STORAGE STABILITY OF TAGATOSE IN BUFFER SOLUTIONS OF VARIOUS COMPOSITION Except where reference is made to the work of others, the work described in this thesis is my own or was done in collaboration with my advisory committee. This thesis does not include proprietary or classified information. __________________________ Cathleen M. Dobbs Certificate of Approval: __________________________ ____________________________ Robin B. Fellers Leonard N. Bell, Chair Associate Professor Professor Nutrition and Food Science Nutrition and Food Science __________________________ ____________________________ Sareen S. Gropper George T. Flowers Professor Dean Nutrition and Food Science Graduate School STORAGE STABILITY OF TAGATOSE IN BUFFER SOLUTIONS OF VARIOUS COMPOSITION Cathleen M. Dobbs A Thesis Submitted to the Graduate Faculty of Auburn University in Partial Fulfillment of the Requirements of the Degree of Master of Science Auburn, Alabama December 19, 2008 iii STORAGE STABILITY OF TAGATOSE IN BUFFER SOLUTIONS OF VARIOUS COMPOSITION Cathleen M. Dobbs Permission is granted to Auburn University to make copies of this thesis at its discretion, upon request of individuals or institutions at their expense. The author reserves all publication rights. ________________________ Signature of Author ________________________ Date of Graduation iv THESIS ABSTRACT STORAGE STABILITY OF TAGATOSE IN BUFFER SOLUTIONS OF VARIOUS COMPOSITION Cathleen M. Dobbs Master of Science, December 19, 2008 (B.S., Auburn University, 2006) 106 Typed Pages Directed by Leonard N. Bell Tagatose, an epimer of fructose, is a minimally absorbed monosaccharide that has been shown to function as a prebiotic in the human body. For this prebiotic effect to be achieved, tagatose in food and beverage products must not be lost during their processing and storage. However, data on the storage stability of tagatose is lacking. The objective of this study was to evaluate the storage stability of tagatose in solutions as affected by buffer type, buffer concentration, pH and temperature. Tagatose solutions (0.05 M or about 1%) were prepared in 0.02 and 0.1 M phosphate and citrate buffers at pH 3 and 7. Triplicate vials were stored at 20, 30 and 40?C. These eight solutions were prepared again with the addition of 0.05 M v glycine. Aliquots were analyzed at regular time intervals for nine months. Tagatose analysis occurred via reverse-phase HPLC while browning was measured using a spectrophotometer at 420 nm. In the solutions with no added glycine, no tagatose loss or browning was observed in 0.02 M phosphate and citrate buffers at pH 3. In 0.1 M buffers at pH 3 and 40?C, 5-10% tagatose was lost over nine months and slight browning occurred. Tagatose loss was enhanced at pH 7. Tagatose degraded in a biphasic manner, with a rapid initial decrease followed by a plateau. The pseudo-first order rate constants (k obs ) for the initial tagatose degradation at pH 7 were greater in phosphate buffer than citrate buffer. Higher buffer concentrations also increased k obs . In phosphate buffers at pH 7, browning accompanied the tagatose loss, increasing to a maximum and then decreasing as tagatose loss plateaued. In solutions with added glycine, tagatose also degraded faster at pH 7 than pH 3. However, glycine did not enhance tagatose loss. Glycine did enhance browning as compared to solutions without glycine. Tagatose degradation and browning occurred faster at higher phosphate buffer concentrations and at higher temperatures. To deliver the prebiotic effect from tagatose, shelf-stable beverages should be formulated to the lowest buffer concentration and pH possible to optimize tagatose?s stability. vi ACKNOWLEDGEMENTS The author would like to express tremendous gratitude to Dr. Leonard Bell for his support, dedication, and guidance throughout the preparation of this thesis, and throughout completion of the graduate program. She would also like to express enormous appreciation to her thesis committee, Dr. Sareen Gropper and Dr. Robin Fellers, for review and recommendations of this thesis, as well as their continued support and advice. Lastly, the author would like to thank her family and friends for their unwavering support and love throughout her educational experience at Auburn University. vii Style manual of journal used: Journal of Food Science Computer software used: Microsoft Word, Microsoft Excel viii TABLE OF CONTENTS LIST OF TABLES .............................................................................................................. x LIST OF FIGURES .......................................................................................................... xiii CHAPTER 1. INTRODUCTION ........................................................................................ 1 CHAPTER 2. REVIEW OF LITERATURE ....................................................................... 3 Nutraceuticals .............................................................................................. 3 Definition of Prebiotics and Probiotics ....................................................... 4 Prebiotic Examples and Effects ................................................................... 5 Effect on Mineral Absorption .......................................................... 7 Effect on Immune System ............................................................... 9 Miscellaneous Human Studies ........................................................ 9 Tagatose ..................................................................................................... 10 Chemical Properties ....................................................................... 10 Manufacturing ............................................................................... 11 Health Benefits .............................................................................. 12 Adverse Side Effects ..................................................................... 14 Monosaccharide Reactions and Stability ................................................... 15 Monosaccharide Degradation ........................................................ 15 Alkaline Degradation ......................................................... 15 Acidic Degradation ............................................................ 18 Maillard Reaction .......................................................................... 19 Basic Overview ................................................................. 19 pH Effect ........................................................................... 20 Buffer Effect ...................................................................... 21 Justification and Objective ........................................................................ 21 CHAPTER 3. MATERIALS AND METHODS ............................................................... 23 Sample Preparation .................................................................................... 23 Sampling Procedure ................................................................................... 25 Tagatose Analysis ...................................................................................... 25 Browning Analysis .................................................................................... 27 Data Analysis ............................................................................................. 27 ix CHAPTER 4. RESULTS AND DISCUSSION ................................................................ 28 Tagatose Degradation ................................................................................ 28 Effect of pH ................................................................................... 33 Effect of Buffer Type and Concentration ...................................... 35 Effect of Temperature .................................................................... 36 Participation of Tagatose in Maillard Reaction ......................................... 37 Effect of pH ................................................................................... 42 Effect of Buffer Type and Concentration ...................................... 43 Effect of Temperature .................................................................... 44 Browning ................................................................................................... 45 Comparing Tagatose Reactivity with and without Glycine ...................... 47 CHAPTER 5. SUMMARY AND CONCLUSIONS ......................................................... 51 REFERENCES .................................................................................................................. 52 APPENDIX A ................................................................................................................... 57 APPENDIX B .................................................................................................................... 66 APPENDIX C .................................................................................................................... 75 APPENDIX D ................................................................................................................... 84 x LIST OF TABLES TABLE 4.1. Pseudo-First Order Rate Constants (d -1 ) with 95% Confidence Limits for the Initial Loss of Tagatose in Solution ............................................... 33 TABLE 4.2. Predicted Percent Loss of Tagatose in Solution at 100 days ........................ 33 TABLE 4.3. Activation Energies (E A ) for the Initial 100 d of Tagatose Degradation in Solution at pH 7 ................................................................ 37 TABLE 4.4. Pseudo-First Order Rate Constants (d -1 ) with 95% Confidence Limits for the Initial Loss of Tagatose in Solution also containing 0.05 M Glycine ......................................................................................... 42 TABLE 4.5. Predicted Percent Loss of Tagatose in Solutions containing 0.05 M Glycine at 100 days ................................................................................... 42 TABLE 4.6. Activation Energies (E A ) for Tagatose Degradation in 0.05 M Glycine Soltuions at pH 7 ....................................................................................... 44 TABLE A1. Tagatose Degradation in 0.02 M Phosphate Buffer at pH 3 as Affected by Temperature ...................................................................... 58 TABLE A2. Tagatose Degradation in 0.02 M Phosphate Buffer at pH 7 as Affected by Temperature ...................................................................... 59 TABLE A3. Tagatose Degradation in 0.1 M Phosphate Buffer at pH 3 as Affected by Temperature ...................................................................... 60 TABLE A4. Tagatose Degradation in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature ...................................................................... 61 TABLE A5. Tagatose Degradation in 0.02 M Citrate Buffer at pH 3 as Affected by Temperature ...................................................................... 62 TABLE A6. Tagatose Degradation in 0.02 M Citrate Buffer at pH 7 as Affected by Temperature ...................................................................... 63 xi TABLE A7. Tagatose Degradation in 0.1 M Citrate Buffer at pH 3 as Affected by Temperature ...................................................................... 64 TABLE A8. Tagatose Degradation in 0.1 M Citrate Buffer at pH 7 as Affected by Temperature ...................................................................... 65 TABLE B1. Tagatose Degradation in 0.02 M Phosphate Buffer (with 0.05 M Glycine) at pH 3 As Affected by Temperature ......................................... 67 TABLE B2. Tagatose Degradation in 0.02 M Phosphate Buffer (with 0.05 M Glycine) at pH 7 As Affected by Temperature ......................................... 68 TABLE B3. Tagatose Degradation in 0.1 M Phosphate Buffer (with 0.05 M Glycine) at pH 3 As Affected by Temperature ........................................................ 69 TABLE B4. Tagatose Degradation in 0.1 M Phosphate Buffer (with 0.05 M Glycine) at pH 7 As Affected by Temperature ........................................................ 70 TABLE B5. Tagatose Degradation in 0.02 M Citrate Buffer (with 0.05 M Glycine) at pH 3 As Affected by Temperature ........................................................ 71 TABLE B6. Tagatose Degradation in 0.02 M Citrate Buffer (with 0.05 M Glycine) at pH 7 As Affected by Temperature ........................................................ 72 TABLE B7. Tagatose Degradation in 0.1 M Citrate Buffer (with 0.05 M Glycine) at pH 3 As Affected by Temperature ........................................................ 73 TABLE B8. Tagatose Degradation in 0.1 M Citrate Buffer (with 0.05 M Glycine) at pH 7 As Affected by Temperature ........................................................ 74 TABLE C1. Browning of 0.05 M Tagatose in 0.02 M Phosphate Buffer at pH 3 as Affected by Temperature ...................................................................... 76 TABLE C2. Browning of 0.05 M Tagatose in 0.02 M Phosphate Buffer at pH 7 as Affected by Temperature ...................................................................... 77 TABLE C3. Browning of 0.05 M Tagatose in 0.1 M Phosphate Buffer at pH 3 as Affected by Temperature ...................................................................... 