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Changes in the Intensities of Volatile Aroma Compounds Contributing to the Off-Flavors in Liquid Cottage Cheese Whey

Permanent Link: http://ufdc.ufl.edu/UFE0041318/00001

Material Information

Title: Changes in the Intensities of Volatile Aroma Compounds Contributing to the Off-Flavors in Liquid Cottage Cheese Whey
Physical Description: 1 online resource (73 p.)
Language: english
Creator: Anguswamy, Uma
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: cheese, intensities, volatiles, whey
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Considerable effort has been devoted over several decades to finding the least costly method of the disposal of liquid whey and to identify new outlets for whey utilization, preventing the loss of potentially valuable nutrients and reducing environmental pollution (Gonzales and others 2002). Although, whey has been used in the form of whey solids, including whey protein concentrates and isolates, direct utilization of liquid whey without fractionation and dehydration represents a more economical alternative, because of the elimination of the cost of evaporation or ultrafiltration (Ryder 1980). However, liquid whey, particularly liquid acid whey, due to its high acid and ash content is associated with undesirable flavors and is less palatable compared to sweet whey (Mc Gugan and others 1979). Processing treatments and drying conditions have been reported to affect the composition of whey thereby affecting the flavor of whey solids (Morr and Foegeding 1990). Similarly, whey handling practices and storage conditions may affect the flavor of liquid acid whey. A better understanding of the nature of the off-flavor compounds in liquid acid whey as affected by whey handling and storage conditions may help in minimizing its wastage and in controlling the handling methodologies that are detrimental to flavor of liquid acid whey. The main objectives of this study were to identify the intensities of the volatile aroma compounds contributing to off-flavors in liquid cottage cheese whey (LCW) and to evaluate the impact of storage and mishandling conditions (e.g. temperature fluctuations and foaming) on the intensities of the off-flavor compounds. The volatiles were extracted using solid phase microextraction (SPME). The intensities of off flavor compounds in LCW were evaluated by three panelists using Gas chromatography-olfactometry (GCO). Statistical differences between samples were determined by using a two-way analysis of variance (ANOVA) test with replications. The most aroma intense compounds detected in liquid cottage cheese whey were butanoic acid (rancid), methylbutanoic acid (cheesy), 3-nonenal (fatty), 2,6 nonadienal (cucumber), methional (cooked potato), dimethyltrisulfide (cabbage). Odor intensities of above-mentioned compounds varied upon storage, temperature fluctuations and foaming of the LCW. The results of this study lead to the conclusion that the volatile aroma compounds contributing to off-flavors in LCW were influenced by the whey handling and storage conditions.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Uma Anguswamy.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Schmidt, Ronald H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041318:00001

Permanent Link: http://ufdc.ufl.edu/UFE0041318/00001

Material Information

Title: Changes in the Intensities of Volatile Aroma Compounds Contributing to the Off-Flavors in Liquid Cottage Cheese Whey
Physical Description: 1 online resource (73 p.)
Language: english
Creator: Anguswamy, Uma
Publisher: University of Florida
Place of Publication: Gainesville, Fla.
Publication Date: 2010

Subjects

Subjects / Keywords: cheese, intensities, volatiles, whey
Food Science and Human Nutrition -- Dissertations, Academic -- UF
Genre: Food Science and Human Nutrition thesis, M.S.
bibliography   ( marcgt )
theses   ( marcgt )
government publication (state, provincial, terriorial, dependent)   ( marcgt )
born-digital   ( sobekcm )
Electronic Thesis or Dissertation

Notes

Abstract: Considerable effort has been devoted over several decades to finding the least costly method of the disposal of liquid whey and to identify new outlets for whey utilization, preventing the loss of potentially valuable nutrients and reducing environmental pollution (Gonzales and others 2002). Although, whey has been used in the form of whey solids, including whey protein concentrates and isolates, direct utilization of liquid whey without fractionation and dehydration represents a more economical alternative, because of the elimination of the cost of evaporation or ultrafiltration (Ryder 1980). However, liquid whey, particularly liquid acid whey, due to its high acid and ash content is associated with undesirable flavors and is less palatable compared to sweet whey (Mc Gugan and others 1979). Processing treatments and drying conditions have been reported to affect the composition of whey thereby affecting the flavor of whey solids (Morr and Foegeding 1990). Similarly, whey handling practices and storage conditions may affect the flavor of liquid acid whey. A better understanding of the nature of the off-flavor compounds in liquid acid whey as affected by whey handling and storage conditions may help in minimizing its wastage and in controlling the handling methodologies that are detrimental to flavor of liquid acid whey. The main objectives of this study were to identify the intensities of the volatile aroma compounds contributing to off-flavors in liquid cottage cheese whey (LCW) and to evaluate the impact of storage and mishandling conditions (e.g. temperature fluctuations and foaming) on the intensities of the off-flavor compounds. The volatiles were extracted using solid phase microextraction (SPME). The intensities of off flavor compounds in LCW were evaluated by three panelists using Gas chromatography-olfactometry (GCO). Statistical differences between samples were determined by using a two-way analysis of variance (ANOVA) test with replications. The most aroma intense compounds detected in liquid cottage cheese whey were butanoic acid (rancid), methylbutanoic acid (cheesy), 3-nonenal (fatty), 2,6 nonadienal (cucumber), methional (cooked potato), dimethyltrisulfide (cabbage). Odor intensities of above-mentioned compounds varied upon storage, temperature fluctuations and foaming of the LCW. The results of this study lead to the conclusion that the volatile aroma compounds contributing to off-flavors in LCW were influenced by the whey handling and storage conditions.
General Note: In the series University of Florida Digital Collections.
General Note: Includes vita.
Bibliography: Includes bibliographical references.
Source of Description: Description based on online resource; title from PDF title page.
Source of Description: This bibliographic record is available under the Creative Commons CC0 public domain dedication. The University of Florida Libraries, as creator of this bibliographic record, has waived all rights to it worldwide under copyright law, including all related and neighboring rights, to the extent allowed by law.
Statement of Responsibility: by Uma Anguswamy.
Thesis: Thesis (M.S.)--University of Florida, 2010.
Local: Adviser: Schmidt, Ronald H.

