Chemical and Physical Stability of Powdered Tagatose as Affected by Temperature and Relative Humidity by Lenese D. Grant A thesis submitted to the Graduate Faculty of Auburn University in partial fulfillment of the requirements for the Degree of Master of Science Auburn, Alabama December 13, 2010 Keywords: tagatose, prebiotic, deliquescence, stability Approved by Leonard N. Bell, Chair, Professor, Professor of Poultry Science Oladiran Fasina, Associate Professor of Biosystems Engineering Tung-Shi Huang, Associate Professor of Poultry Science ii Abstract Tagatose is a reduced-calorie monosaccharide that displays prebiotic properties. Water can interact with powdered tagatose to varying extents, depending upon the storage environment. Adsorbed water can impact the physical and chemical stability of tagatose, altering its functionality and usability as an ingredient. Therefore, the objective of this study was to evaluate the physical and chemical stability of bulk tagatose powder as a function of relative humidity (RH) and temperature. Saturated salt solutions were used to create environments having RH values of 33%, 54%, 75% and 85% at 20?C. Tagatose (0.3-0.5 g) was placed in vials and stored in desiccators (i.e., relative humidity chambers) at 20?C, 30?C and 40?C. Duplicate vials were removed at regular time intervals for 12 months. Moisture contents (MC) and physical characteristics were monitored monthly. Samples were dissolved in water and analyzed using HPLC to quantify tagatose degradation. Early stages of browning were measured at 280 nm, whereas brown pigment formation was measured at 420 nm. Critical relative humidity was determined at 20, 30 and 40?C. Using saturated tagatose solutions, the critical RH associated with deliquescence (RH0) was 85% at 20?C. MC values below RH0 were all less than 2% (db). The average MC at 85%RH ranged from 53-80% (db), increasing as temperature decreased. At 33%RH/20?C tagatose remained free flowing. As either temperature or RH increased, varying degrees of physical caking occurred. At 85%RH, tagatose deliquesced at all temperatures. Browning occurred in all samples at 40?C. iii Despite physical caking and browning, tagatose degradation was only observed in the deliquesced sample at 85%RH/40?C, where a 20% loss occurred during the study. Although RH and temperature must become extreme for tagatose degradation to occur, intermediate RHs and temperatures promote physical caking and deliquescence, which create handling problems during product formulation. Tagatose should be stored in water impermeable packaging at 20?C rather than bulk storage bins in an uncontrolled environment. iv Acknowledgments I would like to thank God for allowing me to advance this far in my education. Sincere gratitude is expressed to my major advisor, Dr. Leonard Bell, for his patience, guidance and expertise. I appreciate the time, direction, and knowledge of my committee members, Drs. Oladiran Fasina and Tung-Shi Huang. Gratitude is extended to the advisors of the Bridge to the Doctorate program, Drs. Overton Jenda and Florence Holland. They continue to encourage me to reach my goals. I would like to thank my immediate family as well as my extended family and friends for their encouragement and motivation. Special thanks to David Grant (father), Fagale Grant (mother), David M. Grant (brother), Loretta Grant (sister), Hurdis Milner-Bozeman, Joyce Hall, Jesse Hall, Justin Vaughner, James Miller, Coretta Collins, Tamisha Jones, Ashley Bryant-Massey, Miniayah DeBruce, Shannon Coleman and Lauren Parker. Appreciation is expressed to my former lab mate Katie Luecke for her kindness and assistance with HPLC. To my uncle (Dr. James Shuford) who introduced me to food science and set an example of academic success, thank you. Although these two persons are no longer with me, they inspired me greatly; thank you Cora Shuford (grandmother) and Charlena Miller (aunt and kindergarten teacher). Lastly, I am grateful for funding from the National Science Foundation and the Alabama Agricultural Experiment Station as well as tagatose from Arla Food Ingredients. v Table of Contents Abstract...............................................................................................................................ii Acknowledgments..............................................................................................................iv List of Tables.....................................................................................................................vii List of Figures.....................................................................................................................ix Chapter 1. Introduction.......................................................................................................1 Chapter 2. Literature Review..............................................................................................3 Water activity, moisture content and relative humidity...........................................3 Properties that affect water activity.........................................................................5 Chemical stability of food systems..........................................................................6 Physical stability of food systems............................................................................8 Methods for measuring the critical relative humidity............................................11 Deliquescence........................................................................................................12 Tagatose properties and applications.................................................................... 22 Tagatose as a prebiotic...........................................................................................24 Tolerance of tagatose.............................................................................................29 Metabolism of tagatose..........................................................................................32 Tagatose in foods...................................................................................................34 Stability of tagatose in solution.............................................................................37 Chapter 3. Materials and Methods....................................................................................41 Sample preparation................................................................................................ 41 Particle size characterization..................................................................................41 Preparation of saturated salt solution..................................................................... 42 Experimental Overview......................................................................................... 43 Physical stability................................................................................................... 44 Moisture content .................................................................................................. 45 Photographs........................................................................................................... 45 Critical relative humidity determination............................................................... 46 Chemical degradation analysis............................................................................. 46 Browning of tagatose............................................................................................ 48 vi pH..........................................................................................................................49 Chapter 4. Results and Discussion....................................................................................50 Moisture sorption...................................................................................................50 Physical stability....................................................................................................53 Critical relative humidity.......................................................................................56 Tagatose browning.................................................................................................57 Chemical stability..................................................................................................63 Chapter 5. Summary and Conclusion...............................................................................69 References..........................................................................................................................70 Appendix A........................................................................................................................77 Appendix B........................................................................................................................91 Appendix C........................................................................................................................93 Appendix D........................................................................................................................97 vii List of Tables Table 3.1 Average particle size distribution of food grade tagatose as a percentage with standard deviation..................................................................42 Table 3.2. Saturated salt solution relative humidity as a function of temperature (Bell and Labuza 2000)...................................................................................................44 Table 4.1. Actual RH values associated with temperatures and the notation that will be used throughout this section.............................................................................50 Table 4.2. Physical characteristics of tagatose as affected by RH and temperature after 12 months and time at which final physical state was observed......................54 Table 4.3. Environmental RH in which tagatose deliquescence was observed and RH0 of tagatose at 20, 30 and 40?C.............................................................................57 Table 4.4. Pseudo-zero order rate constants (OD/g/d) with 95% CL times 104 for browning of tagatose at 20, 30 and 40?C..........................................................60 Table A1. Moisture content, physical appearance, optical density and tagatose remaining of samples stored over drierite at room temperature........................................78 Table A2. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 33% RH at 20?C...............................................................79 Table A3. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 33% RH at 30?C...............................................................80 Table A4. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 33% RH at 40?C...............................................................81 Table A5. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 54% RH at 20?C...............................................................82 Table A6. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 54% RH at 30?C...............................................................83 Table A7. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 54% RH at 40?C.............................................84 viii Table A8. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 75% RH at 20?C...........................................85 Table A9. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 75% RH at 30?C...........................................86 Table A10. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 75% RH at 40?C...........................................87 Table A11. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 85% RH at 20?C...........................................88 Table A12. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 85% RH at 30?C...........................................89 Table A13. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 85% RH at 40?C...........................................90 Table B1. Duplicate pH values for tagatose at time 6, 9 and 12 months.........................92 ix List of Figures Figure 2.1. Moisture sorption data of sucrose, citric acid anhydrous and 50/50 mix of both components at 25?C. The RH0 is determined where the arrows meet. Adapted from Salameh and Taylor (2006b)..................................................12 Figure 2.2. Depiction of deliquescence process. Adapted from Van Campen and others (1983).................................................................................................13 Figure 4.1. Moisture adsorption profile of samples stored at 85% RH at 20, 30 and 40?C........................................................................................................52 Figure 4.2. Moisture sorption isotherm for tagatose at 20, 30 and 40?C after exposure for 12 months.................................................................................................52 Figure 4.3. Tagatose at 20?C after 12 months, left to right, 33, 54, 75 and 85% RH......54 Figure 4.4. Tagatose at 30?C after 12 months, left to right, 33, 54, 75 and 85% RH......55 Figure 4.5. Tagatose at 40?C after 12 months, left to right, 33, 54, 75 and 85% RH......55 Figure 4.6. Early stages of browning in tagatose at 40?C as affected by RH..................59 Figure 4.7. Brown pigment formation in tagatose at 40?C as affected by RH................60 Figure 4.8. Figure 4.8. The effect of time on browning of tagatose at 40?C, left to right, after 6 months, 9 months and 12 month. Tagatose at 33, 54, 75 and 85% RH top to bottom.................................................................................................61 Figure 4.9. Rate constants for tagatose browning (420 nm) as a function of %RH at 40?C. 62 Figure 4.10. Chemical degradation of powdered tagatose at 40?C as affected by RH......64 Figure C1. Duplicate pictures of tagatose at time 6.........................................................94 Figure C2. Duplicate pictures of tagatose at time 9.........................................................95 Figure C3. Duplicate pictures of tagatose at time 12.......................................................96 Figure D1. Moisture content (db) vs. time for particles < 250 nm at 85% RH/20?C.....100 x Figure D2. Moisture content (db) vs. time for particles > 500 nm at 85% RH/20?C.....100 1 Chapter 1: Introduction The food industry utilizes a large number of food ingredients that come in bulk powdered forms. These ingredients may include flour, powdered supplements, confectionery sugar and many other items. There are many advantages to using dry ingredients, such as a longer shelf life due to a lower inherent water activity than liquid ingredients. However, what happens when the environmental storage conditions become unfavorable for powdered ingredients? This is likely to occur in the confectionery industry and bakeries where thermal processes may cause an increase in the temperature of the environment. These food manufacturing facilities often do not control their environmental conditions due to economical purposes. Therefore, precautions must be taken to prevent or limit the exposure of ingredients which are susceptible to caking and deliquescence (liquification) to extreme environmental conditions, such as elevated temperatures and relative humidities. More importantly, adverse physical changes, such as caking and deliquescence may increase the risk of chemical degradation, causing loss of beneficial properties. One powdered ingredient that may be susceptible to caking and deliquescence and subsequent chemical degradation is tagatose, because sugars are known to deliquesce (Hancock and Shamblin 1998; Salameh and Taylor 2006a) Tagatose is a monosaccharide that has a structure similar to fructose, with the exception of the hydroxyl group that is inverted on carbon 4 (Levin 2002). Studies have shown that tagatose displays prebiotic properties (Venema and others 2005; Laerke and others 2000). Prebiotics are important because they facilitate the formation of 2 beneficial bacteria in the intestines. In addition to its prebiotic properties, tagatose is approximately 92% as sweet as sucrose, but has a lower caloric value (Levin 2002). Therefore, it may have many applications in foods as a nutritional additive or sweetener. However, limited research has been conducted that addresses how bulk powdered tagatose reacts to various environmental conditions that could be encountered in an industrial setting. Due to the versatility and novelty of tagatose, the effect of environmental storage conditions should be investigated. The acceptability of tagatose will be jeopardized if environmental storage conditions affect its storage stability. Therefore, the purpose of the research was to investigate how environmental conditions affect tagatose physical and chemical stability. 3 Chapter 2: Literature Review Water plays a critical role in foods that can affect their palatability, shelf life, processing and storage conditions. Water activity, moisture content and relative humidity are related, as will be explained later. After extensive research, it has been established that water activity, in particular, significantly affects the microbial, physical and chemical stability of various foods. This section will focus primarily on the food?s physical and chemical attributes. Water Activity, Moisture Content and Relative Humidity Water activity (aw) may be defined in terms of vapor pressure, chemical potential and availability. One definition of water activity is ?the ratio of the equilibrium partial vapor pressure of water in the system (pw) to the equilibrium partial pressure (pow) of pure liquid water at the same temperature? (Reid 2007). Water activity is also a thermodynamic property, based on the chemical potential of water in a food system. At equilibrium, the chemical potential of water in the food is equal to the chemical potential of the surrounding water in the atmosphere. Consequently, the vapor pressure of water in a food is equal to the vapor pressure of water vapor in the surroundings (Roos 2007). Due to its thermodynamic properties, aw determines the direction of moisture movement until equilibrium is obtained; however, the equilibration rate is unknown (Bell 2007). Water activity may also be described as the energy status of water and its availability to 4 behave as a solvent and reactant, causing changes in chemical and physical stability (Chirife and Fontana 2007). Water activity and relative humidity (RH) are quite similar terms, where %RH is equal to the product of aw and 100. When the water in food has equilibrated with the surrounding environment, the percent relative humidity is termed the equilibrium relative humidity (ERH). The aw can be determined by using the following equation: aw = %ERH/100 (Bell and Labuza 2000). Moisture content also has a dramatic effect on the stability of food. Moisture content is usually expressed on a dry basis as a percentage or x grams of water/ 100 g solids (food). Dry basis moisture content may be plotted against water activity creating a moisture sorption isotherm. A moisture sorption isotherm is an illustration that shows ?the steady state amount of water held by the food solids as a function of water activity or storage % relative humidity at constant temperatures? (Labuza and Alrunakar 2007). Moisture adsorption isotherms are critical to understanding the stability of foods because they allow predictions to be made about reactions that will occur at specified moistures. Moreover, isotherms also allow modifications of aw to be made with the selection of appropriate ingredients (Labuza and Alrunakar 2007). Although moisture content is important, it may be difficult to make predictions regarding stability since it encompasses all water, not just water that is available for the reaction. Therefore, aw provides means for a more suitable prediction of chemical and physical stability as it relates to water behaving as a solvent and reactant (Bell 2007). 