78 TABLE C4. Browning of 0.05 M Tagatose in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature ...................................................................... 79 TABLE C5. Browning of 0.05 M Tagatose in 0.02 M Citrate Buffer at pH 3 as Affected by Temperature ...................................................................... 80 xii TABLE C6. Browning of 0.05 M Tagatose in 0.02 M Citrate Buffer at pH 7 as Affected by Temperature ...................................................................... 81 TABLE C7. Browning of 0.05 M Tagatose in 0.1 M Citrate Buffer at pH 3 as Affected by Temperature ...................................................................... 82 TABLE C8. Browning of 0.05 M Tagatose in 0.1 M Citrate Buffer at pH 7 as Affected by Temperature ...................................................................... 83 TABLE D1. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.02 M Phosphate Buffer at pH 3 as Affected by Temperature .............................................. 85 TABLE D2. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.02 M Phosphate Buffer at pH 7 as Affected by Temperature .............................................. 86 TABLE D3. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.1 M Phosphate Buffer at pH 3 as Affected by Temperature .............................................. 87 TABLE D4. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature .............................................. 88 TABLE D5. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.02 M Citrate Buffer at pH 3 as Affected by Temperature .............................................. 89 TABLE D6. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.02 M Citrate Buffer at pH 7 as Affected by Temperature .............................................. 90 TABLE D7. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.1 M Citrate Buffer at pH 3 as Affected by Temperature .............................................. 91 TABLE D8. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.1 M Citrate Buffer at pH 7 as Affected by Temperature .............................................. 92 xiii LIST OF FIGURES FIGURE 2.1. Structural Representation of Inulin (n=27-29) and Fructooligosaccharides (n=2-10) ................................................................. 6 FIGURE 2.2. Structure of ?-D-Tagatose ........................................................................... 11 FIGURE 3.1. Sample HPLC Chromatogram for the Analysis of Tagatose in Buffer with 0.05 M Glycine using a Luna Amino Column (Phenomenex, Torrence, CA) and Mobile Phase Consisting of 85/15 (v/v) Acetonitrile/Water Running at 2.5 mL/min. Tagatose Elutes at Approximately 4.9 min and Glycine at 28.7 min ...................................... 26 FIGURE 3.2. Sample Calibration Curve for Tagatose Analysis by HPLC ....................... 27 FIGURE 4.1. Tagatose Loss in 0.1 M Buffer Solutions at 40?C as Affected by pH ........ 28 FIGURE 4.2. Tagatose Loss in 0.02 M Buffer Solutions at 20?C as Affected by pH ...... 29 FIGURE 4.3. Tagatose Loss in Solution at pH 3 and 40?C as Affected by Buffer Type and Concentration ................................................................. 29 FIGURE 4.4. Tagatose Loss in Solution at pH 7 and 40?C as Affected by Buffer Type and Concentration ................................................................. 30 FIGURE 4.5. Tagatose Loss in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature ...................................................................... 30 FIGURE 4.6. Tagatose Loss in 0.02 M Citrate Buffer at pH 3 as Affected by Temperature ...................................................................... 31 FIGURE 4.7. Initial Loss of Tagatose in 0.1 M Phosphate Buffer at pH 7 Modeled using Pseudo-First Order Kinetics as Affected by Temperature ............... 32 FIGURE 4.8. Tagatose Loss in 0.1 M Buffer Solutions also Containing 0.05 M Glycine at 40?C as Affected by pH ........................................................... 38 FIGURE 4.9. Tagatose Loss in 0.02 M Buffer Solutions also Containing 0.05 M Glycine at 20?C as Affected by pH ........................................................... 39 xiv FIGURE 4.10. Tagatose Loss in Solution at pH 3 and 40?C also Containing 0.05 M Glycine as Affected by Buffer Type and Concentration ........................... 39 FIGURE 4.11. Tagatose Loss in Solution at pH 7 and 40?C also Containing 0.05 M Glycine as Affected by Buffer Type and Concentration ........................... 40 FIGURE 4.12. Tagatose Loss in 0.1 M Phosphate Buffer also Containing 0.05 M Glycine at pH 7 as Affected by Temperature ............................................ 40 FIGURE 4.13. Tagatose Loss in 0.02 M Citrate Buffer also Containing 0.05 M Glycine at pH 3 as Affected by Temperature ............................................ 41 FIGURE 4.14. Initial Loss of Tagatose in 0.1 M Phosphate Buffer also Containing 0.05 M Glycine at pH 7 Modeled using Pseudo-First Order Kinetics as Affected by Temperature ...................................................................... 41 FIGURE 4.15. Browning of Tagatose in 0.1 M Buffer Solutions at 40?C as Affected by Buffer Type and pH .............................................................................. 45 FIGURE 4.16. Browning of Tagatose and Glycine in 0.1 M Buffer Solutions at 40?C as Affected by Buffer Type and pH .......................................................... 46 FIGURE 4.17. Effect of Glycine on Tagatose Loss in 0.1 M Phosphate Buffer at pH 7 and 40?C ....................................................................................... 47 FIGURE 4.18. Effect of Glycine on Tagatose Loss in 0.1 M Citrate Buffer at pH 7 and 40?C ....................................................................................... 48 FIGURE 4.19. Effect of Glycine on Tagatose Browning in 0.1 M Phosphate Buffer at pH 7 and 40?C ....................................................................................... 48 FIGURE 4.20. Effect of Glycine on Tagatose Browning in 0.1 M Citrate Buffer at pH 7 and 40?C ....................................................................................... 49 1 CHAPTER 1: INTRODUCTION Nutraceuticals are substances found in foods that have been shown to positively impact human health by preventing diseases or improving physiological function. The nutraceutical classification includes vitamins, minerals, genestein, lycopene, flavonoids, inulin, and fructooligosaccharides, among others (Wildman and Kelley 2007). For nutraceuticals to be beneficial in the human body, they must not be degraded during food processing and storage. Significant data have been collected regarding the beneficial health effects of nutraceuticals; however, less data about their stability in foods exists. The nutraceuticals inulin and fructooligosaccharide are also classified as prebiotics. Prebiotics are food components that stimulate the growth and/or activity of beneficial bacteria in the colon, and thus improve host health. Most prebiotics are not digested or absorbed in the small intestine, but pass into the large intestine where they are then fermented into short chain fatty acids by intestinal microflora. The short chain fatty acids help promote the growth of beneficial intestinal bacteria while inhibiting pathogenic bacteria. Tagatose is a monosaccharide that also falls into both the nutraceutical and prebiotic categories. An epimer of fructose, tagatose displays a sweetness and bulk comparable to sucrose (Levin 2002). Tagatose obtained GRAS (generally 2 recognized as safe) status in 2000 in the United States (Levin 2002), and has also been approved for use in Australia, New Zealand, South Korea, Brazil, South Africa and the United Kingdom (FSANZ 2004; Skytte 2006; FSA 2005). Tagatose is currently found in several products in Europe; these include a cocoa drink, a chocolate-hazelnut spread, diet jams, and chocolate bars (Damhert 2008). One reason tagatose may be utilized is for its positive health effects. It contributes less than 1.5 kcal/g, which is far less than other carbohydrates at 4 kcal/g (Levin 2002). Tagatose also does not elicit a glycemic response in diabetes mellitus patients (Donner and others 1999). It does not promote tooth decay (Levin and others 1995). Tagatose has also been shown to contribute positively to human health by increasing the production of short chain fatty acids in the intestines (Bertelsen and others 1999). It is this prebiotic effect that is most significant. For the prebiotic effect of tagatose to be achieved, it must not be degraded during food processing and storage. The storage stability of tagatose has not been widely studied. Therefore, the objective of this study was to evaluate the stability of tagatose in solution under various conditions, such as buffer type and concentration, pH, and temperature. 3 CHAPTER 2: REVIEW OF LITERATURE The literature review addresses nutraceuticals and then more specifically prebiotics. After discussing examples and effects of prebiotics, the properties and utilization of tagatose will be presented. Monosaccharide stability will then be addressed. The objectives of the current study will conclude this chapter. Nutraceuticals Over the past several years, numerous biologically active substances have been shown to contribute positively to human health; these substances have been designated ?nutraceuticals? or ?functional foods?. Nutraceuticals have been defined as ?chemicals found as a natural component of foods or other ingestible forms that have been determined to be beneficial to the human body in preventing or treating one or more diseases or improving physiological performance? (Wildman 2001). Nutraceuticals include an assortment of substances, from vitamins and minerals to antioxidants. Some examples of nutraceuticals are lycopene, flavonoids and tocotrienols. Lycopene, produced by some fruits and vegetables, has been shown to prevent free radical damage in the body (Bruno and Wildman 2001). Flavonoids, found in plants, also protect the body against oxidative damage (DiSilvestro 2001). Tocotrienols, naturally occurring analogues of tocopherol, function as antioxidants and have been shown to have anticancer and cholesterol lowering properties (Guthrie and Kurowska 2001). For these and any other nutraceutical to have a 4 healthful benefit, the substances must not be degraded during food processing or product storage. Significant data have been collected on the health effects of nutraceuticals; however, less information exists about their behavior and stability in foods. Definition of Prebiotics and Probiotics Another group of substances classified as nutraceuticals are the prebiotics and probiotics. Gibson and Roberfroid (1995) defined a prebiotic as ?a nondigestible food ingredient that beneficially affects the host by selectively stimulating the growth and/or activity of one or a limited number of bacteria in the colon, and thus improves host health?. Prebiotics are metabolized by intestinal microorganisms and help those organisms already present in the gut to grow. Many foods are marketed as containing probiotics, which are not the same as prebiotics. Fuller (1989) defined the term probiotic as ?a live microbial feed supplement which beneficially affects the host animal by improving its intestinal microbial balance?. The main difference between a prebiotic and probiotic is that the latter includes living microorganisms that are found in certain foods. Probiotics can be one or two different microorganisms in foods that help the body. Species of lactobacilli and bifidobacteria are commonly used probiotics that are components of fermented milk products, such as yogurt and sour cream (Gibson and Roberfroid 1995). It has been shown that probiotics have a role in alleviating lactose intolerance, enhancing the immune system, and in lowering cholesterol (Roberfroid 2000). 5 Prebiotic Examples and Effects Prebiotics encompass a wide range of substances, including ?-glucans, pectin, gums and resistant starch 3. Two of the more commonly studied prebiotics are inulin and fructooligosaccharides (Figure 2.1). Inulin is a carbohydrate composed of ?-(2-1)-fructans where the degree of polymerization ranges from 2 to 60, but most commonly from 27-29 with a terminal glucose (Franck and De Leenheer 2002). The most common sources of inulin are wheat, onions and bananas. Inulin-type fructans can be used as sugar and fat replacers, and as a way of providing texture, improving mouth feel, and stabilizing foams. It has been suggested that inulin-type fructans can help alleviate constipation and diarrhea as well as reduce the risk of osteoporosis, atherosclerotic cardiovascular disease and obesity (Roberfroid 2000). Fructooligosaccharides consist of a terminal glucose molecule with 2 to 10 fructose units attached by a ?-(2-1)-glycosidic linkage (Niness 1999). They occur naturally in plants such as onion, wheat, rye, asparagus and triticale and can be produced industrially through an enzymatic process (Bornet 1994). Fructooligosaccharides function mainly as sugar replacers. The primary difference between inulin and fructooligosaccharides is the degree of polymerization (Figure 2.1). Most prebiotics that are ingested are not digested or absorbed in the small intestine. Molis and others (1996) determined that 89 ? 