Record Information

Source Institution: UFRGP
Rights Management: Applicable rights reserved.
Classification: lcc - LD1780 2010
System ID: UFE0041318:00001


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1 CHANGES IN THE INTENSITIES OF VOLATILE AROMA COMPOUNDS CONTRIBUTING TO THE OFF -FLAVOR S IN LIQUID COTTAGE CHEESE WHEY By UMA DEVI ANGUSWAMY A THESIS PRESENTED TO THE GRADUATE SCHOOL OF THE UNIVERSITY OF FLORIDA IN PARTIAL FULFILLMENT OF THE REQUIREMENTS FOR THE DEGREE OF MASTER OF SCIENCE UNIVERSITY OF FLORIDA 2010

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2 20 10 Uma Devi Anguswamy

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3 To my mother, with love

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4 ACKNOWLEDGMENTS I would like to express my sincere gratitude to my committee chair, Dr. Ronald H. Schmidt, for his guidance, encouragement, patience and support. It was a great learning experience. To my committee members, Dr. Maurice R. Marshall and Dr. Kathryn R. Willia ms, I would like to extent my gratitude for their advice and assistance. I also thank Dr. Bruce Welt for providing the gas chromatograph for my experiments. I am grateful to Dr. Kurt Schulbach for his assistance and valuable input to begin this study. I t hank my friends Bhuvaneshwari Govindarajan and Siva Kumar for all their help. Thanks go to my family for their support. To my mother, I would like to extend a special gratitude for her kindness and constant support. Finally, I would like to thank my friend s and family for their love and support. A special thanks to my husband, Suresh Babu, for his patience and sacrifices through the long years of graduate school, and for his support and help in all the endeavors I have chosen to pursue.

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5 TABLE OF CONTENTS page ACKNOWLEDGMENTS .................................................................................................................... 4 LIST OF TABLES ................................................................................................................................ 7 LIST OF FIGURES .............................................................................................................................. 8 ABSTRACT .......................................................................................................................................... 9 CHAPTER 1 INTRODUCTION ....................................................................................................................... 11 2 LITERATURE REVIEW ........................................................................................................... 13 Whey: Nature, Types and Composition ..................................................................................... 13 Whey Disposal Problem ............................................................................................................. 14 Whey Utilization ......................................................................................................................... 15 Utilization of the Whey So lids ................................................................................................... 17 Utilization of Raw Unprocessed Liquid Whey ......................................................................... 19 Flavor as a Limiting Factor in Whey Utilization ...................................................................... 20 Factors Affecting the Flavor of Whey ....................................................................................... 21 Lipolysis Reaction ............................................................................................................... 21 Lipid Oxidation Reaction .................................................................................................... 22 Mechanism of Lipid Oxidation ........................................................................................... 22 Other Factors ............................................................................................................................... 24 Flavor Compounds i n Whey ....................................................................................................... 25 Acidic Compounds .............................................................................................................. 25 Aldehydes ............................................................................................................................. 26 Ketones ................................................................................................................................. 28 Sulfur Containing Compounds ........................................................................................... 29 Nitr ogen Containing Compounds ....................................................................................... 30 Alcohols ................................................................................................................................ 30 Phenolic Compounds ........................................................................................................... 30 3 MATERIALS AND METHODS ............................................................................................... 42 Liquid Cottage Cheese Whey ..................................................................................................... 42 Equipm ents .................................................................................................................................. 42 Solid Phase Microextraction (SPME) ................................................................................ 42 Gas Chromatography -Flame Ionization Detector (GC O) ................................................ 42 Whey Treatments ........................................................................................................................ 43 Fresh Whey (FW) ................................................................................................................ 43 Temperature Fluctuated Whey (TFW): .............................................................................. 43

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6 Low Temperature Whey (LTW): ........................................................................................ 43 Foamed Whey (FOW): ........................................................................................................ 44 Volatile Flavor Compound Analysis .......................................................................................... 44 Optimization of the Headspace SPME Method: ............................................................... 44 Panelist ................................................................................................................................. 44 Training of Panelist for GC O ............................................................................................ 45 Identification of Odorants ................................................................................................... 45 Statis tical Analysis ............................................................................................................... 46 4 RESULTS AND DISCUSSION ................................................................................................ 47 Volatile Aroma Compounds contributing to the Off Flavors in Fresh whey (FW) or Control ...................................................................................................................................... 48 Fatty Acids ........................................................................................................................... 48 Sulfur Containing Compounds ........................................................................................... 49 Aldehydes and Ketones ....................................................................................................... 50 Other Compounds ................................................................................................................ 50 Effect of Low Temperature Treatment on the Off-Flavor Compounds of Whey ................... 51 Acidic Compounds .............................................................................................................. 51 Aldehydes and Ke tones: ...................................................................................................... 52 Sulfur and Nitrogen Containing Compounds ..................................................................... 52 Other Compounds ................................................................................................................ 53 Effect of Temperature Fluctuations on the Off-Flavor Compounds of Whey ........................ 53 Acids ..................................................................................................................................... 53 Sulfur Containing Compound s ........................................................................................... 54 Aldehydes and Ketones ....................................................................................................... 54 Nitrogen Containing Compounds ....................................................................................... 55 Effect of Foaming on the Off-Flavor Compounds of Whey .................................................... 55 Acids ..................................................................................................................................... 55 Aldehydes and Ketones ....................................................................................................... 56 Sulfur and Nitrogen Containing Compounds ..................................................................... 57 5 SUMMARY AND CONCLUSION ........................................................................................... 65 LIST OF REFERENCES ................................................................................................................... 66 BIOGRAPHICAL SKETCH ............................................................................................................. 73

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7 LIST OF TABLES Table page 2 1 General Composition of D ry W heys. .................................................................................... 33 2 2 Estimated U.S T otal L iquid W hey and L iquid Cottage Cheese W hey ............................... 33 2 3 Lipid Oxidation Products and Related Odor Compounds Identified in Whey................... 34 2 4 Flavor compounds Originating from the Amino acids with their Odor Properties ............ 34 2 5 Predominant Fatty Acids Identified in Whey ....................................................................... 35 2 6 Major Ketones Identified in Whey and their Flavor Characteristics .................................. 35 2 7 Major Sulfur Compounds Identified in Whey and their Flavor Characteristics ................ 36 2 8 Formation of Aldehydes via amino acid degradation .......................................................... 36 2 9 Formation of Aldehydes via Lipid Oxidation ...................................................................... 36 4 1 Descriptors used by each panelist during free choice profiling of the odors contributing to the off -flavors in the fresh and treated liquid cottage cheese whey .......... 59 4 2 Aroma intensities based on the normalized peak heights of samples analyzed using DB 5 column. Descriptors are listed in ascending order of LRI ......................................... 60 4 3 Aroma active peaks with tentative identification based on aroma attributes and retention characteristics for liquid cottage cheese wh ey samples as detected by GC O on a non polar columns .......................................................................................................... 64