5 Properties that Affect Water Activity The properties that will be discussed have a lowering effect on aw. They include the capillary effects, colligative properties, and surface interactions. Foods, including powders, may contain capillaries, which are small pores and channels where water may exist. The curved liquid meniscus in the pores causes a lowering effect in the vapor pressure above this area compared to that of pure water. This is due to changes in hydrogen bonding directly related to the surface curvature. Increased water molecules are able to interact with each other on the curved capillary surface, which depresses the water activity. Foods generally contain pores ranging in radius from 0.1 to 300 ?m, with most foods having pores between 10 ?m and 300 ?m (Farkas and Singh 1990; Xiong and others 1991; Hicsasmaz and Clayton 1992). Generally, larger capillaries do not have a pronounced effect on aw; however, smaller capillaries (less than 0.1 ?m) have a greater effect on lowering aw (Bell and Labuza 2000). The second factor that plays a role in aw lowering is surface interaction. Surface interaction affects water activity because water interacts with other chemical molecules through hydrogen bonding, dipolar-ionic interactions, dipole-dipole forces, ionic bonding, and van der Waals interactions. Because of the interaction between water and other molecules, water molecules need additional energy to go from the liquid to vapor phase. Consequently, the water molecules are less able to move into the vapor phase, causing a depressed aw. Another concept that is associated with surface interaction is the monolayer value. The monolayer value is the moisture content at which the food?s surface contains a single layer of water molecules. The monolayer is significant because 6 it often correlates with optimum chemical stability for low moisture products (Bell and Labuza 2000). The last property that affects water activity is the colligative property of vapor pressure lowering. Colligative properties include freezing point depression, boiling point elevation, vapor pressure lowering and osmotic pressure lowering. This section will focus primarily on vapor pressure lowering. As mentioned earlier, water activity is defined as the partial vapor pressure of water in the food divided by the vapor pressure of pure water at constant temperature. Therefore, vapor pressure is directly related to water activity. When a solute interacts with water through ionic, dipolar or hydrogen bonding, the escaping tendency of water as well as the chemical potential of water is reduced, resulting in decreased water activity. The water/solute ratio, molecular weight and degree of interaction also play a role in aw lowering by solutes (Bell and Labuza 2000). Chemical Stability of Food Systems Stability is characterized by the ?ability of a substance to resist change over a specific period of time? (Bell 2007). The length of storage time a product remains acceptable is referred to as the storage stability or shelf life of the product. Various environmental conditions, such as relative humidity and temperature, affect the chemical and physical stability of food products. Chemical instability may alter the nutritional value, color, and flavor of foods. In addition, changes in chemical stability may alter the pH or cause degradation of food products (Bell 2007). Bell (2007) mentions a number of chemical reactions that alter the chemical stability of food products causing deterioration. Hydrolysis or cleavage with the addition of water is one example. This phenomenon occurs in diet carbonated beverages; due to 7 the acidic environment, aspartame hydrolysis occurs causing a loss of sweetness. Oxidation is another chemical change that may affect oils and vitamins causing changes in sensory characteristics, appearance and nutritional value. Chemical browning may occur due to enzymatic reactions, nonenzymatic reactions, or the Maillard reaction (Bell 2007). Browning primarily increases with increasing aw. One browning reaction that is common in the food industry is caramelization. Caramelization is a nonenzymatic reaction that involves the breakdown of monosaccharides by heat, generating a caramel? like flavor and a brown pigment (Troller and Christian 1978). Sometimes these reactions may occur simultaneously resulting in increased deterioration of food quality and shelf life. One factor that plays a large role in chemical stability is temperature. Chemical reactions may accelerate when temperatures are increased and decelerate when temperatures decrease (Bell 2007). For example, refrigeration is often used to prevent certain chemical reactions from occurring. Temperature has one of the most pronounced effects on chemical stability. The effect of temperature on chemical stability can be modeled by the Arrhenius equation or shelf life plots (Bell 2007). Water activity and moisture content may also contribute to the chemical stability of foods. Water is required for the dissolving, diffusing and reacting of chemicals in foods. Moisture affects the viscosity of the reaction medium as well as reactant mobility; for example, a less viscous medium may increase degradation by reactants becoming more mobile. When water activity and moisture content continue to increase above the amount necessary for complete reactant dissolution, reactants become diluted, causing the reaction rate to decrease. Consequently, chemical reactivity and aw typically have a 8 proportional relationship, increasing until maximum reactivity occurs, followed by a decrease in reactivity (Bell 2007). As mentioned previously, most reactions cease at the monolayer due to low reactant dissolution and restricted reactant mobility. Physical Stability of Food Powders Like chemical stability, physical stability is also impacted by water activity, moisture content, temperature and time. The glass transition temperature is one factor that impacts physical stability. The glass transition is defined as ?a relaxation process occurring in food solids during transformation of noncrystalline solids to a more liquid- like supercooled state? (Roos 2007). More simply stated, glass transition is the change of an amorphous material from a glassy state to a rubbery state. Water can behave as a plasticizer, which allows for better movement of components in the material (Roudaut 2007). Plasticization can cause amorphous glassy foods to convert into the amorphous rubbery state. A significant decrease in viscosity and increase in molecular mobility also occurs (Bell 2007). These phase conversions are important in understanding the physical stability of foods because changes in temperature or water activity can change the physical state of the product, thus altering its physical stability. For powders, physical stability at the macroscopic level may be described in terms of caking, stickiness, collapse and deliquescence (liquification of a solid due to exposure at very high RH). While there are other occurrences, such as fat melting, surface crystals solubilization and electrostatic attraction that may contribute to caking, this literature review will focus primarily on moisture-induced caking. Caking occurs when individual particles of a free flowing powder stick to each other causing a larger mass to be formed (Roudaut 2007). Moreover, caking may also be described as the 9 conversion of free flowing powder to a substance with aggregates and lumps, causing difficulty in mechanical handling (Salameh and Taylor 2006a). Stickiness is defined in terms of the sticky point of the substance, which is ?the temperature at which the power needed to stir the powder in a tube increases sharply? (Roudaut 2007). Maintaining a free flowing product is important for numerous reasons including, functionality, storage, formulation and mixing (Adhikari 2001). Caking occurs through a variety of mechanisms including the following: 1) formation of liquid bridges, which may form solid bridges, 2) electrostatic or van der Waals forces, 3) mechanical shape interlocking, and 4) particle surface sintering (Adhikari and others 2001; Salameh and Taylor 2006a). Foods that typically experience caking and structural collapse are powdered products with elevated amounts of minerals, soluble sugars or protein hydrolysates. The extent of caking varies from easily breakable aggregates to extremely hard lumps. The amount of caking also varies from a small lump to the entire powder being caked. As a result of physical changes in powders, physical properties such as flowability, water dispersibility, handling properties, appearance and solubility are adversely affected (Roudaut 2007). Moisture-induced caking may occur as a result of drying or storage. Powders may become hydrated through accidental wetting, water sorption, or moisture condensation, which causes the particle surface to become plasticized allowing contact with neighboring particles through interparticle liquid bridging. Water may contact particle surfaces through capillary condensation or adsorbed mono/multilayers (Adhikari and others 2001). Caking occurs in several stages causing a free flowing powder to eventually turn into a solid mass. The beginning stages of caking occur when fine 10 particles develop into larger particles through agglomeration. The initial stages of caking may be desirable because handling and wetability may improve. As the powder becomes more hydrated, the particle bridges thicken and porosity decreases, causing the powder to become compact and collapse. Caking may be quantified by using a caking index, which corresponds to the percentage of sample retained by a specified mesh (Roudaut 2007). The caking kinetics of fish hydrolysates were investigated by storing the sample at humidities of 0.75, 0.63, 0.53 and 0.44. The higher the humidity or temperature was, the higher the caking index (Aguilera and others 1995). A fundamental property that influences caking is glass transition, which is the temperature at which change from a glassy phase to a rubbery phase takes place. Lowering the glass transition temperature below ambient temperatures by plasticization of amorphous food powders produces optimum conditions for caking and sticking (Adhikari and others 2001). Although sugars appear to be in crystalline form at room temperature, they are usually labeled amorphous due to the effect of size reduction operations (Kelley and others 1974). In the anhydrous state, amorphous sugars have glass transition temperatures between 5-100 ?C, depending on the type, that may be further lowered due to water plasticization. Sugars with low molecular weight are more susceptible to caking and stickiness because the glass transition temperature decreases with decreasing molecular weight. Moreover, if the glass transition temperature of a powder is below the storage temperature, caking, stickiness and agglomeration are sure to occur (Adhikari and others 2001). Particle size also has a dramatic effect on caking mechanisms. Powders with a particle size greater than 200 ?m are generally free flowing, while smaller particles may 11 experience cohesion and their flowability significantly affected. Cohesion is an internal property of a powder and a measure of the force binding two particles together. Smaller particles (less than or equal to 1 ?m) are subject to electrostatic or molecular forces which may significantly deform the particles creating a larger contact area (Adhikari and others 2001). The larger surface area promotes faster moisture adsorption, which may lead to extensive caking and deliquescence. Methods for Measuring the Critical Relative Humidity In order to understand deliquescence or a solid liquefying at elevated RH levels, the concept of critical relative humidity must first be addressed. The critical relative humidity, RH0, is the humidity at which deliquescence begins. Critical relative humidity is material specific and often times temperature sensitive. Two methods are generally used to determine the critical relative humidity (RH0) of a material. The first method involves measuring the water activity of a saturated solution of the material using a water activity meter. In theory, when deliquescence takes place, the solid particles are surrounded by a saturated solution at the critical relative humidity. When this method is used, the equilibrium water activity of the saturated solution may be referred to as RH0. The other method that may be used to determine RH0 involves moisture sorption data. A series of saturated salt solutions, with increasing aw, are used to get a representative number of moisture sorption data points at constant temperature. The samples are weighed at various time points to ensure equilibration and monitor weight gain (or moisture uptake). Following equilibration, a graph is generated with moisture content (db) or change in weight as a function of water activity. The steep increase in weight change is where the deliquescence occurs (Mauer and Taylor 2010). The linear portions 12 before and after the deliquescence are extrapolated to obtain the RH0 at the intersection. Figure 2.1 shows moisture sorption data used to obtain RH0 (Salameh and Taylor 2006b). When obtaining RH0, these two methods usually agree (Salameh and Taylor 2006b). Figure 2.1. Moisture sorption data of sucrose, citric acid anhydrous and 50/50 mix of both components at 25?C. The RH0 is determined where the arrows meet. Adapted from Salameh and Taylor (2006b). Deliquescence Although extensive amounts of work have been conducted on various aspects of water activity, few studies have correlated water activity or caking with the phenomenon of deliquescence. Deliquescence is a ?water solid interaction that is known to cause vapor condensation in highly water-soluble and highly crystalline solids at water activities less than 1 or relative humidities less than 100%? (Salameh and Taylor 2006a). In other 13 words, deliquescence is the dissolution of a material due to water adsorption at high water activities. Figure 2.2 shows a schematic of the deliquescence process. When the environmental RH is less than RH0, the powder remains in solid form. As the environmental RH increases, water condenses around the solid. When the RH0 exceeds the environmental RH, complete dissolution or deliquescence of the solid occurs. Caked powders that continue to absorb water may eventually undergo deliquescence. Figure 2.2. Depiction of deliquescence process. Adapted from Van Campen and others (1983). Liquid bridges contribute to deliquescence and may be created by two mechanisms. Liquid bridges are formed when neighboring particles interact with another surface that has come into contact with water. In the first mechanism, elevated RH values allow increased water vapor sorption, causing increased surface wetting and plasticization of the solid regions of the powder. The second mechanism is also caused by a high RH, which allows deliquescence at the surface of the solid and development of a concentrated thin film of solution at the particle surface (Salameh and Taylor 2006b). Increasing RH RH RH 0 Solid Particles Solution Particles and Solution 14 Even though certain powders are deliquescent, when they are held at low RHs, they absorb small amount of water through hydrogen bonding. However, when the critical RH (RH0) is exceeded, the solid dissolves in the condensate film to produce a saturated solution (Salameh and Taylor 2006a). The RH0 is material and temperature specific and is the RH at which deliquescence is initially observed (Hancock and Shamblin 1998). As the RH continues to increase, more solid dissolution occurs due to vapor condensation, ultimately leading to complete deliquescence (Salameh and Taylor 2006a). Hancock and Shamblin (1998) reported that crystalline sugars and sugar derivatives may experience deliquescence at RHs higher than 65%. Deliquescence may accelerate chemical reactions and physical changes in powdered systems (Hancock and Shamblin 1998). Hiatt and others (2008) conducted a study examining the chemical stability of vitamins B1, B6, and C in powdered blends. Exploring powdered blends is important because deliquescence lowering occurs, meaning the critical RH of the mixture (RH0mix) is lower than RH0 of the individual components, so deliquescence occurs more readily. Consequently, this may also increase chemical reactivity. The study explored single, binary, ternary and quaternary combinations of thiamin HCl, fructose, sodium ascorbate, and pyridoxine. Environmental chambers and salt solutions were used to create RHs of 43%, 54%, 59%, 64%, 85% and 98%. Powders at the various RHs were stored for 1, 2, 4, 8, and 12 weeks. Critical relative humidities were determined using moisture sorption isotherms and saturated salt solutions, and powders were stored above and below these RHs. Physical characteristics and moisture contents were monitored. Chemical 15 degradation was determined using high-performance liquid chromatography (HPLC) (Hiatt and others 2008). Following 12 weeks at 98% RH, thiamin HCl was fully deliquesced and sodium ascorbate deliquesced to a dark brown solution with some precipitate. Both powders experienced some