THERMAL STABILITY OF TAGATOSE IN SOLUTION 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. ____________________________ Katherine J. Luecke Certificate of Approval: _____________________________ Tung-Shi Huang Associate Professor Nutrition and Food Science _____________________________ Oladiran Fasina Associate Professor Biosystems Engineering _____________________________ Leonard N. Bell, Chair Professor Nutrition and Food Science _____________________________ George T. Flowers Dean Graduate School THERMAL STABILITY OF TAGATOSE IN SOLUTION Katherine J. Luecke 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 August 10, 2009 iii THERMAL STABILITY OF TAGATOSE IN SOLUTION Katherine J. Luecke 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 THERMAL STABILITY OF TAGATOSE IN SOLUTION Katherine J. Luecke Master of Science, August 10, 2009 (B.S., Fontbonne University, 2007) 109 Typed Pages Directed by Leonard N. Bell Tagatose is a minimally absorbed monosaccharide that has prebiotic properties. To achieve this prebiotic benefit, tagatose in foods and beverages must not be lost during processing. However, data on the thermal stability of tagatose are lacking. The objective of this study was to evaluate the thermal stability of tagatose in solutions. 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. A second set of similar solutions was also made, only differing from the first set in that they contained 0.05 M glycine. All of the solutions were held at 60, 70, and 80?C for a minimum of 5 hours. At least 11 samples were removed at regular time intervals during the study for analysis. Tagatose analysis occurred via reverse-phase HPLC while browning was measured using a spectrophotometer at 420 nm. v In the solutions without glycine, minimal tagatose was lost at 60-80?C in citrate and phosphate buffers at pH 3. For these solutions, slight browning was observed at all temperatures. At pH 7, tagatose loss was enhanced. The pseudo-first-order rate constants (kobs) for tagatose degradation at pH 7 were greater in phosphate buffer than citrate buffer. Higher buffer concentrations and higher temperatures also increased kobs. Enhanced browning accompanied the tagatose degradation in all buffer solutions at pH 7. In the solutions containing tagatose as well as glycine, tagatose degraded faster at pH 7 than pH 3. Tagatose degradation was again greater in phosphate buffer than citrate buffer, and at the higher buffer concentration. Temperature also affected tagatose degradation, with faster tagatose loss occurring as the temperature increased. With glycine present in the solutions, enhanced browning occurred, but tagatose degradation rates were similar to those of the solutions without glycine. For both the solutions containing tagatose as well as those containing tagatose and glycine, the most reactive solution was 0.1 M phosphate at pH 7. Using the activation energies for tagatose degradation, it was predicted that less than 0.5 and 0.02% tagatose would be lost during basic vat and HTST pasteurization, respectively, regardless of whether or not glycine was present. Based on the results from this study, it was determined that although tagatose does breakdown at elevated temperatures, the amount of tagatose lost during the times and temperatures associated with typical thermal processing conditions would be virtually negligible. Due to minimal tagatose degradation during typical thermal processing techniques, the majority of tagatose would remain present in a beverage after pasteurization, allowing its presence to provide the consumer with prebiotic benefits. vi ACKNOWLEDGMENTS This project would not have taken place without the knowledge, guidance, and support of Dr. Leonard Bell. For his dedication and expertise I am tremendously grateful. I would also like to express sincere gratitude to my thesis committee members, Dr. Tung-Shi Huang and Dr. Oladiran Fasina. Their advice and reviews are greatly appreciated. Finally, I would like to thank my family and friends for their relentless encouragement and immeasurable love during the course of this project. 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 ........................................................................................................ xiv CHAPTER 1. INTRODUCTION ...................................................................................... 1 CHAPTER 2. LITERATURE REVIEW ........................................................................... 3 Chemistry and Properties of Tagatose ....................................................... 4 Tagatose Production................................................................................... 5 Digestion and Absorption .......................................................................... 6 Caloric Value ........................................................................................... 13 Tolerance.................................................................................................. 15 Tagatose as a Prebiotic............................................................................. 16 Health Effects of Tagatose ....................................................................... 17 Diabetes........................................................................................ 17 Plasma Uric Acid Levels ............................................................. 18 Tagatose in Food ...................................................................................... 21 Tagatose Stability..................................................................................... 24 CHAPTER 3. MATERIALS AND METHODS ............................................................. 29 Sample Preparation .................................................................................. 29 Experiments ............................................................................................. 31 Sample Analysis....................................................................................... 34 Tagatose Degradation .................................................................. 34 Brown Pigment Formation ........................................................... 36 Data Analysis ........................................................................................... 36 CHAPTER 4. RESULTS AND DISCUSSION ............................................................... 39 Tagatose Degradation .............................................................................. 39 Effect of pH.................................................................................. 39 Effect of Buffer Type and Buffer Concentration ......................... 45 Effect of Temperature .................................................................. 47 ix Tagatose and the Maillard Reaction ........................................................ 50 Effect of pH.................................................................................. 51 Effect of Buffer Type and Buffer Concentration ......................... 52 Effect of Temperature .................................................................. 55 Brown Pigment Formation ....................................................................... 57 Effect of Glycine on Tagatose Degradation and Brown Pigment Formation................................................................................ 63 CHAPTER 5. SUMMARY AND CONCLUSIONS ....................................................... 66 REFERENCES ................................................................................................................ 68 APPENDIX A .................................................................................................................. 73 APPENDIX B .................................................................................................................. 78 APPENDIX C .................................................................................................................. 83 APPENDIX D .................................................................................................................. 88 APPENDIX E .................................................................................................................. 93 x LIST OF TABLES TABLE 3.1. Length (h) of Tagatose Experiments ........................................................... 33 TABLE 3.2. Length (h) of Tagatose-Glycine Experiments ............................................. 33 TABLE 4.1. Pseudo-First-Order Rate Constants (h-1) with 95% Confidence Limits for the Loss of Tagatose in Solution............................................. 41 TABLE 4.2. Decrease in the pH of Tagatose Solutions During Storage at 60 C ........... 43 TABLE 4.3. Activation Energies (EA) for Tagatose Degradation at pH 7 ...................... 50 TABLE 4.4. Pseudo-First-Order Rate Constants (h-1) with 95% Confidence Limits for the Loss of Tagatose in Solution Containing 0.05 M Glycine ........................................................................................ 53 TABLE 4.5. Activation Energies (EA) for Tagatose Degradation in Solutions Containing 0.05 M Glycine...................................................................... 57 TABLE 4.6. Pseudo-Zero-Order Rate Constants (OD/h) for the Formation of Brown Pigment in Tagatose Solutions ..................................................... 60 TABLE 4.7. Pseudo-Zero-Order Rate Constants (OD/h) for the Formation of Brown Pigment in Solutions Containing Tagatose and Glycine ............. 61 TABLE 4.8. Activation Energies (kcal/mol) for Brown Pigment Formation in Tagatose and Tagatose-Glycine Solutions at pH 7 .............................. 63 TABLE A1. Tagatose Degradation in 0.02 M Phosphate Buffer at pH 3 as Affected by Temperature .................................................................... 74 TABLE A2. Tagatose Degradation in 0.02 M Phosphate Buffer at pH 7 as Affected by Temperature ..................................................................... 74 TABLE A3. Tagatose Degradation in 0.1 M Phosphate Buffer at pH 3 as Affected by Temperature ..................................................................... 75 xi TABLE A4. Tagatose Degradation in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature ..................................................................... 75 TABLE A5. Tagatose Degradation in 0.02 M Citrate Buffer at pH 3 as Affected by Temperature ..................................................................... 76 TABLE A6. Tagatose Degradation in 0.02 M Citrate Buffer at pH 7 as Affected by Temperature ..................................................................... 76 TABLE A7. Tagatose Degradation in 0.1 M Citrate Buffer at pH 3 as Affected by Temperature ..................................................................... 77 TABLE A8. Tagatose Degradation in 0.1 M Citrate Buffer at pH 7 as Affected by Temperature ..................................................................... 77 TABLE B1. Tagatose Degradation in 0.02 M Phosphate Buffer (with 0.05 M Glycine) at pH 3 as Affected by Temperature ................... 79 TABLE B2. Tagatose Degradation in 0.02 M Phosphate Buffer (with 0.05 M Glycine) at pH 7 as Affected by Temperature ................... 79 TABLE B3. Tagatose Degradation in 0.1 M Phosphate Buffer (with 0.05 M Glycine) at pH 3 as Affected by Temperature ................... 80 TABLE B4. Tagatose Degradation in 0.1 M Phosphate Buffer (with 0.05 M Glycine) at pH 7 as Affected by Temperature ................... 80 TABLE B5. Tagatose Degradation in 0.02 M Citrate Buffer (with 0.05 M Glycine) at pH 3 as Affected by Temperature ................... 81 TABLE B6. Tagatose Degradation in 0.02 M Citrate Buffer (with 0.05 M Glycine) at pH 7 as Affected by Temperature ................... 81 TABLE B7. Tagatose Degradation in 0.1 M Citrate Buffer (with 0.05 M Glycine) at pH 3 as Affected by Temperature ................... 82 TABLE B8. Tagatose Degradation in 0.1 M Citrate Buffer (with 0.05 M Glycine) at pH 7 as Affected by Temperature ................... 82 TABLE C1. Browning of 0.05 M Tagatose in 0.02 M Phosphate Buffer at pH 3 as Affected by Temperature ..................................................................... 84 TABLE C2. Browning of 0.05 M Tagatose in 0.02 M Phosphate Buffer at pH 7 as Affected by Temperature ..................................................................... 84 xii TABLE C3. Browning of 0.05 M Tagatose in 0.1 M Phosphate Buffer at pH 3 as Affected by Temperature ..................................................................... 85 TABLE C4. Browning of 0.05 M Tagatose in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature ..................................................................... 85 TABLE C5. Browning of 0.05 M Tagatose in 0.02 M Citrate Buffer at pH 3 as Affected by Temperature ..................................................................... 86 TABLE C6. Browning of 0.05 M Tagatose in 0.02 M Citrate Buffer at pH 7 as Affected by Temperature ..................................................................... 86 TABLE C7. Browning of 0.05 M Tagatose in 0.1 M Citrate Buffer at pH 3 as Affected by Temperature ..................................................................... 87 TABLE C8. Browning of 0.05 M Tagatose in 0.1 M Citrate Buffer at pH 7 as Affected by Temperature ..................................................................... 87 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 ........................... 89 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 ........................... 89 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 ........................... 90 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 ........................... 90 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 ................................. 91 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 ................................. 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 ................................. 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 ................................. 92 TABLE E1. Average Temperature of Tagatose Buffer Solutions ................................... 94 xiii TABLE E2. Average Temperatures of Tagatose-Glycine Buffer Solutions ................... 94 xiv LIST OF FIGURES FIGURE 2.1. Structure of ?-D-Tagatose ........................................................................... 4 FIGURE 3.1. Sample HPLC Chromatogram for the Analysis of Tagatose in a 0.1 M Phosphate Buffer Solution at pH 3 containing 0.05 M Glycine. Tagatose Elutes at Approximately 7 minutes ............................ 35 FIGURE 4.1. Tagatose Loss in 0.02 M Phosphate Buffer at pH 7 and 60 C 40 FIGURE 4.2. Pseudo-First-Order Plot of Tagatose Loss in 0.02 M Phosphate Buffer at pH 7 and 60 C .......................................................................... 40 FIGURE 4.3. Tagatose Loss in 0.1 M Buffer Solutions at 80 C as Affected by pH .................................................................................... 42 FIGURE 4.4. Tagatose Loss in Solution at pH 7 and 80 C as Affected by Buffer Type and Concentration .......................................................... 46 FIGURE 4.5. Tagatose Loss in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature ..................................................................... 48 FIGURE 4.6. Pseudo-First-Order Plot of Tagatose Loss in 0.02 M Phosphate Buffer with 0.05 M Glycine at pH 7 and 70?C ........................................ 51 FIGURE 4.7. Tagatose Loss in 0.1 M Buffer Solutions Containing Tagatose and Glycine at 80 C as Affected by pH ................................................... 54 FIGURE 4.8. Tagatose Loss in Solution Containing Tagatose and Glycine at pH 7 and 80 C as Affected by Buffer Type and Concentration .......... 54 FIGURE 4.9. Tagatose Loss in 0.1 M Citrate Buffer Solutions Containing Tagatose and Glycine at pH 7 as Affected by Temperature .................... 56 FIGURE 4.10. Browning in 0.1 M Phosphate Buffer Containing Tagatose and Glycine at pH 7 and 80?C ................................................................. 58 FIGURE 4.11. Brown Pigment Formation in 0.1 M Buffer Solutions Containing Tagatose at 80?C ................................................................... 59 xv FIGURE 4.12. Brown Pigment Formation in 0.1 M Buffer Solutions Containing Tagatose and Glycine at 80?C ............................................... 59 FIGURE 4.13. Brown Pigment Formation in 0.02 M Phosphate Buffer at pH 7 and 80?C ...................................................................................... 