Abstract. ZEVCHAK, SARAH E. Impact of Agglomeration on Flavor and Flavor Stability of Whey Proteins. (Under the direction of Dr. MaryAnne Drake.

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1 Abstract ZEVCHAK, SARAH E. Impact of Agglomeration on Flavor and Flavor Stability of Whey Proteins. (Under the direction of Dr. MaryAnne Drake.) Descriptive sensory analysis of freshly produced WPC80 and WPI has documented a variety of flavors in these products including sweet aromatic, cardboard/wet paper, pasta water, brothy, cucumber, and soapy flavors, astringent mouthfeel and bitter taste. Concurrent volatile analysis has revealed an array of heatinduced and lipid and protein oxidation compounds. The purported shelf life of WPC80 and WPI varies from 12 to 18 months depending on the supplier. However, to our knowledge, no studies have addressed stability of WPC80 or WPI or the impact of agglomeration on the flavor and flavor stability of WPC80 and WPI. In this study, agglomerated (re-wet and single pass) and non-agglomerated samples of WPC80 and WPI from different facilities were analyzed for flavor and selected physical properties over the course of fifteen months. Descriptive sensory analysis and volatile analysis were conducted every 2 months. Samples were tested every six months for solubility, bulk volume, dispersibility, moisture, and color (L,a,b). Proximate analysis was conducted at time zero. Consumer acceptance tests were conducted on representative samples after 0, 6, 9, 12 and 15 months storage using vanilla protein shakes and peach flavored beverages with WPC80 and fruit flavored clear acidified beverages with WPI. Agglomerated powders displayed higher bulk volume and dispersibility than their non-agglomerated counterparts. Solubility, bulk volume, dispersibility, moisture and color did not significantly change with storage time. Higher intensities of lipid oxidation

2 flavors (cardboard, raisin/brothy, cucumber, and fatty) were noted in agglomerated powders compared to control powders (p<0.05). Sensory results were confirmed by volatile analysis results which showed increased formation of aldehydes and ketones in agglomerated products compared to control powders (p<0.05). Acceptance tests with protein beverages revealed few differences in consumer acceptance between agglomerated and non-agglomerated whey proteins or between fresh versus stored whey proteins although trained panelists documented consistent differences. Agglomeration or agglomeration with lecithin decreased the storage stability of whey proteins. These results indicate that the optimum shelf life at 21C for non-agglomerated powders is between months and 8-10 months for agglomerated powders.

3 The Impact of Agglomeration on Flavor and Flavor Stability of Whey Proteins by Sarah E. Zevchak A thesis submitted to the Graduate Faculty of North Carolina State University in partial fulfillment of the requirements for the Degree of Master of Science Food Science Raleigh, North Carolina 2007 Approved by: Dr. MaryAnne Drake, Committee Chair Dr. E. Allen Foegeding, Committee member Dr. Lynn Turner, Committee member

4 Biography Sarah Elizabeth Zevchak was born on May 24, 1983, in Kalamazoo, Michigan, to Constance and David Zevchak. She has one sister, Amy Ranew, who lives with her husband Jason Ranew in Orlando, Florida. Her parents currently reside in South Lyon, Michigan. Sarah graduated from South Lyon High School in June 2001, and began her Bachelor of Science degree at Ohio State University in September of In the summer of 2004, she did an internship with Ross Products Division of Abbott Laboratories in Columbus, Ohio, where she worked on the development of infant formula. In March 2005, Sarah graduated with a Bachelor of Science degree in Food Science and Nutrition from Ohio State University. She interned with Nestle in the summer of 2005, in Marysville, Ohio, where she worked on the development of flavored milks. Sarah decided to continue her education and pursue a Masters degree with the Department of Food Science at North Carolina State University in the fall of ii

5 Acknowledgements To my parents, my steady source of love comfort, thank you for always listening to me and encouraging me. I could not have made it where I am today without you. To Raphael, my rock and source of so much happiness, thank you for being the bright light at the end of the graduate school tunnel. To my advisor, Dr. MaryAnne Drake, thank you for believing in me, even at the times when I did not believe in myself. To Dr. Foegeding and Dr. Sanders, thank you for taking the time to serve on my committee. Thank you to all the members of the Drake lab for tasting, assisting, and answering my many questions. Lastly, thank you to Dairy Management Inc., for funding this research. iii

6 Table of Contents Page List of Tables v List of Figures vi Chapter 1: Literature Review Definition of Whey Processing of Whey Products Agglomeration Functionality and Utilization of Whey Proteins Nutritional Properties of Whey Flavor Properties of Whey References Chapter 2: Manuscript: The impact of agglomeration on flavor and functional stability of whey proteins Abstract Introduction Materials and Methods Results and Discussion Conclusions Acknowledgements References iv

