent the formation of lumichrome. Further investigation using pure standard or other instrumentations might be needed to clarify the identity of this compound.

50

Figure 13. LS formulation chromatogram for sample stored at room temperature with light exposure

51

Table 10. Degradation rate of different compounds from LS formulations stored at different storage conditions for 15 weeks Treatments at Degradation rate2 Treatments at Degradation rate2 Treatments at Degradation rate2 Compounds 37C, dark1 (g/L per day) RT, light1 (g/L per day) RT, dark1 (g/L per day) cap on bottlea 0.06 cap on bottlea 0.03 cap on bottlea 0.04 cap onlyab 0.04 cap onlya 0.04 cap onlya 0.03 Riboflavin hot fillb 0.02 hot fillb 0.48 hot filla 0.04 premixedb 0.02 premixedb 0.57 premixeda 0.05 a a a Niacinamide cap on bottle 0.52 cap on bottle 0.49 cap on bottle 0.40 cap onlya 0.52 cap onlya 0.61 cap onlya 0.50 52 hot filla 0.36 hot filla 0.39 hot filla 0.41

premixeda 0.40 premixeda 0.46 premixeda 0.45 cap on bottlea 0.023 cap on bottlea 0.001 cap on bottlea -3 cap onlya 0.007 cap onlya 0.01 cap onlya -3 Pyridoxine hot filla 0.01 hot fillb 0.08 hot filla 0.005 premixeda 0.002 premixedb 0.09 premixeda 0.002 cap on bottlea -3 cap on bottlea -3 cap on bottleab -3 cap onlya -3 cap onlya 0.2 cap onlya -3 Caffeine hot filla 2.46 hot fillb 8.94 hot fillb 3.63 premixeda 1.32 premixedb 8.08 premixedb 4.16 RT= room temperature 1 Values in each column with same letter are not significantly different (α=0.05) 2 Degradation rate was obtained from the negative slope value of linear regression analysis 3 Indicates positive slope 52

4.6 Pyridoxine and pyridoxal phosphate stability

Two different forms of vitamin B6, pyridoxine and pyridoxal phosphate,

were monitored for their stability in LS and VH formulations. The degradation trend

of pyridoxal phosphate from LS formulation can be seen in figure 14.

Statistically, there was an overall different degradation rate for different

treatments (p-value< 0.01) even though individual tests could not detect the

significant difference. As seen in figure 14, pyridoxine in LS formulation remained

stable and there was no difference between treatments when stored at 37˚C and at

room temperature without light exposure. However, faster degradation was clearly

observed when the compound was dissolved in water and exposed to light (table 10).

At the end of week 15, an average of 46% pyridoxine has lost in hot-fill processed

treatment and 62% lost in premixed beverage samples.

Several studies (Gregory and Kirk, 1978 and Evans et al., 1981) that support

our finding had actually been done to evaluate the factors that affect vitamin B6

stability. They demonstrated the degradation of vitamin B6vitamers at 37˚C using a

first-order kinetic model and calculated the rate constant of pyridoxine degradation at

elevated temperature. One factor that accelerates the degradation was light exposure,

which was also shown in figure 14.

53

.

0.004 a b c

0.0035

0.003 0.0025 0.002 0.0015

54 0.001

concentration (mg/ml) concentration 0.0005 0 0 5 10 15 0 5 10 15 0 5 10 15 week week weel

Figure 14. Pyridoxine degradation in LS formulation stored at a) room temperature, dark; b) 37˚C incubator, dark; c) room temperature, light exposure

54

Instead of pyridoxine, pyridoxal phosphate was listed on the VH formulation nutritional fact table. However, as seen in figure 15, the presence of pyridoxine was detected in all treatments and pyridoxal phosphate was only found when the ingredients were dissolved in water (figure 16). Since pyridoxal phosphate can be produced by oxidation of B6vitamers (Merrill and Henderson, 1990), pyridoxine, as a more stable compound (Saidi and Warthesen, 1983) might be added to prevent degradation prior to consumption. Water seemed to have effect on the formation of pyridoxal phosphate. However, certain time might be required for the activation of pyridoxal since no or low pyridoxalwas detected at week 0.