8.3% of the ingested fructooligosaccharides in healthy humans was never absorbed. Elleg?rd and others 6 O HO OH O CH 2 OH O HO OH OHOH 2 C CH 2 O OH OH OH CH 2 OH HOH 2 C n Figure 2.1. Structural Representation of Inulin (n=27-29) and Fructooligosaccharides (n=2-10). (1997) demonstrated that 88% of inulin and 89% of oligofructose that was ingested by ileostomy patients was recovered intact. These prebiotics then pass to the large intestine where microflora ferment them. Kleessen and others (1997) examined the effect of inulin ingestion on fecal microflora in humans. Inulin was given in doses of 20 g/d for the first eight days, gradually increased to 40 g/d for two days, and then set at 40 g/d for the last seven days. Bifidobacteria in the feces increased from 7.9 ? 0.4 log 10 /g before inulin administration to 9.2 ? 0.5 log 10 /g at the end of the study (Kleessen and others 1997). 7 Gibson and others (1995) demonstrated the laxative effect of prebiotics. When humans were supplemented with 15 g of oligofructose, stool output increased from 135.8 ? 22.8 g/d to 154.1 ? 22.9 g/d. By increasing stool output, oligofructose is acting like other non-digestible carbohydrates (i.e., fibers). The increase in stool output can be attributed to an increase in biomass, which was supported by an increase in nitrogen excretion. Prebiotics can influence many different aspects of human health. The effect of prebiotic consumption on mineral metabolism, the immune system, and on humans in general will be discussed. Effect on Mineral Absorption Ohta and others (1994) examined phosphorus and magnesium balance in rats fed a diet containing different levels of fructooligosaccharides (FO). No difference in phosphorus balance in rats was found when they were fed 1% or 5% FO. The rats that were fed a control diet containing no FO had a phosphorus absorption of 80.1%. The rats that were fed 1% FO had a phosphorus absorption of 81.8%. Similarly, the rats that were fed 5% FO had a phosphorus absorption of 78.1%. Contrary to the phosphorus balance, Ohta and others found that magnesium absorption increased in rats that were fed a diet containing fructooligosaccharides. The rats fed a control diet (no FO) had a magnesium absorption ratio of 54.4% whereas the rats supplemented with 1% FO had an absorption ratio of 62.3%. A more significant increase was shown with the rats fed 5% FO. Their magnesium absorption ratio increased to 71.8%. Rats supplemented with FO showed an 8 increase in magnesium absorption while phosphorus absorption stayed relatively constant. Delzenne and others (1995) showed that absorption of calcium, magnesium, iron and zinc all significantly improved when rats were fed a diet containing 10% fructooligosaccharides versus ones who were not fed fructooligosaccharides. Ohta and others (1995) demonstrated in rats that fructooligosaccharides improved recovery from anemia. This recovery was attributed to increased iron absorption, as shown by Delzenne and others (1995). Scholz-Ahrens and Schrezenmeir (2002) also evaluated the effects of prebiotics on mineral metabolism in rats. To evaluate calcium balance, ovariectomized rats were fed a diet containing 0.5% calcium plus 0, 25, 50 or 100 grams (g) oligofructose/kg diet, or diets containing 1.0% Ca plus 0 or 50 g oligofructose/kg diet for 16 weeks. After 4, 8 and 16 weeks, there was a positive trend for increased calcium retention with oligofructose consumption. Consistent with other studies, they also found that phosphorus absorption and retention were unaffected by 0.5% or 1.0% dietary oligofructose (Scholz-Ahrens and Schrezenmeir 2002). While several studies have determined the effect of prebiotics on mineral metabolism in rats, one study used humans as their subjects. Coudray and others (1997) evaluated the effect of inulin supplementation on the absorption of several minerals in humans. Inulin significantly increased calcium absorption (control, 21.3 9 ? 12.5%; inulin, 33.7 ? 12.1%), but did not affect magnesium, iron, and zinc absorption (Coudray and others 1997). Effect on Immune System Gibson and Roberfroid (1995) demonstrated that inulin and oligofructose fermentation increased the production of short chain fatty acids, mainly acetate, butyrate and propionate in the gut. As mentioned previously, inulin and oligofructose increase the number of beneficial bacteria in the gut, which can help the body ward off infection (Kleessen and others 1997; Gibson and others 1995). Short chain fatty acid production may reduce the need for glutamine by the epithelial cells, which allows the cells in the immune system to use the glutamine (Jenkins and others 1999). Pratt and others (1996) showed that the natural killer cells in rats fed total parenteral nutrition supplemented with short chain fatty acids had significantly higher cytotoxic activity than the rats fed only the total parenteral nutrition. Therefore, it appears the fermentation of prebiotics into short chain fatty acids may improve the immune system. Miscellaneous Human Studies Cummings and Macfarlane (2002) reviewed the effect of oligofructose in humans. No change in blood glucose or insulin levels were observed when 25 g of oligofructose were fed to healthy subjects. With regards to fermentability, in pH controlled co-cultures of B. infantis, E. coli and C. perfringens with oligofructose being the only carbohydrate substrate, the bifidobacteria grew well and displayed an inhibitory effect on the growth of E. coli and C. perfringens. Human feeding 10 studies confirmed the effect of bifidobacteria on the inhibition of harmful bacteria (Cummings and Macfarlane 2002). Saavedra and others (1999) evaluated the effects of supplementing infants with oligofructose. Infants (n=123) aged 4 to 24 months were divided into control and experimental groups. The control group received a commercially available infant cereal, while the experimental group received the same cereal supplemented with oligofructose at a concentration of 0.55 g per 15 g of dry cereal. The experimental group received a daily average of 1.1 g oligofructose. No significant differences were observed between the two groups in stool frequency and consistency or the occurrence of flatulence. The experimental group exhibited a significantly lower frequency of emesis, discomfort with bowel movements, and regurgitation. During a diarrheal episode, the supplemented group did not run a fever as often as the control group [8.25 and 21.4 (frequency per subject-year), respectively]. Saavedra and others (1999) concluded that supplementation with oligofructose resulted in a decrease in severity of symptoms with diarrheal disease. Tagatose In addition to the afore-mentioned prebiotic substances, there are other novel ingredients with prebiotic properties. One such ingredient is the carbohydrate, tagatose. Chemical Properties Tagatose is a monosaccharide that differs from fructose only in the positioning of the hydroxyl group on the fourth carbon (Levin and others 1995). 11 The structure of tagatose is shown in Figure 2.2. Tagatose is 92% as sweet as sucrose and has a similar bulk. Tagatose is a white anhydrous crystalline solid that has no odor. Its solubility in water is approximately 58% at 21?C, while its pH stability range is 2-7. It is found naturally in trace amounts in dairy products and some fruits (Levin 2002; Skytte 2006). O OH OH OH OH CH 2 OH Figure 2.2. Structure of ?-D-Tagatose. Manufacturing Tagatose must be able to be produced economically for it to be used as an ingredient in food. Although tagatose does occur in trace amounts in dairy products, its extraction would not be a practical option. A possible starting material for the production of tagatose is whey powder. Whey powder contains lactose, which could be hydrolyzed to release galactose. Galactose can then be converted into tagatose. The conversion of galactose into tagatose can occur one of two ways: using a chemical process (Beadle and others 1992) or an enzymatic process (Kim and others 2003). The chemical process involves two main steps: isomerization and neutralization. Isomerization is where galactose reacts with a basic metal hydroxide (preferably calcium hydroxide) in the presence of a catalyst at a low temperature to form an intermediate reaction product. The intermediate metal hydroxide-tagatose 12 complex is then neutralized with an acid to yield tagatose and a salt during the second step of the chemical process (Beadle and others 1992). The enzymatic process involves immobilizing a thermostable L-arabinose isomerase, Gali152, in alginate. The galactose isomerization reaction conditions were optimized for the conversion of galactose to tagatose (Kim and others 2003). Production of tagatose could be practical since it utilizes whey, a major byproduct of cheese manufacturing. Health Benefits The main health benefit of tagatose is that it is considered a prebiotic. Tagatose is not digested and is absorbed only minimally in the small intestine; the unabsorbed tagatose is fermented in the intestines to short chain fatty acids. Bertelsen and others (1999) determined that colonic butyrate increased in pigs fed a diet containing tagatose. The effect of tagatose consumption on colonic microflora has also been evaluated in humans. Colonic butyrate increased significantly in subjects after they consumed 10 g doses of tagatose three times a day. Also, the number of lactic acid bacteria increased while the number of coliform bacteria decreased (Bertelsen and others 1999). Prebiotics? role in improving immune function (Schley and Field 2002) may have aided in the reduction of coliform bacteria. Tagatose provides several other health benefits. Tagatose has been proven to contribute less than 1.5 kcal/g, which is far less than sucrose at 4 kcal/g (Levin 2002). Livesey and Brown (1996) determined that the caloric value of tagatose in 13 rats was -2.2 kJ/g, which meant that tagatose basically had a zero energy value. Arla Foods had the caloric value of tagatose estimated using a pig model and factorial method; results ranged from 1.1-1.4 kcal/g. Therefore, Arla Foods requested that the U.S. Food and Drug Administration approve a conservative caloric value of 1.5 kcal/g (Levin 2002). The FDA responded with a ?no objection? letter (Hoadley 1999). This officially set the legal caloric value of tagatose at 1.5 kcal/g, although the actual caloric value may be lower. Donner and others (1999) demonstrated that tagatose does not elicit a glycemic response. After an oral loading of 75 g, tagatose was shown not to increase glucose or insulin concentrations in normal and diabetes mellitus patients. In diabetes mellitus patients, increases in blood glucose were lessened significantly by pretreatment with 75 g of tagatose before an oral loading of 75 g of glucose at 60 min, 120 min and 180 min. Because of its chemical properties discussed earlier, tagatose could possibly function as a sugar replacer. This function would be especially beneficial in foods for diabetic patients since tagatose does not cause a rise in blood sugar after consumption. Tagatose obtained GRAS (generally recognized as safe) status in the United States for use as a sweetener in drugs and cosmetic products in 2000, and later obtained that status for use in foods and beverages (Levin 2002). Tagatose has been approved at maximum concentrations of 1% in diet carbonated beverages, 3% in light ice cream, 15% in regular and diabetic hard candies and 30% in icings or glazes used on baked goods in the U.S. (Rulis 2001). Tagatose use has also been 14 approved in Australia and New Zealand, as well as South Korea, Brazil, and South Africa (FSANZ 2004; Skytte 2006). In June 2004, the Joint Expert Committee of Food Additives approved an ADI of ?not specified? for tagatose (JECFA 2004). The United Kingdom, and consequently the European Union, approved tagatose for use in foods in December 2005 (FSA 2005). Tagatose is currently found in several food products in Europe; these include a cocoa drink, chocolate-hazelnut spread, diet jam and chocolate bars (Damhert 2008). Tagatose appears to have potential as a sugar replacer and prebiotic in foods. However, tagatose must not break down during processing and storage to achieve these health benefits. Adverse Side Effects Donner and others (1999) evaluated the gastrointestinal effects of tagatose. None of 10 diabetes mellitus subjects had any side effects after consuming 10 g of tagatose. When they were given 20 g of tagatose, only one person experienced nausea. They found that 100% of subjects experienced adverse side effects after an oral load of 75 g of tagatose. Symptoms included diarrhea (81%), nausea (44%), flatulence (19%), bloating (31%), abdominal pain (25%) and headache (12%). The diarrhea occurred most commonly 2-3 hours after tagatose consumption and sudsided after approximately 4 hours. Bloating and nausea were reported to begin as early as 30 minutes after consumption. Buemann and others (1999) also determined human gastrointestinal tolerance to tagatose. Seventy-three healthy males aged 21-40 years were given a piece of cake with 30 g of tagatose in the afternoon. Symptoms rated included 15 heartburn, stomach rumble, nausea, vomiting, stomach ache, flatulence and diarrhea. Most symptoms were reported to be light or moderate. No subjects reported vomiting. Nausea was reported to begin 1 to 2 hours after ingestion, lasting for 1 to 2 ? hours. Flatulence was observed during a 4 to 5 hour period after