caking at 54% RH. Pyridoxine did not experience deliquescence even when stored at 94% RH whereas fructose deliquesced at 64% RH. The mixtures deliquesced at RHs below the RH0 values of the separate powders. Thiamin and ascorbate acid experienced the greatest degradation above the RH0, while pyridoxine experienced minimum degradation (Hiatt and others 2008). Deliquescence lowering in binary mixtures has also been observed in pharmaceutical ingredients (Salameh and Taylor 2005). Salameh and Taylor (2006a) looked at caked-induced deliquescence in various single and binary mixtures of different particle sizes (ground and unground). Caked-induced deliquescence can be attributed to the formation of liquid bridges that occur due to partial deliquescence. Following partial deliquescence, recrystallization can occur that causes solid bridge formation and caking. Citric acid, fructose and glucose were stored at RHs below and above their critical relative humidities as single components and as binary mixtures. Relative humidities of 33%, 54%, 65%, 71%, 81%, 85%, and 94% were created using saturated salt solutions and designated powders were exposed to each condition. Various cycles were used to expose samples to selected RHs, with one cycle storing the sample above the RH0mix for 3 days and below the RH0mix for 3 days. A total of three different blends of ground and unground samples were used and they included the following: 50:50 ratio of unground 16 compounds, 50:50 ratio of ground compounds, and a 50:50 ratio of unground and ground compounds. Cake mechanical strength was tested using a three point beam-bending method. Scanning electron microscopy was used to observe deliquescence-induced caking. Critical relative humidities were established through vapor sorption profiles and using a water activity meter (Salameh and Taylor 2006a). Like the previous study discussed, mixed powders (fructose and citric acid) began deliquescing at a lower RH (RH0mix) than the critical relative humidities of either powder. A considerable amount of caking was observed in both single and binary powdered systems. The control mixtures that were stored below the RH0mix remained free flowing and free of caking. Single powder controls that were stored above and below RH0mix, but not above RH0,, formed fragile cakes. The unground and ground particles had minimum differences in vapor sorption below the RH0 where deliquescence occurs. The physical mixtures that experienced caking were cycled at an RH above and below RH0mix, but not above RH0 of individual components. Cycling was identified as a key factor in cake formation. The ground citric acid/glucose mixture experienced increased caking. This phenomenon could be possibly explained by smaller particle sizes (sucrose) having higher water vapor sorption, which results in an increased tendency to cake (Roge and Mathlouthi 2003; Mathlouthi and Roge 2003). Due to the reduction in particle size, there is a larger surface area which leads to more surface water adsorption. Another possible mechanism is an increased number of liquid bridges formed, following by recrystallization and formation of solid bridges which strengthen caking. Like the previous study, the RH0mix was an indicator of stability, most importantly physical stability (Salameh and Taylor 2006a). 17 The previous study focused on deliquescence in relation to physical stability. Salameh and Taylor (2006b) also studied how deliquescence affects chemical stability. Similar methods were used as stated in the previous section to determine critical relative humidities of crystalline sucrose, citric acid, ?-D-fructose and ?-glucose monohydrate. Saturated salt solutions were used to create RHs of 43, 54, 65, 71, 75 and 85%. A 50:50 ratio of sucrose and citric acid was placed in 20-mL vials and stored at the previously mentioned RHs. Sucrose/citric acid solutions at concentrations of 10% w/v and 33% w/v were utilized to study sucrose inversion kinetics. Sucrose served as a control and was stored at 71 and 85% RH. Polarimetry was used to measure sucrose inversion kinetics. Deliquescence was also visually examined using a microscope. Moisture was monitored by preparing sucrose samples and mixtures of sucrose and citric acid, and placing them at all RHs, and at various time points weighing them (Salameh and Taylor 2006b). The results from the study revealed that all RH0 values obtained using single powders by the moisture sorption data extrapolating method were close to the equilibrium water activities of saturated solutions obtained from the water activity meter. The sucrose inversion reaction occurred more rapidly in solution than in powdered form. For example, the citric acid/sucrose powder at 85% RH took 24 d for complete hydrolysis compared to 9 d in the 33% w/v solution. The visual microscopy study provided additional evidence that deliquescence took place in the 72% RH mixture of citric acid and sucrose. Samples that were stored at 65% RH or above were completely deliquesced, and sucrose was completely inverted. The moisture study revealed that as time increased, so did moisture content in sucrose/citric acid mixtures stored at 65% RH and above. 18 Conversely, control samples and mixtures stored below 65% RH had a moisture content of less than 0.04% w/w (Salameh and Taylor 2006b). Salameh and others (2006) conducted a study to investigate deliquescence lowering in food ingredients while comparing two different methods of measuring RH0 (water activity of equilibrated saturated solutions vs. moisture sorption isotherm). The food ingredients included the following: sodium citrate tribasic dehydrate, sodium ascorbate, citric acid anhydrous and crystalline sugars with the exception of lactose (Salameh and others 2006). The dynamic moisture sorption method required samples that ranged from 20-25 mg in weight. The samples were prepared by geometrically mixing the individual ingredients. Geometrically mixing involves mixing the minor component with the major component to achieve a 50/50% w/w premix. Next, an equal amount of the remaining major component is added and mixed to the premix. This method is replicated until the entire quantity of the major component is utilized and the final composition is obtained. Samples containing the hydrate form were not dried in the sorption analyzer. However, samples that contained anhydrous components were dried at 50?C in the sorption analyzer prior to conducting the experiment. The equilibrium criterion set for the experiments was 0.01% w/w in 2 minutes, with a drying time of no greater than 60 minutes. The samples were exposed to increasing RH, with 2-3 data points collected above and below RH0. At each step in the isotherm, samples must equilibrate to ?0.01% w/w in 15 minutes with a maximum step time of 90 minutes (Salameh and others 2006). RH0 was determined using the isotherm generated. Based on extrapolating the linear 19 portions of the curve before and after deliquescence, the intersection value was determined and identified as RH0 (Salameh and others 2005). The second method used to determine the RH0 of single and multi-component samples was water activity measurements. Water activity was measured in duplicate using the AquaLab 3TE (Decagon, Pullman, WA., U.S.A). Approximately 4 g of the physical mixtures were mixed with 250 to 500 ?L of double distilled water and samples were allowed to equilibrate in the water activity meter for 24 hours before data were collected. Optical microscopy was also used to visually examine deliquescence in solid mixtures at 25?C (Salameh and others 2006). Results for the water vapor sorption isotherm for sucrose showed that the RH0 was approximately 85%. Agreement between the two methods (moisture sorption isotherm and water activity meter) used to determine RH0 values of individual ingredients were fairly consistent, with the exception of lactose anhydrous and citric acid. These compounds have the ability to form hydrates, which may cause an increase in aw. Spectroscopy showed that during the measurement period, a phase conversion to a monohydrate occurred. Because the hydrate form was less soluble than the anhydrate form, a spike in aw was observed. The sorption isotherm for the anhydrous citric acid, fructose and 3 mixtures of 10:90, 50:50 and 90:10 w/w blends showed that the mixtures deliquesced at a significantly lower RH than the individual components, thus displaying deliquescence lowering. Deliquescence lowering occurred in binary, tertiary and quaternary blends of the various food ingredients (P<0.01) (Salameh and others 2006). Optical microscopy was carried out using one crystal of citric acid anhydrous and fructose. The two crystals were placed in contact with each other to mimic a mixture at 20 56% RH and 25?C. Obvious signs of deliquescence were observed after 60 minutes. Individual crystals were also stored at the same conditions with no contact. These crystals showed no signs of deliquescence. The RH0mix for the citric acid/fructose mixture was 48% RH. A similar procedure was used for sucrose and citric acid anhydrous. The RH0mix was 64% RH. The individual RH0 values of sucrose, fructose and anhydrous citric acid are 85%, 62% and 75% RH, respectively. This result clearly shows that deliquescence lowering occurred when the ingredients were combined. There was some disagreement between the two methods used to determine critical relative humidity as the number of components increased in the mixture (Salameh and others 2006). The last study focused on the combined effect of temperature and RH on stability. Hiatt and others (2010) investigated the effect of temperature and RH on chemical stability of two types of vitamin C. Vitamin C samples were obtained and stored at the following RHs: 54%, 64%, 75%, 85%, and 98% using desiccators. Drierite (0% RH) was used for the control. The desiccators were placed at temperatures including the following: 4, 25, 35, and 40?C. A microplate reader was used to analyze amount of ascorbate remaining following the treatment period. A moisture sorption isotherm was used to determine the RH0 of the vitamin C at 25 and 40?C. A kinetic study was also conducted on vitamin C in solution (Hiatt and others 2010). The RH0 at 25?C for ascorbic acid and sodium ascorbate was 98% RH and 85% RH, respectively. Increasing the temperature to 40?C resulted in decreased RH0 values of 86% RH (ascorbic acid) and 82% RH (sodium ascorbate). At 40?C below RH0, ascorbic acid was generally stable, but when RH was increased above RH0 there was significant degradation. Following 8 weeks of storage at 98% RH/25?C, ascorbic acid had some 21 yellow colored crystals with moisture present, but was not fully deliquesced. At 98% RH/40?C, the ascorbic acid was a dark brown liquid. Likewise, sodium ascorbate was also stable at RH values below RH0 at 25?C. However, after eight weeks, complete degradation was observed at RH values near 85% RH and above. Room temperature did not have a large impact on degradation until values near RH0 were approached or exceeded (Hiatt and others 2010). Sodium ascorbate and ascorbic acid were stable at 0% RH at all temperatures. At 75% RH/4?C and 75% RH/25?C, sodium ascorbate was stable, but at 35 and 40?C it had almost undergone complete degradation after 8 weeks. Overall, increased moisture adsorption led to greater vitamin instability. As temperature and RH increased, the rate constants generally increased, with 98%RH/40?C having the highest rate constant for degradation. The study showed that both, temperature and RH may have a pronounced effect on chemical stability. At times, they may behave synergistically. For example, a higher temperature may lower the RH0, therefore causing the material to become more susceptible to instability (Hiatt and others 2010). The previous articles demonstrate how deliquescence may alter chemical and physical stability in powdered systems. Information regarding the structures and moisture contents were obtained. Physical caking observed before the onset of deliquescence may be an indication of deteriorating chemical stability. Deliquescence lowering was explored by combining various powdered ingredients, which caused the RH0mix to become lower than the critical RHs of the individual ingredients. Lastly, the synergistic effect of temperature and RH on physical and chemical stability was investigated. 22 The studies demonstrate the importance of dry ingredient physical and chemical stability to the food industry as well as other industries. The bakery, vitamin, pharmaceutical, and spice industries are drastically affected if functionality of dry ingredients fails. Dry ingredients may be susceptible to caking, stickiness and agglomeration and eventually deliquescence. Consequently, problems in product formulation, storage and mixing may occur. Therefore, care must be taken to limit the exposure of dry ingredients to harsh environmental conditions, such as heat and moisture. Taking time to investigate proper storage environments for food powders will save money, time and resources. Another powdered ingredient that may be affected adversely by harsh environmental conditions and undergo deliquescence is tagatose. Although numerous studies have been conducted studying the health benefit, tolerance, and food application of tagatose, there have been limited studies examining the stability of tagatose. Tagatose may be incorporated into various food, hygienic and beauty products. In addition, tagatose may potentially have a large impact on health. It appears to have prebiotic properties, which promote satisfactory colon health. Due to these attributes, it is necessary to investigate how environmental factors may impact the bulk storage of powdered tagatose. Tagatose Properties and Applications D-Tagatose is a naturally occurring six carbon monosaccharide that is an epimer of D-fructose (i.e. the hydroxyl group inverted at carbon 4). Trivial amounts of tagatose may be found in dairy products, including infant formula, certain cheeses, yogurt and various processed milk. The amounts range from 4 mg/kg in infant formula to 6500 23 mg/kg in medications (Levin 2002). It was determined to be 92% as sweet as sucrose when both tagatose and sucrose were tested in 10% aqueous solutions. It is a white anhydrous crystalline powder that has a melting point of 134?C. Research has shown that tagatose is a full bulk, low calorie sweetener with a caloric value of 1.5 kcal/g (Levin 2002). Tagatose has a wide range of applications in a number of industries because of its highly desirable attributes. Tagatose may be used as a flavor enhancer in dairy products (yogurt), bakery products and confectionery products. Preliminary studies indicate that tagatose may be used to replace sucrose (1:1 ratio) in chocolates and gum; the sensory and physical properties were similar to the target product (Levin 2002). Moreover, since it is a low calorie sweetener, it may be used in low carbohydrate diets, soft drinks, cereals and health bars (Oh 2007). Some of the nonfood uses for tagatose include using it as a sweetener in nonchronic prescription medication for children and adults. Moreover, tagatose may be used in the cosmetic industry as a sweetener in lipstick, mouthwash and toothpaste (Levin 2002). Tagatose is mass produced using lactose in a two step method. First, lactose is enzymatically hydrolyzed into galactose and glucose. The second step is performed under alkaline conditions using calcium hydroxide and involves isomerization of galactose to D-tagatose. Calcium hydroxide causes a shift in the isomerization equilibrium between D-tagatose and galactose, favoring D-tagatose and consequently forming an insoluble complex with calcium hydroxide due to the high pH. Sugars usually experience side reactions due to the elevated pH, however because D-tagtose is in the complex it is protected and does not react. Lastly, the suspension is treated with carbon dioxide, which 24 precipitates the calcium in the form of calcium carbonate, thus releasing tagatose (Bar 2004). The tagatose may be further treated by purifying, crystallizing and drying. Other potential manufacturing techniques include using L-arabinose isomerase to catalyze the conversion of galactose to tagatose. However, this technique is challenging due to enzyme activity, yield and shelf life (Lu and others 2008). Tagatose as a Prebiotic The term ?functional food? has created quite a stir among consumers and food scientists alike. Due to illnesses, such as diabetes, hypertension and cancer, consumers have become more educated on foods that may promote better health. In addition, food scientists have begun to incorporate functional foods in products and label packaging with identifying words like ?antioxidants? or ?prebiotics.? Functional foods describe foods that offer an added health benefit in addition to the nutritional value (Siro 2008). Functional foods may include both probiotics and prebiotics. Probiotics are defined as live microbial food substances that have health benefits. Two of the most common genera that are referred to as probiotics are lactobacilli and bifidobacteria. These organisms are commonly found in fermented dairy products, including yogurt. After the probiotics are passed through the stomach and small intestines, some cultures survive and briefly interact with the large intestine. Consequently, the colon?s fermentation ability is affected, and there are an increased number of bacteria in human feces. Some benefits of ingesting foods that contain probiotics include: improved lactose tolerance, immune enhancement, cholesterol reduction and decline in mutagenicity and enzymes (Roberfroid 2000). Moreover, research has provided evidence that probiotics may reduce the risk of 25 colon cancer by reducing the number of aberrant crypt foci (colon cancer marker) (Roberfroid 2000). Prebiotics are described as ?nondigestible food ingredients that benefit the host by selectively stimulating the growth or activity of one or a limited number of bacteria in the colon? (Roberfroid 2000). Research has shown that native or enzymatically hydrolyzed inulin, synthetic fructooligosaccharides and hydrolyzed oligofructose have prebiotic properties. Prebiotics are usually not digested and have been referred to as colonic food, which means they function as a substrate for inherent bacteria and provide energy and metabolic substrates (Roberfroid 2000). Tagatose has been labeled as a prebiotic due to its ability to undergo fermentation by intestinal bacteria and produce short chain fatty acids as well as serve as a substrate for some beneficial bacteria, such as lactic acid bacteria. One study that looked at the prebiotic attributes of tagatose was conducted by Venema and others (2005). There have been a number of studies conducted in vitro on the prebiotic effect of tagatose (Laerke and Jenson 1999; Laerke and others 2000; Bertelsen and others 2001), however limited human studies have been conducted. Venema and others (2005) utilized an in vitro and human study to investigate the effect of tagatose on the production of microbial metabolites and composition of microbiota using a large intestinal model. Moreover, tagatose was compared with subjects receiving fructooligosaccharides and sucrose. A mixture of tagatose and fructooligosaccharides (FOS) was also investigated for prebiotic properties. A total of 18 healthy women and 13 healthy men participated in the study for approximately four months. The study was carried out as a double-blind, cross-over, randomized, reference substance-controlled 26 design. The study contained five treatment periods lasting 14 days, which were separated by 14 day wash-out periods. Participants consumed 30 g of raspberry jam daily for breakfast containing two different doses, one being a high dose (12.5 g) and one a low dose (7.5 g). Participants also consumed 7.6 g of tagatose and 7.5 g of FOS in a mixture, sucrose alone or fructooligosaccharides alone. Following each treatment period, stools were collected in a box that maintained anaerobic conditions and processed for microbial analyses. Fasted urine and blood samples were collected and gastrointestinal (GI) symptoms were monitored using questionnaires. The composition of the fecal matter was determined using a variety of agar plates. Other data was also gathered from the fecal matter such as pH, dry matter content and short chain fatty acid composition (Venema and other 2005). The results showed that tagatose and the tagatose/FOS combination caused the most GI symptoms. There was no significant difference in the number of stools in the first week, however; in the second week the number of stools was significantly higher in the tagatose and tagatose + FOS treatments compared to the negative reference (sucrose). Blood work and fasting serum lipids were unaffected by the treatment. The participants that received the high dose had a larger number of lactobacilli (7.66 ? 