0.006

0.005

0.004

0.003

0.002 concentration (mg/ml) concentration 0.001

0 0 5 10 15 20 25 week

Figure 15. Pyridoxine stability in VH formulation stored at 37˚C incubator, dark

55

The degradation rate of pyridoxal phosphate for each treatment was analyzed from week 5 (figure 16, table 9). Statistically, the degradation rate of a hot fill sample and a premixed sample was significantly different. Storing the samples at room temperature also resulted in significantly lower degradation rates as compared to samples stored at 37˚C. This finding supported a previous study (Saidi and

Warthesen, 1983) that stated increasing temperature would decrease the half life of pyridoxal.

56

0.045 a b c 0.04 0.035 0.03 0.025 0.02

0.015

concentration (mg/ml) concentration 0.01

57 0.005

0 0 10 20 30 0 10 20 30 0 10 20 30 week week week

Figure 16.Pyridoxal degradation in VH formulation stored as a) hot-filled process sample, dark; b) premixed sample; c) cap sample

57

4.7 Niacinamide stability

Niacinamide concentration was observed during 30-day storage study (LL

formulation) and 15-week storage study (LS formulation) under different

environment storage conditions.

0.045

0.04

0.035

0.03

0.025

0.02

0.015

concentration (mg/ml) concentration 0.01

0.005

0 0 10 20 30 day Figure 17.Niacinamide degradation in LL formulation stored at 37˚C incubator for 30 days

58

0.018 cap on bottle, 37C dark cap only, 37C dark 0.016

hot fill, 37C dark 0.014 premixed, 37C dark 0.012 cap on bottle, RT light 0.01 cap only, RT light 0.008 hot fill, RT light 0.006 premixed, RT light 0.004 cap on bottle, RT dark concentration (mg/ml) concentration cap only, RT dark 0.002 hot fill, RT dark 0 premixed, RT dark 0 5 10 15 week Figure 18. Niacinamide degradation in LS formulation stored in different environments for 15 weeks

Niacinamide (vitamin B3) was expected to be stable. It is commonly not affected by light or heat treatment during processing. However, when dissolved in acid or alkaline condition and exposed to heat, niacinamide was converted to nicotinic acid with the same vitamin activity (Gregory III, 2007). This structure conversion might have occurred during the short term (figure 17) and long term storage study (figure 18) because there was a significant degradation of niacinamide over time (p-value< 0.01) in all treatments. Different storage conditions applied did not affect the degradation rate among treatments (table 8 and10). Niacinamide degradation rate followed first-order kinetic model and ranged from 0.4 mg/L to 0.6 mg/L per day. Nicotinic acid concentration needs to be quantified to evaluate the overall vitamin B3 stablity in the beverages.

59

4.8 Caffeine stability

0.7

0.6

0.5

0.4

0.3

0.2 concentration (mg/ml) concentration

0.1

0 0 10 20 30 day Figure 19. Caffeine degradation in GR formulation stored at 37˚C incubator for 30 days

0.35 cap on bottle, 37C dark 0.3 cap only, 37C dark

0.25 hot fill, 37C dark premixed, 37C dark 0.2 cap on bottle, RT light

0.15 cap only,RT light hot fill, RT light 0.1 premixed, RT light 0.05 cap on bottle, RT dark cap only, RT dark 0 0 5 10 15 20 hot fill, RT dark

Figure 20. Caffeine degradation in LS formulation stored in different environments for 15 weeks 60

Previously reported literature confirmed the stability of caffeine at

temperature, pH, and salt concentrations normally applied in food processing

(Graham, 1978). In agreement with this finding, no significant differences among

treatments were observed and caffeine remained stable during the 1 month storage

study (p-value> 0.1, figure 19). Besides that, different storage temperature applied in

the 15 weeks storage study also did not affect the stability of caffeine. However,

exposure to light accelerated caffeine degradation when it was pre-dissolved in water,

both with and without heat treatment (figure 20, table 10). A similar finding was

found by Anan and Nakagawa (1974) in observing the effect of light on caffeine

concentration in tea seedlings growth. They found that shade treatment depressed

caffeine degradation while faster degradation was observed in tea exposed to normal

daylight.

4.9 Color stability

Besides stability of phytonutrients, color appearance was also affected by

the processing and storage conditions applied. Color changes in beverages were

observed on the 30-day storage study. However, no analytical measurement was done

to quantify color differences. Figure 17 shows a difference in color observed in the

BR formulation after storage of premixed beverages at37˚C incubator for 21 days. Heat

seemed to affect the stability of pigment in aqueous solution.