ingestion. Lee and Storey (1999) compared the side effects of tagatose and sucrose consumption in humans. Subjects received either a chocolate bar containing 20 g of tagatose or sucrose, and were instructed to eat two a day. The mean frequency of bathroom visits to pass watery feces was higher after consumption of the tagatose- containing chocolate bars than the sucrose chocolate. However, the increase was not statistically significant. Also, consumption of the tagatose chocolate was linked with significantly more subjects experiencing thirst, appetite loss, nausea, bloating, and flatulence. Monosaccharide Reactions and Stability Monosaccharide Degradation Alkaline degradation Monosaccharides participate in several rearrangements when placed in an aqueous alkaline solution. After going through ionization, mutarotation, enolization and isomerization, one monosaccharide transforms into another. In addition, monosaccharide degradation can occur. The first transformation a monosaccharide goes through is ionization. Ionization is the removal or addition of an H + to change the molecule from an anion 16 to a cation or vice versa. The second rearrangement a monosaccharide participates in is mutarotation. This is simply a transition between the different hemiacetals of the monosaccharide. To achieve mutarotation, a sugar undergoes fast ionization, which is then followed by the opening of the acetal ring (de Bruijn and others 1986). Enolization and isomerization follow ionization and mutarotation. These last two transformations involve three features. There must be a fast equilibrium between the cyclic sugar anions and their pseudo-cyclic carbonyl structures. Next, the formation of the enediol anion must occur. The formation of this anion occurs via an intramolecular proton shift from C 2 to pseudo-cyclic (Z)-enediol anions. The last feature to be observed is the reversal of the formation of the enediol anion, leading to isomerization (de Bruijn and others 1986). The reaction product is a different monosaccharide. The starting intermediate in monosaccharide alkaline degradation reactions is the enediol anion. This intermediate will go through several reaction pathways before obtaining the final degradation products, carboxylic acids. The 1,2-enediol anion may participate in ?-elimination as the first of five pathways leading to carboxylic acids. By undergoing ?-elimination, the dicarbonyl compound 3-deoxy- erythro-hexosulose is formed. The dicarbonyl compound is unstable in basic conditions and may undergo either a benzilic acid rearrangement or a cleavage reaction. The benzilic acid rearrangement yields metasaccharinic, isosaccharinic and saccharinic acid. The cleavage reaction moves toward producing a carboxylic 17 acid and an aldehyde. The 1,2-enediol anion may also go through a retro-aldol reaction, producing two triose moieties. The trioses may participate in ?- elimination, ultimately producing lactic acid, acetic acid and formic acid. Lastly, the dicarbonyl compound may undergo aldolization reactions (de Bruijn and others 1986). As mentioned previously, monosaccharide degradation produces carboxylic acids. The majority of the monosaccharides are converted into low molecular weight carboxylic acids. These acids are composed of the same or smaller number of carbons as the original sugar. The low molecular weight acids are classified as ? C 6 -carboxylic acids. Although monosaccharides primarily degrade into ? C 6 - carboxylic acids, they may degrade into larger molecular weight acids referred to as > C 6 -carboxylic acids. Monosaccharides may also degrade into miscellaneous products, but only a trace amount of them are formed (< 1%). These miscellaneous products include volatile non-acidic compounds and cyclic unsaturated aldehydes or ketones (de Bruijn and others 1986). There are several kinetic features that are characteristic of monosaccharide alkaline degradation. The alkaline degradation reaction is considered a first order reaction. The hexose decomposition rate constant depends on the temperature and is proportional to the hydroxyl ion activity. Another feature notes the fact that divalent cations speed up the decomposition of monosaccharides and can possibly influence the composition of the final products (de Bruijn and others 1986). 18 There is a variety of reaction variables that may influence product formation. These include hydroxyl ion concentration, nature of the base and the concentration of the monosaccharide. When hydroxyl ion concentration is increased, there is an increase in lactic acid selectivity, while the total amount of formic, acetic, glycolic, glyceric and saccharinic acids decrease. It has also been noted that the cation of the base may influence the degradation pattern. The last variable that may influence product formation is the concentration of the monosaccharide. When a monosaccharide is diluted in solution, there is an almost complete conversion into ? C 6 -carboxylic acids. Contrary to this, there is significant formation of > C 6 -carboxylic acids when the monosaccharide is concentrated in solution. One reaction variable, temperature, has been found to have no influence on product formation. It was determined that glucose degradation at 5?C and 80?C lead to the same product composition (de Bruijn and others 1986). Acidic degradation While monosaccharides may degrade in alkaline solution, they also undergo a set of very different reactions in acidic solution. Dehydration occurs when a monosaccharide is heated in a strong acidic solution. This dehydration reaction results in the formation of furfural compounds. Anhydro products may be formed in dilute acid solution (Wong 1989). Furfural compounds form through a series of reactions. First, the carbonyl oxygen is protonated, followed by enolization to form the 1,2-enediol anion. Elimination at carbon three then leads to the formation of the enol form of 3- 19 deoxyglycosulose. This step is assisted by the protonation of the hydroxyl group at carbon three and nucleophilic addition at carbon one. Elimination at carbon four occurs next. This forms 3,4-unsaturated glycosulose. The conjugated system is extended by the protonation of the second carbon?s carbonyl group, followed by enolization. Lastly, cyclodehydration of the oxygen at carbon two and five produces the furfural compound (Wong 1989). A much simpler reaction occurs in dilute acid solution. When an aldohexose loses a water molecule, anhydro products are formed. The most common of these products are 1,6-anhydro sugars (Wong 1989). Maillard Reaction The Maillard reaction is a very common chemical reaction that occurs in food to produce brown discoloration, known as nonenzymatic browning. Foods that undergo this type of browning can also exhibit off-flavors and aromas. Understanding this reaction is critical to prevent it from occurring in foods. Tagatose is a reducing sugar, meaning it will react with amino acids to participate in the Maillard reaction and cause nonenzymatic browning. Tagatose is also lost during this reaction. Basic overview There are several steps that comprise the Maillard reaction. The first step to occur is the formation of glycosylamine. This proceeds via a Schiff-base formation between the amino group of the amine and the carbonyl group of the reducing sugar. The next step to occur is the Amadori rearrangement of the glycosylamine. 20 This compound is converted into a ketoseamine. The process by which this happens involves the nitrogen of the glycosylamine accepting a proton to form an amine salt. Rearrangement of the amine salt produces the enol form, which then may tautomerize to yield the keto form of the Amadori compound. The enol form undergoes elimination of the hydroxyl group at carbon three to form the 2,3-enol. The hydroxyl group at carbon four undergoes elimination to yield an unsaturated glycosulos-3-ene. This compound, glycosulos-3-ene, undergoes cyclodehydration to yield furaldehyde (Wong 1989). The production of furaldehyde leads to the formation of off-flavors associated with nonenzymatic browning. The brown coloration is developed from polymerization of the furaldehyde and copolymerization with amino compounds (Labuza and Baisier 1992). Many factors affect the Maillard reaction. The two factors relevant to the current study are pH and buffer properties. pH effect Ashoor and Zent (1984) determined the effect of pH on the intensity of nonenzymatic browning. Phosphate buffer solutions (0.05 M) at pH 6.0 and 7.5 and carbonate buffer solutions (0.05 M) at pH 8.0, 9.0, 9.5, 10.0, 11.0 and 12.0 were used in the study. The amino acids chosen to show the effect of pH were L-lysine, placed in the high browning group, L-alanine, from the intermediate browning group, and L-arginine, representing the low browning group. The sugars chosen were D-glucose, D-fructose and ?-lactose. It was found that as the pH of the amino acid-sugar solution increased, the intensity of the Maillard browning increased. 21 This occurred to a maximum pH of 10.0, and then decreased at higher pH values. These results were similar for both D-glucose and D-fructose. Amino acids need to be unprotonated to react with the reducing sugars; a lesser extent of protonation occurs at the higher pH. Buffer effect Buffer type and concentration may also affect the Maillard reaction. As shown by Bell (1997), various buffers have differing effects on brown pigment formation. Solutions containing 0.1 M glucose and 0.1 M glycine in citrate and phosphate buffers at pH 7 and 25?C were analyzed. The buffer concentrations were 0.02, 0.05, 0.2 and 0.5 M. Results indicated that the rates of glycine loss and brown pigment formation increased with increasing phosphate buffer concentrations. Contrary to this, reaction rates did not differ significantly (P > 0.05) from zero in citrate buffer. Buffer concentration did not affect either the rate of glycine loss or the rate of brown pigment formation in the citrate buffer. Bell concluded that both rates were enhanced in phosphate buffer, while neither were affected in citrate buffer. These results indicate that buffer type and concentration can affect the rate of the Maillard reaction. Justification and Objective Nutraceuticals and prebiotics have made headlines in recent years for their health benefits. Tagatose is a monosaccharide that falls into both of these categories. For its prebiotic effect to be achieved, tagatose in food products must not be lost during their distribution and storage. However, data on the storage 22 stability of tagatose are lacking. Therefore, the objective of this study was to evaluate the storage stability of tagatose in various solutions as affected by pH, buffer type, buffer concentration and temperature. 23 CHAPTER 3: MATERIALS AND METHODS Sample Preparation Phosphate and citrate buffers were used in the experiment. Four different sodium phosphate buffer solutions were prepared: 0.02 M sodium phosphate monobasic, 0.02 M sodium phosphate dibasic, 0.1 M sodium phosphate monobasic and 0.1 M sodium phosphate dibasic. Prepared in the same manner, a set of four bulk citrate buffer solutions were made: 0.02 M sodium citrate, 0.02 M citric acid, 0.1 M sodium citrate and 0.1 M citric acid. Tagatose was added to each of these eight solutions at a concentration of 0.05 M (or about 1%). To evaluate the effect of the Maillard reaction, these eight buffer solutions were prepared again, containing 0.05 M tagatose and 0.05 M glycine. Aliquots of the 0.02 M sodium phosphate monobasic and dibasic solutions containing 0.05 M tagatose were mixed to give a 0.02 M buffer solution at pH 7. This protocol was repeated for the 0.1 M phosphate buffer, yielding a 0.1 M buffer solution at pH 7. To obtain a 0.02 M phosphate buffer solution at pH 3, 0.02 M phosphoric acid was required. However, it was not desirable to add tagatose to this phosphoric acid solution. Therefore, the phosphoric acid solution was mixed with a 0.02 M phosphate monobasic solution to raise the pH to approximately 2.5. Tagatose was added to the pH 2.5 buffer solution. The phosphate monobasic solution and the pH 2.5 buffer solution, both containing 0.05 M tagatose, were then 24 mixed to obtain a final pH of 3. This procedure was repeated for the 0.1 M phosphate buffer at pH 3, with 0.1 M phosphoric acid replacing the 0.02 M phosphoric acid. These phosphate buffer solutions were prepared again containing the tagatose and glycine. The 0.02 M and 0.1 M citrate buffer solutions were obtained similarly by mixing the appropriate citric acid and sodium citrate solutions to yield 0.02 M and 0.1 M citrate buffers at pH 3 and 7. Eight buffer solutions containing tagatose resulted consisting of two buffer types, two buffer concentrations and two pH values. Also, eight buffer solutions containing tagatose and glycine resulted, consisting of two buffer types, two buffer concentrations and two pH values. Each of these 16 solution types was to be stored at three temperatures to give a total of 48 experiments. A great amount of care was taken to prevent contamination of the solutions. Certified contaminant-free clear vials with septum-containing caps (I-Chem, Chase Scientific Glass, Rockwood, TN) were used in this study. A small amount (i.e., around 5 mL) of the sample solution was placed in a sterile syringe, with a sterile 0.20 ?m nylon filter and sterile needle. The solution was injected into the vial via the septum-containing cap. The vial was then shaken to rinse away any potential microorganisms. The liquid was removed via the syringe and needle and then discarded. After rinsing, approximately 40 mL of each sample solution was transferred into triplicate vials using a sterile syringe, filter and needle. The resulting 144 vials (48 experimental solutions in triplicate) were placed in 20?, 30? 