7 List of Tables v Page Table 1.1. Concentration of Major Proteins in Milk Table 1.2. Typical Nutrient Contents of Various Whey Products.. 3 Table 2.1 Key to facility, treatement and whey source of sample codes. 40 Table 2.2. Sensory language for whey proteins (WPC80 and WPI).. 42 Table 2.3 Vanilla Protein Shake Formulation Table 2.4 Peach Flavored Whey Protein Beverage Formulation.. 46 Table 2.5 Clear acidified protein drink formulation Table 2.6 Selected flavor notes in WPC80 samples across storage.. 52 Table 2.7 Selected flavor notes in WPI samples across storage.. 53 Table 2.8 Concentrations of selected compounds (ppb) in WPC80 samples from facility 4 across storage Table 2.9 Concentrations of selected compounds (ppb) in WPC80 samples from facility 2 across storage Table 2.10 Concentrations of selected compounds (ppb) in WPC80 samples from facility 1 across storage Table 2.11 Concentrations of selected compounds (ppb) in WPI samples from facility 4 across storage Table 2.12 Concentrations of selected compounds (ppb) in WPI samples from facility 3 across storage Table Average bulk density, insolubility index, dispersibility, and color values of samples across storage Table 2.14 Proximate analysis results for fresh WPC80 and WPI powders. 73 Table 2.15 Mineral analysis results for fresh WPC80 and WPI powders. 74 Table Average moisture analysis results of samples across storage. 75 Table Selected trained panel attributes in Vanilla Shakes containing WPC Table Selected trained panel attributes in Peach Flavored Beverages containing WPC Table Selected trained panel attributes in Clear Acidified Beverages containing WPI Table Mean Flavor Liking Scores from Consumers (n=75) using WPC80 Product in Ingredient Applications Table Mean Flavor Liking Scores from Consumers (n=75) using WPI Product in Ingredient Applications Table 2.22 Consumer liking and acceptance scores for vanilla protein shakes containing WPC Table 2.23 Consumer liking and acceptance scores for peach beverages containing WPC Table 2.24 Consumer liking and acceptance scores for clear acidified beverages containing WPI

8 List of Figures Page Figure 1.1. Processing of Whey Protein Concentrate... 5 Figure 1.2. Processing of Whey Protein Isolate Figure 1.3. Essential Amino Acids in Whey Protein versus Nutritional. 6 Requirements of Children and Adults Figure 2.1. PCA plot of selected flavor notes in nonagglomerated and agglomerated WPC80 from Facility 1 over time using descriptive analysis. 91 Figure 2.2. PCA plot of selected flavor notes in nonagglomerated and agglomerated WPC80 from Facility 2 over time using descriptive analysis.. 92 Figure 2.3. PCA plot of selected flavor notes in nonagglomerated and agglomerated WPC80 from Facility 4 over time using descriptive analysis.. 93 Figure 2.4. PCA plot of selected flavor notes in nonagglomerated and agglomerated WPI from Facility 3 over time using descriptive analysis. 94 Figure 2.5. PCA plot of selected flavor notes in nonagglomerated and agglomerated WPI from Facility 4 over time using descriptive analysis. 95 Figure 2.6. PCA plot of selected compounds in nonagglomerated and agglomerated WPC80 from Facility 1 over time using solid phase microextraction Figure 2.7. PCA plot of selected compounds in nonagglomerated and agglomerated WPC80 from Facility 2 over time using solid phase microextraction Figure 2.8. PCA plot of selected compounds in nonagglomerated and agglomerated WPC80 from Facility 4 over time using solid phase microextraction Figure 2.9. PCA plot of selected compounds in nonagglomerated and agglomerated WPI from Facility 3 over time using solid phase microextraction 99 Figure PCA plot of selected compounds in nonagglomerated and agglomerated WPI from Facility 4 over time using solid phase microextraction 100 vi

9 Chapter 1 Literature Review Definition of Whey Whey is defined as the watery component removed after the setting of the curd in cheese manufacture. The two main types of whey are sweet whey and acid whey. Acid whey is produced from cheese that is directly acidified with a mineral acid or lactic acid, whereas sweet whey is the result of starter culture acidification of cheese (Bordenave- Juchereau et al., 2005). Sweet whey generally has a titratable acidity between %, a ph of and is derived from rennet coagulated cheeses like Cheddar (coagulation of milk casein by rennet). Medium acid whey has a titratable acidity of %, ph value of and is derived from fresh acid cheeses like ricotta or cottage cheese. Acid whey has a titratable acidity of 0.4%, a ph less than 5.0 and is derived from fresh acid cheeses (acid coagulation of milk casein). Because of the different treatments, sweet and acid whey have different mineral, protein, and lactose properties as well as different functional properties (Bordenave-Juchereau et al., 2005). On average, whey contains 65g dry matter per kilogram which consists of 50g lactose, 6g protein, 6g ash, 2g non-protein nitrogen and 0.5g fat. The protein consists of 50% β-lactoglobulin, 25% ά-lactalbumin, and 25% of other proteins including immunoglobulins, proteose-peptone, bovine serum albumin and β-casein (Varnam and Sutherland, 1994). A breakdown of the components in whey protein is shown in Table 1. There are several products that can be obtained through the processing of liquid whey 1

10 including concentrated whey, whey powder, lactose, lactalbumin, whey protein fractions, and whey protein concentrates and isolates. Table 1.1. Concentration of Major Proteins in Milk Concentration Protein (g/l) Approximate % of Total Protein Caseins Alpha-casein Beta-casein Kappa-casein Gamma-casein Whey proteins Beta-lactoglobulin Alpha-lactalbumin Proteosepeptones Serum albumin Immunoglobulins Total 100 (Fennema, 1996) Whey powder is the product of dried fresh whey, containing all of the components found in whey except the water. Reduced lactose whey is whey in which the lactose is reduced to less than 60% (USDEC, 2004). Whey protein concentrates result from removing non-protein components in order to achieve a certain protein content. They can vary in protein content depending on the processing method, but can contain no less than the percentage indicated in its name. For example, WPC34 must contain no less than 34% protein. The most common types are WPC34, WPC50, WPC60, WPC75, and WPC80. Whey protein isolate (WPI) results from removing non-protein components in order to achieve a 90% protein content. According to the FDA (1999), whey protein concentrate is the substance obtained by the removal of sufficient nonprotein constituents from whey so that the finished dry product contains not less than 25% protein but no more than 89.9% protein. It cannot 2