64 1 CHAPTER 1: INTRODUCTION Tagatose is an emerging sweetener with many desirable attributes that may lend it to possible applications, such as a sugar substitute or use as a dietary supplement. Tagatose is a low-calorie sweetener with a level of sweetness similar to that of sucrose. It is found naturally in some dairy products, but is commercially manufactured from lactose (Oh 2007). Tagatose is classified as a monosaccharide, with a structure similar to that of fructose, only differing in the position of the hydroxyl group on the fourth carbon. Because tagatose is only minimally absorbed in the upper gastrointestinal tract, the majority of it travels to the colon where it is fermented by indigenous microflora. Carbohydrate fermentation in the intestinal tract leads to the production of short chain fatty acids (SCFAs), carbon dioxide, hydrogen, and methane. SCFAs are believed to be important because of their role in the welfare of the colonic mucosa. Due to the fact that tagatose is fermented to provide SCFAs and provides minimal to no calories, it is classified as a prebiotic. Along with the prebiotic benefit of tagatose, it does not promote dental caries (Arla Foods 2002), and it does not significantly increase blood glucose or insulin levels (Donner and others 1999). Tagatose can be used in various products as a bulk sweetener, humectant, texturizer, stabilizer, and it may act as a flavor enhancer in products like mint and lemon flavored chewing gum and mints (Calorie Control Council 2007; Rulis 2001; Skytte 2006). In October 2001, the FDA agreed with Arla?s findings that tagatose is Generally 2 Recognized as Safe (GRAS) when used in specific beverages and food products up to certain maximum allowable levels (Rulis 2001; Skytte 2006). Outside of the United States, many other countries have also approved tagatose as a food ingredient (Skytte 2006), leading to the addition of tagatose into various food products (Damhert 2008). While the properties and behavior related to the use of tagatose as a food ingredient have been studied quite thoroughly, other aspects remain relatively unexplored. Tagatose appears to have many of the attributes that would allow it to be a successful substitute for commonly used sugars, specifically sucrose. However, there are still properties of tagatose that have not been studied thoroughly. The stability of tagatose under thermal processing conditions and the ability of tagatose to cause browning have only undergone minimal evaluation (Ryu and others 2003). Because tagatose must be present in food to offer any prebiotic benefit and foods are generally processed at high temperatures, studying high temperature stability is important. Browning is desirable in some applications, while undesirable in others, so determining the relationship between tagatose and browning is vital in the production of high quality products containing tagatose. The objective of this study is to determine the effect of pH, temperature, buffer concentration, and buffer type on the thermal stability of tagatose. The extent of browning will also be evaluated. 3 CHAPTER 2: LITERATURE REVIEW In a world where trends and ideals are constantly changing, the food industry is expected to develop and discover new ingredients and food products to keep up with the current demands of today?s society. It is apparent through television commercials, magazine articles, and food labels that consumers are becoming more health-conscious. As the link between diet and disease becomes more prominent, consumers have become increasingly concerned about what they are consuming and the effects it may have on their health. One area that has been receiving a lot of attention lately is nutraceuticals. Nutraceuticals are, ?foods, or parts of foods, that provide medical or health benefits, including the prevention and treatment of disease? (National Nutraceutical Center 2005). Categories of nutraceuticals include dietary supplements (vitamins, minerals, ginseng, gingko biloba) and functional foods. Functional foods are foods that exert a specific beneficial effect on health beyond their basic nutritional properties. They include foods such as tomatoes (contains lycopene which contributes to prostate health), wheat bran (contains insoluble fiber which helps maintain a healthy digestive tract), and foods classified as prebiotics (IFIC 2006). Prebiotics are beneficial to the body since they selectively stimulate the favorable growth or activity of certain indigenous bacteria (Reid and others 2003). Tagatose, a relatively new sweetener, has been shown to display prebiotic characteristics (Bertelsen and others 1999). 4 Chemistry and Properties of Tagatose Tagatose is a monosaccharide that has a structure similar to that of fructose, only differing in the position of the hydroxyl group on the fourth carbon. Acyclic D-fructose is oriented with a hydroxyl group on the right side of the fourth carbon, whereas this hydroxyl group is on the left side of the fourth carbon on acyclic D-tagatose. Figure 2.1 shows the ?-pyranose form of tagatose. This slight difference in structure only allows tagatose to be absorbed minimally in the upper gastrointestinal tract, resulting in a caloric value of less than 1.5 kcal/g (Levin 2002; Levin and others 1995; Livesey and Brown 1996). The tagatose that is not absorbed in the upper gastrointestinal tract travels to the colon where it is fermented to produce short chain fatty acids (SCFAs) including propionate and butyrate (Bertelsen and others 1999; Bertelsen and others 2001; Laerke and Jensen 1999; Laerke and others 2000; Venema and others 2005). Tagatose is therefore classified as a prebiotic, which makes it a very attractive food ingredient for companies looking to boost the overall health content of their product. O O H C H 2 O HO H O H O H Figure 2.1. Structure of -D-Tagatose Although tagatose has a structure similar to fructose, its taste is comparable to that of sucrose, with no cooling effect, aftertaste, or off-flavors (Levin and others 1995). A standard taste panel test on 10% aqueous solutions of tagatose showed it to be 92% as 5 sweet as sucrose (Levin and others 1995). With a sweetness level and bulking ability similar to that of sucrose, tagatose may also have potential as a low calorie substitute for sucrose. Research has also found that tagatose blunts the rise in blood glucose and insulin typically observed after glucose or sucrose loading, and it produces exceptionally low glycemic and insulinemic responses, which links tagatose to possibly being beneficial in products geared towards individuals with diabetes (Gaio 2003; Lu and others 2008). Even though tagatose is a relatively new sweetener, a moderate amount of research has been done to evaluate the properties and applications of this monosaccharide. A look at numerous past studies on tagatose will provide a better understanding of this sugar. Tagatose Production While tagatose is found naturally in a variety of dairy products including sterilized cow?s milk, hot cocoa, powdered cow?s milk, and certain yogurts, the amount of tagatose in these products is so minimal that for economic purposes it is must be manufactured by chemical or biological methods (Levin and others 1995; Oh 2007). Using the chemical method, tagatose is produced from lactose through a two-step process, where initially lactose is split into glucose and galactose; the galactose is then isomerized to create tagatose by adding calcium hydroxide (Beadle and others 1992). The biological method of tagatose production involves converting galactose to tagatose using an immobilized L-arabinose isomerase enzyme (Oh 2007). Upon completion of either the chemical or biological method, tagatose appears as a white, odorless, crystalline product. 6 Digestion and Absorption Numerous studies, mainly done on animals, led researchers to determine that only about 15-20% of tagatose is absorbed in the upper gastrointestinal tract (Laerke and Jensen 1999; Saunders and others 1999b). One study that helped researchers come to this conclusion involved 11 rats (Saunders and others 1999b). For this study, each of the rats was treated with two of the following treatment variations: conventional (normal levels of gut microflora), germ-free (no gut microflora), adapted (fed a diet containing tagatose for 28 days prior to the study), or unadapted to tagatose. Out of the 11 rats involved in the study, four were adapted conventional, three were unadapted conventional, two were unadapted germ-free, and all three groups were all dosed orally. The final two rats were unadapted conventional, and were dosed intravenously. All of the rats received a single dose (220-380 kBq) of tagatose before being placed in a metabolism chamber where samples of carbon dioxide, urine, and feces were taken at regular intervals. Analysis of the results concluded that the intestinal absorption of tagatose in the rat was approximately 20%. Since carbon dioxide is one of the products of carbohydrate fermentation, and the percentage of carbon dioxide produced by the conventional rats was greater than the carbon dioxide produced by the germ-free rats, the researchers were able to conclude that the tagatose was being fermented by bacteria in the large intestine. Also, there was 93% less tagatose in the feces of the adapted conventional rats, compared to the rats that were unadapted conventional, demonstrating the importance of adaptation in the microflora?s ability to ferment tagatose (Saunders and others 1999b). The low digestibility of tagatose in the small intestine was also observed in two similar studies done on pigs (Laerke and Jensen 1999; Laerke and others 2000). Both 7 studies followed the same method, while focusing on different variables including tagatose absorption, adaptation, and SCFA production. In both studies, eight pigs were fed a low fiber diet with 15% of the caloric value of the diet coming from sucrose. A second group of eight pigs was fed a similar diet to the first group of pigs except that a majority of the sucrose was replaced with tagatose. Also, a portion of the pigs in the second group underwent a two-day adaptation period to tagatose prior to the study. The pigs were fed their respective diets for 18 days, and on the 18th day they were killed, and their gastrointestinal contents were analyzed. In the studies, it was found that tagatose was digested and absorbed very minimally in the small intestine of pigs (Laerke and Jensen 1999), and no microbial fermentation of tagatose took place in the stomach or small intestine (Laerke and others 2000), but instead took place in the cecum and the colon (Laerke and Jensen 1999; Laerke and others 2000). The fermentation of tagatose led to the production of many beneficial SCFAs, in particular, formate, acetate, propionate, butyrate, valerate, and caproate (Laerke and Jensen 1999; Laerke and others 2000). The amount of microbial fermentation of tagatose in the cecum and colon was dependent on the amount of tagatose in the diet (Laerke and others 2000). The rate of microbial fermentation of tagatose was much higher in the pigs that were adapted to tagatose before starting the study (Laerke and Jensen 1999; Laerke and others 2000). The colon of the adapted pigs was shown to produce proportions of butyric and valeric acid that were two to three times higher than those seen in the colon of unadapted pigs (Laerke and others 2000). A study done on the small intestine absorption of tagatose in ileostomy subjects resulted in conflicting findings to the other results on tagatose absorption in the small 8 intestine (Normen and others 2001). In this study, six individuals, who had well- functioning ileostomies with less than 10 cm of their terminal ileum removed, were studied to determine the absorption of tagatose in the small intestine. The subjects in the study were fed a specific diet for two periods of two days during three consecutive weeks. In one of the periods, 15 g of tagatose were added to the daily diet of the subjects. Ileostomy effluents were freeze-dried and analyzed to determine the tagatose absorption. Contrary to other studies (Laerke and Jensen 1999; Laerke and others 2000), it was found that 81% of tagatose was absorbed in the small intestines of the subjects in this study. While the results in this study differ dramatically from the results of the previous two studies, fermentation of tagatose by the indigenous microflora, which was not analyzed in this study, cannot be ruled out as a factor for such high absorption values (Normen and others 2001). Since tagatose is only absorbed minimally in the small intestine, the majority of it enters the colon where it is fermented by saccharolytic bacteria, producing SCFAs, carbon dioxide, methane, and hydrogen. SCFAs have been associated with the reduced risk of some diseases, including irritable bowel syndrome, inflammatory bowel disease, cardiovascular disease, and cancer (Hijova and Chmelarova 2007). Upon absorption of the SCFAs in the cecum and colon, they are mainly metabolized in three sites in the body. One site in the body where SCFAs can be metabolized is the cells of the ceco- colonic epithelium. This site uses butyrate as a major substrate for the maintenance of energy producing pathways. A second site where metabolism of SCFAs occurs is in the liver cells, where butyrate and propionate are used for gluconeogenesis. Finally, the muscle cells are able to metabolize acetate and generate energy from its oxidation 9 (Hijova and Chmelarova 2007). Roles of SCFAs include being modulators of colonic and intracellular pH, cell volume, and other functions associated with ion transport. They are also nutrients for the colonic epithelium, and they act as regulators of proliferation, differentiation, and gene expression (Hijova and Chmelarova 2007). SCFAs have many important functions throughout the body, and tagatose is beneficial to the body since it promotes the production of these SCFAs. Various studies have been conducted in order to determine the specific SCFAs that are produced through the fermentation of tagatose (Bertelsen and others 1999; Bertelsen and others 2001; Laerke and Jensen 1999; Laerke and others 2000; Venema and others 2005). In one study (Bertelsen and others 1999), a group of eight pigs was fed a standard diet plus 15% sucrose (unadapted pigs), while a second group of eight pigs was fed a standard diet plus 5% sucrose and 10% tagatose (adapted pigs). All 16 pigs were fed their specified diets for a total of 17 days, and on the 17th day the pigs were killed 3 hours after their morning feed, and their entire gastrointestinal tract was sectioned. Only contents from the mid-colon were used for in vitro fermentation assays. The contents of the intestinal tract were diluted with water to create 20% slurries. After the slurries were incubated at 37 C under anaerobic conditions for four hours, with or without 1% tagatose added, it was found that the colon contents from the adapted pigs fermented tagatose and produced a low acetate, high butyrate, high valerate environment. This differed from the unadapted pigs whose colon contents produced a SCFA profile that was more normal, containing 20% less butyrate and a higher level of acetate (Bertelsen and others 1999). 