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8 LIST OF FIGURES Figure page 2 1 Whey Solids Production ........................................................................................................ 37 2 2 Lipolysis .................................................................................................................................. 38 2 3 Formation of Flavor compounds by Lipid Oxidation .......................................................... 39 2 4 Maillard Reaction: Flavor Compound Formation Pathways and Products ....................... 40 2 5 Formation of Methylketones ................................................................................................. 41 2 6 Formation of Predominant Sulfur Compounds in Whey ..................................................... 41 4 1 Partial FID (top) and Aromagram (bottom) overlay of fresh liquid cottage cheese whey analyzed on DB 5 column ........................................................................................... 58 4 2 Aroma intensitie s for the FW and LTW on DB 5 column. Intensities were normalized peak height based on butanoic acid, which had the highest peak height. Peak numbers correspond to those listed in T able 4 3. ....................................................... 61 4 3 Aroma intensities for the FW and TFW on DB 5 column. Intensities were normalized peak height based on butanoic acid, which had the highest peak height. Peak numbers correspond to those listed in T able 4 3. ...................................................... 62 4 4 Aroma intensities for the FW and FOW on DB 5 column. Intensities were normalized peak height based on butanoic aci d, which had the highest peak height. Peak numbers correspond to those listed in T able 4 3. ....................................................... 63

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9 Abstract of Thesis Presented to the Graduate School of the University of Florida in Partial Fulfillment of the Requirements for the Degree of Ma s ter of Science CHANGES IN THE INTENSITIES OF VOLATILE AROMA COMPOUNDS CONTRIBUTING TO THE OFF -FLAVOR S IN LIQUID COTTAGE CHEESE WHEY By Uma Devi Ang uswamy May 2010 Chair: Dr. Ronald H. Schmidt Major: Food Science and Human Nutrition Considerable effort has been devoted over several decades to finding the least costly method of the disposal of liquid whey and to identify new outlets for whey utiliza tion, preventing the loss of potentially valuable nutrients and reducing environmental pollution (Gonzales and others 2002). Although, whey has been used in the form of whey solids, including whey protein concentrates and isolates, direct utilization of li quid whey without fractionation and dehydration represents a more economical alternative, because of the elimination of the cost of evaporation or ultrafiltration (Ryder 1980). However, liquid whey, particularly liquid acid whey, due to its high acid and a sh content is associated with undesirable flavors and is less palatable compared to sweet whey (Mc Gugan and others 1979). Processing treatments and drying conditions have been reported to affect the composition of whey thereby affecting the flavor of whe y solids (Morr and Foegeding 1990). Similarly, whey handling practices and storage conditions may affect the flavor of liquid acid whey. A better understanding of the nature of the off -flavor compounds in liquid acid whey as affected by whey handling and s torage conditions may help in minimizing its wastage and in controlling the handling methodologies that are detrimental to flavor of liquid acid whey. The main objectives

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10 of this study were to identify the intensities of the volatile aroma compounds contri buting to off flavors in liquid cottage cheese whey (LCW) and to evaluate the impact of storage and mishandling conditions (e.g. temperature fluctuations and foaming) on the intensities of the off flavor compounds. The volatiles were extracted using solid phase microextraction (SPME). The intensities of off flavor compounds in LCW were evaluated by three panelists using Gas chromatographyolfactometry (GCO). Statistical differences between samples were determined by using a two way analysis of variance (AN OVA) test with replications. The most aroma intense compounds detected in liquid cottage cheese whey were butanoic acid (rancid), methylbutanoic acid (cheesy), 3 -nonenal (fatty), 2,6 nonadienal (cucumber), methional (cooked potato), dimethyltrisulfide (cab bage). Odor intensities of above -mentioned compounds varied upon storage, temperature fluctuations and foaming of the LCW. The results of this study lead to the conclusion that the volatile aroma compounds contributing to off -flavors in LCW were influenced by the whey handling and storage conditions.

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11 CHAPTER 1 INTRODUCTION Whey is the liquid by-product obtained by separating the coagulum from milk/cream and or skim milk in cheese making. Whey is generally classified as sweet or acid whey. Sweet whey is obtained from the manufacture of natural enzyme produced cheeses, whereas acid whey is obtained from the manufacture of fresh acid cheeses such as quarg and cottage cheese. Liquid sweet whey is generally fractionated and dehydrated and is mainly utilized in the form of whey powders, such as whey protein concentrates and isolates which are used in the health promoting food and drinks. Liquid acid w hey is a difficult substance to dry by traditional dehydration methods, and, therefore, it is substantially underutilized compared to sweet whey. Attempts to utilize acid whey in the liquid form have not been successful due to its high transportation cost, susceptibility to deterioration in storage, and lack of appropriate mechanism for utilization. Also, the high acid and ash content of liquid acid whey contributes to functional and flavor problems. When used as a food ingredient, whey is expected to have bland and delicate flavor. However, acid whey is often implicated to have unappealing sensory characteristics, which makes it even more difficult in food applications. As a result, acid whey is mostly used in animal feeds, dumped into municipal sewage syst ems, and spread on land as fertilizer, in spite of its nutritional, functional and biological value. Although research has been conducted on the sensory descriptors and the flavor compounds of dry whey and single type cheese whey, no research has been cond ucted on the volatile aroma compounds contributing to off -flavors in liquid acid whey (cottage cheese whey) as affected by whey handling and storage conditions. A better understanding of the nature of the off -flavor compounds in liquid acid whey may help ( a) in the production of food and beverages based on liquid acid whey, (b) to minimize wastage of liquid acid whey (c) to control the flavor compounds of products made from liquid

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12 acid whey, and (d) to control the handling and processing methodologies those are detrimental to flavor of liquid acid whey. Hence the objectives of this study were to identify the off -flavor compounds in liquid cottage cheese whey and to evaluate the changes in the intensities of the off-flavor compounds upon storage, temperature fluctuations and foaming of the liquid cottage cheese whey.