25 and 40?C incubators to determine the storage stabilities of tagatose as affected by buffer type, buffer concentration, pH and temperature. Sampling Procedure Aliquots of the experimental solutions were removed from storage for tagatose analysis 11-13 times over a nine month period. For example, the tagatose solutions in citrate buffer were removed for analysis at time intervals of 0, 20, 44, 55, 73, 95, 118, 153, 189, 224 and 265 days. The tagatose solutions in phosphate buffer at pH 7 were removed for analysis at time intervals of 0, 12, 20, 34, 44, 55, 73, 95, 118, 153, 189, 224 and 265 days. All three vials were sampled at time zero. Then, at each time, a 2-3 mL aliquot was removed from two of the three bottles using a sterile needle and syringe. Sampling rotated between the three vials. The sample was then passed through a 0.45 ?m nylon filter and transferred into cryogenic vials. The vials were labeled with a code that consisted of buffer type, buffer concentration, pH, temperature and data point number. For example, ?02P-3- T20-A1? meant that the sample was 0.02 M phosphate buffer at pH 3 and 20?C. The ?T? stood for ?tagatose?, the ?A? signified which vial in the triplicate series (A, B, or C) it was, and the ?1? represented the particular data point. ?TG? was used for tagatose/glycine solutions. Tagatose Analysis The tagatose concentrations of the solutions were analyzed using reverse- phase high performance liquid chromatography (HPLC). The column used was a 250 x 4.6 mm LUNA 5? Amino column (Phenomenex, Torrance, CA). The mobile 26 phase consisted of a 85/15 (v/v) acetonitrile/deionized water solution flowing at 2.5 mL/min. Tagatose was detected using refractive index measurements. Data were integrated by a Hewlett-Packard integrator. Using tagatose standard curves, the concentrations of tagatose in the experimental solutions were determined. A sample chromatogram is shown in Figure 3.1 while Figure 3.2 shows a typical calibration curve. Figure 3.1. Sample HPLC Chromatogram for the Analysis of Tagatose in Buffer with 0.05 M Glycine using a Luna Amino Column (Phenomenex, Torrance, CA) and Mobile Phase Consisting of 85/15 (v/v) Acetonitrile/Water Running at 2.5 mL/min. Tagatose Elutes at Approximately 4.9 min and Glycine at 28.7 min. 27 Figure 3.2. Sample Calibration Curve for Tagatose Analysis by HPLC. Browning Analysis Brown pigmentation was measured via spectrophotometry. Aliquots (1 mL) of pre-filtered samples were placed in semi-micro methacrylate cuvettes. Absorption was determined at 420 nm. Data Analysis Because the kinetic data did not follow a definite kinetic model, it was necessary to evaluate initial reaction rates. Therefore, the loss of tagatose for the initial 100 days was modeled using pseudo-first order kinetics. Rate constants with 95% confidence limits were calcuated using computerized least-squares analysis (Labuza and Kamman 1983). The same model was used whether or not glycine was present. 0 0.2 0.4 0.6 0.8 1 1.2 1.4 1.6 2040608010 Tagatose ? Concentration ? (g/100 ? mL) Peak?Area 28 CHAPTER 4: RESULTS AND DISCUSSION Tagatose Degradation The degradation of tagatose in solution is affected by several variables, including pH, buffer type and concentration, and temperature. Degradation profiles were created to demonstrate these effects. Figures 4.1 and 4.2 illustrate the effect of pH on tagatose degradation in various phosphate and citrate buffers. Figures 4.3 and 4.4 show the effect of buffer type and concentration on the loss of tagatose in solutions at pH 3 and 7, respectively. The effect of temperature on tagatose degradation in solutions is shown in Figures 4.5 and 4.6. Figure 4.1. Tagatose Loss in 0.1 M Buffer Solutions at 40?C as Affected by pH. 0 0.01 0.02 0.03 0.04 0.05 0.06 0 50 100 150 200 250 300 Tagatose ? Concentration ? (M) Time?(days) Phosphate,?pH?3 Citrate,?pH?3 Phosphate,?pH?7 Citrate,?pH?7 29 Figure 4.2. Tagatose Loss in 0.02 M Buffer Solutions at 20?C as Affected by pH. Figure 4.3. Tagatose Loss in Solution at pH 3 and 40?C as Affected by Buffer Type and Concentration. 0.035 0.04 0.045 0.05 0.055 0 50 100 150 200 250 300 Tagatose ? Concentration ? (M) Time?(days) Phosphate,?pH?3 Citrate,?pH?3 Phosphate,?pH?7 Citrate,?pH?7 0.035 0.04 0.045 0.05 0.055 0 50 100 150 200 250 300 Tagatose ? Concentration ? (M) Time?(days) 0.1?M?Phosphate 0.02?M?Phosphate 0.1?M?Citrate 0.02?M?Citrate 30 Figure 4.4. Tagatose Loss in Solution at pH 7 and 40?C as Affected by Buffer Type and Concentration. Figure 4.5. Tagatose Loss in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature. 0 0.01 0.02 0.03 0.04 0.05 0.06 0 50 100 150 200 250 300 Tagatose ? Concentration ? (M) Time?(days) 20?deg?C 30?deg?C 40?deg?C 31 Figure 4.6. Tagatose Loss in 0.02 M Citrate Buffer at pH 3 as Affected by Temperature. As shown in these figures, especially in phosphate buffer at pH 7, the degradation of tagatose appeared to plateau at approximately one hundred days of storage. Measuring the pH of select solutions indicated a drop in pH had also occurred. This drop in pH could explain the plateau, as will be discussed later. However, the existence of the plateau made traditional kinetic modeling difficult. Therefore, the initial loss of tagatose (i.e., the first 100 d) was modeled using pseudo-first order kinetics to determine degradation rate constants. Figure 4.7 illustrates the initial loss of tagatose over 100 days modeled using pseudo-first order kinetics. The slopes of these plots are equivalent to the pseudo-first order rate constants (k obs ). Pseudo-first order rate constants for the initial loss of tagatose in 0.04 0.045 0.05 0.055 0 50 100 150 200 250 300 Tagatose ? Concentration ? (M) Time?(days) 20?deg?C 30?deg?C 40?deg?C 32 solution were calculated using least-squares analysis and are shown in Table 4.1. Using the pseudo-first order kinetic model, ln (% tagatose remaining) = -k obs (time), and the data in Table 4.1, the predicted percent losses of tagatose in solution after 100 d of storage were calculated. These percentages are listed in Table 4.2. Figure 4.7. Initial Loss of Tagatose in 0.1 M Phosphate Buffer at pH 7 modeled using Pseudo-First Order Kinetics as Affected by Temperature. ?4 ?3.8 ?3.6 ?3.4 ?3.2 ?3 ?2.8 0 2040608010 ln ? (Tagatose ? Concentration) Time?(days) 20?deg?C 30?deg?C 40?deg?C 33 Table 4.1. Pseudo-First Order Rate Constants (d -1 ) with 95% Confidence Limits for the Initial Loss of Tagatose in Solution. Sample Temperature 20?C30?C40?C pH 3 0.02 M Phosphate 1.41 ? 1.01* 1.39 ? 1.76 # 1.37 ? 1.99 # 0.1 M Phosphate 0.35 ? 1.07 # 2.13 ? 1.62 3.27 ? 1.68 0.02 M Citrate 0.76 ? 2.03 # 1.96 ? 1.22 2.97 ? 1.23 0.1 M Citrate 1.65 ? 2.04 # 2.61 ? 1.92 5.47 ? 1.28 pH 7 0.02 M Phosphate 8.22 ? 1.38 10.5 ? 2.48 16.7 ? 4.63 0.1 M Phosphate 19.0 ? 1.92 38.9 ? 5.40 83.8 ? 11.6 0.02 M Citrate 2.29 ? 2.50 # 4.89 ? 1.50 10.9 ? 1.56 0.1 M Citrate 4.10 ? 2.18 7.39 ? 2.81 21.0 ? 4.55 *Rate constants (? 95% CL) have been multiplied by 10 4 . # Not different from zero. Table 4.2. Predicted Percent Loss of Tagatose in Solution at 100 days. Sample Temperature 20?C30?C40?C pH 3 0.02 M Phosphate 1.4% 1.4% 1.4% 0.1 M Phosphate 0.4% 2.1% 3.2% 0.02 M Citrate 0.8% 1.9% 2.9% 0.1 M Citrate 1.6% 2.6% 5.3% pH 7 0.02 M Phosphate 7.9% 10.0% 15.4% 0.1 M Phosphate 17.3% 32.2% 56.7% 0.02 M Citrate 2.3% 4.8% 10.3% 0.1 M Citrate 4.0% 7.1% 18.9% Effect of pH The effect of pH on tagatose degradation is exemplified in Figures 4.1 and 4.2 and quantified in Tables 4.1 and 4.2. Tagatose degradation was very slow in all solutions at pH 3. At pH 7, faster degradation was observed. For example, in 0.1 M phosphate buffer at pH 7 and 40?C, 56.7% of tagatose was lost at 100 d compared 34 to only 3.2% at pH 3. Similar results were seen for tagatose loss in citrate buffer as well. In 0.02 M citrate buffer at pH 7 and 20?C, 2.3% of tagatose was lost in comparison to 0.8% at pH 3. The pH effect was more dramatic in phosphate buffer at elevated temperatures. At pH 7, tagatose is probably undergoing a mild alkaline degradation reaction that would not occur at pH 3. As described by de Bruijn and others (1986), monosaccharides placed in an aqueous alkaline solution undergo various rearrangements before degradation takes place. Through the processes of ionization, mutarotation, and enolization, an enediol anion is formed. The enediol intermediate goes through several reaction pathways before obtaining the final degradation product, carboxylic acids (de Bruijn and others 1986). These acidic products decrease the pH of the solution, and cause the tagatose degradation rate to slow down. As mentioned previously, the pH of some solutions initially at pH 7 decreased during storage. In Figures 4.1 and 4.2, the plateau after storing the pH 7 solutions for 100 d could be explained by the decreased pH associated with the formation of acidic products reducing subsequent tagatose loss. Monosaccharides can also degrade in acidic solutions. When a monosaccharide is heated in a strong acid solution, dehydration occurs and furfural compounds are formed (Wong 1989). Under the milder conditions of this study, such a reaction was minimal. The greatest loss of tagatose at pH 3 was in 0.1 M citrate buffer at 40?C, where about 5% was lost after 100 days. 35 Effect of Buffer Type and Concentration Figures 4.3 and 4.4 illustrate the effect of buffer type and concentration on tagatose degradation. At pH 3, both buffer types and concentrations showed similar results. The tagatose in the 0.1 M citrate buffer solution degraded slightly more than the other solutions, as shown in Figure 4.3. A greater effect of buffer type was seen at a higher pH. At pH 7 and 40?C, tagatose degraded more in the 0.1 M phosphate buffer solution with 56.7% lost at 100 d in comparison to 18.9% lost in the 0.1 M citrate buffer. More tagatose was lost at a higher buffer concentration as well. In the 0.1 M phosphate buffer at pH 7 and 30?C, 32.2% of tagatose was lost at 100 d as compared to 10.0% loss in 0.02 M phosphate buffer at the same conditions. These results are similar to those reported by Bell and Wetzel (1995) and Pachapurkar and Bell (2005) for other reactions. Bell and Wetzel (1995) showed that the rate of aspartame degradation increased as buffer concentration increased. Aspartame in phosphate buffer was found to degrade faster than in citrate buffer at the same conditions. Also, degradation occurred faster at pH 7 rather than pH 3. The fastest degradation was in 0.1 M phosphate buffer at pH 7, which is similar to the tagatose data. Pachapurkar and Bell (2005) found that thiamin also degraded fastest in 0.1 M phosphate buffer at pH 7. Similar to the findings of Bell and Wetzel (1995), Pachapurkar and Bell (2005) found that the rate of degradation increased as the buffer concentration increased. Thiamin degradation also occurred faster in phosphate buffer than citrate buffer at pH 7. 36 The mechanisms of both aspartame and thiamin degradation at pH 7 involve the transfer of protons. The primary component in phosphate buffer at pH 7 is the dibasic anion (HPO 4 -2 ), which appears to be much more efficient at the required proton transfers than citrate anions. Thus, the reactions proceed faster in phosphate buffer than citrate buffer at pH 7. Similarly, based on the discussion of glucose degradation (de Bruijn and others 1986; Wong 1989; Robyt 1998), the initial ionization and mutarotation of tagatose requires the removal of a proton from the hydroxyl at carbon 2 to form the anion. The subsequent opening of the ring (i.e., mutarotation) requires moving a proton from carbon 3 to the oxygen on carbon 6. The resulting product is the enediol anion, which then proceeds into additional degradation pathways. In the current study, the phosphate dibasic anion at pH 7 is a better facilitator of the intramolecular proton shift than the citrate anion, leading to the faster formation of the enediol anion and the degradation that follows. Effect of Temperature The effect of temperature on tagatose degradation is shown in Figures 4.5 and 4.6. Both figures show that the rate of tagatose degradation increases as temperature is increased. In 0.02 M phosphate buffer at pH 7, 7.9% of tagatose is lost at 20?C, 10.0% at 30?C and 15.4% at 40?C at 100 d of storage. The effect of temperature on degradation is less noticeable at pH 3 due to very little tagatose loss and the accompanied scatter of the data. Although not as noticeable, the higher temperature does result in more tagatose degradation. 