1.14 log cfu/g), compared to the low dose group (6.95 ? 1.16 log cfu/g), but not significantly. Following test tube incubation of fresh fecal matter in sucrose, tagatose, FOS or no substrate, the SCFAs were significantly higher after two weeks in all samples except for the positive reference (fructo-oligosaccharides). Butyric acid production was higher after high tagatose, tagatose + FOS and low tagatose treatments compared to the negative reference (sucrose). Higher propionic acid contents were also observed in the high tagatose 27 treatment group compared to FOS. There were no differences among the treatments in lactic acid, valeric acid, iso-valeric acid and iso-butyric acid (Venema and other 2005). The result of the in vitro (large intestine model) experiment showed that the concentration of the majority of microbial groups (Bacteroides, Bifidobacterium, Enterobacteriaceae, Enterococcus, sulphite-reducing Clostridium) did not increase with the exception of lactobacilli, which increased. The human study revealed that tagatose in the allotted dosages did not alter the amounts of butyrate and total SCFAs in the feces of women or men. An increase in butyrate and total SCFAs was only observed after incubation of the fecal matter with tagatose. Similarly, the in vitro study showed an in increased butyrate production. Due to the outcome of this study, tagatose may be regarded as a prebiotic substrate (Venema and other 2005). Laerke and others (1999; 2000) also observed an increase in butyrate, propionate and valerate in a digestability study which suggests that tagatose may potentially have health benefits. Laerke and others (2000) conducted an in vitro study in pigs. The study had three objectives: to investigate the effect of tagatose on SCFA production and lactic acid production, to approximate degradation of tagatose and fermentation products, and to determine the amount of tagatose-degrading bacteria in the feces of the pigs. The study also aimed to understand the unadapted vs. adapted gut microbiota as it related to tagatose degradation (Laerke and others 2000). The study included 16 pigs, 8 each for the control and experimental groups. At 7 day intervals, each group was given low-basal diet with 150 g/kg sucrose (control) or 50 g/kg + 100 g/kg of tagatose (experimental). The pigs had a 2 day (day-4 and day-3) adaptation period that consisted of consuming a traditional Danish diet followed by 2 28 days (day-2 and day-1) of a 1:1 mixture of the experimental diet and standard feed. Next the pigs were fed the experimental diet twice a day for 18 days, (day 0 to day 17). Fecal matter was obtained on days -3, 1, 8 and 15 of the study to determine total anaerobic bacteria, pH, dry matter and D-tagatose-degrading bacteria. On day 17, the pigs were killed using a lethal injection of pentobarbital sodium. The gastrointestinal tract was removed and divided into segments according to Laerke and Jensen (1999). A 20 g/100 g slurry of gastrointestinal contents in 100 mmol/L Na-phosphate buffer was used to determine the production rate of lactic acid and SCFA in the gastrointestinal tract. The rate and composition of fermentation products were determined by exposing a slurry of bacteria from the pig?s stomach to tagatose. Remaining tagatose was quantified using HPLC. The total number of anaerobic and tagatose-degrading bacteria was determined using the most probable number technique (MPN). SCFA and lactic acid were also determined (Laerke and others 2000). The data was analyzed using SAS. Results showed that the quantity of tagatose- degrading bacteria in the feces of the pigs fed the tagatose diet was ten times higher compared to the control diet. In both the control and experimental groups, the fermentation products produced in the stomach and distal small intestines (SI) were primarily lactic acid, acetic acid, and formic acid. The SCFA that were produced from the cecal material of the pigs fed the tagatose diet included the following (least to greatest): formic, valeric, butyric, acetic and propionic acids. Propionic production in the cecum of the pigs fed tagatose was more than twice as high compared to the control. The overall amount of SCFA produced in the cecum was 10.7?2.1 mmol/h and 3.0?2.7 mmol/h for the tagatose and control groups, respectively (P=0.06). Exposing the tagatose 29 to bacterial slurries from the stomach and SI caused no increase in SCFA levels, showing that these portions of the GI tract were unable to degrade tagatose in vitro. Due to the small amount of degradation, energy values for the unadapted microbiota were not determined. Compared to the control, hydrogen production was increased due to the in vitro incubation of tagatose with bacteria taken from the cecum and colon of the tagatose- fed pigs. Likewise, methane production (mid-colon) was also higher in the tagatose-fed pigs. This suggests that some of the energy was lost as gases (Laerke and others 2000). This study showed a higher level of tagatose-degrading bacteria as well as SCFA, which provides additional evidence that tagatose displays prebiotic properties. Tolerance of Tagatose Many studies have examined the consumption of tagatose at various levels to determine the acceptable intake as well as tolerance. Buemann and others (1999) conducted three mini studies to look more closely at how the body responds to tagatose. The studies included a screening study, an adaptation study and a metabolic study. Human subjects consumed 30 g of tagatose in each study and reported symptoms, such as increased flatulence, nausea and headache. In the screening study, 73 males were given cake that contained the tagatose in the afternoon followed by a three hour fasting period. Subjects recorded symptoms and severity of symptoms for the remainder of the day as well as the next day. The screening study was conducted to eliminate subjects from the adaptation and metabolic studies who displayed strong gastrointestinal symptoms. Subjects were responsible for recording and rating the severity of symptoms during the day of consumption and the subsequent day. The main goal of the adaptation study was to identify possible metabolic effects of tagatose consumption by 24 h indirect 30 calorimetry before and after a 2 week adaption period. The report did not present results of the metabolic study but focused only on tagatose tolerance. During the adaptation test, sucrose (control) and tagatose were administered in a randomized, blind crossover study. More than 2 months separated the treatments. Subjects recorded symptoms at specified times during days of indirect calorimetry. Most symptoms were moderate to light and no vomiting occurred. The authors explained that such symptoms may occur due to unabsorbed sugars that ferment in the large intestines. Although no analysis was performed on the data, the adaptation study revealed there was not an improvement in tolerance to tagatose, just adaptive changes in the microbiology of the colon. In the screening study, more symptoms were recorded where as in the adaptation (2 week) and metabolic studies less were recorded. In the screening test a 15.1% incidence of nausea was reported and a 31.5% incidence of diarrhea was reported. The author concluded that less than 30 g of tagatose should be consumed per eating occasion and tolerance varies (Buemann and others 1999). Another study was conducted to compare gastrointestinal disturbances of sucrose, tagatose and lactitol when incorporated into chocolate (Lee and Storey 1999). Two 40 g chocolate candy bars containing 20 grams of each ingredient were randomly given to 50 unadapted subjects (25 male and 25 female). The experiment was designed as a double- blind, controlled, crossover study. Twenty-five of the subjects consumed the chocolate bars in the following order: day 1-lactitol bar, day 9-sucrose bar and day 17-tagatose bar. The additional subjects had the tagatose bar on day 1, sucrose bar on day 9 and lactitol bar on day 17. Subjects were given sheets to record gastrointestinal symptoms (incidence and extent), such as colic, bloating, flatulence, thirst, nausea, etc on a scale of 0 (normal) 31 to 3 (debilitating) during the 24 hour period after consumption. Toilet visits and consistency of bowel movements were also monitored (Lee and Storey 1999). When compared to lactitol, tagatose chocolate had no significant differences in frequency of symptoms, except for an increased amount of thirst. When tagatose was compared to sucrose, significantly more subjects felt symptoms, such as thirst, appetite loss, nausea, bloating and borborygmi. Also compared to sucrose, individuals who consumed tagatose chocolate passed more watery bowel, but not more than those who consumed lactitol. Only one subject recorded a debilitating symptom after consumption of lactitol or tagatose. This study indicated that a 20 gram dose of tagatose may be better tolerated than the same dosage of lactitol (Lee and Storey 1999). Buemann and others (2000) completed another study examining the acute effect of tagatose on ad libitum food intake. Twenty male subjects were required to undergo a pre-screening test in which they had to tolerate 30 g of tagatose without significant symptoms. The tests were separated by 4 days or more. Following a 12 h fast, the breakfast was consumed in 20 min. The subjects received a 1.6 MJ breakfast, which had either a 29 g dose of tagatose or sucrose. After breakfast, a duration of four hours passed in which subjects were not allowed to eat or drink. Subsequently, lunch was served ad libitum and plates were weighed to monitor intake. After lunch subjects were allowed to leave, but were supplied with lunchboxes. They were instructed to eat at will but to save leftovers for weighing. Dinner was served 5 h later and subjects were allowed to eat ad libitum. Similarities were observed in energy intake of the tests at lunch and snack, but a lower energy intake (15%) was observed during dinner in the tagatose group (Buemann and others 2000). 32 The results of the study revealed tagatose did not affect total macronutrient composition of total post lunch food. However, tagatose caused a lower fat intake (% of energy intake) at dinner. The liquid intake of lunch and snack combined was 11% higher after tagatose partially due to a lower intake at dinner. Two cases of nausea and one case of strong flatulence were reported with tagatose intake (Buemann and others 2000). Buemann and others (1999) reported that symptoms such as vomiting, nausea and perceived distention could be caused by an osmotic effect in the small intestine of unabsorbed tagatose. Moreover, the heightened flatulence may be attributed to the fermentation of tagatose in the large intestine. Diarrhea may be caused by osmotic effects in the colon from tagatose that was not digested. In addition, diarrhea may also be caused by the increased fermentation producing poorly absorbed short chain fatty acids (Buemann and others 1999). Metabolism of Tagatose Livesey and Brown (1996) examined the thermic effect of tagatose and determined it supplied zero net metabolizable energy (NME). This study utilized rats that consumed sucrose (control) and tagtatose (1.8 grams) incorporated in a basal diet to investigate the thermic effect of tagatose. The NME value of tagatose was calculated using equations based on the influence of body makeup. All substrate-induced energy losses, such as feces and urine, were accounted for. No symptoms, such as diarrhea, were observed in the rats as reported later in human studies conducted by Buemann and others (1999). The tagatose did not supply significant amounts of beneficial energy?the calculated metabolizable energy value was -0.4 ?2.2 kJ/g (Livesey and Brown 1996). Moreover, Moyer and Roden (1993) also found that sugars, such as tagatose, have 33 diminished net energy contents because of poor digestibility. Although this study (Livesey and Brown 1996) confirmed the thermogenic effect of tagatose, Buemann and others (1998) conducted a study that was not able to confirm that tagatose minimal net energy is a result of a thermogenic effect. Although Livesey and Brown (1996) determined tagatose had a substantially lower caloric content, Levin (2002) reported that the caloric content may range from 1.1 to 1.4 Kcal/g. Differences in caloric content maybe attributed to experimental procedures, variables and result interpretation. Therefore, the Food and Drug Administration approved the caloric content of tagatose as 1.5 Kcal/g (Levin 2002). Another study examined the digestibility of tagatose in pigs (Laerke and Jensen 1999). This study was designed to investigate the ileal and fecal digestibility of tagatose in pigs. In addition, this study looked at the effect of ileal and fecal digestion on macronutrients and changes in microbial activity, pH, and concentration of short chain fatty acids (SCFAs) in the gut contents. Seven days separated two experiments that were performed using two groups of eight pigs. There was an adaptation period of two days, two days of consuming a 1:1 ratio of a mixture of control and experimental diet and eighteen days of the experimental diet. The control group consumed a low fiber diet with sucrose (150 g/kg) and the experimental group consumed a low fiber diet with tagatose (100 g/kg) and sucrose (50 g/kg). Both diets also contained chromic oxide, which served as a digestibility marker. Analysis was conducted on the upper gut (stomach and three equal segments of small intestine [SI]) and the lower gut (cecum and three equal segments of the colon) (Laerke and Jensen 1999). 34 The digestibility of tagatose in the distal portion of the small intestine measured by chromic oxide was 25.8 ? 