10 Bertelsen and others (1999) had another part to their study. In this study, there were three groups containing two pigs each; the first group of pigs received a standard diet plus 20% sucrose, the second group of pigs received a standard diet plus 10% sucrose and 10% tagatose, while the third group of pigs was fed a standard diet plus 20% tagatose for a total of 33 days. Six hours after their morning feed on the 33rd day, the pigs were killed and their entire gastrointestinal tract was divided into eight segments and each segment was analyzed for the concentrations of various SCFAs. Contents from each of the eight segments were also used for 12 hour in vitro incubations at 37 C under anaerobic conditions. The researchers found that at the time of slaughtering, butyrate was only present in the hind gut, and that the more tagatose the pigs were fed, the more butyrate was found in the hind gut. After 12 hours, it was also found that there was a direct relationship between tagatose consumption and butyrate production. The more tagatose the pigs were fed, the more butyrate was produced in the colon. Acetate production was found to decrease in the gastrointestinal tract of the pigs that consumed tagatose, with more of a reduction occurring in the pigs that consumed larger amounts of tagatose (Bertelsen and others 1999). After the in vitro studies done on pigs resulted in findings that were very similar, the researchers looked at the in vivo absorption of SCFAs in the blood of pigs (Bertelsen and others 1999). Blood samples were taken from the same five pigs, 12 hours after morning feeding, on three different occasions. The first blood sample was taken after seven days of adaptation to a standard pig diet plus 20% sucrose. The second blood sample was taken on the first day the pigs were switched to a standard pig diet plus 20% tagatose (unadapted pigs). The third and final blood sample was taken after the pigs had 11 adapted to a pig diet plus 20% tagatose for seven days (adapted pigs). In order to determine the intestinal absorption of SCFAs, the researchers measured the difference between the SCFA content in the samples taken from blood found in the portal vein and those taken from the arterial blood. The results from this study showed that when the pigs were on the 20% sucrose diet, the butyrate concentrations were very low. The butyrate concentrations increased slightly when the pigs first began the 20% tagatose diet, but increased dramatically after the pigs had been on the 20% tagatose diet for seven days. Intestinal absorption of acetate and propionate did not increase when tagatose was added to the diet of the pigs (Bertelsen and others 1999). Pigs were used in the previous studies because their digestive tracts are similar to those of humans, and they both have qualitatively the same types of indigenous intestinal bacteria. In order to get a better idea of how tagatose reacts in humans, in vitro studies were carried out where tagatose was added to human faecal slurries (Bertelsen and others 1999). In this study, there were 16 human volunteers who provided the researchers with faecal samples before consuming tagatose, as well as 14 days after consuming 10 g of tagatose three times a day. The study participants maintained a controlled diet for four days before each faecal sample was taken. The faecal slurries were incubated at 37 C under anaerobic conditions for either 4 or 48 hours. The faecal slurries analyzed after 4 hours showed that the samples taken after the humans had consumed tagatose in their diet for 14 days contained much higher amounts of total SCFAs and butyrate than the samples taken before tagatose consumption. After 48 hours, there was no longer any difference in the amount of SCFAs produced, and the butyrate production was high in both the samples taken before tagatose consumption as well as those taken after tagatose 12 consumption. Also, the number of lactic acid bacteria were significantly higher in the faecal samples collected from humans who had consumed tagatose for 14 days, compared to the faecal samples taken from humans before tagatose consumption (Bertelsen and others 1999). A second study on humans also looked at how tagatose consumption affected the production of SCFAs in the colon (Venema and others 2005). In the in vivo part of this study, 12 men and 18 women consumed 30 g of raspberry jam containing either 7.5 g or 12.5 g tagatose, 7.8 g fructooligosaccharides, 7.6 g tagatose plus 7.5 g fructooligosaccharides, or 15.1 g sucrose at breakfast for two weeks each, in varying orders. After each two-week period, test-tube incubations of faecal slurries were processed for microbiological analyses within two hours after voiding. There was also an in vitro part to the study that involved a model simulating the large intestine to observe the mechanistic effect of tagatose on SCFA production. Results from both the in vitro study and the in vivo study indicated that there was an increase in butyrate production after all treatments with tagatose (Venema and others 2005). In another study, 45 isolates of normal or pathogenic human enteric bacteria were chosen for fermentation of tagatose in vitro (Bertelsen and others 2001). Also, 107 lactic acid bacteria strains were used for fermentation of tagatose in vitro. All samples were incubated for 48 hours in order to determine which intestinal bacteria were able to ferment tagatose. It was found that the fermentation of tagatose is not very widespread among the different genera of human enteric bacteria. Only five of the 45 human enteric bacteria studied were able to ferment tagatose. The five human enteric bacteria that were able to ferment tagatose include two strains of Lactobacillus, two strains of Clostridium, 13 and Enterococcus faecalis. A majority of the lactic acid bacteria showed tagatose fermentation, with strong fermentation in 61 strains, and in 38 of these strains the fermentation of tagatose was very extensive (Bertelsen and others 2001). From the research, it was determined that only about 15-20% of tagatose is absorbed in the upper gastrointestinal tract (Laerke and Jensen 1999; Saunders and others 1999b). The remaining tagatose makes its way into the colon where it is able to undergo fermentation by indigenous microflora, in particular strains of Lactobacillus, Clostridium, and Enterococcus faecalis (Bertelsen and others 2001; Laerke and Jensen 1999; Laerke and others 2000). The fermentation of tagatose leads to the production of SCFAs, specifically butyrate and valerate, which are beneficial to our colonic health (Bertelsen and others 1999; Bertelsen and others 2001; Laerke and Jensen 1999; Laerke and others 2000; Venema and others 2005). Caloric Value Due to the minimal absorption of tagatose in the small intestine, researchers have found tagatose offers the body fewer calories than typical carbohydrates, including sucrose (Livesey and Brown 1996). Tagatose is estimated to provide less than 1.5 kcal/g, whereas sucrose provides 4 kcal/g. In one study (Livesey and Brown 1996), two groups of 30 rats were fed a diet containing either sucrose or tagatose for 21 days prior to the study in order to allow for an adaptation period to the sugar, either sucrose or tagatose. After the 21 day adaptation period, the 30 rats from each group were broken into three groups containing ten rats each. One group immediately underwent body composition analysis, the second group had the test (or reference) carbohydrate removed from their diets for about 6 weeks, and the third group continued to receive a daily diet 14 supplemented with 1.8 g of the test carbohydrate, either sucrose or tagatose, for about 6 weeks. The rats that consumed the diet supplemented with sucrose showed an increase in growth, protein, and lipid deposition, while the rats consuming a diet supplemented with tagatose did not show any measurable changes in those three parameters. Based on the results, the researchers were able to calculate that tagatose contributed -3 14% of its heat of combustion to net metabolizable energy, therefore tagatose effectively had a zero energy value (Livesey and Brown 1996). Arla Foods, the company that distributes tagatose, used a different technique to determine the caloric value of tagatose (Levin 2002). Using a factoral method, they estimated that the metabolizable energy provided by tagatose ranged from 1.1 to 1.4 kcal/g. Depending on the experimental procedure, the interpretation of results, and the factors applied, it could be even lower (Levin 2002). Because tagatose provides the body with few to no calories, one would expect weight loss to occur in subjects consuming a diet where a portion of the sucrose is substituted with tagatose. Levin and others (1995) conducted a study where rats in groups of six each were fed a standard diet plus 15% tagatose for an adaptation period of 14 days. After those 14 days one group of rats continued with the previous diet, while the other group of rats was fed a standard diet plus sucrose at an equal weight basis as the tagatose fed to the first group of rats. After undergoing a 60 day comparison between the tagatose-fed rats and the sucrose-fed rats, it was found that the sucrose-fed rats showed a greater than 7% weight gain than the tagatose-fed rats (Levin and others 1995). A second study looked at the effect of tagatose on food intake of human subjects (Buemann and others 2000c). In this study, the food intake patterns of 19 normal-weight 15 men were studied after 29 g of tagatose or 29 g of sucrose were substituted into their breakfast meal. Following the consumption of either tagatose or sucrose at breakfast, the food intake of the study subjects was evaluated at lunch, afternoon snack, and supper of the same day. Researchers found that the energy intake at lunch and afternoon snack was comparable after intake of the two sugars, but at supper the food intake was 15% lower after consuming tagatose at breakfast (Buemann and others 2000c). While there are a lot of factors that can influence an individual?s food intake, this study does bring up the possibility of tagatose acting as some type of appetite suppressant. Tolerance Tagatose provides only a minimal amount of calories since it is not fully digested or absorbed in the small intestine, which leads to concerns about tolerance levels and possible negative side effects from the overconsumption of this sugar. Incomplete absorption of nutrients has been linked to gastrointestinal discomforts such as cramping, bloating, flatulence, and diarrhea. To determine how humans react to high levels of tagatose consumption, Lee and Story (1999) studied how the gastrointestinal tract of 50 subjects responded to 40 g of chocolate containing 20 g sucrose, lactitol, or tagatose. It was found that lactitol consumption was linked to a significant increase in the frequency of passing feces and the number of subjects passing watery feces, whereas tagatose consumption did not provoke either of these side effects. Tagatose consumption and lactitol consumption both resulted in significant increases in colic, flatulence, borborygmi (rumbling sounds caused by gas moving through the intestines), and bloating, but a majority of these symptoms were described as only ?slightly more than usual? by the study participants. Also, a significant number of subjects reported nausea after 16 consuming the chocolate containing tagatose. While minor side effects were seen after tagatose consumption, in comparison to lactitol, it was found that 20 g of tagatose was well tolerated among the subjects (Lee and Storey 1999). In a second study done on human gastrointestinal tolerance to tagatose, 73 healthy male subjects consumed cake containing 30 g of tagatose (Buemann and others 1999). Following tagatose consumption, the study participants reported any side effects using a questionnaire which explicitly referred to common symptoms such as heartburn, nausea, vomiting, stomach ache, flatulence, and diarrhea. Nausea was reported in 15.1% of the subjects, and 31.5% of the subjects suffered from diarrhea after consuming tagatose. An increase in flatulence was commonly reported and did not decline over a 15 day period where tagatose was consumed in one 30 g dose daily. While the symptoms were generally mild or moderate, this study showed that consuming 30 g of tagatose at one time may be more than the recommended limit (Buemann and others 1999). Tagatose as a Prebiotic Prebiotics are defined as ?nondigestible substances that provide a beneficial physiological effect on the host by selectively stimulating the favorable growth or activity of a limited number of indigenous bacteria? (Reid and others 2003). Prebiotics differ from probiotics, because probiotics are ?live microorganisms which, when administered in adequate amounts confer a health benefit on the host? (Reid and others 2003). As discussed earlier, the majority of tagatose consumed is not absorbed in the small intestine, thus allowing a substantial portion of tagatose to enter the large intestine where it increases the production of certain beneficial bacteria, ultimately leading to an increase in SCFA production. These qualities allow tagatose to be classified as a prebiotic. 17 Besides tagatose, other common carbohydrate sources of prebiotics include fructooligosaccharides, inulin, galactooligosaccharides, and lactulose. Prebiotics offer the body many benefits, including the ability to strengthen the immune system, inhibit the growth of harmful bacteria in the intestines, reduce the risk for colon cancer, and aid in digestion and laxation (Kraft 2007). While it is known that prebiotics are beneficial to the body, the amount required to gain the beneficial effects has not yet been determined. Based on studies mainly done on fructooligosaccharides, inulin, galactooligosaccharides, and lactulose (Macfarlane and others 2006), experts estimate an intake of 5-15 g of prebiotics daily is needed to see beneficial effects (Reid and others 2003; Tuohy and others 2005). More than 15 g/day may lead to undesirable side effects such as flatulence, bloating, and abdominal cramps (Kraft 2007; Saavedra and Tschernia 2002). Because tagatose is also a carbohydrate- based prebiotic, it may behave physiologically in a similar fashion to fructooligosaccharides, inulin, lactulose, and galactooligosaccharides. Health Effects of Tagatose Diabetes With carbohydrates being the main nutrient of concern when referring to individuals with diabetes and because diabetes is very prevalent in today?s society, tagatose was tested in order to determine how its consumption affected blood glucose levels (Donner and others 1999; Gaio 2003; Lu and others 2008). The glycemic and insulinemic responses of tagatose were tested by Sydney University?s Glycaemic Index Research Service (SUGiRS) (Gaio 2003). After 12 people consumed 50 g portions of tagatose and glucose dissolved in 200 mL of water, on different mornings, changes in 18 their blood glucose levels and insulin levels were measured over a two-hour period. While glucose had glycemic and insulinemic responses of 100%, tagatose only produced glycemic and insulinemic responses of 3% (Gaio 2003). In a second study, the glycemic effects of oral tagatose alone or in combination with oral glucose were studied in humans with and without type 2 diabetes mellitus (Donner and others 1999). This study was divided into two parts. In the first part of the study, there were eight participants with type 2 diabetes mellitus and eight participants without diabetes. On separate occasions, the participants consumed 75 g glucose, 75 g tagatose, or 75 g tagatose 30 minutes prior to a 75 g glucose load. Between test days, the participants received 10 g tagatose daily. In the second part of this study, ten participants with diabetes mellitus received separate 0, 10, 15, 20, and 30 g of tagatose prior to a 75 g oral glucose tolerance test. From this study, it was concluded that oral tagatose can significantly blunt the rise in blood glucose typically seen after oral glucose consumption in patients with diabetes mellitus. The ability of tagatose to reduce the rise in blood glucose occurs in a dose-dependant manner without significantly affecting insulin levels (Donner and others 1999). Plasma Uric Acid Levels Although only a small amount of tagatose is absorbed in the body, the part that is absorbed follows a metabolic pathway in the liver similar to that of fructose. The similarities in the way tagatose and fructose are metabolized concerned researchers because high oral doses of fructose are linked to increased uric acid levels in the blood, otherwise known as hyperuricemia. Individuals who suffer from fructose intolerance syndrome, which is a hereditary genetic defect in the aldolase B enzyme responsible for 19 cleaving fructose-1-phosphate into D-glyceraldehyde and dihydroxyacetone phosphate, experience hypoglycemia and hyperuricemia after the consumption of moderate amounts of fructose (Buemann and others 2000a). Even individuals without fructose intolerance syndrome may experience a slight hyperuricemic effect after large oral doses of fructose (Buemann and others 2000a). The possibility of increased uric acid in blood due to the consumption of tagatose can be understood by following the metabolic pathway of tagatose in the liver. Tagatose is initially phosphorylated to D-tagatose-1-phosphate by the enzyme fructokinase. At this point, the enzyme aldolase cleaves D-tagatose-1-phosphate to D-glyceraldehyde and dihydroxyacetone phosphate. Since D-tagatose-1-phosphate is cleaved at a slower rate than fructose-1-phosphate, Pi (inorganic phosphate) is complexed by tagatose for a longer time than by fructose, which is likely to cause a reduction in hepatic Pi (Saunders and others 1999a). Since Pi is an inhibitor of adenosine deaminase, a rate-limiting enzyme in the degradation of adenosine monophosphate (AMP), a reduction of Pi in the liver may lead to increased degradation of purine nucleotides, which can result in a greater amount of uric acid being released from the liver (Saunders and others 1999a). Since