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13 CHAPTER 2 LITERATURE REVIEW Whey: Nature, Types and Composition Whey is the liquid by-product obtained by separating the coagulum from milk/cream and or skim milk in cheese making (USDA 2000). De pending on the type of cheese being manufactured, the liquid whey is termed as: (a) "sweet whey (pH 5.8), obtained from the manufacture of natural enzyme -produced cheeses (e.g., Cheddar, Edam), (b) medium acid whey (pH 5.0 to 5.8), obtained from the manufacture of some fresh acid cheeses (e.g., danbo, queso blanco), (c) acid whey (pH < 5.0), obtained from the manufacture of fresh acid cheeses (e.g., quarg, cottage), and (d) acid casein whey, obtained from the manufacture of acid casein by acidification of skimmed milk(Kosikowski and others 1997; Zadow 2003). Composition of whey at dairy plants mainly depends upon the processing methods used for casein removal from liquid milk. Typically cheese whey contains about 85 to 90% of the volume of the milk used for makin g cheese, and it retains about 55% of the original nutrients in milk. The general composition of commercially produced sweet and acid type dry whey is presented in Table 2 1. Lactose, the major constituent in whey, is generally lower in acid whey than in m ilk due to the fermentation process during cheese production, where some of the lactose is converted to lactic acid. The total protein (Table 2 1) averaged 1.3% less in acid whey compared to sweet whey, which was 10% of the protein. Fat content of the acid whey is lower than sweet whey. The difference averaged 0.55 %, which was 5.3 % of fat. The use of skim milk in the production of acid whey may explain the lower fat content of acid whey than sweet whey. The total ash content is 20% higher is acid whey tha n sweet type whey. Additionally, there is more lactic acid, calcium and phosphorous in acid whey (Glass and Hedrick 1977). The fat and lactose concentration in whey are susceptible to deterioration and may be very important in controlling the flavor stabil ity

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14 of whey. The levels of lactose and total lipids concentration have been associated with the high intensities of off -flavor in whey (Morr and Foegeding 1990). Whey Disposal Problem Whey has been considered as a waste by -product by the dairy industry. The carbohydrate and protein content of whey contribute a biological load susceptible to deterioration and thus create disposal and environmental problems. Some of the procedures that have been employed or have been suggested for the disposal of whey are a s follows: (1) draining into the sewage: This procedure is not practical if the quantity of whey is large or the whey is not diluted to a great extent. Otherwise, when the sewage is treated, an excessive load is placed on the treating plant. Since the biol ogical oxygen demand (BOD) is very high, approximately 40,000 mg/kg, and the amount of whey is usually large, this procedure is not feasible in most cases. (2) Draining into the stream: Any quantity of whey running into the stream will cause enough polluti on to kill fish and to produce deleterious odors in stagnant areas. (3) Discarding in abandoned mines or quarries for disposal purposes: In most instances, the high transportation cost of whey to abandoned mines or quarries will be a limiting factor. In a ddition, the decomposing whey will be objectionable unless the place of disposal is at a considerable distance from the cheese plant and from living areas. (4) Dumping in prepared lagoons: Similar problems in discarding the whey in abandoned mines or quarr ies apply to lagooning or spreading on fields (5) Treating in a sewage disposal: The cost of a disposal plant for whey will be irrationally high because of the BOD of the whey. (6) Producing fuel gas by anaerobic fermentation: Digestion tanks of about 30 times the volume of whey would be required in the cheese plants to produce the heat source for the fuel gas production. (7) Returning the whey to farmers for feeding: The large volume of the whey may not be transported to the farms in the cans used for br inging milk to the plants. This discourages the farmers from using whey for feeding. Furthermore, the farmers having pigs to

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15 feed usually are not the same ones as those delivering milk to the cheese plant. (8) Evaporating or drying for food or feed use: T his procedure appears to be practically feasible when affordable heat is available and the volume of whey is high enough to investment in evaporating or drying equipment. (9) Manufacturing of products such as whey protein, whey cheese, whey lactose, alcoho l, and food specialties: This method is the most desirable from a nutritional standpoint and usually can be operated at the least net cost, and frequently at a profit. The special equipment required would be expensive (Byron and others 1948). Whey Utiliza tion In search of the alternative whey disposal methods by the dairy industry, whey utilization has served as an exciting opportunity for its financial benefits. Since utilization of raw unprocessed whey has several limitations such as high transportation cost, susceptibility to deterioration storage and lack of appropriate mechanism for utilization, the focus was much on maximizing the value of whey solids through greater utilization of whey components. The following steps are involved in the production of whe y solids from unprocessed liquid whey: The defatted whey from the cheese vat is pumped to a holding tank and then to a centrifugal separator. The fat is removed in the centrifugal separator to produce whey cream, which is used in butter manufacture. The w hey from the centrifugal separator is next pumped to holding tanks. The holding tanks are connected in series, to maintain a constant flow to the evaporators and to enable the operator to test the quality of the whey from different sources. The whey is nex t preheated to 220 F. and pumped into three or four -effect evaporators. The whey is concentrated in the evaporators in stages where the whey containing 6% solids is condensed to about 40 to 70% solids.

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16 The condensed or the concentrated whey is cooled to about 35 F., pumped into tanks, and transferred to the final drier. Lactose is stabilized in the whey concentrate to the monohydrate stage during the drying process because the anhydrous product is very hygroscopic. Whey solids are obtained by drying t he concentrated whey using one of the following methods: Tunnel drying: In tunnel -drying, the concentrated whey, containing 70% solids, flows from the vacuum pan into a vat, where it is seeded with a small amount of dried whey from a previous batch and hel d not more than 24 hr. to allow the lactose to crystallize. Then it is spread on mesh screen trays and dried in a tunnel where air circulates at from 140 to 180 F. At the higher temperatures, drying may be complete in 3 to 4 hr. The dried whey is relatively nonhygroscopic. Drum drying: In the drum -drying process, the whey is concentrated to 50% solids and flows to a series of drum driers. The whey dries in light -brown sheets, which are ground and packaged. The resulting product varies in its hygroscopic property. There are several variations of this process. Spray drying: Spray drying of concentrated whey is accomplished with equipment used to dry milk. The lactose should be crystallized before drying; otherwise, a very hygroscopic product will result. The whey is preheated to 180 F. for 20 minutes, and then concentrated to 40% solids. This concentrate is sprayed into the drying chamber at 285 F. The finished product is amorphous and somewhat hygroscopic. The whey proteins are soluble. There are also va riations of the spray drying process. Dried whey products are marketed as commodity ingredients for a variety of food applications. Table 2 2 shows dry whey production from 20042007.