5.6%. No significant differences between groups in the SI digestibilites of macronutrients were found, but there was a lower digestibility of sucrose in the pigs fed the tagatose diet. Significant differences were observed in the total microbial activity in the cecum and colon of the control and test pigs. An increase in butyrate, propionate and valerate were observed in the test pigs, which suggests that tagatose may potentially have health benefits. Tagatose in the large intestine of adapted pigs was completely fermented?this played a role in the overall energy balance with a high production of SCFAs (Laerke and Jensen 1999). Buemann and others (1998) also suggested that some tagatose is not absorbed, but fermented in the large intestine. In the small intestine, absorption and digestion of tagatose is minimal (approximately 25%), with the large majority of tagatose being fermented in the large intestines (Laerke and Jensen 1999). Although metabolism is similar to other monosaccharides, it may occur at a slower rate (Levin 2002). Moreover, the study was also comparable to a study that used rats with radiolabeled tagatose where only 15-20% of tagatose was absorbed across the small intestine (Saunders 1999). Tagatose in Foods For decades, consumers as well as the food industry have searched for a solution to enjoy palatable foods, but without the added calories and sugar. Tagatose is a prime candidate and is currently being incorporated into various food products. Currently, marketed products that have tagatose include the following: Shurgr by Swiss Diet, Pasco Light and Tasty Juice, 7-Eleven?s Diet Pepsi Slurpee, SweetFiber by Dr. Murray Natural 35 Living, Miada Chocolite, and Therasweet by Living Fuel (Wise 2008). Some of the geographical locations where these products are found include the United States, European Union, South Korea, Australia and New Zealand (Peckenpaugh 2006). The Food and Drug Administration maximum specification levels for tagatose include the following: diet and/or sugar free carbonated soda and teas-1%, sugar free gum -60%, icings and glazes- 30%, diet soft candies and low fat and reduced fat energy/nutrient bars-10%, powdered products made with milk-5 g/per serving, diabetic and regular hard candies-15%, frozen novelties- 3% and ready-to-eat cereals- 3 g/serving (FDA 2001). Taylor and others (2008) conducted a study examining the physical properties and consumer preference of cookies formulated with tagatose. In addition to control cookies prepared with 100% sucrose, eight experimental formulations were prepared using fructose and tagatose at 25%, 50%, 75% and 100%. The cookies were prepared according to McWatters and others (2003). Textural properties of the cookie dough were quantified using the Texture Analyzer. Physical properties of the baked cookies were also monitored. They included height and diameter, color and hardness. A total of 53 untrained panelists evaluated 3 different formulations (100% tagatose, 100% sucrose and 50/50 blend of tagatose and sucrose) on texture, color, sweetness and overall liking (Taylor and others 2008). The Texture Profile Analysis revealed results concerning the resilience, hardness, adhesiveness and cohesiveness of the cookie dough. There was no significant difference in resilience between the 100% tagatose cookie dough and control (100% sucrose). Moreover, there were no significant differences in hardness, adhesiveness and cohesiveness of doughs made with 100% sucrose and 100% tagatose. The rheological 36 properties of the dough that was made with tagatose were similar to that made with sucrose. In contrast, the dough made with fructose was softer and stickier and did not closely resemble the rheological properties of the dough made with sucrose. The diameter of cookies made with fructose and tagatose were significantly smaller than the cookies made with sucrose. Compared to the control, cookies made with 100% tagatose were significantly harder. Cookies made with tagatose and fructose were darker in color compared to the control. The sensory evaluation of the cookies revealed that overall acceptance of the 100% sucrose cookie was significantly higher than that of the 100% tagatose cookie, but not significantly different from the 50/50 tagatose/sucrose formulation. Based on the study, it appears possible to formulate an acceptable cookie made with tagatose, but not by completely replacing sucrose with tagatose (Taylor and others 2008). Armstrong and others (2009) investigated whether 1% and 2% tagatose affected the flavor of bakery products when determined by an untrained sensory panel. Cinnamon muffins, chocolate cakes and lemon cookies with 1-2 % tagatose were formulated and prepared. Standard recipes were used for the cinnamon muffins and lemon cookies, whereas a premixed formula was used for the chocolate cake. Control bakery products were prepared with sucrose using the same concentrations. Untrained panelists performed a triangle test and hedonic test. The triangle test revealed panelists could not differentiate between the formulations at the 1% and 2% levels. Therefore, incorporating tagatose into the bakery products does not significantly impact the flavor profile. The hedonic scores indicated the panelists liked the tagatose and control products similarly (Armstrong and others 2009). 37 Stability of Tagatose in Solution A few studies have investigated the stability of tagatose in solution (Ryu and others 2003; Dobbs and Bell 2010; Luecke and Bell 2010). One study that investigated the effect of temperature and pH on the non-enzymatic browning reaction involving tagatose was conducted by Ryu and others (2003). The effect of temperature on non- enzymatic browning was investigated by heating a solution of 0.2 M tagatose and 0.2 M glycine in a water bath for 5 hours at 70, 80, 90 and 100?C with no pH control. To determine the effect of pH on non-enzymatic browning, tagatose/glycine solutions ranging from pH 3 to 7 were heated at 100?C. Two other studies looked specifically at temperature and pH effects on tagatose only. A 5% solution of tagatose was heated to 100?C for 5 hours at pH 3, 4 and 5. The effect of temperature on tagatose was investigated using a 10% tagatose solution and heating it to 100?C for 5 hours. In all studies, samples were removed hourly for analyses, including HPLC, spectroscopy, and color measurements. The study found that the rate of browning increased as temperature increased. Generally, as pH increased, browning also increased, except at pH 6 where a slight decrease in browning was observed. Tagatose alone did not experience any browning or degradation. However, when combined with glycine both were observed; this result was attributed to the Maillard reaction (Ryu and others 2003). Dobbs and Bell (2010) investigated the storage stability of tagatose in buffer solutions at 20, 30 and 40?C. Tagatose was placed into two buffer (citrate and phosphate) solutions at a concentration of 0.05 M or 1% and adjusted to a pH of 3 and 7, respectively. The concentrations of the buffer were 0.02 and 0.1 M. Each sample was 38 stored at 20, 30 and 40?C in triplicate. Over the nine month period, 11-13 samples were obtained (Dobbs and Bell 2010). Concentrations of tagatose were analyzed using reverse phase high performance liquid chromatography (HPLC), whereas brown pigmentation was determined using spectrophotometry (wavelength = 420 nm). Results indicate that pH, temperature, buffer type and concentration all impacted tagatose degradation. Tagatose degradation rate was fastest at the greater pH (i.e., pH 7) whereas at pH 3 the tagatose degradation rate was not significantly different from zero. The tagatose solution consisting of 0.1 M phosphate buffer at pH 7 and 40?C experienced a 29% loss of tagatose in 30 days. Tagatose also degraded faster in phosphate buffer than in citrate buffer. In addition, the higher the buffer concentration, the more rapid was tagatose degradation. At pH 7, the tagatose degradation rate constants for 0.1 M phosphate buffer were 2.5-4.8 times higher than the lower buffer concentration (0.02 M). As expected, the rate constants increased as temperature increased. Browning was also most pronounced at the highest temperature (40?C). In summary, this study found that tagatose degradation was highest at higher temperatures, pHs, and buffer concentrations. The study suggests that tagatose may be permitted in mildly acidic products such as fruit juices and yogurts. Moreover, beverages that take advantage of tagatose should be exposed to low temperatures, low buffer concentrations and low pHs to minimize tagatose degradation and browning (Dobbs and Bell 2010). Luecke and Bell (2010) conducted a similar study that investigated the thermal stability of tagatose when exposed to temperatures of 60, 70 and 80?C. Similar methods were used for this study as the previous study. Citrate and phosphate buffers were 39 prepared at 0.02 M and 0.1 M concentrations and adjusted to pHs 3 and 7. A total of eight buffer solutions contained 0.05 M tagatose. The kinetic studies were carried out in a water bath that maintained temperatures of 60, 70, and 80?C. The tagatose solutions were transferred to NMR tubes, which were placed in the water bath and reached the specified temperature within a minute. Tubes were moved at regular time intervals and transferred to an ice bath to halt further reaction (Luecke and Bell 2010). Tagatose analysis was similar to previous study and was carried out via HPLC and spectroscopy (Dobbs and Bell 2010). Pseudo-first-order kinetics were used to model tagatose degradation. Rate constants as well as 95% confidence intervals were determined. Moreover, activation energies were determined for each experiment using the rate constants and average temperature. The effect of pH, buffer type and concentration, and temperature on tagatose was determined. Similar results were noted when compared to the previous study. Tagatose degradation occurred slower at pH 3 than pH 7. The concentration of tagatose decreased by 26% when stored at the following conditions for 7.5 hours: 0.1 M citrate buffer, pH 7 and 80?C (Luecke and Bell 2010). Tagatose degradation was also more pronounced in the phosphate buffer when stored at the higher pH. The phosphate buffer accelerated tagatose degradation as compared to the citrate buffer. Buffer concentration also impacted tagatose loss, with greater loss being observed at higher buffer concentrations. With a pH of 7, the rate constant for tagatose loss in a 0.02 M phosphate buffer solution was five times less (0.00566?0.00071 h-1) than in 0.1 M phosphate buffer (0.0301?0.0022 h-1). Likewise, as temperature increased from 60? to 80?C, degradation also increased. Pseudo-zero-order rate constants were calculated for browning of the tagatose solutions. Browning was faster at higher 40 temperatures and higher pHs. Browning was also faster at increased buffer concentrations and in phosphate buffer. Based on this study, tagatose may be used in applications, such as beverages, which are subjected to pasteurization without the loss of its prebiotic property or browning due to the very short time of thermal exposure (Luecke and Bell 2010). Often times, food manufacturing facilities are not air conditioned and relative humidity is not controlled. Consequently, extreme environmental conditions may occur, causing powdered ingredients to be adversely affected. As discussed previously, water can impact powdered ingredients, like tagatose, to varying extents causing caking, clumping, loss of flow ability and even deliquescence. Although some studies have focused on the chemical stability of tagatose in solution, none have addressed the physical and chemical stability of powdered tagatose. Therefore, the purpose of this study was to investigate the physical stability (caking, deliquescence) and chemical stability (degradation, browning) of tagatose powder subjected to various temperatures and relative humidities. 41 Chapter 3: Materials and Methods Sample Preparation Food grade tagatose was obtained from Arla Food Ingredients (Basking Ridge, N.J., USA). According to the product specification sheet, it had a purity of greater than 99%. Ash (<0.1%), water (<0.5%) and, non-tagatose monosaccharides (<0.4%) made up the remainder of the percentage. Approximately 150 g tagatose were placed in beakers over anhydrous calcium sulfate indicating Drierite (W.A. Hammond Drierite Company, LTD.) for one month under vacuum to remove any residual moisture present. Three 400- mL beakers contained about 50 g tagatose each to ensure efficient distribution. Following the one month drying time, 0.3-0.5 g of tagatose were analytically weighed into 338 20-mL glass vials making certain to distribute tagatose evenly over the entire surface area of the vial. A total of 26 vials were placed in each of 13 desiccators (environmental chambers) prepared for the experiment. The chambers were vacuum sealed to maintain a constant environment. One desiccator served as the control, and contained Drierite. The remainder of the desiccators contained one of four saturated salt solutions in triplicate, with one of each being stored at 20, 30 and 40?C. Particle Size Characterization Some studies show that particle size influences moisture adsorption and deliquescence (Mathlouthi and Roge 2003; Salameh and Taylor 2006a; Kwok and others 2010); therefore the particle size of tagatose was investigated. Tagatose samples were 42 placed in a Sieve Shaker (CSC Scientific Company, Inc, Fairfax, VI) for 5 min. Three trials were done to determine the average particle size of the tagatose available to the food industry. A total of 29-34 g of tagatose were used for each trial. To characterize the powdered tagatose sample, its particle size distribution was analyzed using the following sieve sizes: 1) 250 ?m, 2) 355 ?m 3) 500 ?m and 4) 710 ?m. The amount retained by each sieve was weighed. Particles that remained at the top of the 710 ?m sieve were a particle size greater than 710 ?m. Likewise, those particles that were sieved through the 250 ?m sieve were less than 250 ?m. The distribution of the particle sizes is given in Table 3.1. Particle size may influence powder physical stability; therefore, a small side study was conducted to investigate this possibility. Further detailed information about this side study appears in Appendix D. Table 3.1. Average particle size distribution of food grade tagatose as a percentage with standard deviation. Particle size % < 250 ?m 7.1?0.8 250-355 ?m 39.8?1.4 355-500 ?m 46.1?1.8 500-710 ?m 6.6?0.5 >710 ?m 0.3?0.1 Preparation of Saturated Salt Solutions Laboratory grade certified A.C.S. salts ( Fisher Scientific, Fair Lawn, N.J.) magnesium chloride (MgCl2) crystals, magnesium nitrate [Mg(NO3)2], sodium chloride (NaCl) and potassium chloride (KCl) were obtained to make saturated salt solutions, which created relative humidities of 33%, 54%, 75% and 85% at 20?C. A 1000-mL 43 beaker with a stir bar was filled with approximately 500 mL of distilled water. The beaker was placed on a hot plate/stir plate and monitored with a thermometer until it reached a temperature ranging from 55-60?C. Upon reaching the desired temperature, the specified salt (i.e., MgCl2, Mg(NO3)2, NaCl or KCl) was stirred into the water in increments. A critical amount of crystals was stirred into the beaker to create a saturated slurry of salt crystals in water. The salt solutions were allowed to cool and equilibrate in a sealed jar at the desired temperature (20, 30, 40?C) for approximately one month. All saturated salt solutions were transferred into desiccators, which were returned to the appropriate incubator and allowed to re-equilibrate for approximately one week. The relative humidities of the salt solutions were verified using an AquaLab (Decagon, Pullman, WA, USA) water activity meter and compared to literature values. Table 3.2 shows the salt solutions that were used and the relative humidity as a function of temperature. Experimental Overview Following the placement of all tagatose samples in the desiccators, they were allowed to equilibrate to the specified %RH for one month (time 0). After the equilibration period, 2 vials from each desiccator at each temperature were removed monthly for 12 months. The end of the equilibration time was termed time 0 and each month thereafter was termed time 1 through time 12. Desiccators were checked weekly to ensure they remained vacuum sealed. Following sample removal, physical descriptors and photographs were used to characterize the powdered tagatose. The physical descriptors included the following: free flowing, partially caked, fully caked, partially deliquesced and fully deliquesced. In addition, the tagatose was weighed to determine 44 water loss/gain. After assessing the physical appearance and morphology of the tagatose, 20 mL of distilled water was volumetrically added. The vials were vortexed and stored at 4?C for 4-5 days to allow complete dissolution of the tagatose before chemical analyses. The concentration of tagatose and brown pigment formation were determined using these solutions. The pHs of the solutions were also determined using a pH meter (model 920A, Orion Research Inc, Boston, MA) at times 6, 9 and 12 months. Throughout the study care was taken to vacuum seal each desiccator after sample removal and place it back in the proper environmental storage conditions. Table 3.2. Saturated salt solution relative humidity as a function of temperature (Bell and Labuza 2000). Salt Solution 20?C 30?C 40?C MgCl2 33.1 32.4 31.6 Mg(NO3)2 54.4 51.4 48.4 NaCl 75.5 75.1 74.7 KCl 85.1 83.6 82.3 Physical Stability Based on the movement and physical state of the samples, they were characterized as either free flowing, partially caked, fully caked, partially deliquesced or fully deliquesced. Upon removing the samples from the desiccators at each time period, they were visually inspected and lightly moved around to characterize the powder. If the powder moved freely throughout the vial, it was considered free flowing. If the powder was caked, but had still had some portions of free flowing powder, it was considered partially caked. If there was no movement in the vial, the powder was labeled fully 45 caked; this powder was fixed to the bottom of the vial. If the sample had some crystals that were dissolved and others that were still in crystal formed, it was labeled partially deliquesced. The sample was very similar to a ?slurry.? It contained some solids (tagatose crystals) and some liquid (deliquesced tagatose crystals). Lastly, if the sample was fully liquefied (deliquesced), it was termed fully deliquesced. There were no crystals present. Moisture Content Moisture content was monitored to give an indication of when the vials containing tagatose equilibrated in the desiccators. An initial weight of the vials was obtained before placement in the desiccators. Each month (time 0 ? time 12), 2 vials were weighed upon removal from each desiccators to obtain moisture gain or moisture loss. The following equation (3.1) was used to calculate moisture content (M) as a percentage on a dry weight basis: i if w wwM ??? 