the metabolism of tagatose could potentially lead to increased uric acid levels, studies were conducted to observe how varying levels of tagatose would affect the uric acid levels in the blood (Buemann and others 2000a; Buemann and others 2000b; Saunders and others 1999a). In one study, eight normal human subjects and eight subjects with type 2 diabetes mellitus were adapted to tagatose for three days (given 5, 10, and 25 g single doses of tagatose for three consecutive days), and then on the fourth day they were given a 75 g, 20 3-hour oral tagatose tolerance test (Saunders and others 1999a). The results from this portion of the test revealed that for both the normal and diabetic subjects, plasma uric acid levels on average peaked 60 minutes into the tagatose tolerance test, although these increases were not statistically significant (Saunders and others 1999a). In the second part of this study, the eight normal subjects were randomly selected to receive either 75 g tagatose or 75 g sucrose daily for eight weeks. The eight diabetic subjects were given either 75 g tagatose or no sugar supplementation for eight weeks. The researchers found that no significant changes occurred in the fasting plasma uric acid levels within each treatment group over the period of the study (Saunders and others 1999a). A second study compared the differences between how serum uric acid concentrations were affected by either 30 g of tagatose or 30 g fructose (Buemann and others 2000b). The uric acid concentrations of the eight male subjects that participated in the study were observed for seven hours after being administered either 30 g of tagatose or 30 g of fructose orally. Four hours and fifteen minutes after the respective sugar was consumed, the test subjects ingested a fixed lunch in attempt to observe any possible influence tagatose may have on the normal glucose and insulin responses seen during a typical meal. It was found that both the peak concentration and 4-hour area under the curve of serum uric acid were significantly higher after tagatose consumption than they were with either 30 g of fructose or plain water. Also, it was found that tagatose lessened the glycemic and insulinemic responses caused by the meal that was consumed (Buemann and others 2000b). A third study on this topic attempted to clarify if the acutely increased blood concentration and renal excretion of uric acid can be associated with an accumulation of 21 D-tagatose-1-phosphate and alterations in ATP in the liver of normal human subjects (Buemann and others 2000a). For this study, five male subjects consumed 30 g tagatose or fructose orally, and were then studied by 31P-magnetic resonance spectroscopy (31PMRS). Blood and urine samples were also taken to observe any increase in uric acid production. Roughly 30 minutes after the tagatose had been administered to the subjects, a peak assigned as D-tagatose-1-phosphate equivalent to roughly 1 mmol/L was apparent on the spectrum for each one of the individuals, and it took about two hours for the baseline to return to normal. Also, around 30 minutes after tagatose consumption, ATP was reduced by roughly 12%. Fifty minutes after tagatose consumption, serum uric acid concentrations increased by 17%, and did not return to the baseline level for the entirety of the experiment (230 minutes after the load). There were no changes in the 31PMRS spectra or serum uric acid concentrations after fructose consumption, leading to the conclusion that moderate tagatose consumption may affect liver metabolism (Buemann and others 2000a). Tagatose in Food In 2001, the United States Food and Drug Administration (FDA) agreed with Arla Food Ingredients, and found tagatose to be Generally Recognized as Safe (GRAS) (Rulis 2001). Maximum levels of tagatose allowed in specific products were outlined by the FDA: 1% in carbonated beverages, 1% in ready-to-drink teas presweetened with low calorie sweeteners, 60% in chewing gum, 30% in icing or glazes used on baked goods, 15% in hard candies, 10% in dietetic soft candies, 3 grams per serving in ready-to-eat cereals, 5 grams per serving in powdered products prepared with milk, 10% in low fat, reduced fat, diet, energy, or nutrient fortified bars, and 3% in light ice cream, frozen milk 22 desserts, low-fat and non-fat frozen yogurt and related frozen novelties (Rulis 2001). Tagatose has also been approved in Australia, New Zealand, and Korea without a specified acceptable daily intake (ADI) (Skytte 2006). In 2005, Brazil and South Africa approved tagatose as a food ingredient and the European Union approved tagatose as a Novel Food Ingredient (Skytte 2006). Tagatose approval is underway in Canada, Japan, and Mexico (IFT 2005; Skytte 2006). After a thorough evaluation process by the Joint FAO/WHO Expert Committee on Food Additives, tagatose was found to be safe as a food ingredient, and due to only minor negative side effects being associated with tagatose consumption, the committee did not specify an ADI for the sugar (WHO 2005). Although the approval of tagatose by numerous regulatory committees has become fairly widespread throughout the world, the addition of tagatose to food products has been fairly small. Nutrilab, a subsidiary of the Belgian company Damhert, has recently begun using tagatose in chocolate, spreads, cookies, and jams (Damhert 2008). In addition, they are selling tagatose as a home sugar replacer under the name Tagatesse (Damhert 2008). Other products containing tagatose are 7-Eleven?s Diet Pepsi Slurpee, Miada Chocolite , Pasco Light and Tasty Juice, Shugr by Swiss Diet, SweetFiber by Dr. Murray Natural Living, and Therasweet by Living Fuel (Wise 2008). Along with the introduction of tagatose into foods found on the retail market, researchers have begun replacing a portion of sucrose with tagatose in bakery products to determine consumer likeability of the products. Two of these studies were conducted by Taylor and others (2008) and Armstrong and others (2009). 23 In the first study, a cookie recipe was formulated with various levels of sucrose replaced by tagatose to determine the physical properties and consumer likeability of cookies containing tagatose (Taylor and others 2008). The amount of tagatose substituted for sucrose ranged from 25 to 100% in the cookies. When compared to the control cookies (made with 100% sucrose), it was found that the cookies containing tagatose were harder, darker in color, and had less spread. Evaluation of the cookies by 53 untrained panelists showed the panelists liked the color of the 100% tagatose cookies better than the color of the control cookies, but did not like the sweetness of the 100% tagatose cookies. When only half of the sucrose was replaced by tagatose, the overall likeness scores were the same between the experimental cookies and the control cookies. Not only were the physical properties acceptable in cookies where tagatose partially replaced sucrose, but the cookies containing 50% tagatose were liked by panelists (Taylor and others 2008). A second study looked at adding tagatose to cinnamon muffins, lemon cookies, and chocolate cakes, at levels of 1 and 2%, to study the effect of tagatose on their flavors, and to evaluate consumer likeability of the baked goods (Armstrong and others 2009). In this study, each of the three baked goods mentioned above were prepared twice, once with 1% tagatose and once with 2% tagatose, and compared to products made without tagatose. The products were evaluated by untrained panelists, using triangle tests to study the flavor differences and a hedonic scale to measure how much the panelists liked the products. Upon the conclusion of the experiment, it was found that the panelists were unable to differentiate between baked goods made with and without tagatose, and the mean likeness scores were not significantly different between baked goods made with or 24 without tagatose. The results show the ability for low levels of tagatose to be added to bakery products without affecting their flavor (Armstrong and others 2009). Tagatose Stability Because tagatose is a reducing sugar, it is able to participate in the Maillard reaction in the presence of an amino acid or protein. The Maillard reaction is a type of non-enzymatic browning that occurs between the carbonyl group of an acyclic reducing sugar and the unprotonated amine group, found on either an amino acid or a protein. These complex series of reactions are responsible for the formation of a brown color, as well as changes in flavor and loss of nutritional value (Brands and others 2000). While a large portion of the Maillard reaction is not clearly understood, it is generally accepted that upon interaction between the reducing sugar and the unprotonated amine, a glycosylamine is formed, which then undergoes the Amadori rearrangement to produce a 1-amino-2-keto sugar. This product then participates in a series of reactions to ultimately produce melanoidin pigments, which are responsible for the brown appearance of foods (Daniel and others 2007). After participating in the Maillard reaction, the amino acid is no longer available as a nutrient, causing the product to lose some of its nutritional value (Daniel and others 2007). There are many factors that influence the Maillard reaction, including temperature, pH, and reactant concentration (Brands and others 2002). While the Maillard reaction generally occurs at higher temperatures associated with thermal processing conditions (60-80?C), it has even been found to occur in products stored at room temperature. Also, the Maillard reaction occurs more rapidly at higher pH values, where more unprotonated amines are available. The reaction rate is also directly 25 proportional to the reactant concentration. As the reactant concentration increases, the Maillard reaction generally increases since more of the reactant is available to participate in the reaction, and there is a greater likelihood that the reactive form of the sugar or amino acid will be present (Daniel and others 2007). While the browning and flavors caused by the Maillard reaction are desirable in some products, such as baked bread and roasted peanuts, in other products, like dried milk, the reaction is undesirable. Understanding the Maillard reaction and the factors that affect it is important when studying the reactivity of a reducing sugar, such as tagatose. Besides the Maillard reaction, monosaccharides are prone to degradation reactions influenced by the pH of the solution in which they are held. Under alkaline conditions, monosaccharides undergo a series of rearrangements, including ionization, mutarotation, enolization, and isomerization to form an enediol anion species. After undergoing several other reactions, the enediol anion species is ultimately degraded into carboxylic acids (De Bruijn and others 1986). The formation of carboxylic acids causes the pH of the solution to decrease, ultimately slowing down monosaccharide degradation reactions that had been taking place under the initial alkaline conditions. Under acidic conditions, monosaccharides undergo a set of degradation reactions that differ from the reactions that are seen in alkaline solutions. When heated in a strongly acidic solution, dehydration occurs and furfural compounds are formed (Wong 1989). In dilute acid solutions, aldohexoses lose a water molecule to form anhydro products, most commonly the 1,6-anhydro sugar (Wong 1989). Although through different reaction pathways, both aqueous alkaline and acidic solutions can cause the degradation of monosaccharides. Since a large portion of tagatose needs to remain intact 26 in order to provide the prebiotic benefit, studying the stability of tagatose at different pH values under typical processing temperatures is critical. Ryu and others (2003) conducted a study to observe the effects of pH and temperature on the non-enzymatic browning reaction between tagatose and glycine in aqueous solutions. To observe the temperature effect on non-enzymatic browning, solutions containing 0.2 M tagatose and 0.2 M glycine were heated without pH control in a water bath for 5 hours at 70, 80, 90, or 100 C. The effect of pH on non-enzymatic browning was determined by heating tagatose-glycine solutions with a pH of 3, 4, 5, 6 or 7 in a 100 C water bath for five hours. The acid stability of tagatose was studied by heating a 5% tagatose solution at 100 C for 5 hours, at pH 3, 4, and 5. The heat stability of tagatose was tested by heating a 10% tagatose solution for 5 hours at 100 C, with no pH control. For each test, one sample was pulled from the water bath each hour for analysis. The samples were analyzed using a spectrophomoter, Hunter color measurements, and HPLC. Overall, the researchers found that the rate of browning was dependent on the temperature (as the temperature increased, so did the browning). In regards to pH, they found that browning increased as the pH increased from 3-7, with a slight decrease in browning occurring at pH 6. They also found that there was no browning or decomposition of tagatose under any of the temperature or pH conditions studied in this experiment, unless glycine was present (Ryu and others 2003). Based on their data, they inferred the browning was caused by tagatose and glycine undergoing the Maillard reaction (Ryu and others 2003). The storage stability of tagatose has recently been studied by Dobbs (2008). In this study, buffered solutions at either pH 3 or 7 were held at 20, 30, and 40 C for 265 27 days. It was found that the greatest tagatose loss occurred in solutions containing higher buffer concentrations (0.1 rather than 0.02 M) at higher pH levels (7 rather than 3). Also, phosphate buffer solutions promoted the breakdown of tagatose more than solutions buffered with citrate. When held at 20, 30, and 40 C, tagatose degradation increased with the storage temperature. After being held under the appropriate conditions for a period of 100 days, tagatose degradation ranged from 0.4 to 56.7% with the greatest loss taking place in 0.1 M phosphate solutions at pH 7 and 40 C. It was also found that at increased levels of tagatose loss, a greater extent of brown pigment formation was observed. The study by Dobbs (2008) indicates that tagatose degradation does occur under storage conditions based on temperature, pH, buffer type, and buffer concentration. This leads to the possibility that tagatose degradation may also occur under processing conditions. The experiment carried out by Ryu and others (2003), provides a basic understanding of how tagatose might react under elevated temperatures, but there is a need for a more thorough investigation of this topic. More reliable kinetic data, regarding the breakdown of tagatose over a period of time, can be achieved by carrying out the experiments for a greater length of time than five hours. Also, in the study conducted by Ryu and others (2003), the researchers controlled the pH of their solutions by using hydrochloric acid or sodium hydroxide. However, most foods contain citrate or phosphate buffers to regulate pH, so it would be beneficial to look at the influence of buffer type and concentration on the stability of tagatose and the ability of tagatose to cause browning at elevated temperatures. More research in this area would be beneficial 28 in providing a better understanding of tagatose and its interactions under various conditions that may be encountered during food processing techniques. The objective of this study was to determine the effect of pH, temperature, buffer concentration, and buffer type on the thermal stability and browning of tagatose. Corresponding to temperatures used in processing techniques such as pasteurization, solutions containing about 1% tagatose, with or without glycine, were held at 60, 70, and 80 C for a period of time ranging from 5-216 hours. In order to create an environment similar to that which is found in common foods, such as fruit juice and milk, pH 3 and 7 were used in this experiment. Citrate and phosphate buffers were also added to the solutions at levels of 0.02 M and 0.1 M, since they are commonly found in foods to maintain a desired pH. The results from this experiment will provide insight on the stability of tagatose at processing temperatures and the ability of tagatose to cause brown discoloration under various conditions. 