37 The activation energy (E A ) gives an indication of the sensitivity of the reaction rates to temperature. A higher E A value means reaction rates change more with temperature whereas a lower E A value means rates will change less with temperature. Because tagatose loss was minimal at pH 3 (Tables 4.1 and 4.2), these activation energies may be quite error prone and were therefore not calculated. The greater tagatose loss at pH 7 makes these activation energies more reliable. Table 4.3 lists the activation energies for the initial loss of tagatose in pH 7 buffer solutions. Most activation energies for the initial loss of tagatose at pH 7 ranged from 13.5-14.8 kcal/mol. It is interesting that the E A value for tagatose loss in 0.02 M phosphate buffer was much lower at 6.4 kcal/mol. The reason for this lower E A value is unclear and warrants further investigation. Table 4.3. Activation Energies (E A ) for the Initial 100 d of Tagatose Degradation in Solution at pH 7 Sample E A (kcal/mol) 0.02 M Phosphate 6.4 0.1 M Phosphate 13.5 0.02 M Citrate 14.2 0.1 M Citrate 14.8 Participation of Tagatose in Maillard Reaction Degradation profiles were created to demonstrate the effects of pH, buffer type and concentration, and temperature on tagatose degradation in the presence of added glycine. Figures 4.8 and 4.9 illustrate the effect of pH on tagatose degradation. Figures 4.10 and 4.11 show the effect of buffer type and concentration 38 on the loss of tagatose. The effect of temperature on tagatose degradation is seen in Figures 4.12 and 4.13. Again, due to the plateau that was observed after 100 d, only the data for the initial 100 d was used to model the loss via pseudo-first order kinetics. An example of the pseudo-first order plot is shown in Figure 4.14, the rate constants are listed in Table 4.4, and values for percent loss are listed in Table 4.5. Figure 4.8. Tagatose Loss in 0.1 M Buffer Solutions also containing 0.05 M Glycine at 40?C as Affected by pH. 0 0.01 0.02 0.03 0.04 0.05 0.06 0 50 100 150 200 250 300 Tagatose ? Concentration ? (M) Time?(days) Phosphate,?pH?3 Phosphate,?pH?7 Citrate,?pH?3 Citrate,?pH?7 39 Figure 4.9. Tagatose Loss in 0.02 M Buffer Solutions also containing 0.05 M Glycine at 20?C as Affected by pH (No phosphate pH 3 due to mold). Figure 4.10. Tagatose Loss in Solution at pH 3 and 40?C also containing 0.05 M Glycine as Affected by Buffer Type and Concentration. 0.035 0.04 0.045 0.05 0.055 0 50 100 150 200 250 300 Tagatose ? Concentration ? (M) Time?(days) Phosphate,?pH?7 Citrate,?pH?3 Citrate,?pH?7 0.035 0.04 0.045 0.05 0.055 0 50 100 150 200 250 300 Tagatose ? Concentration ? (M) Time?(days) 0.02?M?Phosphate 0.1?M?Phosphate 0.02?M?Citrate 0.1?M?Citrate 40 Figure 4.11. Tagatose Loss in Solution at pH 7 and 40?C also containing 0.05 M Glycine as Affected by Buffer Type and Concentration. Figure 4.12. Tagatose Loss in 0.1 M Phosphate Buffer also containing 0.05 M Glycine at pH 7 as Affected by Temperature. 0 0.01 0.02 0.03 0.04 0.05 0.06 0 50 100 150 200 250 300 Tagatose ? Concentration ? (M) Time?(days) 0.02?M?Phosphate 0.1?M?Phosphate 0.02?M?Citrate 0.1?M?Citrate 0 0.01 0.02 0.03 0.04 0.05 0.06 0 50 100 150 200 250 300 Tagatose ? Concentration ? (M) Time?(days) 20?deg?C 30?deg?C 40?deg?C 41 Figure 4.13. Tagatose Loss in 0.02 M Citrate Buffer also containing 0.05 M Glycine at pH 3 as Affected by Temperature. Figure 4.14. Initial Loss of Tagatose in 0.1 M Phosphate Buffer also containing 0.05 M Glycine at pH 7 modeled using Pseudo-First Order Kinetics as Affected by Temperature. 0.035 0.04 0.045 0.05 0.055 0 50 100 150 200 250 300 Tagatose ? Concentration ? (M) Time?(days) 20?deg?C 30?deg?C 40?deg?C ?3.7 ?3.5 ?3.3 ?3.1 ?2.9 0 2040608010120 ln ? (Tagatose ? Concentration) Time?(days) 20?deg?C 30?deg?C 40?deg?C 42 Table 4.4. Pseudo-First Order Rate Constants (d -1 ) with 95% Confidence Limits for the Initial Loss of Tagatose in Solutions also containing 0.05 M Glycine. Sample Temperature 20?C30?C40?C pH 3 0.02 M Phosphate N/A* N/A 1.55 ? 1.31** # 0.1 M Phosphate 1.59 ? 2.30 # 1.29 ? 9.23 # 1.97 ? 2.36 # 0.02 M Citrate 1.02 ? 1.94 # 1.02 ? 2.13 # 1.50 ? 1.72 # 0.1 M Citrate 0.30 ? 1.56 # 0.16 ? 1.93 # 3.07 ? 2.50 pH 7 0.02 M Phosphate 2.97 ? 2.03 8.70 ? 1.64 17.5 ? 3.55 0.1 M Phosphate 8.87 ? 2.65 19.0 ? 3.43 56.4 ? 5.14 0.02 M Citrate 2.00 ? 1.88 4.28 ? 3.64 12.7 ? 1.93 0.1 M Citrate 3.26 ? 1.48 3.89 ? 2.20 21.6 ? 3.79 *N/A: Not available due to mold contamination. **Rate constants (? 95% CL) have been multiplied by 10 4 . # Not different from zero. Table 4.5. Predicted Percent Loss of Tagatose in Solutions containing 0.05 M Glycine at 100 days. Sample Temperature 20?C30?C40?C pH 3 0.02 M Phosphate N/A* N/A 1.5% 0.1 M Phosphate 1.6% 1.3% 2.0% 0.02 M Citrate 1.0% 1.0% 1.5% 0.1 M Citrate 0.3% 0.2% 3.0% pH 7 0.02 M Phosphate 2.9% 8.3% 16.1% 0.1 M Phosphate 8.5% 17.3% 43.1% 0.02 M Citrate 2.0% 4.2% 11.9% 0.1 M Citrate 3.2% 3.8% 19.4% *N/A due to mold contamination. Effect of pH The effect of pH is shown in Figures 4.8 and 4.9 as well as in Tables 4.4 and 4.5. Tagatose degradation in the presence of glycine occurred more rapidly at 43 pH 7 rather than pH 3. In fact, the 95% confidence limits of k obs at pH 3 were larger than the rate constants themselves meaning that these rate constants were not different from zero. This pH effect could be due to a combination of alkaline degradation (discussed previously) and the Maillard reaction. At pH 7, a larger amount of glycine contains the unprotonated amine, which is more reactive with tagatose than the protonated amine at lower pH values. This pH effect on the Maillard reaction is consistent with that reported previously (Labuza and Baisier 1992). Effect of Buffer Type and Concentration Figures 4.10 and 4.11 demonstrate the effect of buffer type and concentration on tagatose degradation. Figure 4.10, Table 4.4, and Table 4.5 show that the degradation rates of tagatose in 0.1 M phosphate and citrate buffers at pH 3 are very similar. Very little degradation is observed at pH 3. Figure 4.11 shows that degradation at pH 7 occurred faster in 0.1 M phosphate buffer. At 100 d of storage, the percent of tagatose loss in 0.1 M phosphate buffer at pH 7 and 40?C was 43.1%, compared to 19.4% in 0.1 M citrate buffer at pH 7 and 40?C. Overall, tagatose degradation occurred faster in 0.1 M phosphate buffer solution. These results are similar to those reported by Bell (1997). The initial step of the Maillard reaction requires proton transfers to form the glycosyl amine, which phosphate dibasic anions do better than citrate anions. 44 Effect of Temperature The effect of temperature on tagatose degradation in the presence of glycine is shown in Figures 4.12 and 4.13. Both graphs show that degradation of tagatose increases as the temperature of the solution is increased. In 0.02 M citrate buffer at pH 7, tagatose loss is 2.0% at 20?C, 4.2% at 30?C and 11.9% at 40?C. This result is consistent with basic principles of reaction kinetics where rates are faster at higher temperatures. To evaluate the temperature sensitivity, the activation energies (E A ) for tagatose loss in the tagatose/glycine solutions were calculated. Because the rate constants for tagatose loss were basically zero at pH 3 (Table 4.4), the activation energies were unable to be calculated with much certainty. At pH 7, all E A values were 16-17 kcal/mol (Table 4.6). Thus, regardless of buffer type or concentration, the temperature sensitivity for tagatose loss is similar at pH 7 in the presence of glycine. These E A values could be used to predict tagatose loss at any other temperature. Table 4.6. Activation Energies (E A ) for Tagatose Degradation in 0.05 M Glycine Solutions at pH 7. Sample E A (kcal/mol) 0.02 M Phosphate 16.2 0.1 M Phosphate 16.8 0.02 M Citrate 16.8 0.1 M Citrate 17.1 45 Browning Both solutions of tagatose alone and with glycine displayed some extent of browning. This browning was most pronounced at 40?C. The effect of buffer type and pH on browning of tagatose solutions is shown in Figure 4.15. Figure 4.15. Browning of Tagatose in 0.1 M Buffer Solutions at 40?C as Affected by Buffer Type and pH. More browning occurred in the 0.1 M phosphate buffer solution at pH 7 and 40?C. There was a steep increase before a decrease and leveling off. The other solutions had virtually no browning (data in Appendix C). The brown pigment formed from tagatose degradation in 0.1 M phosphate buffer at pH 7 is apparently unstable and deteriorates over time. In comparison, the tagatose-glycine solutions showed a more continual increase in browning (Figure 4.16). Similar to the tagatose only solutions, more ?0.05 0 0.05 0.1 0.15 0.2 0.25 0.3 0.35 0.4 0.45 0.5 0.55 0 50 100 150 200 250 300 Absorbance ? at ? 420 ? nm Time?(days) Phosphate,?pH?3 Phosphate,?pH?7 Citrate,?pH?3 Citrate,?pH?7 46 browning was seen in the 0.1 M phosphate buffer solution containing 0.05 M glycine at pH 7 and 40?C. While the other solutions had a gradual increase in browning, the phosphate pH 7 buffer solution increased dramatically before somewhat leveling off. This brown pigment was more stable, remaining for the entire storage period. In the presence of glycine, tagatose participates in a Maillard type reaction leading to brown pigment formation, which apparently is a different type of brown pigment from that produced by tagatose degradation in the absence of glycine. Browning is more favored at pH 7, in phosphate buffer rather than citrate buffer, and at higher rather than lower buffer concentrations. This browning data can be found in Appendix D. These findings are consistent with those reported previously (Bell 1997). Figure 4.16. Browning of Tagatose and Glycine in 0.1 M Buffer Solutions at 40?C as Affected by Buffer Type and pH. ?0.1 0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0 50 100 150 200 250 300 Absorbance ? at ? 420 ? nm Time?(days) Phosphate,?pH?3 Phosphate,?pH?7 Citrate,?pH?3 Citrate,?pH?7 47 Comparing Tagatose Reactivity with and without Glycine An interesting comparison to make is the effect of glycine on the loss of tagatose and the resultant browning. Because tagatose undergoes degradation in buffer solutions at pH 7 in the absence of glycine and because reducing sugars (e.g., tagatose) react with amino acids (e.g., glycine) through the Maillard reaction, it was thought that the addition of glycine to the tagatose solution would enhance its degradation beyond that observed in solutions without glycine. However, as shown in Figures 4.17 and 4.18, the loss of tagatose either remained unchanged in presence of glycine or even decreased. As shown in Figures 4.19 and 4.20, glycine is reacting with tagatose to increase the intensity of the brown pigment over a longer time, but apparently is not increasing the degradation of tagatose itself. Figure 4.17. Effect of Glycine on Tagatose Loss in 0.1 M Phosphate Buffer at pH 7 and 40?C. 0 0.01 0.02 0.03 0.04 0.05 0.06 0 50 100 150 200 250 300 Tagatose ? Concentration ? (M) Time?(days) Tagatose Tagatose/Glycine 48 Figure 4.18. Effect of Glycine on Tagatose Loss in 0.1 M Citrate Buffer at pH 7 and 40?C. Figure 4.19. Effect of Glycine on Tagatose Browning in 0.1 M Phosphate Buffer at pH 7 and 40?C. 49 Figure 4.20. Effect of Glycine on Tagatose Browning in 0.1 M Citrate Buffer at pH 7 and 40?C. The reason why the addition of a known reactant (glycine) would not cause enhanced tagatose degradation is unclear. At pH 7, both the degradation of the monosaccharide and the early steps of the Maillard reaction involve the acyclic sugar derived from mutarotation. The steps leading to the formation of the enediol (alkaline degradation) are reversible as are the steps leading to the glycosylamine (Maillard reaction). Eventually, both reaction pathways continue toward various degradation products and some extent of brown pigment formation. As mentioned, both pathways involve the same intermediate and a series of reversible steps. One possible explanation may involve the manner in which glycine disturbs the equilibrium positioning of these reversible steps. Reaction steps which are reversible can shift in both direction and magnitude by the addition or removal of either reactants or products. Thus, adding glycine as a reactant can 50 alter the equilibrium and direct some of the tagatose toward the Maillard reaction (as seen by enhanced production of browning in Figures 4.19 and 4.20). The combination of re-established equilibrium occurring in competing reaction pathways may yield the net result of similar or slower tagatose loss in the presence of glycine. It is also interesting to note that the most extreme ?slowing down? of tagatose loss in the presence of glycine was in 0.1 M phosphate buffer; tagatose loss in citrate buffer was similar regardless of whether or not glycine was present (Figures 4.17 and 4.18). The importance of phosphate buffer anions as catalysts has been discussed. Perhaps an interaction occurred between the buffer and the glycine, reducing the buffer?s catalytic ability. So while glycine may enhance the reactivity of tagatose via the Maillard reaction, its possible interaction with phosphate anions may decrease the tagatose loss more than the glycine enhances it. This interesting result is worthy of further evaluation. However, regardless of the mechanism, tagatose is being lost both in the presence and absence of glycine at pH 7, but browning is more pronounced when glycine is available. 51 CHAPTER 5: SUMMARY AND CONCLUSIONS Tagatose in solution degrades during storage both with and without glycine. The degradation of tagatose depends upon buffer type, buffer concentration, pH and temperature, with the greatest loss occurring in 0.1 M phosphate buffer at pH 7 and 40?C. The rate of tagatose loss is faster at higher temperatures, higher buffer concentrations, and higher pH values. It is also faster in phosphate buffer than in citrate buffer. The loss of tagatose is accompanied by brown pigment formation. This discoloration occurs both in the presence and absence of glycine. In the solutions with added glycine, browning is more extensive and stable than that produced in the absence of glycine. Without glycine, the brown pigment formed deteriorates over time. These stability issues need to be considered by manufacturers who desire to use tagatose as a prebiotic in their food and beverage products. To obtain the prebiotic effect from tagatose, it should not degrade during storage of the products. Shelf-stable beverages should be made with the lowest buffer concentration and pH possible to deliver the maximum amount of tagatose with the minimal discoloration from browning. 