100 3.1 In the equation, wf refers to the final weight of the tagatose and wi refers to the initial weight of the dried tagatose. Photographs Photographs were taken using a Hewlett Packard Photosmart R725v digital camera (Hewlett-Packard Company, Palo Alto, CA, USA). Photographs of tagatose vials were taken at time 6, 9 and 12 to assess browning as well as morphology of the tagatose. Initially pictures were not taken because browning was not noticeable. Upon removing the vials from the desiccators, triplicate pictures of each vial were taken with no flash. A 46 ring stand was used to make certain the camera was approximately at the same distance to ensure pictures were as comparable as possible; white paper served as the background. The primary lighting consisted of overhead fluorescent light. Due to differences in window lighting, slight differences in lighting occurred. Critical Relative Humidity Determination When determining the deliquescence point of a sample, an assumption is made that the solid particles are surrounded by a saturated solution at the critical relative humidity (RH0). Water activity can be defined as RH/100. Consequently, water activity measurements of saturated solutions can be used to determine RH0, which in turn predicts deliquescence points (Mauer and Taylor 2010). The critical relative humidity (RH0) of tagatose was obtained using a saturated solution of the sugar and distilled water. The tagatose solution and water activity devices were allowed to equilibrate at each temperature for at least 2-3 days before duplicate readings were obtained. To obtain the RH0 at 40?C, the sample was allowed to equilibrate for approximately 24 hours. This action was taken because tagatose degradation was observed at 40?C. Saturated solutions have been used in other studies to determine critical relative humidities (Salameh and Taylor 2005; 2006a; 2006b). Both the AquaLab (Decagon, Pullman, WA, USA) and Rotronic (Rotronic Instruments Corp, Huntington, NY, USA) water activity devices were used to determine RH0 at 20, 30 and 40?C. Saturated salt solutions and water were used to verify accuracy of equipment. Chemical Degradation Analysis Chemical degradation of tagatose was determined by analyzing the tagatose concentration in each sample (i.e., tagatose dissolved in 20 mL H2O) using reverse phase 47 high performance liquid chromatography (HPLC). The mobile phase used was a 91%/9% (v/v) acetonitrile/water solution that had a flow rate of 3 mL/min. Separation was carried out using a 250 x 4.6 mm Luna 5 ? amino column (Phenomenex, Torrance, CA ) in a column oven set at 40?C. Tagatose detection occurred through the use of a refractive index detector RID-10A (Shimadzu, Kyoto, Japan). A Hewlett-Packard Integrator 3395 was used to integrate the data, which allowed the generation of the chromatogram. The tagatose eluted at approximately 7 minutes. A total of seven standard solutions with a known concentration of tagatose were analyzed with the experimental samples at each time period. The peak area from the chromatogram and concentration of the standards were used to generate a linear standard curve. The area was plotted on the x-axis and the concentration was plotted on the y- axis. A linear trend line was obtained from the curve, which allowed the concentration of the experimental samples from each time period to be determined. The area of the tagatose peak was recorded. The concentration of tagatose was determined from the peak area and standard curve. This value was converted into percent recovery, accounting for the initial sample mass and moisture content. The average percent recovery for the control held over drierite was 98.3%, and the coefficient of variation was less than 3% (n=26). This result is similar to that reported by Luecke and Bell (2010). Graphs were generated that plotted percent tagatose remaining versus time (days). No rate constants were determined for tagatose degradation because only one sample (85%RH/40?C) showed substantial degradation, but its loss plateaued at 6 months. Tagatose did not follow the customary degradation kinetic models during the 12 month study. 48 Browning of Tagatose Brown pigmentation was measured monthly for 12 months for all tagatose samples using a spectrophotometer (DU 640, Beckman Instrument Inc, Fullterton, CA). After tagatose was removed from each desiccator and physical appearance was evaluated, 20 mL of distilled water was added to each vial using an electronic pipette. Approximately 1 mL aliquots of each sample were filtered using 5 mL sterile syringes and 0.45 ?m nylon filters, and transferred into disposable methacrylate cuvettes (Fisher Scientific Co LLC, Suwanee, GA ). Distilled water was used as a blank to zero the instrument. Preliminary browning reactions were measured at 280 nm, whereas the later stage brown pigment formation was measured at 420 nm, which has been noted in the literature (Karmas and others 1992). The rate of browning was statistically analyzed using Microsoft? Excel? (Microsoft Corporation, Redmond, WA). Due to the natural off-white color that tagatose displayed, the optical densities (OD) of all samples were corrected using the control samples, which were stored over drierite. The OD/g of these duplicate samples (i.e., stored over drierite) were averaged at each time. The average OD/g values were subtracted from the OD/g of the samples to account for the slight background pigmentation. The resultant OD/g represented the actual discoloration of the tagatose sample associated with a particular relative humidity and temperature. Graphs were generated with time (days) on the x-axis and the corrected absorbance (OD/g) on the y- axis. As outlined by Labuza and Kamman (1983), least squares analysis was used to calculate pseudo zero-order rate constants for tagatose browning with 95% confidence intervals. When select treatments were graphed, some had lag phases. To obtain better 49 R2 values and rate constants, only the linearly increasing portion of the plot was used. Eliminating the lag phase was determined by evaluating the R2 value as well as the appearance of the plot. pH The pH was measured at times 6, 9 and 12 months because when monosaccharide degradation occurs in solution, acidic products are formed (de Bruijn and others 1986). Following HPLC analysis and spectroscopy, a small aliquot of the tagatose solution was transferred to a 20 mL vial and pH measurements were taken using a pH meter with a combination electrode (model 920A, Orion Research Inc, Boston, MA). 50 Chapter 4: Results and Discussion Because relative humidities within the environmental chambers decreased with increasing temperature (Table 4.1), all samples will be identified by the RH at 20?C. This notation will allow for an easier read of this section. For example, 33%RH/30?C refers to an actual RH of 32.4% at 30?C. Table 4.1. Actual RH values associated with temperatures and the notation that will be used throughout this section. Salt Solution 20?C 30?C 40?C Sample Notation Actual RH (%) Sample Notation Actual RH (%) Sample Notation Actual RH (%) MgCl2 33%RH/20?C 33.1 33%RH/30?C 32.4 33%RH/40?C 31.6 Mg(NO3)2 54%RH/20?C 54.4 54%RH/30?C 51.4 54%RH/40?C 48.4 NaCl 75%RH/20?C 75.5 75%RH/30?C 75.1 75%RH/40?C 74.7 KCl 85%RH/20?C 85.1 85%RH/30?C 83.6 85%RH/40?C 82.3 Moisture Sorption Moisture contents were monitored throughout the study to verify that samples equilibrated at each environmental storage condition. The samples stored over MgCl2 solutions (33% RH) adsorbed less than 1% moisture (db) over the 12 month period. The samples stored at 54 and 75% RH adsorbed less than 2% moisture (db) throughout the study. Tagatose stored above KCl solutions (85%) gained substantially more moisture with the amount of moisture decreasing as temperature increased (Figure 4.1). At 20, 30 51 and 40?C tagatose equilibrated at an average of 80%, 66%, and 53% moisture (db), respectively (Figure 4.1). As shown in the figure, the samples stored at 20?C equilibrated at a slower rate than the samples stored at 30 and 40?C. The equilibrium moisture content was plotted as a function of %RH in Figure 4.2. This graph is the moisture sorption isotherm for tagatose. The sudden gain in moisture at 85% RH was due to deliquescence. Salameh and Taylor (2006b) formed moisture sorption profiles for individual solids (fructose, citric acid) and mixtures. The profiles clearly show that minimal moisture is adsorbed prior to the deliquescence point. When the deliquescence point is reached, there is a significant increase in moisture and complete dissolution of the solid (Salameh and Taylor 2006b). Moisture uptake profiles were also generated for a mixture (RH0mix= 64%) of sucrose and citric. For the samples that were stored above or at 65% RH, moisture content increased as a function of time. However, samples stored below 65% RH equilibrated with a moisture content of less than 0.4% w/w (Salameh and Taylor 2006b). Although the tagatose study involved a single component, it followed a similar pattern. Minimal adsorption (< 2%) occurred in all samples stored below 85% RH, with increased moisture adsorption at 85% RH (Figure 4.2). 52 0 10 20 30 40 50 60 70 80 90 0 50 100 150 200 250 300 350 400 % M oi s t ur e ( db ) T i m e ( da y s ) 20 ? C 30 ? C 40 ? C Figure 4.1. Moisture adsorption profile of samples stored at 85% RH at 20, 30 and 40?C. - 5 5 15 25 35 45 55 65 75 85 30 40 50 60 70 80 90 % E MC (d b ) % RH 20 ? C 30 ? C 40 ? C Figure 4.2. Moisture sorption isotherm for tagatose at 20, 30 and 40?C after exposure for 12 months. 53 Physical Stability For a control, tagatose was stored over drierite. The control remained free flowing throughout the study with no noticeable browning or degradation. Depending upon storage relative humidity and temperature, various physical changes in tagatose occurred. Tagatose stored at 33%RH/20?C remained free flowing throughout the study. All other samples experienced some degree of caking and deliquescence. For example, samples stored at 33%RH/30?C were partially caked after 1 month and remained in that state for the duration of the study. At 33% RH, as the temperature of the samples increased, the more the physical stability was affected (Table 4.2). The samples went from being free flowing at 33%RH/20?C to fully caked at 33%RH/40?C after 12 months. All other samples were fully caked, except for samples held at 85% RH. Samples stored at 85% RH at 20, 30 and 40?C were all fully deliquesced after 12 months. However, full deliquescence occurred at various time points including the following: 85%RH/20?C-5 months, 85%RH/30?C-2 months and 85%RH/40?C-1 month. This result was due to the 20?C sample being held slightly below RH0, the 30?C sample being held slightly above RH0, and the 40?C sample being held further above RH0, as will be discussed later. The higher temperature also improves dissolution. Due to the samples being stored at the higher RH, the physical stability was more adversely affected. Table 4.2 shows the physical characteristics of all samples at 12 months. Pictures were also taken and show the physical state of the samples, more specifically deliquescence although some caking is also visible. Figure 4.3 shows caking in the sample stored at 75%RH/20?C for 12 months. Deliquescence is visible in all samples stored at 85% RH (Figure 4.3, 4.4 and 4.5). 54 As mentioned earlier, one of the most common mechanisms through which caking of water soluble solids occurs is particle surface wetting. Particle surface wetting may occur through accidental wetting or condensation of atmospheric moisture. This in turn leads to plasticization of amorphous regions, which facilitate the formation of liquid bridges. The liquid bridges increase the risk of powder stickiness and cohesiveness. Crystallization of the amorphous regions releases moisture and solid bridges are formed, which promote additional caking (Peleg 1983). Furthermore, raising the temperature increases dissolution of particles, which may alter the crystalline form and result in caking and flow problems (Teunou and Vasseur 1996). This mechanism may explain the caking in the tagatose stored below RH0 where there was minimal water adsorption. Table 4.2. Physical characteristics of tagatose as affected by RH and temperature after 12 months and time at which final physical state was observed. RH (%) Temperature (?C) 20?C 30?C 40?C 33% free flowing (12 months) partially caked (1 month) fully caked (1 month) 54% fully caked (10 months) fully caked (3 months) fully caked (9 months) 75% fully caked (1 month) fully caked (1 month) fully caked (1 month) 85% fully deliquesced (5 months) fully deliquesced (1 months) fully deliquesced (1 month) Figure 4.3. Tagatose at 20?C after 12 months, left to right, 33, 54, 75 and 85% RH. 55 Figure 4.4. Tagatose at 30?C after 12 months, left to right, 33, 54, 75 and 85% RH. Figure 4.5. Tagatose at 40?C after 12 months, left to right, 33, 54, 75 and 85% RH. Salameh and Taylor (2006b) investigated caking in single and binary physical mixtures by cycling the mixtures above and below RH0. The study found a large amount of caking occurred for single components and mixtures that were exposed to RH above RH0 and RH0mix and subsequently stored at a lower RH. Control mixtures that were stored below RH0mix remained free flowing. Single components that served as controls were cycled above and below RH0mix, but below the RH0 of the individual components formed fragile cakes (Salameh and Taylor 2006b). Conversely, components that were cycled above RH0 formed strong cakes. This study demonstrated that cycling (exposing ingredients above and below RH0) contributes to caking and the importance of storing components below their RH0 or RH0mix for optimum physical stability. 56 Critical Relative Humidity Because deliquescence was observed, it is desirable to identify the critical relative humidity (RH0) at which tagatose deliquesces. The average RH0 values obtained by measuring the water activity of saturated tagatose solutions at 20, 30, and 40?C were 85.5%, 83.3% and 81.0%, respectively. The actual RHs of the desiccators stored at 20, 30 and 40?C were 85.1, 83.5 and 82.3%, respectively. Samples stored at 85%RH/30?C and 85%RH/40?C were slightly above their RH0s (Table 4.3), causing complete deliquescence to occur faster than at 20?C. Numerous studies have shown that if ingredients are stored below their RH0, they are less susceptible to physical and chemical instability (Salameh and others 2006; Salameh and Taylor 2006a, Mauer and Taylor 2010). Mauer and Taylor (2010) reported that RH0 can vary depending upon temperature. This largely depends on the solubility of the ingredient. For example, the RH0 of sucrose decreases from approximately 86% RH at 20?C to 83% RH at 40?C. Another compound whose RH0 is drastically affected by changes in temperature is ranitidine HCl (active compound in Zantac?). Increasing the temperature from 15 to 40?C caused RH0 to decrease from 79% to 70% (Mauer and Taylor 2010). Both temperature and RH work together to influence the physical stability of powdered ingredients. 57 Table 4.3. Environmental RH in which tagatose deliquescence was observed and RH0 of tagatose at 20, 30 and 40?C. Temperature (?C) RHa RH0b 20 85.1 85.5 30 83.5 83.3 40 82.3 81.0 a from Bell and Labuza (2000) b averages obtained from water activity meters Tagatose Browning During storage, powdered tagatose was observed to brown to varying extents depending upon RH and temperature. Tagatose browning was more extensive at 40?C. Browning was quantified by measuring the optical density of tagatose dissolved in 20 mL water and subtracting the OD/g of the control (i.e., held over drierite). The tagatose stored at 85%RH had the largest absorbance for preliminary browning (280 nm) as shown in Figure 4.6. Tagatose at 75%RH had the largest absorbance for brown pigment formation (420 nm) as shown in Figure. 4.7. The rate constants (OD/g/d) for preliminary activity and brown pigment formation were obtained using the slopes of each line in Figures 4.6 and 4.7. The line for tagatose stored at 85%RH/40?C had the largest slope for preliminary activity, and therefore the greatest rate constant (Figure 4.6 and Table 4.4). Likewise, the slope of the line at 75%RH/40?C was the greatest for brown pigment formation, so the rate constant was the largest (Figure 4.7 and Table 4.4). The rate constants for early stages of browning and brown pigment formation generally increased as temperatures increased, with the highest rate constants at 40?C (Table 4.4). At 40?C, all samples showed visible browning at each RH with pronounced browning at 75 and 85% RH, whereas no visible browning was observed at 20?C. Figure 4.5, presented 58 earlier, shows pictures of the enhanced browning of samples stored at 40?C as well as deliquescence of the samples at 85% RH. When observing the qualitative data, it appears the samples became browner as time increased (Figure 4.8), which is reflected in the quantitative data (Figures 4.6 and 4.7). Results indicated that the rate of brown pigment formation was highest at 75%RH/40?C. Due to the phase change (deliquescence), which was associated with high moisture gain, the reactants leading to brown pigment formation became diluted at 85%RH/40?C. The pH of this sample also decreased (data shown later), which reduces the rate of browning reactions (Hodge 1953). Therefore, the rate constant for brown pigment formation was reduced. Samples stored at 33 and 54% RH at 40?C browned similarly as reflected by their brown pigment formation rate constants (Table 4.4) and pictures (Figure 4.8). However, as the RH continued to increase, samples became noticeably darker with a caramel-like color. When examining the plot for early stages of browning, the lines for 33 and 54% RH are virtually flat with no slope, indicating there was minimal reactivity occurring. Similar results were seen in the plot for brown pigment formation. In nonenzymatic browning at water activity values of 0.5-0.8, the browning rate usually increases with water activity (Labuza and Baiser 1992). Increased aw results in greater mobility as well as dissolution of solutes, which causes the material to become rubbery. However, at low aw the water is bound via hydrogen bonds and reactions do not take place readily (Labuza and Baiser 1992). A maximum is usually obtained followed by a decline due to dilution of reactants (Vaikousi and others 2008). This trend was observed in the current study as shown in Figure 4.9, where a maximum in browning was 59 reached around 75% RH. This maximum was followed by a decline in reactivity around 85% RH. Although Labuza and Baiser (1992) were investigating Maillard browning, a similar trend in browning was observed in the powdered tagatose, which was presumed to be caused by caramelization. - 5 0 5 10 15 20 25 0 50 100 150 200 250 300 350 400 A b s or b an c e at 28 0 n m ( O D /g) T i m e ( d ays ) 3 3 % R H 5 4 % R H 7 5 % R H 8 5 % R H Figure 4.6. Early stages of browning in tagatose at 40?C as affected by RH. 60 - 0 . 1 0 0 . 1 0 . 2 0 . 3 0 . 4 0 . 5 0 . 6 0 . 7 0 . 8 0 . 9 0 100 200 300 400 A b s or b an c e at 420 n m ( O D /g ) T i m e ( d ay s ) 3 3 % R H 5 4 % R H 7 5 % R H 8 5 % R H Figure 4.7. Brown pigment formation in tagatose at 40?C as affected by RH. Table 4.4. Pseudo-zero order rate constants (OD/g/d) with 95% CL times 104 for browning of tagatose at 20, 30 and 40?C. Relative Humidity 20?C 30?C 40?C 280 nma 33% 0c 1.3?0.47 11.5?1.4 54% 0.59?0.39 2.5?0.56 11.1?0.76 75% 2.4?1.1 0c 345?35 85% 2.8?1.3 38.5?10.5 554?40.2 420 nmb 33% 0c 0.63?0.44 2.7?0.45 54% 0c 0c 2.7?0.1 75% 0.94?0.32 0.41?0.38 40.2?3.2 85% 0c 0.48?0.25 20.5?4.8 a= early stages of browning, b= brown pigmentation formation, c= rate constants that were statistically equivalent to zero based on the 95% CL. 61 Figure 4.8. The effect of time on browning of tagatose at 40?C, left to right, after 6 months, 9 months and 12 month. Tagatose at 33, 54, 75 and 85% RH top to bottom. 