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17 On the other hand, prior to the drying process the condensed or the conc entrated whey is demineralized by ion exchange or electrodyalisis to produce demineralized whey. Lactose and whey protein concentrate are produced from the condensed or concentrated whey by two distinctly different processing operations: Manufacture of re fined lactose: Refined lactose is produced by chemically precipitating proteins and minerals in the concentrated whey to insoluble sediments. The insoluble sediments are then separated from the pure lactose serum. This lactose serum is then crystallized, r efined and air dried to produce refined lactose Manufacture of whey protein concentrates and isolates: Processes that are available in the industry for the manufacture of whey protein concentrate are the (1) heat coagulation (2) gel filtration (3) electro dialysis (4) ultrafiltration (5) ion exchange. These processes are either used as single processes or as a sequence to remove low molecular weight materials to obtain whey protein products of higher purity. Figure 2 1 provides an overview of the whey proce ssing industry showing a breakdown of whey solids utilization by process operations (Gillies 1 974) Utilization of the Whey Solids The quality of the fresh cheese whey is maintained for a longer period of time when it is converted into the solid form to fa cilitate manipulation and transport (Byron and others 1948). The principal market for whey solids (condensed whey, acid or sweet whey powders, demineralized whey powder, delactosed whey powder, deproteinized whey, fat enriched whey) is animal feeding and i n mixtures with molasses or soya flour. Smaller quantities may be used in human foods (ice creams, baked goods, cakes, sauces, milky derivatives, and so on). Food applications of whey solids are as follows. Foaming and emulsifying agents: In terms of its f oaming properties, whey protein is often compared with egg albumin. However, it cannot be substituted for egg white in foods where air

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18 must be incorporated by whipping and a firm structure set up by heat coagulation. Stability of foams made from concentrat ed whey may be increased by neutralization or by the addition of small quantities of tannic acid, saponin, or bisulfites (Peter and others 1930). Sweetened condensed whey can be whipped to foam in 4 minutes having 200 per cent overrun and a stability of 15 hours (Ramsdell and others 1938). Soups: Soups that are to be canned and sterilized are made more easily with whey than with milk solids. A tomato soup containing whey solids retains the natural tomato acidity and does not contain clots or lumps of protei n after heating (Webb 1938). Whey protein imparts emulsion stability in soups and sterile creamed soups where the color and acidity are such that the use of whey solids will not mask the desired color and produce a smooth and tender body as well. Cheese Pr oducts: Process cheese foods provide one of the largest uses for whey solids. Bakery products : Lactose is often used at the level of 3 to 4% of the total weight of ingredients to replace sucrose. Lactose offers flavor enhancement, emulsifying action, browning, low sweetness, tenderizing effect, volume improvement and improved quality and shelf life Lactose has absorption -carrier ability and hence retards the loss of flavor, color and aroma during baking. Whey solids can be used for its functional prop erty in bakery products. Bread loaves containing whey solids showed desirable crust color, softness, resilience and shelf life. Ice creams and sherbets: Ice creams can be produced by improved body, flavor and texture by incorporating 25% whey solids, which provides superior emulsifying, and water -holding properties of the whey proteins. Whey solids can replace about 50% of the solids -not -fat. Whey solids are used 100% in sherbets for improved flavor, stability and palatability

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19 Candies: Candies such as fudg e; caramel and taffy can be made with a whey solids content of 14 to 40 % The crystallizing property of whey lactose contributes to the desired texture in fudge making. Whey caramels can be fortified with casein containing milk solids in order to produce the characteristic chewy body. Whipped sweetened condensed whey may be used to incorporate air in special types of candies. Whey solids contribute brown color to the candies due to the low caramelization point of lactose. Spirit vinegar: Spirit vinegar is made from the alcohol produced by whey fermentation. A simple distillation of the fermented whey will yield a dilute alcohol suitable for conversion to vinegar. The dilute alcohol is allowed to trickle over beech shavings or birch twigs impregnated with t he acetic acid -producing organism. A current of air passing upward through the vinegar converter accelerates the fermentation (Webb 1938). Whey butter: This is made from whey cream in accordance with usual butter making procedures. The low ash and the abse nce of casein make possible rapid churning. Hence, a butter of good body and texture can be produced by churning at a lower temperature than customarily is employed in churning cream from whole milk. The churning time of cream from whole milk can be shorte ned somewhat by adding whey cream. Meat products: Utilization of whey solids in comminuted meat products and meat loaves resulted in improved color, moisture retention, less shrinkage and improved palatability (Byron and others 1948). Utilization of Raw Unprocessed Liquid Whey The United States is the worlds largest whey producer. The total U S whey production exceeded over 8.7 billion pounds and the production of cottage cheese whey was over 1.2 billion pounds in 2007 (USDA 2007). Only 20% of the liqui d whey produced in the United States enters into the whey processing industry (Table 2 2). The remaining liquid whey is used in cattle feed,

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20 spread on land as fertilizers or dumped into municipal sewage treatment systems. Attempts to commercialize the utilization of liquid whey have not been successful. BOD of whey presents problems in sewage disposal. Utilization of liquid whey in food applications is limited for the following reasons: high transportation cost, susceptibility to deterioration during stora ge, and lack of appropriate and practical mechanisms for utilization (Coton 1985). Disposal and utilization of acid whey is even more difficult than sweet whey due to its high BOD, high ash content and relatively low solids content. The high acidity and as h content of acid whey makes it a difficult substance to dry by traditional dehydration methods. This is compounded by the lack of uniformity in the raw whey feed stock due to many factors: the varying nature of milk in different regions of the country, h andling and sanitary conditions, and most prevalent, the non standard methodology in the making of fresh acid cheeses (Kosikowski 1979). Equipment and technology currently available on the market are not suitable for the extreme requirements of drying acid whey. Each project should be separately engineered to the particular needs of the plant or number of plants being serviced. The high acidity and ash content of cottage cheese whey is also associated with other functional and flavor problems. However, the most acid -stable proteins are present in acid whey. Functionality of these proteins as a food ingredient can be increased by demineralization and concentration (Kosikowski and Mistry1967). Flavor as a Limiting Factor in Whey Utilization Flavor is generally considered as an important factor affecting consumer acceptance of foods. Whey is often thought to be unpleasant to consumers due to its undesirable flavor, which limits its usage in bland or delicately flavored food (Whetstine and others 2003). Since W he y is mainly used in the form of whey protein concentrates and isolates in human foods, much of the investigations on the flavor of whey have focused on the whey protein concentrates and isolates. Flavor of whey solids has been considered as an important fa ctor limiting its wide spread usage