29 CHAPTER 3: MATERIALS AND METHODS Sample Preparation A total of 16 different solutions were prepared for this experiment. Eight solutions contained 0.05 M tagatose and either citrate or phosphate buffer at levels of either 0.02 M or 0.1 M, and at pH 3 or 7. The other 8 solutions were similar to the previous 8 solutions, but also included 0.05 M glycine. The tagatose used for the experiment was donated by Arla Food Ingredients (Basking Ridge, N.J., USA) and represents the quality of tagatose available to food manufacturers. Product specifications indicated the purity of tagatose was greater than 99% with water, non-tagatose monosaccharides, and ash making up the balance. The sodium phosphate dibasic anhydrous, sodium phosphate monobasic monohydrate, sodium citrate, citric acid anhydrous, 85% phosphoric acid, and glycine were obtained from Fisher Scientific (Pittsburgh, PA). The tagatose solutions containing 0.1 M phosphate buffer at pH 7 were prepared using the following procedure. First, a bulk buffer solution containing 3.45 g of sodium phosphate monobasic monohydrate and 2.25 g of tagatose was mixed in a 250 mL volumetric flask. The flask was filled with enough distilled water to reach 250 mL. The second bulk buffer solution was made by adding 3.55 g of sodium phosphate dibasic anhydrous and 2.25 g of tagatose to a 250 mL volumetric flask. Again, enough distilled water was added to bring the volume to 250 mL. Once the bulk buffer solutions were 30 mixed thoroughly, appropriate volumes of the 2 solutions were mixed together until the solution reached pH 7. The pH of the solutions were determined using a pH meter (model 920A, Orion Research Inc, Boston, MA). The solution was separated into 3, 50 mL aliquots and held in a -80 C freezer until use. The tagatose solutions containing 0.02 M phosphate buffer at pH 7 were prepared in a similar fashion to the 0.1 M phosphate solutions at pH 7, except only 0.690 g of sodium phosphate monobasic monohydrate and 0.710 g of sodium phosphate dibasic anhydrous were included in the bulk buffer solutions. The tagatose solutions containing 0.1 M phosphate buffer at pH 3 were prepared by first adding 6.90 g of sodium phosphate monobasic monohydrate to a 500 mL volumetric flask, and filling the flask with distilled water to reach a volume of 500 mL. After mixing the solution thoroughly, 250 mL of the solution was transferred to a 250 mL volumetric flask containing 2.25 g of tagatose to create the first bulk buffer solution, which had a pH around 4.4. Next, 2.88 g of 85% phosphoric acid were added to distilled water in a 250 mL volumetric flask, and distilled water was added to the flask until the solution reached 250 mL. Part of the 85% phosphoric acid solution was then mixed with the remaining sodium phosphate monobasic monohydrate solution (the one without tagatose), to create a solution with a pH of 2.7. Once the desired pH was reached, 2.25 g of tagatose were added to 250 mL of this pH 2.7 solution to create the second bulk buffer solution. Portions of the two bulk buffer solutions at pH 2.7 and 4.4 were mixed together until pH 3 was achieved. The final solution was then divided into 3 separate 50 mL aliquots, which were stored in a -80 C freezer until use. The tagatose solutions containing 0.02 M phosphate buffer at pH 3 were prepared in a similar fashion to the 0.1 31 M phosphate solutions at pH 3, except only 0.576 g of 85% phosphoric acid and 1.38 g of sodium phosphate monobasic monohydrate were included in the bulk buffer solutions. A citrate buffer was also used in some of the tagatose solutions. Those solutions containing 0.1 M citrate buffer both at pH 3 and pH 7 were prepared by initially adding 7.35 g of sodium citrate and 2.25 g of tagatose to a 250 mL volumetric flask. The flask was then filled with distilled water until the volume reached 250 mL to create a bulk buffer solution. In a second flask, 4.80 g of citric acid and 2.25 g of tagatose were mixed thoroughly with distilled water to create 250 mL of this second bulk buffer solution. Once each of the solutions were mixed thoroughly, both solutions were combined in varying proportions to reach the desired pH (either pH 3 or pH 7). Three, 50 mL aliquots of the pH 3 solution, and 3, 50 mL aliquots of the pH 7 solution were held in a -80 C freezer until use. The tagatose solutions containing 0.02 M citrate buffer at pH 3 and pH 7 were prepared in a similar fashion to the 0.1 M citrate solutions, except only 1.47 g of sodium citrate and 0.961 g of citric acid were included in the bulk buffer solutions. The 8 tagatose-glycine solutions were prepared using the same techniques as those described above for the tagatose solutions, except that each bulk buffer solution also contained 0.05 M glycine (0.938 g of glycine in each 250 mL flask of solution). Experiments The experiments were carried out in a water bath preheated to either 60, 70, or 80 C. While all of the 16 solutions (8 tagatose solutions and 8 tagatose-glycine solutions) were tested at 80 C, only 12 of the solutions were tested at 60 and 70 C. The tagatose and tagatose-glycine solutions with 0.02 M citrate and 0.02 M phosphate buffers at pH 3 were not tested at 60 and 70 C due to extremely low reactivity at 80 C. The duration of 32 the experiments ranged from 5 hours for the fastest reaction to a maximum of 216 hours for the slowest reaction (Tables 3.1 and 3.2). Each of the experiments were carried out under the following procedure. For each experiment, the desired solution was thawed to room temperature and mixed thoroughly with a vortex. Using a 20 mL syringe fitted with a 25 mm, 0.2 m nylon filter and needle, approximately 1.5 mL solution was distributed into eleven to fifteen 7 inch x 5 mm Kontes brand NMR sample tubes obtained from Fisher Scientific. Once filled, the NMR tubes were then fitted with push caps and placed into a rack sitting in the preheated water bath. After being placed in the water bath, it took the solutions inside the tubes approximately 1 minute to reach the temperature of the water bath. To measure the actual temperature of the samples, an NMR tube was filled with distilled water, and a thermocouple sensor (Type K, Fisher Scientific, Pittsburgh, PA) was placed in the tube to provide continuous temperature readings. At regular time intervals, individual tubes were removed from the water bath and immediately placed in an ice bath for approximately 1 minute to cool the solution to below room temperature in order to stop any reactions. For example, at 70 C the 0.1 M phosphate buffer solutions at pH 7 were pulled at 1 hour intervals for a total of 10 hours. The temperature of the water bath and the time at which samples were pulled were recorded. Upon cooling, the solution was transferred from the NMR tube to a 5 mL cryogenic vial using a plastic pipette. Each cryogenic vial was labeled according to the sample it contained. The buffer concentration was denoted as either ?1? meaning 0.1 M or ?02? meaning 0.02 M. Buffer type was indicated by the first letter of the buffer: ?P? meaning phosphate, ?C? meaning 33 Table 3.1. Length (h) of Tagatose Experiments. Sample Temperature (?C) 60 70 80 pH 3 0.02 M phosphate ND ND 10.0 0.02 M citrate ND ND 10.0 0.1 M phosphate 215.8 168.0 10.0 0.1 M citrate 215.8 168.0 10.0 pH 7 0.02 M phosphate 215.8 56.0 10.0 0.02 M citrate 215.8 56.0 10.0 0.1 M phosphate 81.5 10.0 5.0 0.1 M citrate 81.5 10.0 7.5 ND = not determined Table 3.2. Length (h) of Tagatose-Glycine Experiments. Sample Temperature (?C) 60 70 80 pH 3 0.02 M phosphate ND ND 10.0 0.02 M citrate ND ND 10.0 0.1 M phosphate 216.0 156.2 10.0 0.1 M citrate 216.0 156.2 10.0 pH 7 0.02 M phosphate 216.0 60.0 10.0 0.02 M citrate 216.0 60.0 10.0 0.1 M phosphate 82.0 10.0 7.0 0.1 M citrate 82.0 10.0 7.0 ND = not determined 34 citrate. The pH value of the solution, either 3 or 7, was placed on the label, along with whether the sample contained only tagatose or both tagatose and glycine (?T? or ?TG?, respectively). Temperature, in Celsius, was denoted on the label as ?60?, ?70?, or ?80?. For example, 1P7-T80 meant that the vial contained a 0.1 M phosphate solution with only tagatose, at pH 7 and held at 80 C. After placing the sample in the correctly labeled vial and securing the vial with a screw cap, it was placed in the refrigerator until analysis, which generally occurred 1-2 days after the experiment was completed. Stability data from Dobbs (2008) indicated additional tagatose loss during refrigerated storage was minimal (less than 0.4% in 0.1 M phosphate buffer at pH 7 held for 7 d). Sample Analysis Tagatose Degradation Tagatose degradation was determined by analyzing the tagatose concentration in each sample using reverse-phase high performance liquid chromatography (HPLC). A 250 x 4.6 mm Luna 5 amino column (Phenomenex, Torrance, CA) and a 91%/9% acetonitrile/water mobile phase having a flow rate of 3 mL/min were used for analysis. Tagatose detection occurred through the use of a refractive index detector (Shimadzu, Kyoto, Japan). A Hewlett-Packard Integrator was used to integrate the data. Figure 3.1 shows a typical chromatogram where tagatose elutes around 7 minutes. Under the parameters of HPLC analysis for this experiment, the glycine was retained on the column; therefore for those samples that contained glycine, only a tagatose peak was observed on the chromatogram. Six standard solutions with a known concentration of tagatose were analyzed prior to the analysis of the study samples. The area and concentration of the standards 35 were used to create a linear standard curve, from which the concentration of the samples from each experiment could be determined. From the chromatogram, the area of the tagatose peak was recorded and used to determine the concentration of tagatose in each sample from a standard curve. To evaluate the validity of the analytical methods, 10 solutions of tagatose in 0.1 M phosphate buffer at pH 7 were prepared, 5 containing 0.0514 M tagatose and 5 containing 0.0302 M tagatose. Solutions were filtered and analyzed by HPLC, as described previously. The average percent recovery ranged from 99.5-102.6% and the coefficient of variation was less than 2%, indicating a highly reliable method. Figure 3.1. Sample HPLC Chromatogram for the Analysis of Tagatose in a 0.1 M Phosphate Buffer Solution at pH 3 containing 0.05 M Glycine. Tagatose Elutes at Approximately 7 minutes. 36 Brown Pigment Formation The formation of brown pigment in the tagatose and tagatose-glycine samples was measured using a spectrophotometer (DU 640, Beckman Instrument Inc, Fullterton, CA). Approximately 1 mL aliquots of each sample were transferred to methacrylate semimicro style cuvettes. Absorption was read at 420 nm. Data Analysis Microsoft Excel was used to statistically analyze the data collected in this study. Monosaccharide degradation pathways have been studied using a first-order kinetic model (De Bruijn and others 1986; Kelly and Brown 1978/79). Therefore, tagatose degradation was modeled using first order kinetics (Eq. 3.1). The pseudo-first- order rate constants for tagatose loss were determined, along with 95% confidence intervals, using least squares analysis as described by Labuza and Kamman (1983). In the equation, ?C? refers to the tagatose concentration (M) at a specific time, ?C0? is the initial tagatose concentration (M), ?k? is the rate constant (h-1), and ?t? is the time (h). ln CC 0 kt (3.1) After plotting the data using pseudo-first-order kinetics, the tagatose concentrations appeared to level off in some of the experiments, deviating from typical first order behavior. Visual determination as well as comparison of the R2 values with and without the ?plateau? were used to establish its existence and magnitude for each experiment individually. For those experiments where a ?plateau? was observed, only the initial data prior to the ?plateau? were used when determining pseudo-first-order rate constants. For all other experiments, the complete data set was used in the kinetic model. 37 For solutions containing tagatose as well as glycine, the data would typically follow second order kinetics. However, since multiple reaction pathways are occurring simultaneously, it was determined to be more appropriate to model the data using pseudo- first-order kinetics (Eq. 3.1). The formation of brown pigmentation has generally been modeled using pseudo- zero-order kinetics (Baisier and Labuza 1992; Karmas and others 1992; Warmbier and others 1976). Therefore, rate constants for brown pigment formation, as well as 95% confidence intervals, were determined using pseudo-zero-order kinetics (Labuza and Kamman 1983). In Eq. 3.2, ?A? is the absorbance at time (h), ?t?, and ?A0? is the initial absorbance. A kt A 0 (3.2) Upon plotting graphs of absorbance as a function of time for tagatose and tagatose-glycine samples, a lag phase was noticed in some of the experiments. For these experiments, the magnitude of the lag phase was determined individually, based on visual determination and comparison of the R2 values with and without the lag phase. When determining pseudo-zero-order rate constants for those experiments, the lag phase was removed. All other experiments were void of any noticeable lag phase; therefore, all data were used to determine pseudo-zero-order rate constants. Using the rate constants and average temperature for each experiment, activation energies, EA, were determined using Eq. 3.3 (Labuza and Kamman 1983). 38 ln k ln k0 E ART (3.3) In equation 3.3, k is the rate constant (h-1), k0 is the pre-exponential factor (h-1), R is the ideal gas constant (1.987 cal K-1 mol-1), and T is the temperature in Kelvin. Plotting ln k versus 1T gives a straight line, which has a slope equal to EAR . 39 CHAPTER 4: RESULTS AND DISCUSSION Tagatose Degradation Tagatose degradation in solution is affected by many factors including temperature, pH, buffer type, and buffer concentration. Examples of the tagatose degradation profile and the pseudo-first-order plot are shown in Figures 4.1 and 4.2, respectively. As shown in Figure 4.2, the pseudo-first-order model did not fit the entire data set for tagatose degradation in 0.02 M phosphate buffer at pH 7 and 60?C. A leveling off of the tagatose degradation appeared after 120 h, which is likely due to a pH drop. For this particular model system, only the data up to 120 h were used for the kinetic model. This ?plateau? was also noted by Dobbs (2008) for tagatose degradation at 20-40?C. Tagatose loss in the other buffer solutions followed pseudo-first-order kinetics over the duration of the experiment, without a clear ?plateau?. Pseudo-first-order rate constants and 95% confidence limits for tagatose degradation were calculated using least squares analysis and are listed along with the R2 values in Table 4.1. Effect of pH In this study, solutions were held at pH 3 and pH 7 to determine the effect of pH on tagatose degradation. Tagatose degradation occurred faster at higher pH values (Table 4.1). This result is shown graphically in Figure 4.3, where the overall tagatose 40 Figure 4.1. Tagatose Loss in 0.02 M Phosphate Buffer at pH 7 and 60 C. Figure 4.2. Pseudo-First-Order Plot of Tagatose Loss in 0.02 M Phosphate Buffer at pH 7 and 60 C. 41 Table 4.1. Pseudo-First-Order Rate Constants (h-1) with 95% Confidence Limits for the Loss of Tagatose in Solution. Sample Temperature (?C) 60 70 80 pH 3 0.02 M phosphate ND ND 0* 0.02 M citrate ND ND 0* 0.1 M phosphate 0.00021?0.00012 (R2=0.55) 0.00059?0.00016 (R2=0.82) 0* 0.1 M citrate 0.000092?0.00008 (R2=0.32) 0.00063?0.00014 (R2=0.88) 0* pH 7 0.02 M phosphate 0.00170?0.00024 (R2=0.96) 0.00566?0.00071 (R2=0.97) 0.0249?0.0020 (R2=0.99) 0.02 M citrate 0.00082?0.00013 (R2=0.94) 0.00345?0.00047 (R2=0.96) 0.0192?0.0030 (R2=0.96) 0.1 M phosphate 0.00661?0.00044 (R2=0.99) 0.0301?0.0022 (R2=0.99) 0.0756?0.0070 (R2=0.98) 0.1 M citrate 0.00239?0.00071 (R2=0.81) 0.0113?0.0036 (R2=0.84) 0.0358?0.0046 (R2=0.97) ND = not determined due to extremely low reactivity at 80?C *Not different from zero over the 10 h experiment duration based on the 95% confidence limit exceeding the value of the rate constant to encompass zero. 42 Figure 4.3. Tagatose Loss in 0.1 M Buffer Solutions at 80 C as Affected by pH. concentration in 0.1 M citrate buffer at pH 7 and 80 C decreased by approximately 26% from the initial concentration in comparison to tagatose in 0.1 M citrate buffer at pH 3, which showed little to no change in tagatose concentration over the course of the experiment. Similar results were seen in the solutions containing phosphate buffer, in which greater tagatose degradation was also seen at pH 7 than pH 3. Monosaccharides are able to undergo degradation reactions under both alkaline and acidic conditions. When monosaccharides are heated in a strongly acidic solution, dehydration occurs and furfural compounds are formed (Wong 1989). At pH 3, tagatose participated very minimally in the acidic degradation reaction (Table 4.1). A greater extent of tagatose degradation was seen in