61

Figure 21. Color change in BR formulation after 21 days of storage at 37˚C incubator

The effect of light exposure was clearly shown in figure 18 (LS formulation) and 19 (LS formulation). Storage of samples at dark environments did not give significant color changes. Table 11 and 12show CIE L*, a*, b* values of VH and LS formulations when stored at room temperature. L* represents light (L*=100) and dark (L*=0); +a* represents green, -a* represents red; +b* represents blue, and –b* represents yellow.

62

Stored under light exposure

Figure 22. Color change in VH formulation after 25 weeks of storage

Table 11. CIE L*, a*, b* values of VH formulation when stored under light exposure

Week Cap on bottle cap only hot fill Premixed ontents L* (SD) L* (SD) L* (SD) L* (SD)

Abstract0 ...... 76.00 (2.34) ...... 76.00 (2.34) ...... 78.18 (1.70) ...... 76.00 (2.34) ii 5 84.61 (3.78) 76.82 (1.45) 86.68 (2.25) 85.72 (2.72) Acknowledgements...... iv 10 78.71 (2.45) 79.47 (3.63) 90.67 (0.54) 89.93 (1.92) VITA15 ...... 67.72 (10.14) ...... 63.45 (4.16) ...... 91.67 (0.61) ...... 91.97 (1.13).... v 20 68.20 (1.35) 65.39 (3.54) 86.25 (1.02) 84.33 (0.99) List25 of Tables69.34 ...... (10.05) 67.01...... (3.77) ...... 93.11 (0.95) ...... 93.93 (0.69) ix List of Figures ...... a* (SD) ...... a* (SD) ...... a* (SD) ...... a* (SD) x 0 19.06 (2.66) 19.06 (2.66) 27.22 (7.21) 19.06 (2.66) CHAPTER5 1:16.14 INTRODUCTION (2.85) ...... 22.52 (1.74) ...... 9.82 (2.21) ...... 10.83 (3.32) 1 10 21.02 (3.07) 20.26 (4.06) 4.40 (0.47) 4.96 (2.31) CHAPTER 2: LITERATURE REVIEW ...... 3 15 35.38 (14.67) 42.16 (4.81) 3.10 (0.59) 2.67 (1.04) 2.120 Functional35.53 Food (1.33 ...... ) 39.58 (...... 5.30) 7.36...... (1.01) 9.78...... (1.10) 3 25 34.40 (12.73) 36.43 (4.41) 1.86 (0.75) 1.03 (0.54) 2.2 Functionalb Beverages* STDEV ...... b* (SD) ...... b* (SD)...... b* (SD)...... 4 0 17.96 (3.78) 17.96 (3.78) 17.07 (2.32) 17.96 (3.78) 5 2.2.1 Energy10.75 drinks (3.37 ...... ) 16.55 (0.91...... ) 12.99 ...... (2.85) 19.71...... (4.67) 5 10 13.58 (1.15) 12.41 (6.26) 6.97 (0.73) 10.17 (1.71) 2.2.2 Enhanced water ...... 6 15 38.11 (22.53) 48.90 (6.32) 6.36 (1.03) 8.00 (1.88) 2.320 U.S functional37.41 ( beverages1.94) market44.32 ...... (7.54) 15.79...... (1.09) ...... 17.99 (1.70) 7 25 38.30 (17.44) 36.58 (5.69) 4.52 (1.19) 4.60 (1.05) 2.3.1 Market of functional beverages ...... 7 2.3.2 Market for enhanced water and energy drink ...... 8 2.4 Functionality of enhanced water and energy63 drink phytochemicals ...... 9

2.4.1 Vitamins ...... 9 2.4.2 Energy Enhancing Ingredients ...... 10 2.4.3 Weight loss compounds ...... 12 2.4.4 Natural food colorant ...... 13

Stored under light exposure

Figure 23. Color change in LS formulation after 15 weeks

Table 12. CIE L*, a*, b* values of LS formulation when stored under light exposure