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21 in food products (Hugunin and others 1987; Livney and others 1994; Morr and others 1991 1993). The non-standardized processing conditions and the usage of different whey sources in the production of whey solids lead to variation in the sensory properties, which in turn limits its usage as food ingredients (Morr and others 1990). Off -flavors in whey are not acceptable, but desirable flavor attributes are favored if they match the application (Hugunin and others 1987). Info rmation on the flavor of liquid whey is scarce. Typically, liquid whey flavor has slightly dirty -sweet/acidic taste and odor (Bodyfelt and others 1988). Liquid acid whey flavor is considered to be less desirable than sweet whey, due to its high acid and as h content. Acid whey flavor was described as a mixture of brothiness, saltiness, bitterness and diacetyl (Mc Gugan and others 1979). Liquid acid whey, which contains nutritional, biological and functional value similar to that of sweet whey, would greatly be accepted as a food ingredient, if it had bland and delicate flavor. Factors Affecting the Flavor of Whey Whey composition plays a major part in determining its flavor profile. The lactose and fat content of whey are susceptible to deterioration causing off-flavors. The level of lactose and lipid may be an important factor in determining the flavor stability of whey protein concentrates and isolates. High intensities of off -flavor were reported in whey protein concentrates and isolates due to the increas ed lactose and the total lipid content (Morr and others 1990). The chemical reaction pathways that impact the development of off -flavor are lipolysis, lipid oxidation, keto acids and fermentation. Lipolysis Reaction L ipolysis is the enzymatic hydrolysis of the ester linkage between a fatty acid and the glycerol core of the triglyceride, to produce a free fatty acid and a diglyceride. Milk lipase is destroyed during pasteurization leaving the starter culture as the prim ary source of lipolytic

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22 enzymes in whey and cheese (Whetstine 2003; Karagul and others 2003; Mahajan and others 2004). Free fatty acids are not favored in the whey products for two reasons. First, the shorter chain fatty acids have low flavor threshold and contribute to major flavor notes; and second; they can catalyze the oxidation reaction. Fatty acids are catabolized into four classes of compounds: ketoacids, 4 and 5 hydroxyacids, unsaturated fatty acids and free fatty acids (Figure2 ketoacids up on degradation produce methyl ketones and secondary alcohols. Hydroxy acids are into aldehydes, acids, and alcohols. A variety of enzymes cause fatty acids to b e esterified with ethanol to form esters. Lipid Oxidation Reaction Although whey contains a very small amount of fat, lipid oxidation reactions were considered as the cause of the initialization of the off -flavor development in whey based products (Whitfie ld 1992). Volatile lipid oxidation products were reported to be the main cause of off flavors in dairy products including whey (Swaisgood 1996; Lee and Morr 1994; Hidalgo and Kinsella 1989; Mills 1993). Research has shown that starter cultures can form lipid oxidation products (including hexanal) during fermentation (Suriyaphan and others 2001). Lipid oxidation products formed during cheesemaking would be expected in liquid whey because there is some binding of lipid oxidation products by whey proteins ( Kinsella 1989). Lipid oxidation and the subsequent production of short chain aldehydes are main contributors to off -flavors in dairy products (Lee and Morr 1994; Badings 1991). Figure 2 3 illustrates the mechanism of the lipid oxidation reaction. Lipid oxidation products and related odor properties are listed in Table 23. Mechanism of Lipid Oxidation A sequence involving initiation, propagation and termination reactions was proposed t o explain the lipid oxidation reaction:

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23 Initiation: Initiation step is the autooxidation of the fatty acids to form free radicals. In case of mono and poly unsaturated fatty acids in milk lipids, the reaction is initiated by the removal of hydrogen from the carbon atom next to the double bond to produce free radical ( R o) RH R0 Propagation: The free radical (R0) reacts with ground state molecular oxygen to form peroxy free radical (ROO 0). The peroxyl radical (ROO 0) attracks the weak C H bonds on fat (RH) and generates a hydroperoxide ROOH and fatty acid radical (R 0). The R 0 reacts with oxygen (O2) to form another peroxyl radical (ROO 0). The newly formed ROO 0 will enter the reaction cycle again to form more ROOH and R 0. R0 +O2 ROO0 ROO0 +RH ROOH + R0 R0 + O2 ROO0 Termination: Two free radicals can undergo termination reaction to produce a stable and inactive product. R0 + R0 stable end product ROO0 + ROO0 stable end product Proteolysis is another important reac tion that affects the flavor of whey. Inconsistency in the type and level of free amino acids were reported to cause of flavor variability in dried whey products (Mavropoulou and Kosikowski 1972; Mills 1993). Amino acids contribute to the basic tastes. Fu rther, they undergo trasamination to other amino acids, decarboxylation to amines and ketoacids can be converted to aldehydes by the Strecker degradation and further oxidized to acids or r educed to alcohols. Proteolytic enzymes were also thought to promote the degradation of amino acids, Heat, light, metal

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24 leading to off -flavor formation in whey. Proteolytic enzymes, including chymosin, carry over into the whey and may promote the degradation of amino acids, leading to undesirable flavor formation (Holmes and others 1977; Amudson 1984). Proteins may also bind volatile flavor compounds during processing (Stevenson and Chen 1996), contributing to flavor formation in the end product. Several Maillard reaction pro ducts were reported to produce off -flavors in whey products. Amino acids in whey undergo Maillard reaction to produce a variety chemical compound responsible for a range of odors and flavors. The type of amino acid involved in the Maillard reaction determi nes the resulting flavor. Formation of flavor compounds via the Maillard reaction is shown in F ig ure 2 4 Table 2 4 show the flavor compounds and their odor properties along with their amino acid precursors. Other Factors Whey flavor is affected by the mil k source that was used in the cheese making (Tomaino and others 2001) Flavor of whey also depends on the type of cheese being manufactured. M ilky, sweet and bland flavors were exhibited by fresh liquid cheddar whey, Gouda whey and rennet whey respectivel y. Rennet caesin whey was reported to produce sweet, oat like flavor (Gallardo and others 2005) Starter cultures used in the cheese production significantly affect the flavor of whey and several studies have focused on the influence of starter culture on the flavor of whey. Higher levels of total free fatty acids, such as lauric, myristic, and palmitic acids, were produced by Lactococcus lactis subsp. lactis starter culture in liquid Cheddar whey (Tomaino and others 2001). Wide variations were noted in the headspace volatile profile and sensory flavor profiles of liquid cheddar cheese whey produced by different starter culture rotations (Whetstine and others 2003) Other studies have evaluated processing methods that have an effect on the volatile components in whey (Stevenson and Chen 1996). To our knowledge, the effects of whey