11 contain more than 10 percent milkfat or 5% moisture and the ph cannot exceed 7.0 USDA, 2003). Ash content is set at 2-15% while lactose content is a maximum of 60% and the limit on heavy metals is 10ppm (FDA, 1999). The whey used must be derived from pasteurized milk or the whey protein concentrate must be pasteurized (FDA, 1999). The flavor should be bland and clean but some flavors are allowed to a slight degree including sweet, acid, bitter, utensil, feed and weedy. However, fermented, sour, chalky, lipase, and chemical flavors are not allowed (USDA, 2003). Typical nutrient contents of various whey products can be found in Table 2. Table 1.2. Typical Nutrient Contents of Various Whey Products Protein (%) Lactose (%) Fat (%) Ash (%) Moisture (%) Sweet Whey Powder Acid Whey Powder Reduced Lactose Whey Demineralized Whey WPC WPC WPC WPC WPC WPI (USDEC, 2004) Processing of Whey Products In order to convert liquid whey into various end products it must be processed. The first step in the processing of whey is clarification. In this step the whey is screened and/or centrifuged in order to remove the excess curds from cheese production. The curds, if left in solution, would block the channels in the heat exchanger or ultrafiltration system in later steps of processing and would negatively affect the flavor and solubility properties of the end product. Whey that is produced from cheese that contains fat will 3

12 also contains some fat in it. This fat must be removed from the whey for flavor stability and to facilitate membrane filtration. Fat separation is achieved using a separator in which the whey feeds through a disc stack and is separated based on the density of the different components (Zadow, 1992). Following clarification and fat removal, whey should be pasteurized immediately to eliminate pathogens and inactivate cheese starter cultures (Smith, 2004). The heat treatment ranges from 72-78C for seconds (Zadow, 1992). The whey can be concentrated by many methods including reverse osmosis, evaporation, or ultrafiltration. For reverse osmosis concentration, the liquid whey is passed under high pressure through a membrane with a pore size small enough such that only water passes through it. With this method, the whey can be concentrated to 20-22% solids (Zadow, 1992). Evaporation is often achieved under vacuum during which the water can be removed at temperatures lower than 100C, keeping the whey protein from denaturing (Smith, 2004). Sometimes reverse osmosis is used as a pre-concentration step before evaporation (Zadow, 1992). Whey protein concentrates are often produced by ultrafiltration of clarified whey. During ultrafiltration, the whey is passed along a semi-permeable membrane through which water is removed along with some of the lactose and minerals while the larger protein molecules remain (Smith, 2004). In this way, the liquid whey can be concentrated to various concentrations up to 65% dry matter. A great advantage in ultrafiltration is that temperatures do not exceed 55C, avoiding heat damage to the proteins (Smith, 2004). By diluting the retentate with water and using diafiltration, the protein can be further concentrated to 80% (Varnam and Sutherland, 1994). However, some lactose and 4

13 minerals will still remain in the retentate. A combination of ultrafiltration with microfiltration can yield WPC with protein levels up to 99% (Henning et al., 2006). Whey protein concentrate can be produced as either a concentrated liquid or it can undergo further processing to become a dried powder. Figure 1.1. Processing of Whey Protein Concentrate (USDEC, 2004) Manufacture of whey protein isolate involves the use of either microfiltration or an ion exchanger to further purify the whey protein. Whey proteins are amphoteric and thus can act like either an acid or base depending on the ph of the medium it is in. Whey proteins have a net positive charge at ph values below their isoelectric point and a net negative charge above their isoelectric point (Varnam and Sutherland, 1994). In this way, 5

14 the ph can be adjusted so that the proteins will be absorbed onto a proper ion exchanger that has a pore size and surface suitable for the recovery of proteins from dilute solutions (Varnam and Sutherland, 1994). The proteins are recovered from the ion exchanger at around ph 9 and the solution is concentrated by ultrafiltration (Morr 1992). Figure 1.2. Processing of Whey Protein Isolate (USDEC, 2004) Dried whey and whey proteins are desirable for many applications. Spray drying is the most common method for drying whey (Smith, 2004; Zadow, 1992). This process involves spraying droplets of whey concentrate into a stream of hot air, causing the water 6

15 to rapidly evaporate from the whey into the air (Smith, 2004). In a two stage dryer, the powder is discharged from the spray drier at about 5-7% moisture onto a vibrating fluid bed where it is further dried and cooled (Henning et al., 2006). Condensed whey can also be roller dried, though generally this is only done so for use in animal feed since the high heat causes protein denaturation and scorching (Smith, 2004). The whey can also become very sticky during this process unless specialized equipment is used in which one set of drums is used to concentrate the whey from 50% solids to about 80% solids and a second set is used to seed the whey for crystallization and rapidly cool the product. It is then fed into an internal drum and dried to 4% moisture at 54-60C. After any type of drying, the dried whey can be processed through a hammer mill to reduce the particle size. The tendency of the whey to cake during storage can be minimized by methods that maximize the crystallization of lactose and by storing it in a bag with a moisture barrier (Hall and Hedrick, 1971). Agglomeration As the last stage of processing, the whey powder may be agglomerated. The shelf life and convenience of products like liquid whey protein concentrate are preserved through the spray drying of the product into a powder. Droplet size must be small during spray drying in order to evaporate enough liquid to dry the product. This results in very small solid sphere shaped particles which are hollow. These particles that are very fine tend to form lumps when mixed with water (Henning et al., 2006; Peitsch, 2005). Unless rigorous mixing is applied, the powder will not fully reconstitute resulting in powder 7