52 REFERENCES Ashoor SH, Zent JB. 1984. Maillard browning of common amino acids and sugars. J Food Sci 49:1206-7. Beadle JR, Saunders JP, Wajda Jr TJ, inventor; Biospherics Incorporated, assignee. 1992 Jan 7. Process for manufacturing tagatose. 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In: Wildman REC. Handbook of nutraceuticals and functional foods. Boca Raton, FL: CRC Press. p 1-21. Wong DWS. 1989. Mechanism and theory in food chemistry. New York, NY: Van Nostrand Reinhold. p 105-46. 57 APPENDIX A TAGATOSE LOSS IN BUFFER SOLUTIONS WITHOUT GLYCINE 58 Table A1. Tagatose Degradation in 0.02 M Phosphate Buffer at pH 3 as Affected by Temperature Time Tagatose Concentration (M) (days) 20?C 30?C 40?C 0 0.0507 0.0512 0.0513 0 0.0509 0.0510 0.0510 0 0.0511 0.0511 0.0510 12 0.0510 0.0510 0.0509 12 0.0511 0.0509 0.0510 20 0.0510 0.0508 0.0508 20 0.0507 0.0507 0.0507 44 0.0500 0.0508 0.0520 44 0.0504 0.0519 0.0520 55 0.0505 0.0502 0.0502 55 0.0503 0.0504 0.0506 73 0.0502 0.0497 0.0498 73 0.0500 0.0498 0.0500 95 0.0505 0.0508 0.0509 95 0.0507 0.0510 0.0506 118 0.0501 0.0503 0.0498 118 0.0503 0.0502 0.0500 153 0.0498 0.0506 0.0502 153 0.0503 0.0502 0.0501 189 0.0507 0.0507 0.0509 189 0.0497 0.0512 0.0510 224 0.0487 0.0510 0.0482 224 0.0495 0.0500 0.0487 265 0.0501 0.0501 0.0515 265 0.0502 0.0528 0.0505 59 Table A2. Tagatose Degradation in 0.02 M Phosphate Buffer at pH 7 as Affected by Temperature Time Tagatose Concentration (M) (days) 20?C 30?C 40?C 0 0.0511 0.0508 0.0505 0 0.0510 0.0508 0.0507 0 0.0508 0.0510 0.0506 12 0.0504 0.0486 0.0468 12 0.0504 0.0487 0.0469 20 0.0499 0.0480 0.0462 20 0.0499 0.0482 0.0459 34 0.0493 0.0475 0.0455 34 0.0498 0.0472 0.0455 44 0.0497 0.0481 0.0433 44 0.0504 0.0471 0.0443 55 0.0485 0.0467 0.0442 55 0.0483 0.0470 0.0435 73 0.0477 0.0458 0.0428 73 0.0481 0.0470 0.0430 95 0.0472 0.0454 0.0428 95 0.0470 0.0455 0.0430 118 0.0469 0.0447 0.0430 118 0.0469 0.0455 0.0429 153 0.0465 0.0454 0.0421 153 0.0466 0.0449 0.0425 189 0.0469 0.0452 0.0434 189 0.0469 0.0453 0.0438 224 0.0483 0.0434 0.0413 224 0.0470 0.0437 0.0414 265 0.0465 0.0450 0.0433 265 0.0461 0.0463 0.0437 60 Table A3. Tagatose Degradation in 0.1 M Phosphate Buffer at pH 3 as Affected by Temperature Time Tagatose Concentration (M) (days) 20?C 30?C 40?C 0 0.0511 0.0516 0.0516 0 0.0511 0.0516 0.0519 0 0.0509 0.0515 0.0502 12 0.0509 0.0512 0.0508 12 0.0509 0.0510 0.0508 20 0.0513 0.0511 0.0510 20 0.0511 0.0510 0.0511 44 0.0511 0.0510 0.0512 44 0.0509 0.0522 0.0506 55 0.0509 0.0503 0.0505 55 0.0513 0.0503 0.0506 73 0.0504 0.0502 0.0495 73 0.0502 0.0502 0.0494 95 0.0513 0.0509 0.0500 95 0.0511 0.0506 0.0497 118 0.0501 0.0504 0.0489 118 0.0500 0.0501 0.0487 153 0.0513 0.0506 0.0490 153 0.0508 0.0502 0.0490 189 0.0508 0.0510 0.0493 189 0.0508 0.0504 0.0490 224 0.0522 0.0491 0.0484 224 0.0506 0.0495 0.0457 265 0.0508 0.0509 0.0488 265 0.0530 0.0514 0.0489 61 Table A4. Tagatose Degradation in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature Time Tagatose Concentration (M) (days) 20?C 30?C 40?C 0 0.0508 0.0507 0.0506 0 0.0506 0.0506 0.0506 0 0.0506 0.0513 0.0506 12 0.0485 0.0456 0.0432 12 0.0483 0.0458 0.0441 20 0.0481 0.0445 0.0413 20 0.0479 0.0439 0.0408 34 0.0465 0.0407 0.0350 34 0.0469 0.0413 0.0373 44 0.0465 0.0407 0.0291 44 0.0463 0.0408 0.0303 55 0.0442 0.0404 0.0302 55 0.0443 0.0394 0.0283 73 0.0434 0.0360 0.0246 73 0.0436 0.0357 0.0250 95 0.0424 0.0353 0.0241 95 0.0421 0.0346 0.0243 118 0.0418 0.0340 0.0220 118 0.0417 0.0339 0.0209 153 0.0412 0.0330 0.0197 153 0.0406 0.0316 0.0194 189 0.0402 0.0316 0.0175 189 0.0408 0.0310 0.0177 224 0.0389 0.0315 0.0145 224 0.0418 0.0287 0.0154 265 0.0393 0.0327 0.0175 265 0.0397 0.0305 0.0171 62 Table A5. Tagatose Degradation in 0.02 M Citrate Buffer at pH 3 as Affected by Temperature Time Tagatose Concentration (M) (days) 20?C 30?C 40?C 0 0.0509 0.0513 0.0512 0 0.0508 0.0513 0.0516 0 0.0503 0.0508 0.0513 20 0.0504 0.0508 0.0506 20 0.0504 0.0505 0.0505 44 0.0492 0.0507 0.0508 44 0.0516 0.0508 0.05011 55 0.0503 0.0505 0.0502 55 0.0504 0.0502 0.0503 73 0.0501 0.0498 0.0498 73 0.0501 0.0498 0.0497 95 0.0506 0.0504 0.0500 95 0.0502 0.0505 0.0501 118 0.0504 0.0505 0.0497 118 0.0501 0.0500 0.0498 153 0.0499 0.0503 0.0498 153 0.0499 0.0498 0.0502 189 0.0502 0.0499 0.0498 189 0.0505 0.0512 0.0502 224 0.0504 0.0507 0.0482 224 0.0482 0.0499 0.0479 265 0.0500 0.0508 0.0500 265 0.0507 0.0511 0.0507 63 Table A6. Tagatose Degradation in 0.02 M Citrate Buffer at pH 7 as Affected by Temperature Time Tagatose Concentration (M) (days) 20?C 30?C 40?C 0 0.0506 0.0513 0.0515 0 0.0506 0.0513 0.0512 0 0.0507 0.0510 0.0514 20 0.0506 0.0504 0.0498 20 0.0506 0.0504 0.0496 44 0.0506 0.0501 0.0492 44 0.0509 0.0509 0.0493 55 0.0521 0.0498 0.0476 55 0.0494 0.0492 0.0485 73 0.0502 0.0493 0.0477 73 0.0497 0.0492 0.0478 95 0.0493 0.0484 0.0459 95 0.0495 0.0494 0.0460 118 0.0494 0.0491 0.0451 118 0.0493 0.0495 0.0453 153 0.0490 0.0483 0.0441 153 0.0491 0.0485 0.0446 189 0.0501 0.0483 0.0436 189 0.0500 0.0482 0.0433 224 0.0508 0.0480 0.0454 224 0.0494 0.0492 0.0432 265 0.0502 0.0488 0.0420 265 0.0495 0.0500 0.0420 64 Table A7. Tagatose Degradation in 0.1 M Citrate Buffer at pH 3 as Affected by Temperature Time Tagatose Concentration (M) (days) 20?C 30?C 40?C 0 0.0502 0.0506 0.0510 0 0.0503 0.0506 0.0505 0 0.0504 0.0508 0.0510 20 0.0503 0.0503 0.0501 20 0.0502 0.0503 0.0501 44 0.0488 0.0513 0.0497 44 0.0500 0.0509 0.0504 55 0.0490 0.0498 0.0489 55 0.0488 0.0501 0.0490 73 0.0503 0.0491 0.0486 73 0.0497 0.0492 0.0486 95 0.0497 0.0495 0.0484 95 0.0495 0.0499 0.0482 118 0.0493 0.0496 0.0483 118 0.0496 0.0494 0.0479 153 0.0502 0.0492 0.0478 153 0.0496 0.0483 0.0478 189 0.0498 0.0493 0.0477 189 0.0500 0.0491 0.0479 224 0.0496 0.0486 0.0469 224 0.0492 0.0472 0.0469 265 0.0490 0.0500 0.0469 265 0.0501 0.0506 0.0473 65 Table A8. Tagatose Degradation in 0.1 M Citrate Buffer at pH 7 as Affected by Temperature Time Tagatose Concentration (M) (days) 20?C 30?C 40?C 0 0.0517 0.0516 0.0521 0 0.0517 0.0510 0.0521 0 0.0519 0.0520 0.0515 20 0.0511 0.0504 0.0494 20 0.0508 0.0506 0.0492 44 0.0503 0.0505 0.0482 44 0.0503 0.0497 0.0469 55 0.0522 0.0493 0.0457 55 0.0502 0.0487 0.0459 73 0.0506 0.0500 0.0465 73 0.0500 0.0499 0.0467 95 0.0494 0.0475 0.0412 95 0.0495 0.0475 0.0415 118 0.0498 0.0477 0.0405 118 0.0500 0.0475 0.0406 153 0.0485 0.0465 0.0390 153 0.0493 0.0468 0.0379 189 0.0491 0.0477 0.0362 189 0.0496 0.0460 0.0367 224 0.0495 0.0435 0.0313 224 0.0504 0.0441 0.0313 265 0.0496 0.0476 0.0339 265 0.0492 0.0463 0.0347 66 APPENDIX B TAGATOSE LOSS IN BUFFER SOLUTIONS WITH GLYCINE 67 Table B1. Tagatose Degradation in 0.02 M Phosphate Buffer (with 0.05 M Glycine) at pH 3 as Affected by Temperature Time Tagatose Concentration (M) (days) 20?C 30?C 40?C 0 N/A* N/A 0.0507 0 0.0507 0.0503 7 0.0508 0.0511 14 0.0504 0.0502 32 0.0507 0.0503 53 0.0493 0.0494 74 0.0503 0.0519 104 0.0528 104 0.0531 146 0.0492 146 0.0489 180 0.0489 180 0.0488 209 0.0494 209 0.0490 256 0.0490 256 0.0505 *N/A: Not available due to mold contamination. 68 Table B2. Tagatose Degradation in 0.02 M Phosphate Buffer (with 0.05 M Glycine) at pH 7 as Affected by Temperature 20?C 30?C 40?C Time (days) Concentration (M) Time (days) Concentration (M) Time (days) Concentration (M) 0 0.0512 0 0.0512 0 0.0507 0 0.0514 0 0.0517 0 0.0503 0 0.0517 0 0.0514 0 0.0507 7 0.0497 7 0.0510 7 0.0499 7 0.0508 7 0.0511 7 0.0486 15 0.0494 15 0.0497 14 0.0476 15 0.0495 15 0.0497 14 0.0477 25 0.0500 25 0.0491 24 0.0465 25 0.0494 25 0.0488 24 0.0468 33 0.0497 33 0.0493 32 0.0479 33 0.0497 33 0.0492 32 0.0489 49 0.0494 49 0.0486 48 0.0455 49 0.0494 49 0.0482 48 0.0452 75 0.0496 75 0.0478 74 0.0432 75 0.0493 75 0.0479 74 0.0418 105 0.0487 105 0.0470 104 0.0424 105 0.0499 105 0.0464 104 0.0424 155 0.0498 147 0.0474 146 0.0417 155 0.0492 147 0.0482 146 0.0418 181 0.0506 181 0.0457 180 0.0406 181 0.0483 181 0.0473 180 0.0405 210 0.0484 210 0.0470 209 0.0423 210 0.0490 210 0.0467 209 0.0423 257 0.0497 257 0.0481 256 0.0427 257 0.0494 257 0.0458 256 0.0406 69 Table B3. Tagatose Degradation in 0.1 M Phosphate Buffer (with 0.05 M Glycine) at pH 3 as Affected by Temperature 20?C 30?C 40?C Time (days) Concentration (M) Time (days) Concentration (M) Time (days) Concentration (M) 0 0.0507 0 0.0505 0 0.0491 0 0.0506 0 0.0505 0 0.0494 0 0.0505 0 0.0506 0 0.0492 7 0.0519 7 0.0519 7 0.0510 7 0.0518 7 0.0512 7 0.0508 15 0.0503 15 0.0510 14 0.0503 15 0.0503 15 0.0504 14 0.0503 33 0.0504 33 0.0498 32 0.0497 49 0.0503 33 0.0498 32 0.0497 75 0.0501 54 0.0498 53 0.0486 105 0.0503 54 0.0495 53 0.0486 155 0.0503 75 0.0501 74 0.0481 181 0.0515 75 0.0505 74 0.0495 210 0.0503 105 0.0503 104 0.0494 257 0.0508 105 0.0504 104 0.0494 147 0.0503 146 0.0479 147 0.0492 146 0.0475 181 0.0516 180 0.0479 181 0.0507 180 0.0456 210 0.0498 209 0.0454 210 0.0510 209 0.0484 257 0.0497 256 0.0471 257 0.0504 256 0.0478 70 Table B4. Tagatose Degradation in 0.1 M Phosphate Buffer (with 0.05 M Glycine) at pH 7 as Affected by Temperature 20?C 30?C 40?C Time (days) Concentration (M) Time (days) Concentration (M) Time (days) Concentration (M) 0 0.0500 0 0.0500 0 0.0477 0 0.0502 0 0.0502 0 0.0483 0 0.0506 0 0.0502 0 0.0488 7 0.0514 7 0.0504 7 0.0475 7 0.0514 7 0.0500 7 0.0455 15 0.0500 15 0.0483 14 0.0433 15 0.0509 15 0.0482 14 0.0434 25 0.0500 25 0.0472 24 0.0392 25 0.0493 25 0.0453 24 0.0392 25 0.0502 33 0.0484 32 0.0383 33 0.0501 33 0.0491 32 0.0407 33 0.0484 49 0.0448 48 0.0351 49 0.0479 49 0.0448 48 0.0344 75 0.0479 75 0.0435 74 0.0302 105 0.0463 75 0.0438 74 0.0312 155 0.0457 105 0.0404 104 0.0267 181 0.0473 105 0.0418 104 0.0275 201 0.0464 147 0.0424 146 0.0227 210 0.0463 147 0.0410 146 0.0228 257 0.0462 181 0.0398 180 0.0214 181 0.0387 180 0.0218 210 0.0388 209 0.0188 210 0.0383 209 0.0192 257 0.0392 256 0.0178 257 0.0389 256 0.0173 71 Table B5. Tagatose Degradation in 0.02 M Citrate Buffer (with 0.05 M Glycine) at pH 3 as Affected by Temperature 20?C 30?C 40?C Time (days) Concentration (M) Time (days) Concentration (M) Time (days) Concentration (M) 0 0.0503 0 0.0504 0 0.0502 0 0.0506 0 0.0509 0 0.0504 0 0.0507 0 0.0507 0 0.0508 15 0.0511 15 0.0506 14 0.0506 15 0.0507 15 0.0506 14 0.0507 33 0.0506 33 0.0503 32 0.0501 33 0.0506 33 0.0501 32 0.0503 54 0.0493 54 0.0493 53 0.0490 54 0.0491 54 0.0486 53 0.0490 75 0.0497 75 0.0497 74 0.0497 75 0.0498 75 0.0497 74 0.0496 110 0.0504 110 0.0506 109 0.0499 110 0.0507 110 0.0505 109 0.0502 155 0.0499 147 0.0499 146 0.0490 155 0.0504 147 0.0501 146 0.0490 181 0.0510 181 0.0509 180 0.0488 181 0.0499 181 0.0509 180 0.0503 210 0.0503 210 0.0498 209 0.0493 210 0.0503 210 0.0507 209 0.0487 257 0.0509 257 0.0504 256 0.0491 257 0.0508 257 0.0501 256 0.0500 72 Table B6. Tagatose Degradation in 0.02 M Citrate Buffer (with 0.05 M Glycine) at pH 7 as Affected by Temperature 20?C 30?C 40?C Time (days) Concentration (M) Time (days) Concentration (M) Time (days) Concentration (M) 0 0.0507 0 0.0510 0 0.0513 0 0.0507 0 0.0506 0 0.0512 0 0.0507 0 0.0508 0 0.0507 15 0.0501 15 0.0499 14 0.0492 15 0.0499 15 0.0499 14 0.0490 25 0.0496 25 0.0494 24 0.0498 25 0.0496 25 0.0539 24 0.0489 33 0.0513 33 0.0501 32 0.0489 33 0.0503 33 0.0499 32 0.0492 54 0.0493 54 0.0486 53 0.0472 54 0.0492 54 0.0486 53 0.0473 75 0.0497 75 0.0501 74 0.0459 75 0.0498 75 0.0499 74 0.0462 110 0.0503 110 0.0479 109 0.0453 110 0.0487 110 0.0488 109 0.0434 155 0.0495 147 0.0488 146 0.0447 155 0.0498 147 0.0486 146 0.0449 181 0.0514 181 0.0507 180 0.0439 181 0.0512 181 0.0480 180 0.0425 210 0.0513 210 0.0497 209 0.0452 210 0.0486 210 0.0484 209 0.0448 257 0.0509 257 0.0495 256 0.0422 257 0.0506 257 0.0494 256 0.0434 73 Table B7. Tagatose Degradation in 0.1 M Citrate Buffer (with 0.05 M Glycine) at pH 3 as Affected by Temperature 20?C 30?C 40?C Time (days) Concentration (M) Time (days) Concentration (M) Time (days) Concentration (M) 0 0.0504 0 0.0505 0 0.0501 0 0.0509 0 0.0501 0 0.0497 0 0.0502 0 0.0502 0 0.0494 15 0.0505 15 0.0503 14 0.0505 15 0.0510 15 0.0504 14 0.0510 33 0.0501 33 0.0501 32 0.0502 36 0.0501 33 0.0501 32 0.0503 54 0.0499 54 0.0491 53 0.0489 54 0.0494 54 0.0490 53 0.0486 75 0.0505 75 0.0497 74 0.0494 75 0.0497 75 0.0501 74 0.0497 110 0.0506 110 0.0512 109 0.0482 110 0.0507 110 0.0503 146 0.0480 155 0.0496 147 0.0497 146 0.0486 155 0.0498 147 0.0497 180 0.0477 181 0.0499 181 0.0486 180 0.0478 181 0.0492 181 0.0494 209 0.0461 210 0.0499 210 0.0488 209 0.0468 210 0.0505 210 0.0497 256 0.0468 257 0.0507 257 0.0500 256 0.0475 257 0.0499 257 0.0512 74 Table B8. Tagatose Degradation in 0.1 M Citrate Buffer (with 0.05 M Glycine) at pH 7 as Affected by Temperature 20?C 30?C 40?C Time (days) Concentration (M) Time (days) Concentration (M) Time (days) Concentration (M) 0 0.0507 0 0.0503 0 0.0525 0 0.0504 0 0.0506 0 0.0524 0 0.0505 0 0.0505 0 0.0522 15 0.0508 15 0.0499 14 0.0485 15 0.0509 15 0.0506 14 0.0477 25 0.0505 25 0.0506 24 0.0480 25 0.0501 25 0.0500 24 0.0476 33 0.0499 33 0.0499 32 0.0493 33 0.0498 33 0.0507 32 0.0485 