PAGE 25

25 handling methods immediately after curd draining on the whey flavor analysis have not been conducted. Flavor Compounds in Whey In general, flavor of whey is complex with a wide varie ty of chemical compounds Whey flavor is presently understood to arise from a balance of key volatile components rather than from a unique character impact compound. Since very limited studies have investigated the volatile compounds responsible for the fla vor of whey, the literature review section in this study will include the flavor compounds and the flavor formation mechanisms in other dairy products. Acidic Compounds Acidic compounds are found to have impact on the aroma of whey. Free fatty acids are formed as a result of lypolytic reaction, and their presence in excess results in a rancid off -flavor defect (Badings and others 1991). Fatty acids can then be converted into powerful aroma components as esters, alcohols, ketones and lactones. Tomaino and others 2001 attributed the highest level of total free fatty acids to the activity of Lactococcus lactis subsp. lactis starter culture. The study reported that the long chain acids such as dodecanoic, tetradecanoic and hexadodecanoic acids had a minimal a roma impact and therefore assumed to have minimal contribution to the overall flavor. In contrast, the short chain acids such as acetic, butanoic and hexanoic acids were the most aroma intense acidic compounds in liquid cheddar whey responsible for the sour, rancid and sweaty aroma, respectively (Karagul and others 2003). Short chain fatty acids were found in lower concentrations than long chain fatty acids in commercially produced liquid cheddar whey (Whetstine and others 2003). A similar trend was observ ed in milk, where long chain fatty acids are found more than the short chain fatty acids. Long chain fatty acids are cleaved from triglycerides by lypolyic enzymes from the starter culture and are not typically found in milk. (Nawar 1996; Swaisgood 1996; Jensen 1991; Soda and others 1995). Acidic compounds in sweet

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26 whey powder included free fatty acids such as acetic, propanoic, butanoic, hexanoic, heptanoic, octanoic, decanoic, dodecanoic and 9 -decenoic acids. Of all the acids identified, acetic acid was the most prominent, imparting strong vinegar -like odor. Butanoic acid contributed to a strong rancid odor in sweet whey powder. (Mahajan and others 2004). Cheesy off -flavor was reported in whey and whey products and was attributed to 2 and 3 -methyl butyri c acid (Patton 1954). 2and 3 -methyl butanoic acids are branched chain fatty acids, generated by hydrolysis from milkfat or by microbial amino acid degradation. It was found that 3 -methyl butanoic acid contributed a sweaty, cheesy and rancid aroma. The ox idative stability of whey ingredients was related to the free fatty acids. The extremely low fat content of liquid whey, which typically contains less than 0.1% of total fat along with high amount of moisture, hinders many of the lipid extraction technique s thereby causing difficulty in removing the fatty acids which are subjected to deterioration due to oxidation. Some important fatty acids reported in whey and whey products are shown in Table 2 5. Aldehydes In whey, aldehydes are important aroma active v olatiles contributing greatly to the overall flavor. Aldehydes with more than 6 carbon atoms are typical products of lipid oxidation. Aldehydes are formed from the autooxidative mechanism of unsaturated fatty acids (Loury 1972; Paquette and others 1985). A ldehydes have very low thresholds and impart oily, fatty or tallowy odors. Aldehydes are also produced by the amino acid catabolism involving a decarboxylation to amines, followed by oxidation via Strecker degradation to aldehydes. Strecker degradation of amino acid phase of Maillard reaction produces aldehydes, which further undergo condensation and a series of further reactions to form furfurals and polymers that result in off -flavor (Hodge and others 1972; Hammond 1989).

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27 Odorants were evaluated in fresh cheddar cheese whey from 2 processing plants and 4 starter culture rotations by gas chromatography -olfactometry (GCO) and Aroma Extract Dilution Analysis (AEDA). The aroma active aldehydes that were detected in all the whey were hexanal (green), methional (potato), (E, E) 2,4 -decadienal (frying oil), and (E,E) 2,4 -nonadienal (frying oil) were potent neutral/basic aroma active compounds identified in all whey. Odor intensities of hexanal, (E, E) 2, 4 -nonadienal, 2, 3 -butanedione, and (E, E) 2, 4 decadienal were variable (Karagul and others 2003). Mahajan and others 2004 investigated aroma compounds in sweet whey powder by GC O/mass spectrometry. Volatiles were isolated by Solvent assisted Flavor Evaporation. Gas chromatography/mass spectrometry and gas chromatography/olfactometry were used for the identification of aroma compounds. The most aroma intense saturated aldehydes detected were hexanal (grassy), heptanal (sweet), nonanal (floral), phenylacetaldehyde (floral), methional (cooked potato). The same study stated that the most prominent unsatura ted aldehyde to be (E) 2 octenal (roasted peanuts), (Z) 2 -nonenal (pungent), (E, Z) 2,6 nonadienal (cucumber), (E, E) 2,4 nonadienal (green), (E,E) 2,4 octadienal (cucumber), (E,Z) 2,4 -decadienal (baked) contributing to a distinct aroma, collectively re sponsible for the metallic or cardboardy flavor in whey. These compounds were thought to have originated from the original milk generated by the starter culture during cheese making, and compounds or formed during the manufacturing process of whey powder. (Whetstine and others 2005) studied the flavor of whey protein concentrates (WPC 80) and whey protein isolates (WPI) using instrumental and sensory techniques. The key aroma active aldhydes identified were 2 nonenal (fatty/oldbooks), (E, Z) 2, 6 nonadienal (cucumber), and (E,Z) 2,4 decadienal (fatty/oxidized).

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28 Short chain aldehydes primarily produced by lipid oxidation reactions are found to be the main contributors of off -flavors in dairy products (Lee and Morr 1994). For example, unsaturated aldehydes w ere formed from the autooxidation of unsaturated fatty acids (Wadodkar and others 2002). Aldehydes are also formed from amino acid degradation. For example, floral note, produced by phenylacetaldehyde in sweet whey powder, is a product of Strecker degradat ion of phenylalanine (Counet and others 2002). Table 28 and Table 2 9 shows the formation of predominant lipid derived aldehydes and amino acid derived aldehydes respectively. Ketones Methyl ketones, listed in Table 2 6 are formed from free fatty acids. T he free fatty acids ketoacids, which on decarboxylation form ketones (Hawke 1966). Flavor potency of ketones varies with carbon chain length, and 2 -heptanone has the lowest flavor threshold value (700 ppb). Diacetyl, a main aroma c ompound in liquid whey judged to have a cultured buttery sensory note, is formed in dairy products from acetyl lactic acid, which may be formed from lactose or citric acid via pyruvic acid or on pyrolysis of sugars (Maier 1970). Diacetyl (Butanedione) was identified in liquid cheddar whey in high intensities and was related to the buttery aroma of whey (Karagul and others 2003). Diacetyl is believed to undergo reduction reaction to form acetoin, an aromatic hydroxy ketone (Hugenholtz and others 2000). This compound plays a major role in the flavor of butter (Schieberle and others 1993). 1-octen 3 one is another important aroma compound in whey which imparts a characteristic mushroom like odor at a moderate intensity, whereas, 2 -heptanone and 2 nonanone was a ssociated to fatty odor in liquid cheddar whey. The levels of ketones were found to increase largely upon heat. Ketones such as 2 -heptanone, 2 nonanone, 2, 3 butanedione, 2 pentanone, and 2undecanone were found in high concentrations in UHT milk. Increas ed concentration of 2 -pentanone, 2 -hexanone, 2-