16 floating on the liquid surface. Enlarging the particle size of the whey protein concentrate or isolate powder by agglomeration helps to decrease this problem by increasing dispersion ease and decreasing dispersion time (Turchiuli et al., 2005). Powders that reconstitute quickly and thoroughly in liquid are often termed instant products. Most manufacturers have procedures to determine the maximum allowable time it should take for powders to reconstitute. Typically, in warm liquid, dispersion should be complete in a few seconds and within 30 to 60 seconds in cold liquid (Peitsch, 2005). There are many instant products out on the market including milk, tea, coffee, soups and sauces. The term instantized is often synonymous with agglomerated products as agglomeration aids in making a powder more dispersible. However, instantized can also refer to agglomerated product that has been further coated with lecithin to enhance wettability. Four mechanisms occur during the dispersion and dissolution of a powder in a liquid: 1. Penetration of liquids into the dry powder (also called wetting) 2. Submergence of the powder into the liquid (also called sinking behavior) 3. Break-up of powder mass into the primary particles (also called dispersability) 4. If the solid is soluble, dissolution of the primary particles (also called solubility) (Pietsch, 2005) In order for the liquid to penetrate into the dry powder, the powder must have pathways or pores between the particles in which liquid can travel. Agglomeration helps the process of liquid penetration by creating additional pores within the agglomerates. Since agglomerates are several particles linked together, they are larger and heavier than 8

17 non-agglomerated particles, which improves the submergence of the powder (Peitsch, 2005). Thus, agglomeration increases particle size, porosity and decreases density as compared to a non-agglomerated spray dried product. For particles to become agglomerated they must collide with and adhere to each other. This step can be achieved during the actual drying process (single-pass agglomeration) or after drying (re-wet agglomeration) (Henning et al., 2006). In the food industry the most common way to produce instantized products is by spray drying followed by the rewetting and agglomeration of powders in fluidized beds (Pietsch, 2005). Whey protein powder is fluidized on a bed by an upward hot air flow. A solvent, called a binder, is sprayed onto the powder from above or inside of the bed (Turchiuli et al., 2005). The binder can be water, whey solution, or lecithin. This partially dissolves the outer coating of the dried particle, making it sticky and it now acts as a binder with other particles (Peitsch, 2005). Lecithin is used to further increase dispersion properties of the powder. The binder can also be added as a powder and then pure water sprayed on as a solvent. A study by Turchiuli et al. (2005), indicated that agglomerates produced by introduction of a liquid binder were larger, more irregular, and fragile than agglomerates produced through introduction of a powder binder. The particles can then be further dried on a vibrating fluid bed with warm air and then cooled (Henning et al., 2006). Forced agglomeration is a method in which conditions that lead to collisions are provided during the drying of the powder. One way to achieve this is to use a spray dryer with multiple nozzles angled in such a way that the product streams cross each other and particles will collide and stick together resulting in larger particles (Henning et al., 2006; 9

18 Peitsch, 2005). Another method that is used is to increase agglomeration is to recirculate the very small powder particles called fines back into the spraying zone of the dryer where they collide with droplets that wet the surface of the dried particles, making them sticky enough to adhere to other particles (Refstrup, 1992). The agglomeration process is most successful when the powder has a uniform particle size and moisture content (Hall and Hedrick, 1971). These types of agglomeration produce small, loosely bonded agglomerates. This is important as the bonds within the agglomerates must be strong enough to hold the individual particles together but weak enough to quickly and easily break up and disperse in the liquid. However, these agglomerates are somewhat fragile and cannot be stored under a heavy weight load so storage and stacking during shipment or storage is an issue. Additionally, agglomeration increases the bulk volume of the powder, causing storage and shipping costs to be higher. Functionality and Utilization of Whey Proteins Over the past twenty years, whey protein has risen from being a waste product of cheese making, disposed of primarily in animal feed, and emerged into the human market as a valued, nutritious product (Hoolihan, 2005). This resulted partly due to the rise in production of cheese and thus whey as its by-product, partly due to the rising cost of skim milk powder since whey can often be used as a substitute, and partly to new research showing the beneficial properties of whey protein consumption. Whey protein concentrates are value-added dairy products and are profitable for manufacturers to 10

19 include in processed foods (Veith and Reynolds, 2004). Whey proteins are generally recognized as safe (GRAS) for food product applications. They can be incorporated into many processed foods including health foods, dairy products, meat products, frozen foods, and infant formulas (Morr and Foegeding, 1990: Jayaprakasha and Brueckner, 1999). Whey tends to be used to improve the nutritional and functional properties of food. Whey proteins are generally not the major ingredient in food products but rather one of many. Whey is a major ingredient in only in a small number of products, like ricotta cheese, or high-protein shakes and bars. WPC80 and WPI are often chosen for their functional properties. For example, whey protein can be used in baked goods to improve baking qualities or as an egg replacement, in cheeses to increase the yield, in yogurt as a stabilizer, in dips and spreads as a texturizer, in confections as a replacement for egg white and in meat products as an extender (Varnam and Sutherland, 1994). Liquid whey can also be used as a fermentation substrate to produce citric acid, alcohol, and single cell proteins (Varnam and Sutherland, 1994). During the low-carb craze, consumer desire for higher protein foods increased. And even though enthusiasm for the low-carb diet has decreased, the need for high protein foods remains strong. Proteins are known to have many nutritional properties including enhancing the ability to gain muscle mass, improve hair strength and lose weight (Hazen, 2005; Walzem, 2004; Dahm, 2005). The consumer demand for foods high in protein has opened up a need for protein ingredients with a wide variety of applications and a need for new product formulations. Whey proteins are less expensive 11