tagatose solutions held at pH 7, with the fastest tagatose degradation occurring in 0.1 M phosphate buffer at 80 C. Since more tagatose degradation was seen at pH 7 than pH 3, it is apparent that tagatose more readily undergoes the alkaline degradation reaction than the degradation reaction 43 that occurs under acidic conditions. Under alkaline conditions, monosaccharides undergo a series of rearrangements, including ionization, mutarotation, enolization, and isomerization to form an enediol anion species. After undergoing several other reactions, the enediol anion species is ultimately degraded into carboxylic acids (De Bruijn and others 1986). The formation of carboxylic acids can cause the pH of the solution to decrease, which would slow down the rate of the alkaline degradation reaction. This effect was observed in 0.02 M phosphate buffer at pH 7 and 60?C, where tagatose degradation leveled off after 120 h. Dobbs (2008) noted a similar ?plateau? in tagatose degradation after 100 d storage. As presented in Table 4.2, the pH of the tagatose solutions originally at pH 7 decreased over a period of 6-7 days at 60 C, indicating the formation of acidic degradation products. However, a definitive ?plateau? in the degradation profile was only observed for tagatose degradation in 0.02 M phosphate buffer where the pH drop was greatest. Table 4.2. Decrease in the pH of Tagatose Solutions During Storage at 60 C. Sample Initial pH Final pH 0.02 M phosphate 6.93 5.07* 0.02 M citrate 7.04 6.19* 0.1 M phosphate 7.06 6.59** 0.1 M citrate 7.09 6.45** * After 7 days of storage **After 6 days of storage 44 The effect of pH on tagatose degradation, as determined by this study, is consistent with the findings of Dobbs (2008) in the respect that tagatose degradation was faster at pH 7 than pH 3. However, in the study by Dobbs (2008), the degradation of tagatose in most solutions appeared to level off at approximately 100 days when held at ambient storage temperatures (20, 30, and 40?C), especially in phosphate buffer at pH 7. As shown in Figure 4.3, tagatose degradation at high temperatures continued rather consistently without a ?plateau? for all solutions. The only exception was degradation in 0.02 M phosphate buffer at pH 7 and 60?C, as mentioned earlier. The difference between the studies is likely due to differences in temperature and duration. Under the higher temperature conditions used in the current study, sufficient energy was available to compensate for the reduced pH, which maintained the reaction rates. Therefore, over the duration of most experiments in the current study, no ?plateau? in tagatose degradation was seen. A ?plateau? may have appeared if the duration of the experiment was increased, such as in 0.02 M phosphate buffer at pH 7 and 60?C. Ryu and others (2003) also studied the effect of pH on tagatose degradation, however their results appear to conflict with those of the current study. In the study by Ryu and others (2003), unbuffered solutions containing tagatose were held at pH 3, 4, and 5 for a period of 5 hours at 100 C. No tagatose degradation occurred at any of the pH levels in their study. Even though the degradation of tagatose was not as dramatic as that which occurred at pH 7, tagatose degradation was seen in buffered tagatose solutions at pH 3 in the current study. One reason for the conflicting findings between the two studies was that Ryu and others (2003) did not use buffer salts in their solutions. As determined in this study and other studies (Bell and Wetzel 1995; Dobbs 2008; 45 Pachapurkar and Bell 2005), the presence of buffer salts affects the stability of molecules, including tagatose. Reaction rates tend to increase as buffer concentration increases. Another difference between the studies was the duration of the experiments. In the current study, the majority of the experiments were carried out for at least 10 hours, whereas the experiments conducted by Ryu and others (2003) only took place for 5 hours. The shorter experiment length may not have been sufficient for reactions leading to tagatose degradation to have taken place in detectable amounts, especially in the absence of buffer salts. Effect of Buffer Type and Buffer Concentration From the data presented in Table 4.1 and Figure 4.4, it is apparent that the tagatose solutions containing phosphate buffer generally had faster tagatose degradation than the solutions containing citrate buffer, with a more noticeable difference observed at the higher buffer concentration (0.1 M). As shown in Table 4.1, in the tagatose solutions with 0.1 M buffer at pH 7 and 80 C, the solution containing phosphate buffer had a rate constant twice as large as in the solution containing citrate buffer (0.0756?0.0070 h-1 and 0.0358?0.0046 h-1, respectively). Under similar conditions, but at a lower buffer concentration (0.02 M), the rate constants for tagatose degradation in phosphate and citrate buffers were 0.0249?0.0020 h-1 and 0.0192?0.0030 h-1, respectively. Higher buffer concentrations also promoted faster tagatose degradation; tagatose loss in 0.1 M phosphate buffer at pH 7 and 70 C had rate constant of 0.0301?0.0022 h-1 while that in 0.02 M phosphate buffer under the same conditions only had a rate constant of 0.00566?0.00071 h-1. The effect of buffer type and concentration on tagatose degradation are in agreement with the findings of other researchers. 46 Figure 4.4. Tagatose Loss in Solution at pH 7 and 80 C as Affected by Buffer Type and Concentration. Bell and Wetzel (1995) studied aspartame degradation in citrate and phosphate buffer solutions and found that the reaction rates for aspartame degradation were significantly greater in phosphate buffer than citrate buffer at pH 7, with less dramatic differences between the buffer types seen at pH 3. They also found that aspartame degradation increased as the buffer concentration increased. Pachapurkar and Bell (2005) found that thiamin degradation occurred faster in phosphate buffer rather than citrate buffer under similar conditions. Also, a decrease in thiamin stability was observed when the buffer concentration increased. Dobbs (2008), who studied the effect of buffer type and concentration on tagatose degradation at storage temperatures, also found that tagatose degradation increased when held in phosphate buffer rather than citrate buffer. Tagatose degradation rates were also 47 found to increase as the buffer concentration increased. The fastest tagatose degradation was seen in 0.1 M phosphate buffer at pH 7, which is consistent with the data from the current study at high temperatures. As mentioned previously, Ryu and others (2003) found no tagatose loss in unbuffered solutions at 100?C for 5 h, which also indicates the importance of buffer salts on tagatose degradation. The results from this study are consistent with the findings from the literature (Bell and Wetzel 1995; Pachapurkar and Bell 2005; Dobbs 2008). The dibasic anion (HPO4-2), which is the primary component in the phosphate buffer at pH 7, appears to be much more efficient at the required proton transfers for the degradation reaction than citrate anions. Bell and Wetzel (1995) discussed the ability of the small phosphate anions to act as bifunctional catalysts, meaning they can simultaneously donate and accept protons. Bifunctional catalysis by phosphate anions allows for faster proton transfers than by citrate anions, which are not bifunctional catalysts. Proton transfers are required in the alkaline degradation reaction of tagatose in order to form the enediol species, including the removal of a proton from one hydroxyl group during ionization and the transfer of a proton during mutarotation (Wong 1989). Since the phosphate dibasic anion is more efficient at these proton transfers than the citrate anion, tagatose degradation can occur faster in phosphate buffer solutions, as seen in this study. Effect of Temperature The effect of temperature on tagatose degradation is shown in Figure 4.5 and Table 4.1. A proportional relationship exists between temperature and tagatose degradation; as the temperature increased from 60 to 80 C, the rate constants for tagatose degradation also increased. Due to an increase in molecular movement at higher 48 temperatures, it is expected for reactions to occur faster when the temperature is raised. As shown in Table 4.1, the rate constants for tagatose loss in 0.1 M phosphate buffer at pH 7 increased from 0.00661?0.00044 h-1 at 60 C to 0.0756?0.0070 h-1 at 80 C. The results from this study are in agreement with the findings of Dobbs (2008). The study by Dobbs (2008) looked at the stability of tagatose under storage conditions (20, 30, and 40 C), and it was found that the higher temperatures enhanced tagatose degradation. Figure 4.5. Tagatose Loss in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature. The activation energies were calculated and are shown in Table 4.3. A higher activation energy indicates the reaction rate is more sensitive to changes in temperature, whereas a lower activation energy indicates the reaction rate is less sensitive to temperature changes. At pH 3, activation energies were unable to be determined due to 49 only having reliable rate constants at 2 temperatures (Table 4.1). At pH 7, the activation energies ranged from 31.4 to 38.7 kcal/mol. Dobbs (2008) calculated activation energies for tagatose degradation in 0.02 and 0.1 M phosphate and citrate buffers at pH 7 held at storage temperatures (20, 30, and 40?C). The activation energies for tagatose degradation in the solutions held at storage temperatures ranged from 6.4 to 14.8 kcal/mol, which are much lower than the activation energies for the same solutions held under thermal processing temperatures (Table 4.3). An explanation for the difference in the activation energies between the two temperature ranges is not clear, but may be due to different reaction pathways predominating at the lower storage temperatures as compared to the higher processing temperatures. Also, in the experiment conducted by Dobbs (2008), a ?plateau? was seen in the data; therefore only the initial 100 days of storage data were used when determining the rate constants and activation energies. The portion of data containing the ?plateau? in tagatose degradation was not included in the calculations. In the current study, the entire data set was used in calculating the activation energies for all experiments, except 0.02 M phosphate at pH 7 and 60?C. The utilization of initial rates by Dobbs (2008) may have contributed to some of the variation between the activation energies at the different temperature ranges. The activation energies were slightly lower for tagatose degradation in phosphate buffer than citrate buffer (Table 4.3). The catalytic effect of the phosphate buffer, discussed previously, reduced the energy needed for the reaction as compared to citrate buffer. Using the activation energies, the extent of tagatose degradation that may occur under actual thermal processing conditions can be mathematically predicted. Because the 50 Table 4.3. Activation Energies (EA) for Tagatose Degradation at pH 7. Sample EA (kcal/mol) 0.02 M phosphate 33.2 0.02 M citrate 38.7 0.1 M phosphate 31.4 0.1 M citrate 33.9 fastest tagatose degradation was seen in 0.1 M phosphate buffer at pH 7, the activation energy for this solution was used to predict the amount of tagatose degradation that would occur during commonly used pasteurization techniques in the food industry. Under vat pasteurization, liquids are subjected to a temperature of 63?C for 30 minutes, and high temperature short time (HTST) pasteurization consists of heating products for 15 seconds at 72?C. Using the activation energy of 31.4 kcal/mol for 0.1 M phosphate buffer at pH 7, it was calculated that less than 0.5 and 0.02% tagatose would be lost under basic vat and HTST pasteurization conditions, respectively. Under conditions where tagatose is more stable, even less tagatose loss would be expected. Tagatose and the Maillard Reaction Tagatose solutions also containing 0.05 M glycine were observed at the same temperatures, pH values, and buffer types and concentrations as the tagatose solutions without glycine in order to determine the participation of tagatose in the Maillard reaction. As explained previously, typically a solution containing two reactants would follow second order kinetics, however, in this case multiple reactions are occurring simultaneously. When plotted, the data fit well into a first order model, so pseudo-first- order kinetics were appropriate to use. A ?plateau? in tagatose degradation was observed 51 in five tagatose-glycine solutions: 0.02 M phosphate and citrate buffers at pH 7 and 60?C, 0.02 M phosphate buffer at pH 7 and 70?C, and 0.1 M citrate buffer at 60 and 80?C at pH 7. For these solutions only the initial data prior to the ?plateau? were used for the kinetic model. A graphical example of the data modeled by pseudo-first-order kinetics is shown in Figure 4.6. The pseudo-first-order rate constants with 95% confidence limits are presented in Table 4.4. Figure 4.6. Pseudo-First-Order Plot of Tagatose Loss in 0.02 M Phosphate Buffer with 0.05 M Glycine at pH 7 and 70?C. Effect of pH The effect of pH on tagatose degradation in solutions containing 0.05 M glycine is shown in Table 4.4 and Figure 4.7. At pH 7, considerably faster tagatose loss occurred than at pH 3. In the 0.1 M phosphate buffer at pH 3 and 80 C, the rate constant for 52 tagatose degradation was 0.0031?0.0028 h-1, but under the same conditions at pH 7, the rate constant increased to 0.0790?0.0085 h-1. As discussed earlier, greater tagatose degradation at pH 7 could be attributed to tagatose more readily participating in the alkaline degradation reaction than the degradation reaction that can occur under acidic conditions. Also, because these solutions contain glycine, the Maillard reaction is likely taking place between tagatose and glycine. At pH 7, a greater amount of glycine will contain an amine group in the unprotonated form. The unprotonated form of the amine is more reactive in the Maillard reaction than the protonated form, which is in a greater concentration at lower pH values, thus explaining why greater tagatose degradation was seen in the solutions held at higher pH. Dobbs (2008) found similar results when looking at tagatose degradation at storage temperatures. Effect of Buffer Type and Buffer Concentration The buffer type and concentration also played a role in tagatose degradation when glycine was present. The effect is shown in Table 4.4 and Figure 4.8. Tagatose degradation was faster in phosphate buffer than citrate buffer, and the difference between the two buffer types was much more noticeable at pH 7 than pH 3. For example, the rate constant for tagatose loss in 0.02 M phosphate buffer at pH 7 and 80 C was 0.0260?0.0037 h-1, whereas the rate constant for tagatose loss was 0.0183?0.0020 h-1 in the citrate buffer under the same conditions. Also, under similar conditions, the higher buffer concentration (0.1 M) resulted in greater tagatose loss than the lower buffer concentration (0.02 M); the rate constant for tagatose degradation in 0.02 M citrate buffer at pH 7 and 80 C was 0.0183?0.0020 h-1, while the rate constant for tagatose degradation 53 in 0.1 M citrate buffer at pH 7 and 80 C was 0.0381?0.0050 h-1. These results are consistent with the findings of Dobbs (2008) and Bell (1997). Table 4.4. Pseudo-First-Order Rate Constants (h-1) with 95% Confidence Limits for the Loss of Tagatose in Solution Containing 0.05 M Glycine. Sample Temperature (?C) 60 70 80 pH 3 0.02 M phosphate ND ND 0* 0.02 M citrate ND ND 0.0032?0.0022 (R2=0.54) 0.1 M phosphate 0.00014?0.00011 (R2=0.37) 0.00062?0.00009 (R2=0.94) 0.0031?0.0028 (R2=0.41) 0.1 M citrate 0.00015?0.00014 (R2=0.31) 0.00106?0.00026 (R2=0.87) 0* pH 7 0.02 M phosphate 0.00177?0.00020 (R2=0.97) 0.00689?0.00068 (R2=0.98) 0.0260?0.0037 (R2=0.97) 0.02 M citrate 0.00099?0.00014 (R2=0.95) 0.00404?0.00051 (R2=0.96) 0.0183?0.0020 (R2=0.98) 0.1 M phosphate 0.00659?0.0013 (R2=0.91) 0.0280?0.0066 (R2=0.91) 0.0790?0.0085 (R2=0.97) 0.1 M citrate 0.00359?0.00087 (R2=0.88) 0.0144?0.0050 (R2=0.83) 0.0381?0.0050 (R2=0.97) ND = not determined due to extremely low reactivity at 80?C *Not different from zero over the 10 h experiment duration based on the 95% confidence limit exceeding the value of the rate constant to encompass zero. 54 Figure 4.7. Tagatose Loss in 0.1 M Buffer Solutions Containing Tagatose and Glycine at 80 C as Affected by pH. Figure 4.8. Tagatose Loss in Solution Containing Tagatose and Glycine at pH 7 and 80 C as Affected by Buffer Type and Concentration. 