54 0.0508 54 0.0495 53 0.0451 54 0.0505 54 0.0496 53 0.0470 75 0.0502 75 0.0473 74 0.0426 75 0.0491 75 0.0489 74 0.0432 110 0.0488 110 0.0497 109 0.0408 110 0.0485 110 0.0482 109 0.0412 155 0.0490 147 0.0478 146 0.0406 155 0.0498 147 0.0482 146 0.0384 181 0.0492 181 0.0484 180 0.0363 181 0.0489 181 0.0449 180 0.0364 210 0.0487 210 0.0478 209 0.0349 210 0.0493 210 0.0471 209 0.0352 257 0.0502 257 0.0468 256 0.0323 257 0.0497 257 0.0470 256 0.0321 75 APPENDIX C BROWNING OF TAGATOSE IN BUFFER SOLUTIONS WITHOUT GLYCINE 76 Table C1. Browning of 0.05 M Tagatose in 0.02 M Phosphate Buffer at pH 3 as Affected by Temperature Time Absorbance at 420 nm (days) 20?C 30?C 40?C 0 -0.0027 -0.0020 -0.0037 0 -0.0021 -0.0021 -0.0033 0 -0.0014 -0.0020 -0.0034 12 -0.0011 -0.0018 -0.0029 12 0.0002 -0.0018 -0.0028 20 -0.0023 -0.0015 -0.0039 20 -0.0006 -0.0017 -0.0035 44 -0.0018 -0.0020 -0.0034 44 -0.0015 -0.0019 -0.0034 55 -0.0003 -0.0018 -0.0025 55 -0.0015 -0.0015 -0.0025 73 -0.0039 -0.0024 -0.0029 73 -0.0038 -0.0026 -0.0029 95 -0.0038 -0.0021 -0.0020 95 -0.0038 -0.0021 -0.0018 118 -0.0035 -0.0018 -0.0011 118 -0.0035 -0.0018 -0.0013 153 -0.0037 -0.0023 -0.0012 153 -0.0038 -0.0024 -0.0004 189 -0.0034 -0.0018 0.0023 189 -0.0035 -0.0021 0.0030 224 -0.0033 -0.0016 0.0029 224 -0.0034 -0.0019 0.0040 265 -0.0037 -0.0018 0.0032 265 -0.0037 -0.0019 0.0037 77 Table C2. Browning of 0.05 M Tagatose in 0.02 M Phosphate Buffer at pH 7 as Affected by Temperature Time Absorbance at 420 nm (days) 20?C 30?C 40?C 0 -0.0008 -0.0013 -0.0035 0 -0.0002 -0.0018 -0.0028 0 0.0002 -0.0008 -0.0021 12 0.0006 0.0013 0.0112 12 0.0017 0.0022 0.0162 20 -0.0008 -0.0018 0.0108 20 -0.0002 -0.0011 0.0066 34 -0.0014 -0.0001 0.0035 34 0.0029 -0.0003 0.0031 44 0.0018 0.0026 0.0027 44 0.0011 0.0007 0.0058 55 -0.0006 -0.0023 0.0078 55 -0.0022 -0.0016 0.0036 73 0.0002 0.0002 0.0009 73 0.0006 -0.0015 0.0011 95 0.0034 0.0012 0.0042 95 0.0021 0.0006 0.0045 118 0.0022 -0.0001 0.0020 118 0.0013 0.0001 0.0015 153 0.0009 -0.0012 0.0011 153 0.0002 -0.0011 0.0002 189 0.0021 0.0017 0.0056 189 0.0006 -0.0001 0.0048 224 0.0001 -0.0008 0.0048 224 0.0007 -0.0008 0.0039 265 0.0015 -0.0010 0.0025 265 0.0004 -0.0010 0.0042 78 Table C3. Browning of 0.05 M Tagatose in 0.1 M Phosphate Buffer at pH 3 as Affected by Temperature Time Absorbance at 420 nm (days) 20?C 30?C 40?C 0 0.0020 -0.0020 -0.0018 0 0.0068 -0.0010 -0.0012 0 0.0024 -0.0017 0.0020 12 0.0024 -0.0017 -0.0006 12 0.0025 -0.0018 0.0002 20 0.0009 -0.0024 0.0008 20 0.0013 -0.0017 -0.0016 44 0.0006 -0.0019 -0.0001 44 0.0025 -0.0019 0.0001 55 0.0032 -0.0014 0.0034 55 0.0065 -0.0011 0.0016 73 -0.0004 -0.0021 0.0018 73 0.0003 0.0028 0.0127 95 -0.0003 -0.0020 0.0063 95 0.0015 -0.0017 0.0066 118 0.0016 -0.0004 0.0110 118 0.0012 -0.0013 0.0093 153 -0.0009 -0.0022 0.0097 153 0.0001 -0.0019 0.0101 189 -0.0001 -0.0014 0.0269 189 0.0006 -0.0011 0.0271 224 0.0015 -0.0006 0.0332 224 0.0006 -0.0007 0.0343 265 0.0003 -0.0003 0.0391 265 0.0006 -0.0005 0.0419 79 Table C4. Browning of 0.05 M Tagatose in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature Time Absorbance at 420 nm (days) 20?C 30?C 40?C 0 -0.0041 0.0008 0.0093 0 -0.0039 0.0003 0.0062 0 -0.0037 0.0008 0.0087 12 0.0012 0.0416 0.1713 12 0.0025 0.0516 0.1708 20 0.0030 0.0509 0.2503 20 0.0024 0.0322 0.2446 34 0.0024 0.0250 0.2865 34 0.0019 0.0253 0.3429 44 0.0082 0.0229 0.2808 44 0.0065 0.0406 0.4010 55 0.0048 0.0328 0.3898 55 0.0026 0.0184 0.3807 73 0.0034 0.0149 0.1679 73 0.0027 0.0150 0.3097 95 0.0059 0.0158 0.1401 95 0.0200 0.0234 0.2362 118 0.0020 0.0093 0.1201 118 0.0024 0.0100 0.1858 153 0.0038 0.0137 0.1602 153 0.0026 0.0080 0.1516 189 0.0047 0.0070 0.0936 189 0.0031 0.0113 0.1441 224 0.0011 0.0113 0.1269 224 -0.0004 0.0055 0.1230 265 0.0017 0.0051 0.0756 265 0.0023 0.0086 0.1141 80 Table C5. Browning of 0.05 M Tagatose in 0.02 M Citrate Buffer at pH 3 as Affected by Temperature Time Absorbance at 420 nm (days) 20?C 30?C 40?C 0 -0.0032 -0.0012 -0.0006 0 -0.0030 0.0677 -0.0006 0 -0.0020 0.0001 -0.0002 20 -0.0017 0.0003 0.0003 20 -0.0005 0.0019 0.0060 44 -0.0033 -0.0011 -0.0007 44 -0.0030 -0.0006 -0.0008 55 -0.0022 0.0003 0.0025 55 -0.0022 0.0001 0.0007 73 0.0005 0.0016 0.0014 73 -0.0020 0.0004 0.0007 95 -0.0038 -0.0011 -0.0002 95 -0.0031 -0.0008 0.0003 118 -0.0025 0.0001 0.0013 118 -0.0025 0.0002 0.0007 153 -0.0015 0.0042 0.0031 153 -0.0023 0.0018 0.0013 189 -0.0033 -0.0008 0.0023 189 -0.0027 -0.0003 0.0026 224 -0.0019 0.0007 0.0041 224 -0.0020 0.0007 0.0038 265 0.0012 0.0068 0.0071 265 -0.0020 0.0004 0.0062 81 Table C6. Browning of 0.05 M Tagatose in 0.02 M Citrate Buffer at pH 7 as Affected by Temperature Time Absorbance at 420 nm (days) 20?C 30?C 40?C 0 -0.0021 -0.0010 -0.0002 0 -0.0026 -0.0006 0.0006 0 -0.0011 0.0007 0.0011 20 -0.0013 0.0000 0.0004 20 -0.0003 0.0029 0.0016 44 -0.0028 -0.0005 -0.0008 44 -0.0018 -0.0009 0.0000 55 -0.0018 -0.0007 0.0019 55 -0.0006 -0.0003 0.0033 73 -0.0004 0.0002 0.0047 73 0.0008 -0.0009 0.0005 95 -0.0029 -0.0019 0.0008 95 -0.0022 -0.0013 0.0043 118 -0.0023 -0.0011 0.0030 118 -0.0013 -0.0006 0.0022 153 0.0007 0.0007 0.0030 153 -0.0004 0.0005 0.0013 189 -0.0026 -0.0018 0.0009 189 -0.0023 -0.0011 0.0014 224 -0.0013 -0.0010 0.0028 224 -0.0015 0.0046 0.0023 265 -0.0007 0.0024 0.0054 265 0.0035 -0.0007 0.0066 82 Table C7. Browning of 0.05 M Tagatose in 0.1 M Citrate Buffer at pH 3 as Affected by Temperature Time Absorbance at 420 nm (days) 20?C 30?C 40?C 0 -0.0034 -0.0013 -0.0030 0 -0.0029 -0.0010 -0.0031 0 -0.0029 -0.0005 -0.0026 20 -0.0025 -0.0002 -0.0023 20 -0.0026 0.0009 -0.0025 44 -0.0032 -0.0018 -0.0025 44 -0.0029 -0.0011 -0.0025 55 -0.0034 -0.0005 -0.0015 55 -0.0035 0.0003 -0.0015 73 0.0001 0.0011 0.0004 73 -0.0029 -0.0004 0.0036 95 -0.0035 -0.0026 0.0050 95 -0.0035 -0.0022 0.0033 118 -0.0034 -0.0021 0.0074 118 -0.0033 -0.0020 0.0076 153 -0.0027 -0.0018 0.0112 153 -0.0032 -0.0020 0.0098 189 -0.0038 -0.0006 0.0223 189 -0.0037 -0.0007 0.0202 224 -0.0036 -0.0002 0.0272 224 -0.0031 -0.0007 0.0271 265 -0.0023 -0.0003 0.0418 265 -0.0033 -0.0001 0.0373 83 Table C8. Browning of 0.05 M Tagatose in 0.1 M Citrate Buffer at pH 7 as Affected by Temperature Time Absorbance at 420 nm (days) 20?C 30?C 40?C 0 -0.0028 -0.0030 0.0031 0 0.0003 -0.0022 0.0032 0 -0.0014 -0.0023 0.0032 20 -0.0025 -0.0017 0.0026 20 -0.0027 -0.0023 0.0077 44 -0.0004 -0.0009 0.0037 44 0.0014 -0.0025 -0.0017 55 0.0021 0.0003 -0.0010 55 -0.0002 -0.0029 0.0006 73 0.0001 0.0000 -0.0013 73 0.0008 0.0009 0.0001 95 -0.0024 -0.0019 0.0081 95 -0.0024 -0.0011 -0.0001 118 -0.0016 -0.0020 0.0013 118 0.0001 -0.0013 0.0012 153 -0.0021 -0.0007 -0.0011 153 -0.0022 -0.0006 -0.0007 189 -0.0016 -0.0031 -0.0008 189 -0.0037 -0.0036 -0.0013 224 -0.0012 -0.0024 0.0015 224 -0.0008 -0.0020 -0.0005 265 -0.0021 -0.0033 -0.0007 265 -0.0025 -0.0031 -0.0012 84 APPENDIX D BROWNING OF TAGATOSE AND GLYCINE IN BUFFER SOLUTIONS 85 Table D1. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.02 M Phosphate Buffer at pH 3 as Affected by Temperature Time Absorbance at 420 nm (days) 20?C 30?C 40?C 0 N/A* N/A -0.0002 0 -0.0003 -0.0002 7 0.0003 0.0004 14 0.0015 0.0014 32 0.0041 0.0042 53 0.0094 0.0094 74 0.0303 0.0318 104 0.0479 104 0.0479 146 0.0558 146 0.0560 180 0.0653 180 0.0655 209 0.0981 209 0.0968 256 0.0981 256 0.0981 *N/A: Not available due to mold contamination. 86 Table D2. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.02 Phosphate Buffer at pH 7 as Affected by Temperature 20?C 30?C 40?C Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm 0 0.0026 0 0.0019 0 0.0028 0 0.0026 0 0.0020 0 0.0030 0 0.0049 0 0.0024 0 0.0030 7 0.0028 7 0.0041 7 0.0237 7 0.0038 7 0.0043 7 0.0223 15 0.0021 15 0.0077 14 0.0326 15 0.0026 15 0.0073 14 0.0352 25 0.0038 25 0.0106 24 0.0507 25 0.0040 25 0.0104 24 0.0461 33 0.0029 33 0.0123 32 0.0593 33 0.0052 33 0.0122 32 0.0545 49 0.0051 49 0.0150 48 0.0757 49 0.0056 49 0.0151 48 0.0718 75 0.0113 75 0.0196 74 0.0997 75 0.0089 75 0.0185 74 0.0966 105 0.0103 105 0.0230 104 0.1352 105 0.0100 105 0.0239 104 0.1312 155 0.0092 147 0.0274 146 0.1828 155 0.0094 147 0.0268 146 0.1787 181 0.0112 181 0.0314 180 0.2081 181 0.0077 181 0.0299 180 0.2094 210 0.0098 210 0.0346 209 0.2263 210 0.0097 210 0.0356 209 0.2231 257 0.0097 257 0.0404 256 0.2577 257 0.0091 257 0.0384 256 0.2563 87 Table D3. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.1 M Phosphate Buffer at pH 3 as Affected by Temperature 20?C 30?C 40?C Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm 0 0.0001 0 0.0001 0 0.0007 0 0.0008 0 -0.0006 0 -0.0002 0 0.0009 0 -0.0010 0 0.0001 7 0.0005 7 -0.0003 7 0.0009 7 0.0014 7 -0.0005 7 0.0009 15 -0.0003 15 -0.0008 14 0.0033 15 0.0006 15 -0.0010 14 0.0033 33 0.0006 33 -0.0001 32 0.0098 49 0.0007 33 0.0001 32 0.0102 75 0.0002 54 0.0036 53 0.0225 105 0.0016 54 0.0006 53 0.0223 155 0.0028 75 0.0028 74 0.0509 181 0.0048 75 0.0026 74 0.0504 210 0.0021 105 0.0044 104 0.0785 257 0.0018 105 0.0055 104 0.0785 147 0.0081 146 0.1190 147 0.0074 146 0.1166 181 0.0092 180 0.1525 181 0.0087 180 0.1507 210 0.0137 209 0.2031 210 0.0152 209 0.2065 257 0.0152 256 0.2419 257 0.0148 256 0.2384 88 Table D4. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature 20?C 30?C 40?C Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm 0 0.0006 0 -0.0035 0 0.0102 0 0.0005 0 -0.0039 0 0.0100 0 0.0010 0 -0.0036 0 0.0124 7 0.0034 7 0.0116 7 0.0725 7 0.0042 7 0.0109 7 0.0711 15 0.0078 15 0.0157 14 0.1091 15 0.0082 15 0.0155 14 0.1532 25 0.0107 25 0.0194 24 0.1821 25 0.0111 25 0.0214 24 0.2028 25 0.0121 33 0.0224 32 0.2068 33 0.0116 33 0.0228 32 0.1923 33 0.0114 49 0.0269 48 0.2931 49 0.0130 49 0.0279 48 0.2596 75 0.0139 75 0.0338 74 0.3340 105 0.0155 75 0.0364 74 0.3409 155 0.0169 105 0.0459 104 0.4544 181 0.0177 105 0.0492 104 0.4049 201 0.0182 147 0.0639 146 0.5675 210 0.0192 147 0.0671 146 0.5271 257 0.0213 181 0.0776 180 0.5711 181 0.0811 180 0.5856 210 0.0862 209 0.6281 210 0.0914 209 0.5899 257 0.1059 256 0.7052 257 0.1115 256 0.6795 89 Table D5. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.02 M Citrate Buffer at pH 3 as Affected by Temperature 20?C 30?C 40?C Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm 0 -0.0064 0 -0.0003 0 -0.0071 0 -0.0060 0 -0.0005 0 -0.0065 0 -0.0062 0 -0.0007 0 -0.0055 15 -0.0039 15 -0.0001 14 -0.0046 15 -0.0052 15 -0.0001 14 -0.0009 33 -0.0063 33 -0.0005 32 -0.0042 33 -0.0057 33 0.0001 32 -0.0038 54 -0.0057 54 0.0001 53 -0.0003 54 -0.0056 54 0.0005 53 0.0002 75 -0.0049 75 0.0052 74 0.0214 75 -0.0050 75 0.0024 74 0.0193 110 -0.0053 110 0.0026 109 0.0312 110 -0.0053 110 0.0030 109 0.0316 155 -0.0047 147 0.0032 146 0.0430 155 -0.0048 147 0.0042 146 0.0430 181 -0.0048 181 0.0038 180 0.0519 181 -0.0045 181 0.0037 180 0.0517 210 -0.0046 210 0.0070 209 0.0734 210 -0.0050 210 0.0067 209 0.0746 257 -0.0052 257 0.0056 256 0.0788 257 -0.0050 257 0.0058 256 0.0794 90 Table D6. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.02 M Citrate Buffer at pH 7 as Affected by Temperature 20?C 30?C 40?C Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm 0 0.0012 0 -0.0044 0 0.0034 0 0.0011 0 -0.0044 0 0.0105 0 0.0009 0 -0.0038 0 0.0046 15 0.0015 15 -0.0026 14 0.0090 15 0.0017 15 -0.0015 14 0.0087 25 0.0014 25 -0.0033 24 0.0112 25 0.0016 25 -0.0032 24 0.0114 33 0.0014 33 -0.0025 32 0.0148 33 0.0015 33 -0.0017 32 0.0147 54 0.0023 54 0.0004 53 0.0227 54 0.0038 54 -0.0011 53 0.0223 75 0.0027 75 -0.0010 74 0.0369 75 0.0026 75 0.0013 74 0.0302 110 0.0024 110 -0.0008 109 0.0494 110 0.0029 110 0.0008 109 0.0497 155 0.0028 147 0.0043 146 0.0805 155 0.0051 147 0.0035 146 0.0816 181 0.0028 181 0.0026 180 0.1098 181 0.0030 181 0.0020 180 0.1087 210 0.0070 210 0.0047 209 0.1339 210 0.0038 210 0.0051 209 0.1318 257 0.0050 257 0.0080 256 0.1785 257 0.0090 257 0.0071 256 0.1798 91 Table D7. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.1 M Citrate Buffer at pH 3 as Affected by Temperature 20?C 30?C 40?C Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm 0 0.0000 0 -0.0154 0 -0.0065 0 0.0001 0 -0.0156 0 -0.0063 0 -0.0001 0 -0.0154 0 -0.0060 15 0.0001 15 -0.0149 14 -0.0027 15 0.0003 15 -0.0151 14 -0.0021 33 0.0004 33 -0.0153 32 0.0001 36 0.0002 33 -0.0151 32 0.0001 54 0.0003 54 -0.0137 53 0.0085 54 0.0005 54 -0.0130 53 0.0075 75 0.0010 75 -0.0092 74 0.0375 75 0.0011 75 -0.0104 74 0.0365 110 0.0006 110 -0.0080 109 0.0544 110 0.0011 110 -0.0067 109 0.0539 155 0.0010 147 -0.0061 146 0.0778 155 0.0014 147 -0.0065 146 0.0750 181 0.0014 181 -0.0044 180 0.0968 181 0.0017 181 -0.0052 180 0.0979 210 0.0012 210 -0.0017 209 0.1356 210 0.0012 210 -0.0013 209 0.1304 257 0.0012 257 -0.0004 256 0.1658 257 0.0017 257 -0.0005 256 0.1702 92 Table D8. Browning of 0.05 M Tagatose and 0.05 M Glycine in 0.1 M Citrate Buffer at pH 7 as Affected by Temperature 20?C 30?C 40?C Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm Time (days) Absorbance at 420 nm 0 0.0018 0 0.0027 0 0.0052 0 0.0017 0 0.0025 0 0.0057 0 0.0015 0 0.0025 0 0.0054 15 0.0024 15 0.0071 14 0.0182 15 0.0030 15 0.0067 14 0.0184 25 0.0021 25 0.0088 24 0.0215 25 0.0021 25 0.0082 24 0.0213 33 0.0028 33 0.0093 32 0.0262 33 0.0032 33 0.0094 32 0.0264 54 0.0093 54 0.0139 53 0.0413 54 0.0040 54 0.0106 53 0.0415 75 0.0050 75 0.0100 74 0.0552 75 0.0046 75 0.0097 74 0.0555 110 0.0051 110 0.0106 109 0.0915 110 0.0055 110 0.0107 109 0.0914 155 0.0062 147 0.0126 146 0.1493 155 0.0061 147 0.0124 146 0.1510 181 0.0056 181 0.0136 180 0.2097 181 0.0057 181 0.0137 180 0.2104 210 0.0056 210 0.0149 209 0.2591 210 0.0059 210 0.0158 209 0.2560 257 0.0060 257 0.0189 256 0.3461 257 0.0059 257 0.0188 256 0.3536