20 than caseinates or milk proteins and they have a purportedly bland flavor as compared to other proteins like soy, allowing them to be used in many different products (Hazen, 2005). In addition, whey proteins can be processed to contain less than one percent carbohydrates, making it an ideal ingredient for a low-carb, high protein food (Canning, 2004). For example, whey proteins can be added to breakfast cereals to boost the protein per serving (Hazen, 2005). The high solubility of WPCs at a low ph allows them to be used in acid beverages and foods such as fruit juices, soft drinks, milk-based beverages, and yogurts (Morr, 1984). In fact, whey protein concentrates are soluble at low ionic strength over almost the whole ph range used in food applications (Jayaprakasha and Brueckner, 1999). However, solubility is highest between ph 6.0 and 6.6 (Fachin and Viotto, 2004). Solubility decreases at high salt concentrations and with increasing heat treatment, denaturation occurs at 70 C. However, pasteurization of liquid whey did not have a significant effect on the solubility of spray dried whey protein concentrates (Jayaprakasha and Brueckner, 1999). Studies have shown that whey protein solubility is dependent on ph and heat treatment before ultrafiltration (Fachin and Viotto, 2004). Heat treatment disrupts the van der Waals interactions, hydrogen bonds, and electrostatic forces within the protein structure and exposes some hydrophobic protein residues. Exposing a small number of residues can be helpful for protein emulsion functionality but exposing too many can hinder protein solubility (Fachin and Viotto, 2004). Solubility also depends on the ph history of the cheese source (Onwulata et al., 2004). Whey proteins are ideal for use in sports or meal replacement drinks. Gatorade 12

21 and Accelerade both recently introduced whey protein fortified sports beverages targeted to the athlete wanting to build muscle (Dahm, 2005). For a high acid, low ph, clear beverage like a sports drink it is important to use whey protein isolate, which can withstand high heat and low ph while remaining clear in the liquid (Dahm, 2005). A study by Etzel (2004), indicated that ph must be 3.0 or less for a WPI beverage to remain clear after thermal processing at 88C for 120 seconds. For beverages where clarity is not an issue, WPC 80 would work well (Burrington, 2005). Burrington (2005) noted that 40 grams of whey protein is generally the limit in a 20 ounce high acid beverage. Pectin in fruit juices can pose a problem when mixed with whey protein since pectin and whey protein directly interact and can increase viscosity of the liquid. Agglomerated whey protein powders should be used for powdered drinks, allowing for rapid hydration of the product when mixed with water (Hazen, 2005). WPC80 and WPI are often added to nutrition bars, allowing a substantial amount of protein to be added in a small volume. However, high levels of protein can affect the flavor and texture of the bar. One recommendation involves combining the whey protein concentrate or isolate with a hydrolyzed whey protein because it helps to equilibrate moisture in the bar and gives it a more consistent texture over shelf life (Hazen, 2005). Whey protein concentrate is useful in bakery, confectionary, meats and seafood for its production of a high strength gel with good water holding capacity (Veith and Reynolds, 2004). Gels are formed as the result of protein interaction and production of an elastic network (Foegeding et al., 2002). The protein molecules unfold and bind more water. As the proteins interact around the water molecules, a network forms entrapping 13

22 the water molecules and forming a gel (Mangino, 1984). Gel strength depends on ph, ionic strength, protein concentration, and composition (Veith and Reynolds, 2004). Lactoglobulin, a major whey protein, is thought to contribute the most to gelation (Veith and Reynolds, 2004). Whey proteins begin to gel around 65 C. It takes at least 7-8% protein to form gels in aqueous solutions but in food systems as little as 0.5 to 3.0% may be needed (Jayaprakasha and Brueckner, 1999). The ph at gel formation affects the appearance of the gels. Gels formed at high ph values were shown to be elastic and transparent, with greater gel strength whereas gels formed at low ph values were opaque and had more syneresis (Jayaprakasha and Brueckner, 1999). Gel strength increases with increasing sulphhydryl groups (Schmidt et al., 1979). Free calcium concentration is another factor that affects the formation of gels due to the cross-linking properties of calcium (Barbut and Foegeding, 1993) The ability of whey to gel at a low ph makes it a useful ingredient in yogurts and drinkable yogurts (Hazen, 2005). Smoothie type beverages are a fast growing segment of the yogurt market and whey proteins are often added to enhance the nutrient content (Dahm, 2005). WPC34 is often added to yogurt as an emulsifier and water-binding agent, to reduce the defect of syneresis and to produce a firmer texture (Jayaprakasha and Brueckner, 1999). In this way, the addition of stabilizers and skim milk powder can be reduced. WPC34 can also be used to replace some of the milk solids nonfat in ice cream. The flavor, texture, and appearance of the ice cream are the same as standard ice cream if WPC34 replaces 25% or less of the nonfat milk solids (Jayaprakasha and Brueckner, 1999). 14