6 months 9 months 12 months 85% RH 75% RH 54% RH 33% RH 62 0 5 10 15 20 25 30 35 40 45 30 40 50 60 70 80 90 10 4 x R at e C on s t an t ( O D /g/ d ) % R H Figure 4.9. Rate constants for tagatose browning (420 nm) as a function of %RH at 40?C. Hiatt and others (2008; 2010) also captured deliquescence and browning in sodium ascorbate at 85 and 98% RH. In one study (Hiatt and others 2008), a combination of pyridoxine HCl and sodium ascorbate also showed browning at 98% RH after 12 weeks. Binary (thiamin HCl/ sodium ascorbate) and tertiary (thiamin HCl/sodium ascorbate/ pyridoxine HCl) also showed browning at the same conditions. Similarly, browning was observed in tagatose solutions held at various conditions (Dobbs and Bell 2010; Luecke and Bell 2010). Dobbs and Bell (2010) observed enhanced browning at 40?C. Browning of tagatose was most enhanced in the sample with 0.1 M phosphate buffer at pH 7 at 40?C. Luecke and Bell (2010) noticed that as temperature increased, the rate of browning also increased. The rate constants obtained for tagatose browning in 0.1 M phosphate buffer at pH 7 at 60 and 80?C were 0.00661?0.00044 h-1 and 0.0756?0.0070 h-1, respectively. Likewise, faster browning 63 was also observed in the samples exposed to higher concentrations of phosphate buffer at the higher pH (Dobbs and Bell 2010; Luecke and Bell 2010). Since there were no amino acids present in the solutions, the reaction taking place was believed to be a caramelization reaction in both studies (Dobbs and Bell 2010; Luecke and Bell 2010). Conversely, Ryu and others (2004) did not observe any browning or degradation in 10% tagatose solutions heated up to 100?C for 5 hours. However, when combined with glycine, browning and degradation were observed; this was attributed to the Maillard reaction (Ryu and others 2003). Although the powdered tagatose stored at 40?C was generally in a solid form (exception is 85%RH/40?C), it also probably underwent a caramelization reaction. Caramelization can be defined as a ?non-enzymatic sugar browning reaction?which generates a brown color and a caramel-like flavor? (Lee and Lee 1997). Caramelization may be characterized by heat-induced decomposition of monosaccharides. Caramelization includes reactions such as enolization, dehydration, dicarboxylic cleaving and aldol condensation. A release of H+ usually accompanies the reaction, thus decreasing the pH (Kroh 1994). Chemical Stability A combination of high RH and high temperature was required to achieve chemical instability in the tagatose samples. Tagatose degradation only occurred at one environmental condition (85%RH/40?C), which was slightly above RH0. This sample had the largest rate constant for preliminary browning and was fully deliquesced. During the 12 month period, about a 20% loss of tagatose occurred in the sample, but the loss leveled off around 6 months without further degradation. Although all samples 64 deliquesced at 85% RH, only the sample stored at 40?C displayed measurable degradation. Samples stored at 33, 54 and 75% RH at 40?C showed approximately 100% tagatose remaining upon HPLC analysis (Figure 4.10). No loss was observed at 20 and 30?C either. 50 60 70 80 90 100 110 0 50 100 150 200 250 300 350 400 % T a g a t o s e Re ma in in g Ti m e (d a y s ) 3 3 % R H 5 4 % R H 7 5 % R H 8 5 % R H Figure 4.10. Chemical degradation of powdered tagatose at 40?C as affected by RH. A combination of factors caused the dissolution and degradation of tagatose at 85%RH/40?C. The higher temperature contributed to a greater amount of energy, which may have accelerated the reaction. Likewise, the higher RH may have also accelerated the reaction by causing reactants to become more mobile, therefore increasing interaction. Mauer and Taylor (2010) reported that crystalline solids generally have greater chemical stability than amorphous materials or solutions. Mobility is generally limited in crystalline solids, whereas it is higher in solutions (Mauer and Taylor 2010). 65 Furthermore, if a crystalline ingredient, such as tagatose, undergoes deliquescence, chemical stability will most likely be affected adversely. Although the mechanism for degradation of powdered tagatose is unclear, assumptions can be made based on the pH and appearance (caramelization) of the tagatose. In addition, some similarities to tagatose degradation in solution can be identified. The pH values of tagatose dissolved in water were obtained at 6, 9 and 12 months. The pH values of the control samples were 5.8-6.0 at 6 months. The solution pH of tagatose held at 85%RH/40?C was approximately 4 at all time periods. On the other hand, all other samples had pH values ranging from approximately pH 5-6.3. When tagatose stability was investigated in solution, pH decreased from neutral to mildly acidic as tagatose degraded (Dobbs and Bell 2010; Luecke and Bell 2010). The lower pH was attributed to the acidic degradation products and was believed to be responsible for stopping degradation. A plateau was also associated with tagatose loss (Dobbs and Bell 2010), which was also observed in the deliquesced tagatose around 6 months (Figure 4.10). The initial tagatose loss may be the result of a similar alkaline degradation reaction as described by Dobbs and Bell (2010). In solution, monosaccharides undergo a series of rearrangements including, ionization, mutarotation, enolization and isomerization to create an enediol anion species. The species may then participate in several other reactions including ?-elimination, retro-aldol reaction, and aldolization reactions. The series of reactions result in carboxylic acid degradation products, thus lowering the pH and slowing the reaction (de Bruijn and others 1986). It is important to note that caramelization may also decrease pH. 66 Based on the similarities to the studies mentioned above (degradation plateau, pH decrease, brown pigment formation), it can be inferred that the tagatose at 85%/40?C may be participating in a similar alkaline degradation reaction. Tagatose at 85%/40?C was fully deliquesced and brown, so it may very well display behavior similar to alkaline degradation in solution. The behavior is not exactly like the tagatose solutions due to buffers that were present in solution, which allowed the systems to resist changes in pH and also catalyze the reaction (Dobbs and Bell 2010; Luecke and Bell 2010). As mentioned earlier, chemical instability was observed in tagatose solutions (Dobbs and Bell 2010; Luecke and Bell 2010). Tagatose in solution was affected by temperature, buffer type and buffer concentration in both studies. Dobbs and Bell (2010) investigated tagatose loss in solution at 20, 30 and 40?C. Following one month at 40?C, Dobbs and Bell (2010) observed a 29% loss of tagatose in 0.1M phosphate buffer at pH 7 compared to less than 1% loss at pH 3 under the same conditions. The citrate buffer also resulted in less loss compared to the phosphate buffer. Luecke and Bell (2010) investigated tagatose loss at 60, 70 and 80?C and similar results were observed in the study. The interesting observation is that tagatose degradation was favored at the higher pH in higher concentrations of phosphate buffer. Degradation was greatly reduced at lower pH levels and in the absence of buffer. The powdered tagatose, once deliquesced, displayed degradation without buffer present and at relatively low pH levels. Dobbs and Bell (2010) reported the greatest tagatose loss at an acidic condition to be 5% in 6 months (0.1 M citrate buffer at pH 3 and 40?C). The deliquesced tagatose at 85% RH and 40?C experienced approximately 20% loss in 6 months. 67 Hiatt and others (2008) also looked at the effect of deliquescence on the chemical stability of vitamins B1, B6, and C in powder blends. In the study, thiamin HCl (RH0= 89%) degradation ranged from 0-29.8%. Thiamin HCl (vitamin B1) that was stored below the RH0 (54%) had no chemical degradation and showed slight caking. However, samples that were stored above (98% RH) or near the RH0 (85%) displayed significant degradation compared to the samples below RH0. In addition, the thiamin HCl stored at 98% RH deliquesced after 12 weeks (Hiatt and others 2008). Pyridoxine HCl (vitamin B6) degradation ranged from 7-25.3%. The degradation of pyridoxine was not greatly affected by RH because it is less soluble, has a higher RH0 and normally undergoes degradation via light. Salameh and Taylor (2006) also conducted a study looking at the role of deliquescence lowering in increasing chemical reactivity in physical mixtures. The ingredients that were investigated included crystalline sucrose, ?-D-fructose, and ?- glucose monohydrate. The RH0 of mixtures (RH0mix) has been shown to create more chemical instability because when two ingredients are mixed, the RH0mix is even lower than the individual component RH0 values. The RH0mix for a sucrose/citric acid mixture was 64% RH. When the mixture was exposed to a RH at or above 65% RH, complete sucrose inversion occurred. Sucrose inversion is an indication of degradation because the sucrose is breaking down into fructose and glucose. Control samples of sucrose (RH0=85%) alone that were stored at 85 and 71% showed no degradation. Likewise, sucrose/citric acid mixtures stored below the RH0mix at 54 and 43% RH showed no discernible degradation. The previous two studies give a clear indication that if ingredients are stored above the RH0 or RH0mix, chemical degradation can occur. 68 Another explanation for the tagatose degradation is the synergistic effect of RH and temperature. Hiatt and others (2010) conducted a study that looked at the effect of temperature (4, 25, 35 and 40?C) and RH (54, 64, 75, and 85% RH) on vitamin C stability. The study found that both RH and temperature impacted vitamin C stability, but the impact of RH was greater. Temperature and RH behaved synergistically. Higher temperatures caused better vitamin C solubility, which resulted in a reduction in RH0. Consequently, the vitamin C experienced deliquescence, degradation, and browning (Hiatt and others 2010). Temperature and RH also worked synergistically to cause deliquescence, degradation, and browning in tagatose. 69 Chapter 5: Summary and Conclusions Powdered tagatose physical and chemical stabilities were affected to varying extents by RH and temperature. All samples experienced varying amounts of physical instability except for the sample stored at 33% RH/20?C. This sample remained free flowing and was very similar to the control tagatose sample stored over drierite. All samples stored at 85% RH deliquesced and adsorbed large quantities of water. Although samples experienced varying amounts of caking and browning, chemical degradation only occurred in the tagatose sample stored at 85%RH/40?C. This sample had a 20% loss following 6 months of storage. Browning was most pronounced in all samples stored at 40?C. Although no correlation was made between chemical degradation and the prebiotic property of tagatose, it is predicted that the beneficial property will be lessened. It is unlikely that tagatose would be stored for 1 year without use by a food company since materials are ordered as needed and the shelf life is listed as 6 months by the manufacturer. The specifications included with the powdered tagatose stated that it could be stored at 65% RH at 20?C for 6 months. Based on the study conducted, tagatose would cake at this storage condition. The study provides evidence that tagatose should be stored at 33% RH/20?C to remain a free flowing powder with minimal to no chemical degradation. It is also evident that although chemical degradation is not likely to occur, adverse physical changes are probable. Therefore, steps should be taken to keep tagatose stored in a cool, dry location. The shelf life could possibly be extended if stored at these conditions. 70 References Citation Style: Journal of Food Science Adhikari B, Howes T, Bhandari BR, Truong V. 2001. Stickiness in foods: a review of mechanisms and test methods. Int J Food Prop 4(1):1-33. 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Effect of composition and pore structure on binding energy and effective diffusivity of moisture in porous food. J Food Eng 15:187-208. 77 Appendix A Moisture content, physical appearance, optical density and % tagatose remaining of tagatose powder stored at all environmental conditions 78 Table A1. Moisture content, physical appearance, optical density and tagatose remaining of samples stored over drierite at room temperature. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 Drierite 1A 0.03043 free flowing 0.171 0.00487 97.54 0 Drierite 1B 0.09944 free flowing 0.169 0.00365 99.22 28 Drierite 2A 0.2510 free flowing 0.172 0.00628 99.57 28 Drierite 2B 0.2405 free flowing 0.171 0.00601 97.87 56 Drierite 3A 0.2617 free flowing 0.167 0.00552 100.38 56 Drierite 3B -0.02443 free flowing 0.172 0.00562 94.83 84 Drierite 4A 0.3689 free flowing 0.178 0.00851 100.18 84 Drierite 4B 0.2800 free flowing 0.178 0.00653 97.70 112 Drierite 5A 0.2485 free flowing 0.182 0.00963 93.85 112 Drierite 5B -0.2968 free flowing 0.179 0.00759 92.06 140 Drierite 6A 0.09271 free flowing 0.183 0.00618 99.98 140 Drierite 6B 0.3424 free flowing 0.192 0.00871 99.96 168 Drierite 7A 0.3827 free flowing 0.176 0.00902 90.44 168 Drierite 7B 0.4760 free flowing 0.174 0.00254 94.66 196 Drierite 8A 0.3645 free flowing 0.203 0.0129 99.40 196 Drierite 8B 0.5068 free flowing 0.172 0.0101 99.65 224 Drierite 9A 0.6930 free flowing 0.176 0.00181 102.54 224 Drierite 9B 0.7203 free flowing 0.180 0.00345 99.50 252 Drierite10A 0.4956 free flowing 0.157 -0.00204 99.31 252 Drierite 10B 0.7982 free flowing 0.165 0.0128 99.70 282 Drierite11A 0.8173 free flowing 0.164 0.0389 100.12 282 Drierite 11B 0.6422 free flowing 0.171 0.0428 99.69 318 Drierite12A 0.6834 free flowing 0.194 -0.00746 98.51 318 Drierite 12B 0.5270 free flowing 0.188 -0.00620 98.06 350 Drierite13A 0.6878 free flowing 0.193 0.00807 101.37 350 Drierite 13B 0.5159 free flowing 0.180 0.00593 100.25 79 Table A2. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 33% RH at 20?C. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 33-20-1A 0.4268 free flowing 0.187 -0.00711 98.78 0 33-20-1B -0.3216 free flowing 0.181 -0.00397 97.35 28 33-20-2A 0.2613 free flowing 0.188 -0.00402 99.13 28 33-20-2B 0.0377 free flowing 0.198 -0.00264 98.86 56 33-20-3A 0.3212 free flowing 0.187 -0.00505 98.38 56 33-20-3B -2.685 free flowing 0.192 -0.00290 95.53 84 33-20-4A -0.1361 free flowing 0.185 -0.0127 92.85 84 33-20-4B 0.03965 free flowing 0.208 -0.00833 95.63 112 33-20-5A -0.2936 free flowing 0.203 -0.00822 94.40 112 33-20-5B 0.3106 free flowing 0.198 -0.00580 97.26 140 33-20-6A -0.2446 free flowing 0.204 -0.00828 100.52 140 33-20-6B 0.2629 free flowing 0.212 -0.00627 99.17 168 33-20-7A 0.4711 free flowing 0.211 -0.00621 100.84 168 33-20-7B 0.3718 free flowing 0.210 -0.0117 99.92 196 33-20-8A 0.2969 free flowing 0.179 0.00959 96.78 196 33-20-8B 0.4288 free flowing 0.175 0.00453 99.11 224 33-20-9A 0.6711 free flowing 0.176 0.00183 100.98 224 33-20-9B 0.5110 free flowing 0.182 0.00266 101.61 252 33-20-10A 0.3055 free flowing 0.166 -0.00131 98.70 252 33-20-10B 0.5215 free flowing 0.166 -0.000869 98.89 282 33-20-11A 0.5495 free flowing 0.169 0.0132 98.81 282 33-20-11B 0.6721 free flowing 0.161 0.0103 96.76 318 33-20-12A 0.4355 free flowing 0.238 -0.000207 97.63 318 33-20-12B 0.3607 free flowing 0.200 -0.00180 97.68 350 33-20-13A 0.4109 free flowing 0.193 0.00799 100.34 350 33-20-13B 0.3375 free flowing 0.192 0.00675 100.32 80 Table A3. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 33% RH at 30?C. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 33-30-1A -0.1355 partially caked 0.196 -0.00697 98.66 0 33-30-1B 0.04187 partially caked 0.195 -0.0105 98.72 28 33-30-2A -0.02240 fully caked 0.205 -0.0125 98.84 28 33-30-2B -0.09508 fully caked 0.211 0.00837 101.35 56 33-30-3A -0.2052 slightly caked 0.212 -0.0117 96.69 56 33-30-3B 0.0000 slightly caked 0.204 -0.0113 105.28 84 33-30-4A 0.08339 slightly caked 0.209 -0.0136 100.16 84 33-30-4B -0.3167 slightly caked 0.210 -0.0174 100.34 112 33-30-5A 0.1436 free flowing 0.215 -0.00923 97.23 112 33-30-5B 0.0000 free flowing 0.218 -0.0113 95.71 140 33-30-6A -0.6682 slightly caked 0.229 -0.0124 100.60 140 33-30-6B 0.01962 slightly caked 0.231 -0.0129 100.68 168 33-30-7A 0.4324 partially caked 0.243 -0.00827 95.50 168 33-30-7B 0.2383 partially caked 0.239 -0.00814 96.82 196 33-30-8A 0.1434 partially caked 0.212 0.00771 99.61 196 33-30-8B 0.1776 partially caked 0.212 0.00888 99.18 224 33-30-9A 0.3393 partially caked 0.230 0.0132 101.37 224 33-30-9B 0.3537 partially caked 0.219 0.00791 101.61 252 33-30-10A 0.3484 partially caked 0.202 0.00261 99.95 252 33-30-10B 0.2839 partially caked 0.209 0.00454 99.24 282 33-30-11A 0.3387 partially caked 0.240 0.0134 98.60 282 33-30-11B 0.05767 partially caked 0.217 0.00961 98.90 318 33-30-12A 0.2733 partially caked 0.272 0.00605 97.41 318 33-30-12B 0.06333 partially caked 0.260 0.00274 98.28 350 33-30-13A 0.0000 slightly caked 0.260 0.0197 98.63 350 33-30-13B 0.05673 partially caked 0.274 0.0151 101.21 81 Table A4. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 33% RH at 40?C. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 33-40-1A -0.4622 fully caked 0.0624 -0.0153 101.07 0 33-40-1B -0.3610 fully caked 0.211 -0.0214 97.23 28 33-40-2A -0.4404 fully caked 0.257 -0.0167 98.45 28 33-40-2B -0.1940 fully caked 0.245 -0.0199 100.04 56 33-40-3A -0.07278 fully caked 0.267 -0.0138 91.68 56 33-40-3B -0.2684 fully caked 0.266 -0.0137 94.53 84 33-40-4A -0.2178 fully caked 0.320 -0.00139 101.17 84 33-40-4B -0.06306 fully caked 0.342 -0.000210 98.80 112 33-40-5A -0.2400 fully caked 0.358 -0.0122 99.15 112 33-40-5B -0.1930 fully caked 0.359 -0.0113 96.04 140 33-40-6A -0.2877 fully caked 0.414 -0.00310 98.33 140 33-40-6B -0.02470 fully caked 0.406 -0.0109 95.73 168 33-40-7A 0.09706 fully caked 0.451 -0.00461 99.00 168 33-40-7B 0.1419 fully caked 0.453 -0.00497 96.25 196 33-40-8A 0.0000 fully caked 0.446 0.0376 99.99 196 33-40-8B 0.04792 fully caked 0.431 0.0345 99.98 224 33-40-9A 0.1444 fully caked 0.473 0.0390 98.98 224 33-40-9B 0.4352 fully caked 0.452 0.0369 99.05 252 33-40-10A 0.2407 fully caked 0.498 0.0652 99.79 252 33-40-10B 0.3840 fully caked 0.468 0.0420 99.89 282 33-40-11A 0.2687 fully caked 0.512 0.0764 97.02 282 33-40-11B 0.3068 fully caked 0.518 0.0771 98.75 318 33-40-12A -0.07065 fully caked 0.545 0.0440 98.67 318 33-40-12B 0.02430 fully caked 0.596 0.0476 98.43 350 33-40-13A -0.1942 fully caked 0.613 0.0650 98.64 350 33-40-13B -0.1671 fully caked 0.620 0.0683 100.28 82 Table A5. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 54% RH at 20?C. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 54-20-1A -0.6346 fully caked 0.174 0.00562 98.30 0 54-20-1B 0.09198 fully caked 0.156 0.00515 98.50 28 54-20-2A 0.01977 slightly caked 0.152 0.00119 102.31 28 54-20-2B 0.2851 slightly caked 0.187 0.0101 100.06 56 54-20-3A -0.4618 slightly caked 0.184 0.00482 100.46 56 54-20-3B 0.1961 slightly caked 0.177 0.00349 99.81 84 54-20-4A -0.3034 slightly caked 0.170 0.00433 93.33 84 54-20-4B -0.4213 free flowing 0.162 0.00441 96.14 112 54-20-5A -0.4829 slightly caked 0.189 0.00984 95.75 112 54-20-5B -0.2141 slightly caked 0.175 0.00707 101.80 140 54-20-6A 0.3120 slightly caked 0.185 0.00716 98.85 140 54-20-6B -0.5856 slightly caked 0.195 0.00390 99.21 168 54-20-7A -0.2871 partially caked 0.180 0.00390 100.94 168 54-20-7B 0.3696 partially caked 0.181 0.00564 97.97 196 54-20-8A 0.06025 partially caked 0.181 0.00562 99.84 196 54-20-8B -0.3788 partially caked 0.176 0.000421 99.78 224 54-20-9A 0.1482 partially caked 0.187 0.00296 103.60 224 54-20-9B -2.038 partially caked 0.186 0.00530 98.96 252 54-20-10A 0.6647 partially caked 0.174 0.00236 97.15 252 54-20-10B 0.4300 fully caked 0.171 0.000748 98.99 282 54-20-11A 0.2944 fully caked 0.169 0.0269 98.72 282 54-20-11B 0.3030 fully caked 0.180 0.0277 99.13 318 54-20-12A -0.2224 fully caked 0.223 0.00425 98.32 318 54-20-12B 0.2023 fully caked 0.202 -0.00465 98.44 350 54-20-13A 0.08963 fully caked 0.211 0.00605 100.34 350 54-20-13B 0.1116 fully caked 0.196 0.00357 100.93 83 Table A6. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 54% RH at 30?C. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 54-30-1A -0.8316 fully caked 0.151 0.00151 100.84 0 54-30-1B -0.3540 fully caked 0.155 0.00819 98.92 28 54-30-2A 0.0000 fully caked 0.162 0.00275 99.81 28 54-30-2B -0.4003 fully caked 0.153 0.000445 98.62 56 54-30-3A -0.09956 partially caked 0.185 0.00538 96.08 56 54-30-3B -1.331 partially caked 0.156 0.00148 97.58 84 54-30-4A -2.016 fully caked 0.174 0.00450 96.17 84 54-30-4B 1.154 fully caked 0.160 0.00266 93.30 112 54-30-5A -0.7922 fully caked 0.190 0.0136 96.35 112 54-30-5B -0.5736 fully caked 0.189 0.0111 95.67 140 54-30-6A -0.04322 fully caked 0.198 0.00605 96.89 140 54-30-6B -0.01981 fully caked 0.201 0.00436 98.32 168 54-30-7A -0.3956 fully caked 0.231 0.0136 98.06 168 54-30-7B -1.426 fully caked 0.199 0.00643 97.09 196 54-30-8A -0.7758 fully caked 0.243 -0.00446 96.72 196 54-30-8B 0.08198 fully caked 0.209 0.0150 98.95 224 54-30-9A -0.1951 fully caked 0.189 -0.0141 99.88 224 54-30-9B 0.08505 fully caked 0.183 -0.0117 101.34 252 54-30-10A 0.1071 fully caked 0.212 0.00514 97.31 252 54-30-10B -1.041 fully caked 0.210 0.00167 98.17 282 54-30-11A 0.3175 fully caked 0.216 -0.00437 98.50 282 54-30-11B -0.09398 fully caked 0.217 -0.00376 99.05 318 54-30-12A -1.072 fully caked 0.240 -0.00445 96.90 318 54-30-12B -0.07388 fully caked 0.237 -0.00351 97.38 350 54-30-13A 0.0000 fully caked 0.267 0.0128 101.12 350 54-30-13B -0.1447 fully caked 0.273 0.0174 100.52 84 Table A7. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 54% RH at 40?C. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 54-40-1A -0.07455 fully caked 0.183 0.00746 100.61 0 54-40-1B -0.1608 fully caked 0.184 0.00299 96.91 28 54-40-2A -0.1168 slightly caked 0.203 0.00974 98.21 28 54-40-2B -0.1119 slightly caked 0.202 0.00918 98.43 56 54-40-3A -0.3391 partially caked 0.230 0.0143 98.82 56 54-40-3B -0.2393 partially caked 0.203 0.00892 94.13 84 54-40-4A -0.1452 fully caked 0.235 0.0104 101.70 84 54-40-4B -0.2432 fully caked 0.238 0.0107 103.54 112 54-40-5A -0.2962 fully caked 0.270 0.0116 95.38 112 54-40-5B -0.07378 fully caked 0.287 0.0219 98.77 140 54-40-6A -0.07224 fully caked 0.357 0.0330 100.95 140 54-40-6B -0.04912 fully caked 0.344 0.0270 98.43 168 54-40-7A -0.04779 fully caked 0.362 0.0282 93.90 168 54-40-7B 0.04937 fully caked 0.361 0.0281 97.14 196 54-40-8A -0.06745 fully caked 0.380 0.0297 99.22 196 54-40-8B -0.04896 fully caked 0.385 0.0184 99.77 224 54-40-9A 0.2885 slightly caked 0.380 0.0300 101.60 224 54-40-9B 0.4177 slightly caked 0.395 0.0316 101.83 252 54-40-10A 0.3353 fully caked 0.440 0.0309 99.80 252 54-40-10B 0.4292 fully caked 0.443 0.0181 97.46 282 54-40-11A 0.2747 fully caked 0.441 0.0764 96.95 282 54-40-11B 0.4011 fully caked 0.456 0.0750 97.22 318 54-40-12A -0.04898 fully caked 0.538 0.0544 98.62 318 54-40-12B 0.02459 fully caked 0.542 0.0578 98.61 350 54-40-13A -0.1666 fully caked 0.582 0.0664 100.56 350 54-40-13B -0.1160 fully caked 0.604 0.0705 100.92 85 Table A8. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 75% RH at 20?C. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 75-20-1A 0.5000 fully caked 0.204 -0.00860 98.87 0 75-20-1B 0.4958 fully caked 0.205 -0.00873 99.21 28 75-20-2A 0.6419 fully caked 0.225 -0.00553 97.68 28 75-20-2B 0.6198 fully caked 0.220 -0.00826 96.82 56 75-20-3A 0.6264 fully caked 0.234 -0.00304 99.27 56 75-20-3B 0.4172 fully caked 0.242 0.000199 97.51 84 75-20-4A 0.6639 fully caked 0.255 -0.00664 98.63 84 75-20-4B 0.6037 fully caked 0.247 -0.00765 97.93 112 75-20-5A 0.6375 fully caked 0.256 -0.00717 94.12 112 75-20-5B 0.4765 fully caked 0.250 -0.00834 98.15 140 75-20-6A 0.6575 fully caked 0.212 0.0125 97.82 140 75-20-6B 0.5989 fully caked 0.268 -0.00867 100.30 168 75-20-7A 0.7769 fully caked 0.262 -0.0133 97.48 168 75-20-7B 0.7914 fully caked 0.273 -0.0134 98.56 196 75-20-8A 0.8356 fully caked 0.233 0.0190 97.41 196 75-20-8B -0.8554 fully caked 0.249 0.0210 98.59 224 75-20-9A 1.034 fully caked 0.217 0.00823 101.38 224 75-20-9B 0.9259 fully caked 0.224 0.00903 100.89 252 75-20-10A 1.108 fully caked 0.243 -0.000443 99.59 252 75-20-10B 1.000 fully caked 0.247 0.00652 99.14 282 75-20-11A 1.249 fully caked 0.246 0.0450 97.13 282 75-20-11B 0.9599 fully caked 0.250 0.0422 98.60 318 75-20-12A 0.8946 fully caked 0.305 0.0183 98.04 318 75-20-12B 0.7413 fully caked 0.292 0.0160 98.32 350 75-20-13A 0.8357 fully caked 0.387 0.0233 101.06 350 75-20-13B 0.9083 fully caked 0.364 0.0202 100.08 86 Table A9. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 75% RH at 30?C. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 75-30-1A 0.3403 fully caked 0.211 -0.0159 97.74 0 75-30-1B -0.2569 fully caked 0.216 -0.0193 99.93 28 75-30-2A 0.2280 fully caked 0.214 -0.0174 97.84 28 75-30-2B 0.4321 fully caked 0.166 0.00518 97.94 56 75-30-3A 0.3850 fully caked 0.218 -0.0207 94.74 56 75-30-3B -0.2365 fully caked 0.215 -0.0206 95.38 84 75-30-4A 0.3596 fully caked 0.159 -0.0161 97.96 84 75-30-4B 0.3620 fully caked 0.158 -0.0207 98.08 112 75-30-5A 0.3934 fully caked 0.219 -0.0221 94.13 112 75-30-5B 0.5348 fully caked 0.226 -0.0206 95.38 140 75-30-6A 0.3366 fully caked 0.238 -0.0206 97.49 140 75-30-6B 0.4791 fully caked 0.250 -0.0183 99.28 168 75-30-7A 0.6612 fully caked 0.185 0.000735 97.66 168 75-30-7B 0.7384 fully caked 0.238 -0.0174 96.74 196 75-30-8A 0.4010 fully caked 0.211 0.0110 97.24 196 75-30-8B 0.8158 fully caked 0.216 0.00258 97.33 224 75-30-9A 0.7635 fully caked 0.179 -0.0252 101.16 224 75-30-9B 0.9759 fully caked 0.397 0.000413 100.99 252 75-30-10A 0.9098 fully caked 0.199 -0.0215 101.37 252 75-30-10B 0.6388 fully caked 0.220 -0.00793 100.33 282 75-30-11A 0.8168 fully caked 0.203 0.0286 99.01 282 75-30-11B 0.8949 fully caked 0.199 0.0267 98.63 318 75-30-12A 0.7443 fully caked 0.217 -0.0271 95.92 318 75-30-12B 0.6527 fully caked 0.215 -0.0306 95.70 350 75-30-13A 1.135 fully caked 0.253 0.00817 100.63 350 75-30-13B 0.6405 fully caked 0.262 0.00941 100.59 87 Table A10. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 75% RH at 40?C. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 75-40-1A 0.2824 fully caked 0.228 -0.0212 97.25 0 75-40-1B 0.2633 fully caked 0.224 -0.0268 99.57 28 75-40-2A -1.154 fully caked 0.235 -0.0244 98.16 28 75-40-2B 0.4170 fully caked 0.237 -0.0216 99.47 56 75-40-3A 0.4941 fully caked 0.306 -0.0205 96.48 56 75-40-3B 0.4867 fully caked 0.290 -0.0204 96.12 84 75-40-4A 0.7290 fully caked 0.364 -0.00142 96.25 84 75-40-4B 0.7907 fully caked 0.384 -0.00333 96.44 112 75-40-5A 0.7308 fully caked 0.539 0.0127 96.47 112 75-40-5B 0.8800 fully caked 0.539 0.0153 99.22 140 75-40-6A 1.118 fully caked 0.786 0.0474 98.08 140 75-40-6B 0.8525 fully caked 0.846 0.0632 95.16 168 75--40-7A 1.104 fully caked 1.08 0.0888 96.39 168 75-40-7B 1.150 fully caked 1.05 0.0858 94.31 196 75-40-8A 1.231 fully caked 1.54 0.188 97.05 196 75-40-8B 1.093 fully caked 1.51 0.185 99.22 224 75-40-9A 1.518 fully caked 2.44 0.262 101.03 224 75-40-9B 1.394 fully caked 2.50 0.261 101.26 252 75-40-10A 1.578 fully caked 3.18 0.365 98.87 252 75-40-10B 1.488 fully caked 3.15 0.364 98.30 282 75-40-11A 1.659 fully caked 4.12 0.498 98.22 282 75-40-11B 1.745 fully caked 4.17 0.500 97.81 318 75-40-12A 1.250 fully caked 5.60 0.668 96.37 318 75-40-12B 1.410 fully caked 5.81 0.644 96.32 350 75-40-13A 1.957 fully caked 7.60 0.826 96.54 350 75-40-13B 1.721 fully caked 7.22 0.830 99.08 88 Table A11. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 85% RH at 20?C. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 85-20-1A 26.48 partially deliq 0.177 0.00294 100.15 0 85-20-1B 14.31 partially deliq 0.239 0.00233 100.17 28 85-20-2A 22.04 partially deliq 0.148 0.00362 97.60 28 85-20-2B 13.85 partially deliq 0.232 0.0102 98.00 56 85-20-3A 14.72 partial deliq 0.179 0.00340 97.15 56 85-20-3B 80.79 fully deliq 0.147 0.00369 95.61 84 85-20-4A 80.87 fully deliq 0.224 0.00277 99.65 84 85-20-4B 75.81 fully deliq 0.174 -0.000407 100.19 112 85-20-5A 54.97 partial deliq 0.188 -0.00101 99.31 112 85-20-5B 40.14 partial deliq 0.213 -0.000863 96.97 140 85-20-6A 81.38 fully deliq 0.184 0.000820 96.93 140 85-20-6B 77.62 fully deliq 0.257 -0.00134 96.70 168 85-20-7A 80.40 fully deliq 0.242 -0.00366 98.97 168 85-20-7B 82.06 fully deliq 0.246 -0.00425 99.89 196 85-20-8A 76.41 fully deliq 0.225 0.00907 99.42 196 85-20-8B 80.76 fully deliq 0.255 0.00866 99.31 224 85-20-9A 83.80 fully deliq 0.188 -0.00139 100.33 224 85-20-9B 81.38 fully deliq 0.184 0.00158 97.72 252 85-20-10A 80.52 fully deliq 0.269 0.00197 96.55 252 85-20-10B 79.58 fully deliq 0.238 0.00977 96.16 282 85-20-11A 83.05 fully deliq 0.298 0.0280 95.80 282 85-20-11B 77.74 fully deliq 0.282 0.0300 94.51 318 85-20-12A 79.57 fully deliq 0.284 0.00264 96.57 318 85-20-12B 76.10 fully deliq 0.275 0.00392 97.59 350 85-20-13A 81.52 fully deliq 0.254 0.00185 97.34 350 85-20-13B 79.22 fully deliq 0.317 0.000942 100.03 89 Table A12. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 85% RH at 30?C. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 85-30-1A 65.70 fully deliq 0.177 -0.00488 98.73 0 85-30-1B 63.28 almost fully deliq 0.186 -0.00260 99.48 28 85-30-2A 68.70 fully deliq 0.230 -0.00552 102.64 28 85-30-2B 68.56 fully deliq 0.217 -0.00394 98.01 56 85-30-3A 68.51 fully deliq 0.196 -0.00812 96.31 56 85-30-3B 67.75 fully deliq 0.191 -0.00646 91.53 84 85-30-4A 67.55 fully deliq 0.320 -0.00514 98.46 84 85-30-4B 67.75 fully deliq 0.347 -0.00962 96.42 112 85-30-5A 64.79 fully deliq 0.524 -0.00477 96.28 112 85-30-5B 68.67 fully deliq 0.409 -0.00664 96.44 140 85-30-6A 68.09 fully deliq 0.690 -0.00797 97.82 140 85-30-6B 68.11 fully deliq 0.641 -0.00940 97.17 168 85-30-7A 66.10 fully deliq 0.764 -0.00569 97.25 168 85-30-7B 66.91 fully deliq 0.829 -0.00370 98.36 196 85-30-8A 65.68 fully deliq 0.848 0.00793 94.42 196 85-30-8B 65.97 fully deliq 0.829 0.00984 95.91 224 85-30-9A 65.73 fully deliq 1.36 -0.00129 96.70 224 85-30-9B 65.64 fully deliq 1.62 -0.00525 97.91 252 85-30-10A 65.20 fully deliq 1.54 0.00159 93.58 252 85-30-10B 64.35 fully deliq 1.07 0.00107 95.87 282 85-30-11A 66.32 fully deliq 1.59 0.0302 95.33 282 85-30-11B 66.65 fully deliq 1.65 0.0318 93.83 318 85-30-12A 65.60 fully deliq 1.00 0.00680 94.54 318 85-30-12B 65.18 fully deliq 1.02 0.0127 93.74 350 85-30-13A 65.08 fully deliq 0.820 0.00466 94.88 350 85-30-13B 65.02 fully deliq 1.47 0.00536 96.82 90 Table A13. Moisture content, physical appearance, optical density and tagatose remaining of samples stored at 85% RH at 40?C. Time (days) Sample Moisture Content (%db) Physical Appearance OD/g (280 nm) OD/g (420 nm) % Tagatose remaining 0 85-40-1A 61.77 fully deliq 0.294 0.00706 97.86 0 85-40-1B 57.28 fully deliq 0.304 0.00838 95.34 28 85-40-2A 56.36 fully deliq 0.651 0.00468 96.10 28 85-40-2B 55.26 fully deliq 0.819 0.00432 96.43 56 85-40-3A 57.94 fully deliq 1.82 0.0158 91.58 56 85-40-3B 57.53 fully deliq 1.69 0.00745 91.79 84 85-40-4A 52.13 fully deliq 4.10 0.0169 91.43 84 85-40-4B 50.72 fully deliq 3.59 0.0137 90.26 112 85-40-5A 54.27 fully deliq 6.08 0.0312 87.68 112 85-40-5B 53.66 fully deliq 6.29 0.0274 91.25 140 85-40-6A 55.41 fully deliq 7.78 0.0440 86.78 140 85-40-6B 54.89 fully deliq 7.20 0.0435 85.60 168 85-40-7A 56.61 fully deliq 7.22 0.0431 81.96 168 85-40-7B 56.27 fully deliq 7.29 0.0492 81.97 196 85-40-8A 51.71 fully deliq 12.5 0.0518 83.01 196 85-40-8B 49.51 fully deliq 12.4 0.0540 82.68 224 85-40-9A 54.98 fully deliq 12.9 0.0620 86.15 224 85-40-9B 54.95 fully deliq 13.2 0.0750 85.41 252 85-40-10A 53.79 fully deliq 14.3 0.216 83.65 252 85-40-10B 55.70 fully deliq 11.8 0.075 85.96 282 85-40-11A 50.88 fully deliq 15.1 0.287 82.15 282 85-40-11B 50.46 fully deliq 14.6 0.210 81.19 318 85-40-12A 52.09 fully deliq 16.0 0.247 81.26 318 85-40-12B 53.08 fully deliq 16.8 0.268 80.33 350 85-40-13A 49.17 fully deliq 20.3 0.376 81.48 350 85-40-13B 49.81 fully deliq 18.2 0.340 84.45 91 Appendix B pH values of tagatose obtained at time 6, 9 and 12. 92 B1. Duplicate pH values for tagatose at time 6, 9 and 12 months. Sample Time 6 pH Time 9 pH Time 12 pH Drierite 5.755 6.201 5.567 Drierite 6.02 6.137 5.737 33%RH/20?C 4.98 5.626 5.326 33%RH/20?C 5.23 5.53 5.631 33%RH/30?C 5.43 5.029 5.445 33%RH/30?C 5.49 4.945 5.485 33%RH/40?C 5.09 4.894 5.352 33%RH/40?C 5.39 4.967 5.456 54%RH/20?C 5.22 5.121 5.435 54%RH/20?C 4.96 5.01 5.321 54%RH/30?C 4.94 5.055 5.298 54%RH/30?C 4.92 4.917 5.399 54%RH/40?C 5.08 4.945 5.551 54%RH/40?C 5.87 5 5.373 75%RH/20?C 5.59 5.657 5.867 75%RH/20?C 5.47 5.706 5.856 75%RH/30?C 5.29 5.36 5.806 75%RH/30?C 5.51 5.404 5.666 75%RH/40?C 5.65 5.898 5.636 75%RH/40?C 5.87 5.913 5.601 85%RH/20?C 5.52 6.045 6.158 85%RH/20?C 5.89 5.87 6.116 85%RH/30?C 5.28 4.792 5.132 85%RH/30?C 5.53 5.092 5.527 85%RH/40?C 4.06 3.955 4.052 85%RH/40?C 3.98 4.109 4.101 93 Appendix C Pictures of tagatose at time 6, 9 and 12 months 94 Figure C1. Duplicate pictures of tagatose at time 6. 0%RH/RT 0%RH/RT 33%RH/20?C 33%RH/20?C 54%RH/20?C 54%RH/20?C 75%RH/20?C 75%RH/20?C 85%RH/20?C 85%RH/20?C 33%RH/30?C 33%RH/30?C 54%RH/30?C 54%RH/30?C 75%RH/30?C 75%RH/30?C 85%RH/30?C 85%RH/30?C 33%RH/40?C 33%RH/40?C 54 %RH/40?C 54%RH/40?C 75%RH/40?C 75%RH/40?C 85%RH/40?C 85%RH/40?C 95 Figure C2. Duplicate pictures of tagatose at time 9. 0%RH/RT 0%RH/RT 33%RH/20?C 33%RH/20?C 54%RH/20?C 54%RH/20?C 75%RH/20?C 75%RH/20?C 85%RH/20?C 85%RH/20?C 33%RH/30?C 33%RH/30?C 54%RH/30?C 54%RH/30?C 75%RH/30?C 75%RH/30?C 85%RH/30?C 85%RH/30?C 33%RH/40?C 33%RH/40?C 54 %RH/40?C 54%RH/40?C 75%RH/40?C 75%RH/40?C 85%RH/40?C 85%RH/40?C 96 Figure C3. Duplicate pictures of tagatose at time 12. 0%RH/RT 0%RH/RT 33%RH/20?C 33%RH/20?C 54%RH/20?C 54%RH/20?C 75%RH/20?C 75%RH/20?C 85%RH/20?C 85%RH/20?C 33%RH/30?C 33%RH/30?C 54%RH/30?C 54%RH/30?C 75%RH/30?C 75%RH/30?C 85%RH/30?C 85%RH/30?C 33%RH/40?C 33%RH/40?C 54 %RH/40?C 54%RH/40?C 75%RH/40?C 75%RH/40?C 85%RH/40?C 85%RH/40?C 97 Appendix D Particle size study 98 Particle Size Study As mentioned earlier in the Materials and Methods section, studies show that particle size influences moisture adsorption and deliquescence (Mathlouthi and Roge 2003; Salameh and Taylor 2006a; Kwok and others 2010); therefore the effect of particle size on deliquescence of tagatose was investigated. In the primary study, particle size was not taken into account. Therefore, to eliminate any particle size bias on deliquescence, a smaller study was conducted to see if smaller particles of tagatose would deliquesce at a faster rate than larger particles. It is important to note that the tagatose used for the primary study was food grade tagatose and would be the type normally used in an industrial setting. Similar methods were used as mentioned in the particle size characterization section to obtain desired particle sizes. Tagatose that had a particle size greater than 500 ?m was used to study the effect of larger particles on deliquescence. Likewise tagatose particles less than 250 ?m were used to study the effect of smaller crystals on deliquescence. Triplicate vials were placed in four desiccators. The four desiccators were prepared at two different environmental conditions, 75% RH (NaCl) and 85% RH (KCl). Two of each desiccator were placed at 20 and 30?C. The desiccators were first checked daily and then weekly for a total of 17 weeks. Physical descriptions were recorded to monitor how fast samples deliquesced. Physical descriptors included: wet, slurry, partially deliquesced, fully deliquesced, caked and solid. Plots were generated that displayed moisture content (dry basis) vs. time (days). Standard deviations were represented with error bars on each chart to determine if samples of varying particle sizes equilibrated at a faster rate and/or adsorbed more water. 99 There was no conclusive evidence that suggested that smaller or larger particles of tagatose deliquesced at a faster rate. Samples that were stored at 75% RH at 20 and 30?C gained less that 2% moisture (db). Due to no significant water gain, no further analysis was done on those samples. Figures D1 and D2 show average moisture contents of the three samples on the y-axis and time on the x-axis. The plots look very similar. The larger particle six adsorbed water at a faster rate initially. For the most part, the profiles for small and larger particles of tagatose were similar. It was hypothesized that smaller particles would deliquesce at a faster rate. Salameh and Taylor (2006a) investigated moisture sorption of unground and ground powders. Using a higher fraction of fine particles in the glucose/citric acid mixture led to increased cake mechanical strength. Similarly, another study observed increased water sorption in smaller particle sizes of sucrose. This led to increased caking, which was credited to the increased specific solid surface area which allowed more water adsorption (Roge and Mathlouthi 2003; Mathlouthi and Roge 2003). Kwok and others (2010) also explored moisture sorption of various particle sizes including the following: 425?600 mm (large particles), 150?212 mm (medium particles), <53 mm (small particles). Particle size did not have a large impact on the sodium chloride/sucrose system. However, in the fructose/citric acid system, the smallest particles absorbed the most moisture (Kwok and others 2010). Although tagatose particle size does not seem to drastically affect its moisture adsorption and subsequent deliquescence, particle size does have an effect on moisture adsorption for some powdered ingredients. 100 0 20 40 60 80 100 120 0 20 40 60 80 100 120 140 160 % M oi s t ur e ( db ) T i m e ( da y s ) Figure D1. Moisture content (db) vs. time for particles < 250 nm at 85% RH/20?C. 0 20 40 60 80 100 120 0 20 40 60 80 100 120 140 160 % M oi s t ur e ( db ) T i m e ( da y s ) Figure D2. Moisture content (db) vs. time for particles > 500 nm at 85% RH/20?C.