55 As mentioned earlier, phosphate dibasic anions are better than citrate anions at the proton transfers that are required for certain reactions to take place. In the Maillard reaction, a glycosylamine is formed after a number of proton transfers. Since the phosphate anions are more efficient at these proton transfers, tagatose degradation occurs at a faster rate, resulting in an overall greater tagatose loss in phosphate buffer solutions as compared to in citrate buffer solutions. Also, the increased buffer concentration means there are more buffer anions available to transfer protons; therefore, more tagatose degradation is seen in the solutions containing a higher buffer concentration. Effect of Temperature As shown in Table 4.4 and Figure 4.9, the increase in temperature from 60 to 80 C resulted in greater tagatose degradation in the presence of glycine. The rate constant for tagatose loss in 0.1 M citrate buffer at pH 7 was 0.00359?0.00087 h-1 at 60 C, while the rate constant for tagatose loss in this same solution at 80 C was 0.0381?0.0050 h-1. Due to greater molecular movement at higher temperatures, reactions are expected to occur at a greater rate as the temperature increases, as seen in this study. These results are consistent with the study conducted by Dobbs (2008). Activation energies were also calculated for the tagatose solutions that contained glycine (Table 4.5). At pH 3, activation energies were only calculated for tagatose loss in 0.1 M phosphate buffer because the other solutions only had reliable rate constants at 2 temperatures (Table 4.4). At pH 7, the activation energies for tagatose degradation in the presence of glycine range from 27.6 to 33.0 kcal/mol, with lower activation energies occurring in the solutions containing the higher buffer concentration. At pH 3, the activation energy value was 36.0 kcal/mol in 0.1 M phosphate buffer. The slightly higher 56 activation energy for degradation at pH 3 may indicate these pathways require more energy than those at pH 7. For the samples containing 0.05 M glycine, the activation energies calculated in this experiment are considerably higher than those reported under storage conditions by Dobbs (2008). The activation energies for tagatose degradation in 0.02 and 0.1 M phosphate and citrate buffer solutions containing tagatose and 0.05 M glycine held at 20- 40?C ranged from 16.2 to 17.1 kcal/mol (Dobbs 2008). As for the results for tagatose alone, these results suggest that the effect of temperature on various reaction pathways leading to tagatose degradation could be dependent on the temperature ranges. Figure 4.9. Tagatose Loss in 0.1 M Citrate Buffer Solutions Containing Tagatose and Glycine at pH 7 as Affected by Temperature. 57 Table 4.5. Activation Energies (EA) for Tagatose Degradation in Solutions Containing 0.05 M Glycine. Sample EA (kcal/mol) 0.02 M phosphate, pH 7 30.4 0.02 M citrate, pH 7 33.0 0.1 M phosphate, pH 7 29.0 0.1 M citrate, pH 7 27.6 0.1 M phosphate, pH 3 36.0 Using the activation energies, tagatose degradation under vat and HTST pasteurization conditions were able to be predicted. Since tagatose degradation in the presence of glycine occurred the fastest in 0.1 M phosphate buffer at pH 7, the activation energy of 29.0 kcal/mol was used in the calculations. Under vat pasteurization conditions, less than 0.5% tagatose degradation is predicted, and under HTST conditions the amount of tagatose degradation that would be expected is approximately 0.01%. Brown Pigment Formation The majority of solutions containing tagatose alone or tagatose with glycine browned to varying extents depending upon the solution composition and pH. The absorbance at 420 nm was measured for each sample, and these data were used to calculate pseudo-zero-order rate constants and 95% confidence limits for each experiment. The majority of experiments contained a lag phase (Figure 4.10) and based on visual determination as well as comparison of the R2 values, the lag phase for each experiment was determined individually and removed from the rate constant calculations. Browning as affected by buffer type and pH is shown graphically in Figures 4.11 and 58 4.12. Tables 4.6 and 4.7 present the rate constants and 95% confidence limits for brown pigment formation ignoring the initial lag phase. From these tables and figures, the effect of temperature, pH, buffer type, and buffer concentration on the formation of brown pigment can be seen. For both sets of experiments, those solutions containing only tagatose and those containing tagatose and glycine, it is apparent that the amount of brown pigment formed increased as the temperature increased. Also, the browning was faster at pH 7 than pH 3. Tagatose in phosphate buffer resulted in more browning than in citrate buffer solutions, and more browning was seen at the 0.1 M buffer concentration than the 0.02 M buffer concentration. Browning was also generally faster in the presence of glycine. Figure 4.10. Browning in 0.1 M Phosphate Buffer Containing Tagatose and Glycine at pH 7 and 80?C. 59 Figure 4.11. Brown Pigment Formation in 0.1 M Buffer Solutions Containing Tagatose at 80?C. Figure 4.12. Brown Pigment Formation in 0.1 M Buffer Solutions Containing Tagatose and Glycine at 80?C. 60 Table 4.6. Pseudo-Zero-Order Rate Constants (OD/h) with 95% Confidence Limits for the Formation of Brown Pigment in Tagatose Solutions. Sample Temperature (?C) 60 70 80 pH 3 0.02 M phosphate ND ND 0* 0.02 M citrate ND ND 0* 0.1 M phosphate 0.000053?0.000010 (R2=0.92) 0.00030?0.00006 (R2=0.92) 0* 0.1 M citrate 0.000027?0.000008 (R2=0.81) 0.00029?0.00008 (R2=0.89) 0.00014?0.00010 (R2=0.53) pH 7 0.02 M phosphate 0.00051?0.00011 (R2=0.92) 0.00192?0.00041 (R2=0.91) 0.00774?0.0016 (R2=0.94) 0.02 M citrate 0.000053?0.000035 (R2=0.48) 0.00084?0.00037 (R2=0.77) 0.00374?0.00073 (R2=0.96) 0.1 M phosphate 0.00137?0.00038 (R2=0.88) 0.00215?0.00031 (R2=0.97) 0.055?0.018 (R2=0.80) 0.1 M citrate 0.00028?0.00018 (R2=0.53) 0.00099?0.00028 (R2=0.87) 0.0193?0.0022 (R2=0.99) ND = not determined due to extremely low reactivity at 80?C *Not different from zero over the 10 h experiment duration based on the 95% confidence limit exceeding the value of the rate constant to encompass zero. 61 Table 4.7. Pseudo-Zero-Order Rate Constants (OD/h) with 95% Confidence Limits for the Formation of Brown Pigment in Solutions Containing Tagatose and Glycine. Sample Temperature (?C) 60 70 80 pH 3 0.02 M phosphate ND ND 0* 0.02 M citrate ND ND 0.00042?0.00019 (R2=0.74) 0.1 M phosphate 0.00019?0.00003 (R2=0.96) 0.00120?0.00011 (R2=0.98) 0.00118?0.00036 (R2=0.86) 0.1 M citrate 0.00012?0.00002 (R2=0.93) 0.00098?0.00026 (R2=0.90) 0.00054?0.00028 (R2=0.69) pH 7 0.02 M phosphate 0.00200?0.00037 (R2=0.94) 0.0064?0.0011 (R2=0.94) 0.02402?0.0055 (R2=0.94) 0.02 M citrate 0.00086?0.00041 (R2=0.74) 0.00378?0.00032 (R2=0.99) 0.01182?0.0046 (R2=0.84) 0.1 M phosphate 0.0078?0.0014 (R2=0.94) 0.0138?0.0022 (R2=0.97) 0.0647?0.0065 (R2=0.98) 0.1 M citrate 0.00141?0.00023 (R2=0.95) 0.00304?0.00051 (R2=0.97) 0.0187?0.0033 (R2=0.95) ND = not determined due to extremely low reactivity at 80?C *Not different from zero over the 10 h experiment duration based on the 95% confidence limit exceeding the value of the rate constant to encompass zero. In the study by Dobbs (2008), it was found that at the storage temperature of 40 C, the 0.1 M phosphate buffer at pH 7 that contains only tagatose showed a steep increase in browning followed by a decrease and then a leveling off. When glycine was added to this solution, the browning increased dramatically before somewhat leveling off. This browning pattern was not seen under the thermal processing conditions tested in this experiment. Instead, the formation of brown pigment increased steadily over the course of the experiment without any leveling off occurring (Figures 4.11 and 4.12). Other than 62 the differences at 0.1 M phosphate buffer at pH 7 and 80 C, the results from this study are consistent with the findings from the study conducted by Dobbs (2008). Ryu and others (2003) conducted a study looking at the effect of temperature and pH on the non-enzymatic browning reaction that occurs between tagatose and glycine. They found that more brown pigment formed as the temperature increased from 70 C to 100 C, and browning increased as the pH increased from 3 to 7. These results are in agreement with the findings of the current study. When looking at the effect of buffer type and concentration on the Maillard reaction, Bell (1997) found that solutions containing glucose and glycine at pH 7 and held at 25 C participated more readily in the Maillard reaction in phosphate buffer than citrate buffer. While higher phosphate buffer concentrations resulted in faster brown pigment formation, no browning was seen in the solutions containing the citrate buffer. After further investigation, Bell (1997) determined that citrate did not act as an inhibitor to the Maillard reaction, but rather citrate only has minimal effects on the non-enzymatic browning reaction. The higher temperatures in the current study are likely responsible for the presence of brown pigment formation in not only the phosphate buffers but also the citrate buffers. However, the study by Bell (1997) reiterates the point that phosphate anions are more efficient at proton transfers than citrate anions, as explained earlier. Activation energies for the brown pigment formation were also calculated (Table 4.8). At pH 3, activation energies were not calculated due to extremely small and inconsistent rate constants (Tables 4.6 and 4.7). At pH 7, those solutions containing only tagatose had activation energies ranging from 33.6 to 52.1 kcal/mol, while those solutions containing tagatose and glycine had activation energies ranging from 24.7 to 30.1 63 kcal/mol. For both the solutions containing only tagatose, as well as those containing tagatose and glycine, the citrate buffer solutions had higher activation energies than the phosphate buffer solutions at pH 7. Also, the activation energies for brown pigment formation were higher in the solutions containing only tagatose as compared to the solutions containing tagatose and glycine. The contribution of the Maillard reaction pathways to browning is apparently less sensitive to temperature than the browning association with the alkaline degradation reaction of monosaccharides. Table 4.8. Activation Energies (kcal/mol) for Brown Pigment Formation in Tagatose and Tagatose-Glycine Solutions at pH 7. Sample Tagatose Tagatose-Glycine 0.02 M phosphate 33.6 28.1 0.02 M citrate 51.8 29.7 0.1 M phosphate 45.5 24.7 0.1 M citrate 52.1 30.1 Effect of Glycine on Tagatose Degradation and Brown Pigment Formation The addition of an amino acid such as glycine to a solution containing a reducing sugar, like tagatose, would be expected to increase tagatose degradation and brown pigment formation due to the participation of glycine and tagatose in the Maillard reaction. Looking at the results, the addition of glycine to tagatose solutions did enhance the browning (Figure 4.13), but there was not much of an effect on tagatose degradation between the solutions with and without glycine (Tables 4.1 and 4.4). 64 Figure 4.13. Brown Pigment Formation in 0.02 M Phosphate Buffer at pH 7 and 80 C. When comparing the browning seen in solutions containing only tagatose to the solutions which contained tagatose and glycine, it is noticeable from the rate constants that more browning occurred in the solutions containing glycine. In the 0.02 M citrate buffer at pH 7 and 80 C, the pseudo-zero-order rate constant with the 95% confidence limit for brown pigment formation in the solution only containing tagatose was 0.00374?0.00073 OD/h, but the addition of glycine increased the rate constant to 0.0118?0.0046 OD/h. This result is expected since tagatose can interact with glycine to form brown pigment through the Maillard reaction. With an increase in browning in the solutions containing glycine, a correlating increase in tagatose degradation would be expected. However, the rate constants for tagatose degradation are very similar between the samples with and without glycine. This same observation was noted when tagatose degradation was studied under storage 65 conditions by Dobbs (2008). While the reason as to why the addition of glycine would cause an increase in browning but not an increase in tagatose degradation is unclear, Dobbs (2008) suggests that glycine may disturb the equilibrium positioning of reversible steps involved in the alkaline degradation reaction and Maillard reaction: formation of the enediol in the alkaline degradation reaction and the formation of the glycosylamine in the Maillard reaction. The addition of glycine could change the equilibrium and direct some of the tagatose towards the Maillard reaction. The combination of re-established equilibrium occurring in competing reaction pathways may result in similar or slower tagatose loss when glycine is present (Dobbs 2008). This study shows that the addition of glycine to tagatose solutions containing either a phosphate or citrate buffer causes an increase in brown pigment formation, but the tagatose degradation remains relatively unchanged from solutions without glycine. Further investigation is needed in order to determine the exact mechanism of the reactions causing this phenomenon. 66 CHAPTER 5: SUMMARY AND CONCLUSIONS From this study, it was determined that the thermal stability of tagatose, with and without the presence of glycine, was influenced by temperature, pH, buffer concentration, and buffer type. Tagatose degradation was found to increase as the temperature increased, with the greatest tagatose loss seen at 80 C. Also, at pH 7 more tagatose degradation was seen than at pH 3. Buffer type and concentration also affected tagatose degradation, with higher levels of tagatose degradation occurring in phosphate buffer than citrate buffer, and an increase in tagatose loss seen at higher buffer concentrations. Overall, the most reactive solution observed was 0.1 M phosphate buffer at pH 7 and 80 C. Brown pigment formation was found to be higher in the solutions containing glycine than those only containing tagatose. As seen with the tagatose degradation, the brown pigment formation increased as the temperature and pH increased. Also, more browning occurred in the phosphate buffer than the citrate buffer, and the higher buffer concentration resulted in an increase in brown pigment formation. Browning occurred the fastest in the 0.1 M phosphate buffer at pH 7 and 80 C. 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Sixty-third report of the joint FAO/WHO expert committee on food additives. Geneva: World Health Organization. Wise BC. 2008. Try tagatose, an artificial sweetener. Available from: http://www.associatedcontent.com/article/778721/try_tagatose_an_artificial_swee tener.html. Accessed August 26, 2008. Wong DWS. 1989. Mechanism and theory in food chemistry. New York, NY: Van Nostrand Reinhold. p 105-15. 