23 The ability of whey proteins to bind water is used to enhance the texture of some yogurts, soups and sauces (Jayaprakasha and Brueckner, 1999). As whey proteins decrease in solubility they increase in water binding capacity (Jayaprakasha and Brueckner, 1999). Thus, denatured proteins have very low solubility, but high water binding capability. Heating a whey protein solution can cause an increase in viscosity and water binding ability. This is because the heat causes the protein to unfold and expose water binding sites previously hidden, increasing the volume that the protein occupies. Whey can be utilized in processed cheeses to improve water binding and improve mouthfeel. This increase in water binding also improves spreadability in cheese products and can reduce brittleness and increase gloss in processed cheese. Using whey protein concentrate in Ricotta cheese improves the cohesiveness of the curd (Jayaprakasha and Brueckner, 1999). Whey proteins can be utilized in meat products. In comminuted meats, whey protein concentrates serve in fat emulsification, water binding, and improved texture. In minced meat products, whey proteins aid in gel formation, water binding capacity, and texture. Whey protein concentrate can be used as a fat replacer in sausage type products. Adding whey proteins to sausages increases fat binding properties, product firmness, and improves the flavor and texture and increases the juiciness (Jayaprakasha and Brueckner, 1999). At the Ohio State University, a low fat sausage was produced using whey protein and water to replace some of the fat, reducing the calories from 310 to 110. An added benefit was that the protein content increased from 9 grams to 14 grams (Hazen, 2005). Dressings, ice creams, soufflés, and other food emulsions are popular among 15

24 consumers but they must be combined with surface-active agents to be formed and stabilized. Whey protein is a common ingredient chosen for this role (Foegeding et al., 2002). Whey protein concentrate is reported to exhibit emulsion stability and consistency similar to that of whole eggs (Jayaprakasha and Brueckner, 1999). Thus, whey proteins may be a good emulsifier in applications such as salad dressings and spreads. Jayaprakaha and Brueckner (1999) found that when WPC80 was used as the emulsifier in a low calorie butter substitute spread, mouthfeel and spreadability were improved. Whey proteins can be used in sauces as thickeners or emulsifiers. Whey protein can be used to replace fat in low-fat products because the whey proteins help to bind water, creating a gel-like structure and simulating a fat-like mouth feel. It can also be used to replace fat in frozen desserts, dairy products like cheeses and puddings, and baked goods. Whey proteins can play an important role in products requiring the Maillard browning reaction such as caramel, toffee, and chocolate flavored products. In these applications, whey proteins can be used to replace milk solids (Hazen, 2005). Foaming is an important property for many foods including whipped toppings, cakes, milkshakes and frozen desserts. Whey proteins can contribute to foam formation by lowering interfacial tension. In foams, proteins concentrate at the foam cell interface, partially unfold and interact through intermolecular bonding, essentially encapsulating the air bubbles, which stabilizes the foam cells and forms a cohesive film (Jayaprakasha and Brueckner, 1999). Studies have shown that the foaming properties of sweet whey powder have a positive correlation to particle size and lightness value. However increased lipid content in whey protein decreased in foaming abilities (Banavara et al., 16

25 2003). An increase in ph may decrease the calcium activity in some systems and thus increase the foaming capacity. Heat treatment can improve foaming abilities with maximum overrun and stability reached with a heat treatment between 50 C and 60 C (Jayaprakasha and Brueckner, 1999). Fat reduced, undenatured whey proteins show foaming properties similar to that of egg white (Jayaprakasha and Brueckner, 1999). This could make whey protein a good substitute or extender for egg whites in certain products. Defatted whey protein concentrate has also been successfully used to make a meringue product with the same appearance and mouthfeel as those made from egg whites (Jayaprakasha and Brueckner, 1999). Incorporation of whey protein concentrate into a whipped topping resulted in higher overrun and showed that β-lactoglobulin content may contribute to the foaming cababilities of whey proteins (Kim et al., 1987). Whey proteins can add functionality to bakery products through water binding, emulsification, foaming, gelation, viscosity, elasticity, cohesion, and nutritional benefits. Some other important functions they provide is heat stability, aeration, and Maillard browning (Hazen, 2005). They can be used as replacements for eggs in cakes. Whey protein concentrate can improve the color and texture of cookies, and the flavor, toasting and crust-browning qualities of bread (Hazen, 2005). However the use of whey proteins may mean that other ingredients like water in the formulation need to be adjusted because whey absorbs less water than flour. The use of whey can cause crust color to develop faster resulting in decreased baking time and temperature. Food scientists are constantly seeking new ways to utilize whey proteins or transform them into more functional ingredients. In one study, Mishra et al. (2001) 17

26 produced a protein-polysaccharide complex with WPC71 and pectin, mixing the two biopolymers at equal ratios. The complexes showed increased solubility, emulsification, gelation, foam formation, and foam stability as compared to whey protein concentrate alone. This complex may be one way to increase the usability of whey protein concentrates (Mishra et al., 2001). A recent development in improving and creating new functionality of whey protein concentrates and isolates is texturizing them by extrusion processing. Extrusion shears and stretches the proteins, altering the structure and exposing groups that were previously enclosed in the protein, and aligning the proteins in fibrous bundles. Extrusion also heats the protein, further denaturing it. By first texturizing the whey protein through extrusion, researchers were able to show that whey proteins provide functionality in other extruded products as well as in some non-traditional products such as acidified shelf stable beverages (Onwulata et al., 2003). WPC80 has been extruded to form a white to cream texturized product that could be used as a substitute or extender for poultry meats (Jayaprakasha and Brueckner, 1999). Grande Custom Ingredients Group, Utah State University, and DMI created a crispy whey protein product by combining whey protein with a starch and extruding it through a twin-screw extruder, which could be used in granola like bars or products, and in frozen desserts (Canning, 2004). Another recent development is the use of whey proteins to create a clear, glossy, flexible, edible film that could be used to coat peanuts, chocolate, cheese, or packaging materials (Canning, 2004). In a study by Maté et al. (1996), this film delayed oxidative deterioration of dry-roasted peanuts. Another study indicates that using transglutaminase 18