Week Cap on bottle cap only hot fill Premixed L* (SD) L* (SD) L* (SD) L* (SD) 0 83.38 (0.20) 83.38 (0.20) 82.43 (1.01) 83.38 (0.20) 2 83.63 (0.84) 83.14 (0.56) 96.55 (0.07) 96.67 (0.03) 5 83.43 (1.76) 83.35 (1.29) 96.62 (0.04) 96.71 (0.01) 10 82.31 (2.08) 84.78 (1.04) 95.71 (0.06) 95.86 (0.02) 15 82.71 (0.28) 82.23 (0.18) 95.77 (0.05) 95.91 (0.03) a* (SD) a* (SD) a* (SD) a* (SD) 0 21.72 (0.54) 21.72 (0.54) 23.54 (1.55) 21.72 (0.54) 2 21.71 (1.52) 22.82 (0.50) -0.34 (0.12) -0.41 (0.10) 5 22.36 (3.02) 22.00 (2.16) -0.02 (0.04) -0.09 (0.03) 10 22.35 (3.25) 19.04 (1.39) 0.09 (0.08) -0.03 (0.01) 15 21.88 (0.62) 22.74 (0.07) 0.09 (0.04) -0.01 (0.02) b* (SD) b* (SD) b* (SD) b* (SD) 0 1.29 (0.13) 1.29 (0.13) 1.41 (0.10) 1.29 (0.13) 2 1.26 (0.06) 1.40 (0.05) 1.80 (0.43) 1.68 (0.36) 5 1.21 (0.10) 1.12 (0.15) 0.56 (0.14) 0.48 (0.10) 10 1.16 (0.12) 1.24 (0.09) 0.33 (0.03) 0.33 (0.02) 15 1.16 (0.07) 1.17 (0.08) 0.28 (0.02) 0.27 (0.01)

64

No information was reported on the label regarding the source of natural colorants used in the VH and LS formulations. Based on color characteristics, pigments used can be predicted. A basic solvent was added to each formulation to observe any color change. The initial color in VH formulation was red. However, when the pH reached 8.0, the color changed to yellow. In the LS formulation, no color change was observed and maintained its purple color. From the observation above, the colorants used in the VH formulation was might be derived from anthocyanins, while betalains group was used for the LS formulation. Analysis by using HPLC and MS may be needed to further confirm the compounds identity.

Light affects anthocyanins stability. The highest loss (70%) occurred under fluorescent light with slightly higher temperature storage (Palamides and Markakis,

1978). A linear relationship was obtained between the log rate of anthocyanin color degradation in strawberry and storage temperature (Meschter, 1953). A previous study (Markakiset al., 1957) suggested high temperature-short time processing of fruit and vegetables to maintain color stability.

In our study, light exposure, different treatments (water content), and storage temperature affected the color stability in the VH formulation (p-value < 0.1).

Both hot fill and premixed samples in VH showed increasing lightness, reducing +a*

(red) and +b* (yellow) values when stored under light exposure, which could be seen in figure 18 as a faint orange color observed. A slight color change was also observed in cap treatment samples. Visually there was no clear color difference observed between beverages stored at 37˚C and at room temperature. However, based on the 65

color spectrometer measurement, there was a significant difference in color appearance.

A significant effect of light and treatment (water content) in color degradation was observed in the LS formulation (p-value< 0.01). No significant difference observed in the LS formulation color even after 15 weeks of storage under light exposure when the ingredients were stored dry inside a cap. However, after 2 weeks of storage at room temperature under light exposure, a clear solution was obtained from hot fill and premixed beverages, which was instrumentally indicated by decrease in L* value and increase in both a* and b* values.

Absorption of light leads to electron excitation of the betalain chromophore to a higher energy state which will cause higher compound’s reactivity (Jackman and

Smith, 1996). In addition to light exposure, water activity also affects betalain stability. Improved stability can be obtained at aw below 0.63 (Kearsley and

Katsabrakis, 1980) due to low mobility of reactants.

66

CHAPTER 5: CONCLUSION

This study investigated the ability of cap dosing closure to protect ingredients of functional beverages in retaining chemical integrity of phytochemicals as compared to nutrients subjected to the conditions of typical hot fill beverage manufacturing. Exposure to light was the biggest factor that affected compounds stability. As compared to other storage conditions, stability of ascorbic acid, riboflavin, pyridoxine, hydroxycitric acid, and caffeine increased significantly improved when kept dry, even under light exposure.

No significant difference was observed on niacinamide stability among treatments.

Color degradation was also limited when the samples were kept dry in a cap.

Stability of betalains and anthocyanins was significantly affected by light when dissolved in water.

There was no significant difference between the compounds stability when stored in a cap that was attached to bottled water and without any attachment in different storage conditions.

Our results indicate that the delivery of functional ingredients in the dry form inside a cap would improve stability, lower the manufacturing cost due to reduced losses, and provide functional beverages with more consistent nutritional value for customers.

67

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