73 APPENDIX A TAGATOSE LOSS IN BUFFER SOLUTIONS WITHOUT GLYCINE 74 Table A1. Tagatose Degradation in 0.02 M Phosphate Buffer at pH 3 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) ND* ND* ND* ND* 0.0 0.05047 1.0 0.04897 2.0 0.04950 3.0 0.05066 4.0 0.05046 5.0 0.05153 6.0 0.04906 7.0 0.04890 8.0 0.05003 9.0 0.04960 10.0 0.04898 *ND = Not determined due to low reactivity at 80?C. Table A2. Tagatose Degradation in 0.02 M Phosphate Buffer at pH 7 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.04766 0.0 0.04890 0.0 0.04840 12.8 0.04671 4.0 0.04615 1.0 0.04650 24.0 0.04592 8.0 0.04576 2.0 0.04510 36.0 0.04395 12.0 0.04404 3.0 0.04420 47.8 0.04327 16.0 0.04232 4.0 0.04260 60.0 0.04198 28.0 0.04026 5.0 0.04160 72.0 0.04139 32.0 0.03876 6.0 0.04070 84.0 0.04094 36.0 0.03890 7.0 0.03980 96.0 0.04065 40.0 0.03829 8.0 0.03910 120.0 0.03901 48.0 0.03501 9.0 0.03840 144.0 0.03830 52.0 0.03587 10.0 0.03740 168.0 0.03727 56.0 0.03543 192.0 0.03671 215.8 0.03702 75 Table A3. Tagatose Degradation in 0.1 M Phosphate Buffer at pH 3 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.04984 0.0 0.05118 0.0 0.05045 12.8 0.04872 12.0 0.04969 1.0 0.05046 24.0 0.05015 24.0 0.04893 2.0 0.05015 36.0 0.04784 35.5 0.04796 3.0 0.04995 47.8 0.04960 47.8 0.04780 4.0 0.05079 60.0 0.04920 59.3 0.04903 5.0 0.04880 72.0 0.04943 71.8 0.04707 6.0 0.04872 84.0 0.04828 84.0 0.04730 7.0 0.04922 96.0 0.04884 96.0 0.04792 8.0 0.05051 120.0 0.04892 107.8 0.04744 9.0 0.05039 144.0 0.04737 120.0 0.04592 10.0 0.04937 168.0 0.04769 132.0 0.04547 192.0 0.04754 144.0 0.04654 215.8 0.04768 156.0 0.04519 168.0 0.04574 Table A4. Tagatose Degradation in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.04920 0.0 0.04964 0.0 0.04970 5.0 0.04649 1.0 0.04720 0.2 0.04870 9.8 0.04504 2.0 0.04681 0.3 0.04850 15.0 0.04309 3.0 0.04498 0.5 0.04730 24.0 0.04075 4.0 0.04313 0.7 0.04590 29.0 0.03965 5.0 0.04234 0.8 0.04670 34.0 0.03780 6.0 0.04060 1.0 0.04610 38.8 0.03618 7.0 0.03973 1.3 0.04490 48.0 0.03505 8.0 0.03810 2.0 0.04200 53.0 0.03356 9.0 0.03792 2.5 0.04060 58.0 0.03264 10.0 0.03674 3.0 0.03760 62.5 0.03139 3.5 0.03830 72.0 0.02945 4.0 0.03690 77.0 0.03051 4.5 0.03390 81.5 0.02754 5.0 0.03500 76 Table A5. Tagatose Degradation in 0.02 M Citrate Buffer at pH 3 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) ND* ND* ND* ND* 0.0 0.04905 1.0 0.04890 2.0 0.04963 3.0 0.04961 4.0 0.04978 5.0 0.04849 6.0 0.04881 7.0 0.04915 8.0 0.04919 9.0 0.04838 10.0 0.04941 *ND = Not determined due to low reactivity at 80?C. Table A6. Tagatose Degradation in 0.02 M Citrate Buffer at pH 7 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.04860 0.0 0.04964 0.0 0.04940 12.8 0.04721 4.0 0.04844 1.0 0.04795 24.0 0.04695 8.0 0.04707 2.3 0.04610 36.0 0.04645 12.0 0.04662 3.0 0.04580 47.8 0.04646 16.0 0.04541 4.0 0.04480 60.0 0.04569 24.0 0.04421 5.0 0.04310 72.0 0.04574 28.0 0.04451 6.0 0.04320 84.0 0.04459 32.0 0.04245 7.0 0.04299 96.0 0.04321 36.0 0.04277 8.0 0.04260 120.0 0.04218 40.0 0.04221 9.0 0.04060 144.0 0.04233 48.0 0.04144 10.0 0.04020 168.0 0.04155 52.0 0.04059 192.0 0.04201 56.0 0.04092 215.8 0.04034 77 Table A7. Tagatose Degradation in 0.1 M Citrate Buffer at pH 3 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.05011 0.0 0.05158 0.0 0.04960 12.8 0.04917 12.0 0.04956 1.0 0.04970 24.0 0.04965 24.0 0.04907 2.0 0.04850 36.0 0.04955 35.5 0.04810 3.0 0.04910 47.8 0.04848 47.8 0.04905 4.0 0.04930 60.0 0.04891 59.3 0.04738 5.0 0.04920 72.0 0.04864 71.8 0.04774 6.0 0.05000 84.0 0.04842 84.0 0.04725 7.0 0.05060 96.0 0.04873 96.0 0.04716 8.0 0.04950 120.0 0.04807 107.8 0.04636 9.0 0.04940 144.0 0.04891 120.0 0.04691 10.0 0.04980 168.0 0.04878 132.0 0.04595 192.0 0.04876 144.0 0.04644 215.8 0.04861 156.0 0.04532 168.0 0.04512 Table A8. Tagatose Degradation in 0.1 M Citrate Buffer at pH 7 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.05131 0.0 0.05047 0.0 0.04960 5.0 0.04989 1.0 0.04934 0.5 0.04790 9.8 0.04851 2.0 0.04928 1.0 0.04750 15.0 0.04793 3.0 0.04804 1.5 0.04630 24.0 0.04579 4.0 0.04795 2.0 0.04630 29.0 0.04600 5.0 0.04799 2.5 0.04490 34.0 0.04438 6.0 0.04838 3.0 0.04410 38.8 0.04578 7.0 0.04518 3.5 0.04340 48.0 0.04566 8.0 0.04698 4.0 0.04240 53.0 0.04207 9.0 0.04512 5.0 0.04240 58.0 0.04400 10.0 0.04451 6.3 0.04020 62.5 0.04341 7.5 0.03660 72.0 0.04518 77.0 0.03952 81.5 0.04093 78 APPENDIX B TAGATOSE LOSS IN BUFFER SOLUTIONS WITH GLYCINE 79 Table B1. Tagatose Degradation in 0.02 M Phosphate Buffer (with 0.05 M Glycine) at pH 3 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) ND* ND* ND* ND* 0.0 0.04954 1.0 0.05022 2.0 0.04948 3.0 0.05033 4.0 0.04848 5.0 0.05008 6.0 0.04948 7.0 0.04861 8.0 0.04902 9.0 0.04680 10.0 0.04933 *ND = Not determined due to low reactivity at 80?C. Table B2. Tagatose Degradation in 0.02 M Phosphate Buffer (with 0.05 M Glycine) at pH 7 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.04851 0.0 0.04887 0.0 0.05003 12.0 0.04625 4.0 0.04629 1.0 0.04747 24.0 0.04518 7.9 0.04551 2.0 0.04635 35.8 0.04411 12.0 0.04386 3.0 0.04553 48.0 0.04371 16.3 0.04202 4.0 0.04368 60.0 0.04219 24.0 0.04020 5.0 0.04258 72.0 0.04164 28.0 0.03983 6.3 0.04042 83.7 0.04099 31.8 0.03858 7.0 0.04193 95.9 0.03972 36.0 0.03685 8.0 0.03940 120.0 0.03897 39.7 0.03716 9.0 0.03904 144.0 0.03864 48.0 0.03644 10.0 0.03823 168.0 0.03756 52.0 0.03602 192.3 0.03612 56.0 0.03580 216.0 0.03654 60.0 0.03468 80 Table B3. Tagatose Degradation in 0.1 M Phosphate Buffer (with 0.05 M Glycine) at pH 3 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.04974 0.0 0.04665 0.0 0.05023 12.0 0.04955 12.6 0.04687 1.0 0.04866 24.0 0.04930 23.9 0.04647 2.0 0.04996 35.8 0.04936 36.0 0.04570 3.0 0.04904 48.0 0.04916 48.0 0.04560 4.0 0.04922 60.0 0.05027 60.0 0.04543 5.0 0.04898 72.0 0.04966 72.0 0.04481 6.0 0.05020 83.7 0.04944 84.0 0.04430 7.0 0.04903 95.9 0.04944 96.0 0.04375 8.0 0.04838 120.0 0.04827 108.2 0.04404 9.0 0.04784 144.0 0.05010 120.0 0.04356 10.0 0.04830 168.0 0.04938 132.0 0.04405 192.3 0.04831 144.0 0.04252 216.0 0.04740 156.2 0.04261 Table B4. Tagatose Degradation in 0.1 M Phosphate Buffer (with 0.05 M Glycine) at pH 7 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.04908 0.0 0.04728 0.0 0.04886 6.3 0.04429 1.0 0.04594 0.5 0.04501 10.5 0.04647 2.0 0.04340 1.0 0.04312 15.5 0.04517 3.0 0.04420 1.5 0.04151 24.0 0.03815 4.0 0.04296 2.0 0.04025 29.0 0.03526 5.0 0.04053 2.5 0.03788 33.0 0.03539 6.0 0.03836 3.0 0.03649 39.0 0.03397 7.0 0.03758 3.5 0.03474 48.0 0.03531 8.0 0.03567 4.0 0.03461 53.0 0.03330 9.0 0.03751 4.5 0.03521 57.0 0.02916 10.0 0.03689 5.0 0.03128 63.0 0.03182 5.5 0.03066 72.0 0.02934 6.0 0.03127 77.0 0.02963 6.5 0.02821 82.0 0.02798 7.0 0.02577 81 Table B5. Tagatose Degradation in 0.02 M Citrate Buffer (with 0.05 M Glycine) at pH 3 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) ND* ND* ND* ND* 0.0 0.05005 1.0 0.04997 2.0 0.04978 3.0 0.04979 4.0 0.04950 5.0 0.04896 6.0 0.04944 7.0 0.04848 8.0 0.04800 9.0 0.04972 10.0 0.04842 *ND = Not determined due to low reactivity at 80?C. Table B6. Tagatose Degradation in 0.02 M Citrate Buffer (with 0.05 M Glycine) at pH 7 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.04954 0.0 0.04895 0.0 0.04888 12.0 0.04764 4.0 0.04817 1.0 0.04872 24.0 0.04670 7.9 0.04619 2.0 0.04737 35.8 0.04670 12.0 0.04609 3.0 0.04615 48.0 0.04568 16.3 0.04485 4.0 0.04593 60.0 0.04490 24.0 0.04341 5.0 0.04479 72.0 0.04427 28.0 0.04183 6.3 0.04324 83.7 0.04371 31.8 0.04172 7.0 0.04317 95.9 0.04380 36.0 0.04087 8.0 0.04224 120.0 0.04294 39.7 0.04077 9.0 0.04130 144.0 0.04288 48.0 0.03887 10.0 0.04157 168.0 0.04084 52.0 0.03913 192.3 0.03975 56.0 0.03881 216.0 0.04150 60.0 0.03887 82 Table B7. Tagatose Degradation in 0.1 M Citrate Buffer (with 0.05 M Glycine) at pH 3 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.05161 0.0 0.04817 0.0 0.04998 12.0 0.04948 12.6 0.04909 1.0 0.04866 24.0 0.05033 23.9 0.04795 2.0 0.04921 35.8 0.04849 36.0 0.04769 3.0 0.04798 48.0 0.04958 48.0 0.04638 4.0 0.04930 60.0 0.04836 60.0 0.04783 5.0 0.04840 72.0 0.04995 72.0 0.04679 6.0 0.04924 83.7 0.04841 84.0 0.04665 7.0 0.04882 95.9 0.04923 96.0 0.04449 8.0 0.04986 120.0 0.04993 108.2 0.04309 9.0 0.04727 144.0 0.04895 120.0 0.04302 10.0 0.04956 168.0 0.04896 132.0 0.04431 192.3 0.04836 144.0 0.04122 216.0 0.04847 156.2 0.04185 Table B8. Tagatose Degradation in 0.1 M Citrate Buffer (with 0.05 M Glycine) at pH 7 as Affected by Temperature 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.04925 0.0 0.05035 0.0 0.04853 6.3 0.04887 1.0 0.04825 0.5 0.04819 10.5 0.04658 2.0 0.04782 1.0 0.04552 15.5 0.04464 3.0 0.04593 1.5 0.04542 24.0 0.04277 4.0 0.04687 2.0 0.04390 29.0 0.04203 5.0 0.04465 2.5 0.04393 33.0 0.04190 6.0 0.04341 3.0 0.04275 39.0 0.04080 7.0 0.04463 3.5 0.04220 48.0 0.04015 8.0 0.04447 4.0 0.04158 53.0 0.03955 9.0 0.04228 4.5 0.04095 57.0 0.04075 10.0 0.04379 5.0 0.03973 63.0 0.03923 5.5 0.04150 72.0 0.04230 6.0 0.04171 77.0 0.04032 6.5 0.04182 82.0 0.03896 7.0 0.04201 83 APPENDIX C BROWNING OF TAGATOSE IN BUFFER SOLUTIONS WITHOUT GLYCINE 84 Table C1. Browning of 0.05 M Tagatose in 0.02 M Phosphate Buffer at pH 3 as Affected by Temperature 60?C 70?C 80?C Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm ND* ND* ND* ND* 0.0 0.0154 1.0 0.0165 2.0 0.0160 3.0 0.0179 4.0 0.0167 5.0 0.0167 6.0 ND** 7.0 ND** 8.0 0.0156 9.0 0.0168 10.0 0.0171 *ND = Not determined due to low reactivity at 80?C. **ND = Not determined due to insufficient sample volume. Table C2. Browning of 0.05 M Tagatose in 0.02 M Phosphate Buffer at pH 7 as Affected by Temperature 60?C 70?C 80?C Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm 0.0 0.0020 0.0 -0.0001 0.0 0.0156 12.8 0.0076 4.0 0.0068 1.0 0.0194 24.0 0.0115 8.0 0.0266 2.3 0.0217 36.0 0.0288 12.0 0.0184 3.0 0.0263 47.8 0.0390 16.0 0.0230 4.0 0.0339 60.0 0.0519 24.0 0.0268 5.0 0.0442 72.0 0.0630 28.0 0.0460 6.0 0.0400 84.0 0.0466 32.0 0.0607 7.0 0.0542 96.0 0.0754 36.0 0.0636 8.0 0.0592 120.0 0.0830 40.0 0.0857 9.0 0.0707 144.0 0.0848 48.0 0.1168 10.0 0.0872 168.0 0.0890 52.0 0.0858 192.0 0.1212 56.0 0.0989 215.8 0.1154 85 Table C3. Browning of 0.05 M Tagatose in 0.1 M Phosphate Buffer at pH 3 as Affected by Temperature 60?C 70?C 80?C Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm 0.0 -0.0006 0.0 0.0025 0.0 0.0047 12.8 -0.0010 12.0 0.0050 1.0 0.0058 24.0 -0.0012 24.0 0.0060 2.0 0.0058 36.0 -0.0006 35.5 0.0077 3.0 0.0064 47.8 0.0008 47.8 0.0112 4.0 0.0074 60.0 0.0000 59.3 0.0139 5.0 0.0075 72.0 -0.0001 71.8 0.0164 6.0 0.0002 84.0 0.0018 84.0 0.0186 7.0 0.0035 96.0 0.0007 96.0 0.0205 8.0 0.0016 120.0 0.0050 107.8 0.0266 9.0 0.0024 144.0 0.0048 120.0 0.0390 10.0 0.0042 168.0 0.0064 132.0 0.0367 192.0 0.0093 144.0 0.0350 215.8 0.0093 156.0 0.0495 168.0 0.0423 Table C4. Browning of 0.05 M Tagatose in 0.1 M Phosphate Buffer at pH 7 as Affected by Temperature 60?C 70?C 80?C Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm 0.0 0.0014 0.0 0.0001 0.0 0.0000 5.0 0.0027 1.0 0.0029 0.2 0.0058 9.8 0.0070 2.0 0.0040 0.3 0.0063 15.0 0.0110 3.0 0.0052 0.5 0.0154 24.0 0.0179 4.0 0.0065 0.7 0.0243 29.0 0.0226 5.0 0.0114 0.8 0.0246 34.0 0.0299 6.0 0.0098 1.0 0.0184 38.8 0.0474 7.0 0.0137 1.3 0.0360 48.0 0.0438 8.0 0.0161 2.0 0.0766 53.0 0.0560 9.0 0.0193 2.5 0.0663 58.0 0.0585 10.0 0.0230 3.0 0.1764 62.5 0.0572 3.5 0.1643 72.0 0.0683 4.0 0.1685 77.0 0.1115 4.5 0.2986 81.5 0.0953 5.0 0.1922 86 Table C5. Browning of 0.05 M Tagatose in 0.02 M Citrate Buffer at pH 3 as Affected by Temperature 60?C 70?C 80?C Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm ND* ND* ND* ND* 0.0 0.0151 1.0 0.0167 2.0 0.0167 3.0 0.0166 4.0 0.0165 5.0 0.0168 6.0 0.0149 7.0 0.0163 8.0 0.0165 9.0 0.0172 10.0 0.0171 *ND = Not determined due to low reactivity at 80?C. Table C6. Browning of 0.05 M Tagatose in 0.02 M Citrate Buffer at pH 7 as Affected by Temperature 60?C 70?C 80?C Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm 0.0 0.0027 0.0 0.0001 0.0 0.0160 12.8 0.0043 4.0 0.0017 1.0 0.0176 24.0 0.0045 8.0 0.0033 2.3 0.0195 36.0 0.0063 12.0 0.0039 3.0 0.0203 47.8 0.0066 16.0 0.0066 4.0 0.0230 60.0 0.0081 24.0 0.0121 5.0 0.0258 72.0 0.0081 28.0 0.0149 6.0 0.0279 84.0 0.0071 32.0 0.0264 7.0 0.0318 96.0 0.0081 36.0 0.0271 8.0 0.0352 120.0 0.0185 40.0 0.0105 9.0 0.0412 144.0 0.0139 48.0 0.0267 10.0 0.0476 168.0 0.0116 52.0 0.0419 192.0 0.0049 56.0 0.0446 215.8 0.0196 87 Table C7. Browning of 0.05 M Tagatose in 0.1 M Citrate Buffer at pH 3 as Affected by Temperature 60?C 70?C 80?C Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm 0.0 -0.0002 0.0 0.0008 0.0 -0.0001 12.8 -0.0007 12.0 0.0006 1.0 0.0000 24.0 -0.0006 24.0 0.0012 2.0 -0.0001 36.0 0.0002 35.5 0.0020 3.0 0.0005 47.8 0.0017 47.8 0.0050 4.0 0.0007 60.0 0.0021 59.3 0.0049 5.0 0.0010 72.0 -0.0006 71.8 0.0080 6.0 -0.0002 84.0 0.0013 84.0 0.0072 7.0 0.0007 96.0 0.0002 96.0 0.0105 8.0 0.0005 120.0 0.0025 107.8 0.0155 9.0 0.0010 144.0 0.0037 120.0 0.0164 10.0 0.0019 168.0 0.0036 132.0 0.0277 192.0 0.0042 144.0 0.0214 215.8 0.0056 156.0 0.0363 168.0 0.0336 Table C8. Browning of 0.05 M Tagatose in 0.1 M Citrate Buffer at pH 7 as Affected by Temperature 60?C 70?C 80?C Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm Time (h) Absorbance at 420 nm 0.0 0.0013 0.0 0.0003 0.0 -0.0009 5.0 0.0029 1.0 0.0024 0.5 -0.0006 9.8 0.0019 2.0 0.0022 1.0 0.0050 15.0 0.0021 3.0 0.0028 1.5 0.0040 24.0 0.0085 4.0 0.0040 2.0 0.0081 29.0 0.0038 5.0 0.0060 2.5 0.0128 34.0 0.0028 6.0 0.0038 3.0 0.0174 38.8 0.0114 7.0 0.0066 3.5 0.0237 48.0 0.0021 8.0 0.0069 4.0 0.0356 53.0 0.0119 9.0 0.0115 5.0 0.0499 58.0 0.0279 10.0 0.0106 6.3 0.0749 62.5 0.0112 7.5 0.1049 72.0 0.0149 77.0 0.0187 81.5 0.0209 88 APPENDIX D BROWNING OF TAGATOSE AND GLYCINE IN BUFFER SOLUTIONS 89 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 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) ND* ND* ND* ND* 0.0 0.0030 1.0 0.0049 2.0 0.0046 3.0 0.0049 4.0 0.0081 5.0 0.0055 6.0 0.0046 7.0 0.0061 8.0 0.0057 9.0 0.0064 10.0 0.0066 *ND = Not determined due to low reactivity at 80?C. 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 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.0006 0.0 -0.0009 0.0 0.0045 12.0 0.0134 4.0 0.0115 1.0 0.0142 24.0 0.0301 7.9 0.0277 2.0 0.0252 35.8 0.0480 12.0 0.0514 3.0 0.0429 48.0 0.0621 16.3 0.0730 4.0 0.0634 60.0 0.0800 24.0 0.1024 5.0 0.0770 72.0 0.1518 28.0 0.1327 6.3 0.1037 83.7 0.1161 31.8 0.1587 7.0 0.1154 95.9 0.1518 36.0 0.1975 8.0 0.1673 120.0 0.2321 39.7 0.2288 9.0 0.1600 144.0 0.2218 48.0 0.3481 10.0 0.2369 168.0 0.2539 52.0 0.2604 192.3 0.4280 56.0 0.3143 216.0 0.3969 60.0 0.3466 90 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 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 -0.0008 0.0 -0.0007 0.0 0.0042 12.0 -0.0009 12.6 0.0022 1.0 0.0062 24.0 -0.0002 23.9 0.0052 2.0 0.0065 35.8 0.0020 36.0 0.0120 3.0 0.0071 48.0 0.0047 48.0 0.0199 4.0 0.0097 60.0 0.0038 60.0 0.0298 5.0 0.0087 72.0 0.0042 72.0 0.0385 6.0 0.0073 83.7 0.0072 84.0 0.0456 7.0 0.0110 95.9 0.0076 96.0 0.0683 8.0 0.0155 120.0 0.0137 108.2 0.0834 9.0 0.0148 144.0 0.0172 120.0 0.1026 10.0 0.0167 168.0 0.0278 132.0 0.1156 192.3 0.0299 144.0 0.1304 216.0 0.0352 156.2 0.1420 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 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.0012 0.0 -0.0006 0.0 0.0029 6.3 0.0184 1.0 0.0085 0.5 0.0135 10.5 0.0362 2.0 0.0151 1.0 0.0233 15.5 0.0522 3.0 0.0216 1.5 0.0354 24.0 0.1041 4.0 0.0350 2.0 0.0527 29.0 0.1234 5.0 0.0409 2.5 0.0774 33.0 0.1406 6.0 0.0502 3.0 0.0931 39.0 0.2085 7.0 0.0684 3.5 0.1278 48.0 0.3696 8.0 0.0810 4.0 0.1526 53.0 0.3161 9.0 0.1008 4.5 0.1794 57.0 0.3051 10.0 0.1206 5.0 0.2389 63.0 0.3760 5.5 0.2692 72.0 0.5581 6.0 0.3241 77.0 0.4819 6.5 0.3144 82.0 0.5445 7.0 0.3627 91 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 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) ND* ND* ND* ND* 0.0 0.0026 1.0 0.0044 2.0 0.0044 3.0 0.0047 4.0 0.0055 5.0 0.0052 6.0 0.0068 7.0 0.0054 8.0 0.0055 9.0 0.0086 10.0 0.0072 *ND = Not determined due to low reactivity at 80?C. 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 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.0017 0.0 -0.0002 0.0 0.0047 12.0 0.0037 4.0 0.0025 1.0 0.0088 24.0 0.0075 7.9 0.0079 2.0 0.0110 35.8 0.0144 12.0 0.0159 3.0 0.0235 48.0 0.0223 16.3 0.0280 4.0 0.0260 60.0 0.0279 24.0 0.0523 5.0 0.0318 72.0 0.0196 28.0 0.0655 6.3 0.0513 83.7 0.0480 31.8 0.0770 7.0 0.0457 95.9 0.0641 36.0 0.0922 8.0 0.0589 120.0 0.0603 39.7 0.1173 9.0 0.1152 144.0 0.0792 48.0 0.1530 10.0 0.0998 168.0 0.1193 52.0 0.1543 192.3 0.2065 56.0 0.1681 216.0 0.1134 60.0 0.2014 92 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 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 -0.0007 0.0 -0.0002 0.0 0.0029 12.0 0.0001 12.6 0.0026 1.0 0.0050 24.0 -0.0002 23.9 0.0026 2.0 0.0070 35.8 0.0007 36.0 0.0076 3.0 0.0059 48.0 0.0014 48.0 0.0163 4.0 0.0065 60.0 0.0035 60.0 0.0183 5.0 0.0063 72.0 0.0021 72.0 0.0260 6.0 0.0047 83.7 0.0026 84.0 0.0250 7.0 0.0082 95.9 0.0038 96.0 0.0776 8.0 0.0080 120.0 0.0068 108.2 0.0564 9.0 0.0084 144.0 0.0099 120.0 0.0713 10.0 0.0110 168.0 0.0123 132.0 0.0944 192.3 0.0198 144.0 0.0946 216.0 0.0193 156.2 0.1220 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 60?C 70?C 80?C Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) Time (h) Tagatose Concentration (M) 0.0 0.0014 0.0 0.0002 0.0 0.0013 6.3 0.0035 1.0 0.0020 0.5 0.0050 10.5 0.0055 2.0 0.0023 1.0 0.0094 15.5 0.0097 3.0 0.0049 1.5 0.0148 24.0 0.0170 4.0 0.0058 2.0 0.0213 29.0 0.0217 5.0 0.0104 2.5 0.0279 33.0 0.0206 6.0 0.0128 3.0 0.0330 39.0 0.0333 7.0 0.0164 3.5 0.0433 48.0 0.0364 8.0 0.0165 4.0 0.0485 53.0 0.0502 9.0 0.0250 4.5 0.0609 57.0 0.0616 10.0 0.0260 5.0 0.0616 63.0 0.0771 5.5 0.0671 72.0 0.0857 6.0 0.0873 77.0 0.0907 6.5 0.1052 82.0 0.0854 7.0 0.1201 93 APPENDIX E AVERAGE TEMPERATURES OF TAGATOSE AND TAGATOSE-GLYCINE BUFFER SOLUTIONS 94 Table E1. Average Temperature of Tagatose Buffer Solutions Sample Temperature (?C) pH 3 0.02 M phosphate ND ND 81.37 0.02 M citrate ND ND 81.35 0.1 M phosphate 62.26 71.05 81.30 0.1 M citrate 62.26 71.05 80.58 pH 7 0.02 M phosphate 62.26 70.95 81.35 0.02 M citrate 62.26 70.95 81.50 0.1 M phosphate 61.06 71.03 79.39 0.1 M citrate 61.06 71.03 79.82 Table E2. Average Temperatures of Tagatose-Glycine Buffer Solutions Sample Temperature (?C) pH 3 0.02 M phosphate ND ND 81.33 0.02 M citrate ND ND 81.33 0.1 M phosphate 61.32 71.21 81.67 0.1 M citrate 61.32 71.21 81.67 pH 7 0.02 M phosphate 61.32 71.42 82.16 0.02 M citrate 61.32 71.42 82.16 0.1 M phosphate 61.51 71.35 81.72 0.1 M citrate 61.51 71.35 81.72