27 as a cross-linking agent in the films improves the oxygen barrier (Di Pierro et al., 2006). Development is still being done to make this coating more effective in protecting against oxidation (Lin and Krochta, 2005). Nutritional Properties of Whey There are many advantages of using whey protein instead of other proteins to fortify or enhance foods. Whey protein actually consists of several component proteins including beta-lactoglobulin, alpha-lactalbumin, glycomacropeptide, bovine serum albumin, immunoglobulins, and lactoferrin (Canning, 2004). Whey proteins are high quality proteins due to their high cysteine content, and significant quantities of B vitamins (Varnam and Sutherland, 1994). They are also rich in calcium, phosphorous, magnesium and zinc (Hazen, 2005). WPC and WPI typically contains mg calcium per 100g powder (Dirienzo, 2004). Whey proteins are one of the most bioavailable proteins, having an amino-acid profile that is easily digested and allowing the body to utilize the protein faster than rice or soy protein (Hazen, 2005; Canning, 2004). The amino acid profile of sweet whey is very balanced, and each amino acid present exceeds the intake recommendations for both children ages 2-5 and adults as set by the Food and Agricultural Oranization and World Health Organization (Walzem, 2004; Sindayikengera and Xia, 2005). 19

28 Whey Protein Child (2-5 years) Adult Essential Amino Acids Leucine Lysine Threonine Phenylalanine + Tyrosine Isoleucine Valine Methionine + Cystine Tryptophan Histidine Crude Protein mg/g Figure 1.3. Essential Amino Acids in Whey Protein versus Nutritional Requirements of Children and Adults (USDEC, 2004) Whey proteins contain the highest concentration of branched-chain amino acids, 26 grams per 100 grams of protein, compared to any natural food protein source (Hazen, 2005). In particular, whey protein contains high levels of leucine, isoleucine and valine and sulfur containing amino acids, methionine and cysteine (Pasin and Miller, 2004; Sindayikengera and Xia, 2005). These branched-chain amino acids are metabolized directly by the skeletal muscles, promoting protein synthesis and helping to preserve muscle mass and provide energy during prolonged exercise (Hazen, 2005; Walzem, 2004). Research points to leucine a as key amino acid for regulating protein synthesis in order to maintain lean muscle mass (Dahm, 2005). It has also been suggested that whey 20

29 protein may minimize muscle loss associated with aging, a problem affecting many of the elderly in the United States (Hoolihan, 2005). In a study by Paddon-Jones (2005), 15g of daily WPI supplementation was found to adequately stimulate muscle synthesis. There is more recent research indicating that whey as used in a high protein, low carbohydrate diet could contribute to weight loss and a reduction in body fat (Dahm, 2005). This may result from the ability of proteins to moderate energy intake and increase the feeling of fullness (Hoolihan, 2005). Glycomacropeptide (GMP), a component of whey protein, has been found to stimulate production of the hormone cholecystokinin, which sends a signal to the brain that the stomach is full (Canning, 2004). Additionally, dietary supplementation with WPC 80 decreased hepatic fatty acid synthesis and increased production in skeletal muscle much like exercise would (Morifuji et al., 2005). Whey protein could contribute many other health benefits to humans. Peptides in hydrolyzed whey may lower blood pressure and reduce blood cholesterol levels (Dahm, 2005; Canning, 2004). In a study by Nelson et al. (2004) in which human subjects consumed 60 grams WPI per day for 12 weeks, total cholesterol decreased by 15% and LDL cholesterol decreased by 20% for the group. Whey proteins may have a positive effect on blood glucose and insulin levels (Dahm, 2005). In a study by Nilsson et al. (2005), a meal consisting of foods with a high glycemic index and supplemented with 27.6g whey powder stimulated insulin release and improved blood glucose control as compared to the same meal without whey supplementation. They have also been shown to boost glutathione levels in tissues of the body, which functions as an antioxidant to protect cells from free radical damage (Tseng et al., 2005; Hoolihan, 2005). This boost in 21

30 glutathione levels from whey protein supplementation may even decrease co-infections in HIV infected adults (Moreno et al., 2006). Several animal studies have looked at whey as a potential ingredient to protect against cancer. In a study by Dave et al. (2006), whey protein hydrolysate was fed to rats and provided protection against DNA damage. WPC 80 was found to be effective at retarding colon cancer in rats (McIntosh et al., 1998). Several whey proteins components, such as lactoferrin, may even be able to protect the body from infection by inhibiting the activity of certain microorganisms including some foodborne pathogens (Canning, 2004; Walzem, 2004). A study using WPI film containing lysozyme on cold-smoked salmon effectively retarded the growth of L. monocytogenes at 4 and 10C for 35 days, while a WPI film containing lactoperoxidase inhibited the growth of S. enterica and E. coli 0157:H7 on roasted turkey at 4 and 10C for 42 days (Min et al., 2005; 2006) Flavor Properties of Whey The flavor of liquid whey varies depending on the source of cheese, and type of starter culture. Gallardo-Escamilla et al. (2005) reported that fresh, liquid Cheddar, Gouda, and rennet wheys had a bland, sweet, and milky flavor. This flavor was suitable for use in liquid dairy beverages. Rennet whey obtained without the use of a starter culture similarly produced a mild aroma and sweet flavor. Acid casein whey, on the other hand, produced flavors such as bitter, stale, rancid and chemical. For whey retrieved from Mozzarella and Quarg, in which fermentation continued to a low ph, the type of starter culture had a significant impact on the flavor. Rennet casein whey was described 22

31 by a sweet, oat-like flavor (Gallardo-Escamilla et al., 2005). The flavor of liquid whey can vary with milk source, processing and handling, and starter culture blend within a single type of cheese. In a study by Tomaino et al. (2001), liquid Cheddar whey produced with a Lactococcus lactis subsp. lactis starter culture contained higher levels of total free fatty acids, and especially lauric, myristic, and palmitic acids than whey produced from Lactococcus lactis subsp. cremoris. Liquid Cheddar cheese whey that was analyzed from two processing facilities and different starter culture rotations resulted in wide variation in the headspace volatiles and sensory flavor profiles (Carunchia Whetstine et al., 2003). Tomaino et al. (2004), analyzed liquid Cheddar whey over fourteen days at refrigeration temperature using descriptive sensory analysis along with a purge and trap method to evaluate headspace volatiles. They reported an increased cardboardy note with storage time which correlated with an increase in volatile lipid oxidation products such as hexanal. Volatile compound analysis during 14 days of refrigerated storage resulted in a decrease in oleic, linoleic, and palmitic acids, presumeably due to lipid oxidation (Tomaino et al., 2001). These studies reveal that the flavor of liquid whey varies greatly based on the manufacturer, source of milk, and type of starter culture (Carunchia-Whetstine et al., 2003; Tomaino et al., 2001). Fresh whey has a bland, milky, or oat-like flavor with some sweet aromatics (Gallardo-Escamilla et al., 2005). The level of free fatty acids is also variable between samples (Tomaino et al., 2001). In addition, the volatile profile will change over storage with an increase in hexanal and a decrease in free fatty acids (Tomaino et al., 2004). 23

32 Whey powder has a different flavor profile and aroma than liquid whey because of the additional processing steps required to concentrate and dry the liquid (Mahajan et al., 2004). Some work has been done to identify the aroma compounds in sweet whey powder obtained from Cheddar cheese making. The compounds that contributed most to the aroma were free fatty acids including acetic, propanoic, butanoic, hexanoic, heptanoic, octanoic, decanoic, dodecanoic, and 9-decenoic acids. Non-acidic compounds included hexanal, heptanal, nonanal, which generated grassy, green, and floral aromas. Also phenylacetaldehyde, 1-octen-3-one, methional, 2,6-dimethylpyrazine, 2,5- dimethylpyrazine, 2,3-dimethylpyraxine, 2,3,5-trimethylpyrazine, furfuryl alcohol, p- cresol, 2-acetylpyrrole, maltol, furaneol, and several lactones were identified (Mahajan et al., 2004). These are compounds that could arise from milk, starter culture during cheese making, and manufacturing of the whey powder. The most important aroma compounds were short-chain fatty acids, aldehydes, ketones, lactones, sulfur compounds, phenols, indoles, pyrazines, furans, and pyrroles. Among the key compounds, diacetyl, a straight chain ketone, produced a buttery note while 1-octen-3-one had a mushroom-like odor (Mahajan et al., 2004). Also key to the flavor and aroma, the sulfur compounds dimethyl disulfide and dimethyl trisulfide have a cabbage-like aroma while methional has a cooked potato odor. Maillard browning, caramelization of sugar, and lipid oxidation are likely the cause of many of these aroma compounds in whey powder (Mahajan et al., 2004). A study by Sithole et al. (2005) indicated that the flavor of sweet whey powder was characterized by descriptive analysis by sweet aromatic, caramelized, cooked, cardboard, oxidized and barny notes and this flavor did not significantly change over a shelf-life of 24

33 twelve months at 21C. Limited research has also addressed the flavor of whey protein concentrates and isolates. Quach et al. (1999) showed that SPME combined with gas chromatography/mass spectrometry (GC-MS) was an effective method to extract and identify flavor volatiles from solutions of whey protein concentrates, with optimal sampling at 40C for 30 minutes followed by fiber exposure for 30 minutes. In this study, the major volatile components of whey protein concentrates were aldehydes, especially hexanal, carbonyl compounds, acetic acid, octanoic acid and decanoic acid, dimethyl sulfide and methyl ketones (Quach et al., 1999). Fresh WPC80 and WPI were analyzed using descriptive analysis coupled with solvent assisted flavor extraction and gas chromatrography-olfactometry (GC-O) (Carunchia-Whetstine et al., 2005). Sensory differences were found between samples from different manufacturers as well as between WPC80 and WPI. WPC80 was generally characterized by either cardboard/wet paper or pasta water flavors. Flavors unique to WPI were animal/wet dog, cucumber, soapy, and bitter. Flavors common to both WPC80 and WPI included sweet aromatic, brothy, and astringent (Carunchia-Whetstine et al., 2005). These flavors were linked through volatile analysis to aldehydes, ketones, and free fatty acids in both the WPC80 and WPI. Among these key volatile compounds were butanoic acid, 2-acetyl-1-pyrroline, 2-methyl-3-furanthiol, 2,5-dimethyl-4-hydroxy-3- (2H)-furanone, 2-nonenal, (E,Z)-2,6-nonadienal, and (E,Z)-2,4-decadienal (Carunchia- Whetstine et al., 2005). In a separate study, the source of a cabbage off-flavor in WPI was 25