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29 heptanone, 2 -nonanone, 2 undecanone were linked to stale flavor in UHT milk. Methyl ketones although naturally present in raw milk were said to increase after heat treatment by decarboxylation or beta oxidatio n of saturated fatty acids (Nawar 1996) or As shown in F igure 2 5 by decarboxylation of beta -oxidation naturally present in milk fat (Grosch 1982; Jensen and others 1995). Sulfur Containing Compounds Sulfur containing compounds identified in whey are shown in T able 2 7 Formation of methional, an important sulfur containing compound is shown in Figure 2 6 A study conducted on whey protein isolate indicated that the dimethyl trisulfide plays an import ant role in the cabbage off -flavor in whey protein isolate. The same study associated dimethyl disulfide to garlic like aroma. Dimethyl sulfide, dimethyl trisulfide, and methional are the major sulfur containing compounds that have been identified in chees e (Aston and Dulley 1982; Qian and others 2002; Boscaini and others 2003; Qian and Reineccius 2003) and liquid whey (Karagul and others 2003; Whetstine and others 2003; Tomaino and others 2004). Methional is believed to be generated from Strecker degradati on of methionine (Aston and Dulley 1982; Da Silva and others 1993; Fu and others 2002; Boscaini and others 2003). Methionine can degrade to form dimethyl sulfide and methanethiol. Methanethiol can be further oxidized to form dimethyl disulfide and dimethyl trisulfide (Bendall 2001). Dimethyl sulfide can be oxidized to form dimethyl sulfone (Livney and Bradley 1994). Application of heat was found to increase the concentration of hydrogen sulfide, methanethiol, carbon disulfide, dimethyl trisulfide, and dimet hyl sulfoxide. The high odor activity values of methanethiol and dimethyl trisulfide suggested that these 2 compounds could be the most important contributors to the sulfurous note in UHT milk (Vazquez Landaverde and others 2006a).

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30 Nitrogen Containing Compounds Pyrazines are nitrogen -containing heterocyclic compounds, which impart a baked, roasted potato, nut, and meat -like aromas. The aroma active pyrazines are 2,6 -dimethylpyrazine, 2,5 dimethylpyrazine, 2,3 dimethylpyrazine, and 2,3,5 trimethylpyrazine. Most of these pyrazines had moderate Osme values in sweet whey powder except 2,5 dimethylpyrazine with Osme value of 10. Most of these pyrazines have been identified in Parmesan cheese as compounds responsible for the nutty aroma (Qian and Reineccius 2002) 2 -Methoxy3 -isopropylpyrazine and 2 isobutyl 3 -methoxypyrazine have been identified in liquid whey (Karagul and others 2003), Pyrazines are the products of Maillard reaction (Friedman 1996; Alasalvar and others 2003), possibly formed during concentration, spray drying, or other processes involved in the manufacturing of whey powder. Alcohols Maltol and furaneol have been identified in whey powder and gives moderate to large intensity with a burnt sugar like aroma for maltol and a sweet aroma for furaneol Maltol and furaneol are believed to be products of sugar degradation (Da Silva and others 1993; Wadodkar and others 2002). Sotolon gave a spicy note, with high intensity in liquid cheddar. This ketobutyric acid and acetaldehyde (Takahashi and others 1976). Maltol and sotolon have been identified in liquid whey (Karagul and others 2003), and furaneol was found in stored nonfat dry milk (Karagul and others 2001). Fruity odor was associated to propan 1 -ol in fermented whey and whey produced from fermented milk. Phenolic Compounds p Cresol, a phenolic compound with a typical cattle like odor, was at high intensity in sweet whey powder. It has long been known as the factor responsible for the smell of cow urine.

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31 It imparts a barn/medi cine like flavor to milk upon liberation from conjugated precursors (Bendall 2001; Kim 2003). It is evident from the literature that only 20% of the whey produced in the United States is utilized in the form of whey powders, including whey protein concentr ates and isolates (USDA 2007). Direct utilization of the liquid whey without fractionation and dehydration represents a more economical alternative due to the elim in ation of the cost of evaporation or ultrafiltration (Ryder 1980). However, the flavor of l iquid whey is considered as one of the limiting factors in its utilization. There is an indication that liquid whey has a slightly dirty -sweet/acidic taste and odor (Bodyfelt and others 1988). Much of the research has focused on the flavor of whey protein concentrates and isolates compared to raw unprocessed liquid whey. Liquid whey is expected to have a different flavor profile compared to whey powder because of the additional steps involved in the processing of whey powder such as evaporation and ultrafil tration (Mahajan and others 2004). Moreover liquid whey is sometimes pooled from different types of cheese before processing into whey powders leading to different flavor patterns in whey powders. Although McGugan and others 1979, reported that saltiness, bitterness, brothiness and acidity as some of the flavor characteristics of liquid acid whey, to date, no direct information is available on the volatile aroma compounds responsible for the flavor of liquid acid whey. Processing treatments and drying condi tions have been reported to affect the composition of whey thereby affecting the flavor of whey solids (Morr and Foegeding1990). Similarly, whey handling practices and storage conditions may affect the flavor of liquid acid whey. A better understanding of the nature of the off-flavor compounds in liquid acid whey as affected by whey handling and storage conditions may help in minimizing its wastage in controlling the handling methodologies that are detrimental to flavor of liquid acid whey. Hence this study focused on the volatile aroma

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32 compounds responsible for the off -flavors in liquid cottage cheese whey as affected by whey handling and storage conditions.

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33 Table 2 1. General Composition of D ry W heys. Sweet type dry whey Acid type dry whey Mean Range Mean Range Lactose (%) 69.4 56.9 74.6 63.2 58.8 71.7 Total protein (%) 13.0 11.1 16.6 11.7 8.0 12.6 Nonprotein nitrogen (%) 0.50 0.23 0.65 0.58 0.45 0.73 Total ash(%) 8.3 7.1 10.7 10.6 7.3 12.2 Ash Alkalinity(%) 124 54 304 335 214 404 Moisture, (toluene) (%) 5.0 2.7 9.2 6.2 3.6 8.1 Moisture, vacuum(%) 3.0 1.1 6.3 3.1 1.6 5.0 Moisture, Karl fischer(%) 3.7 1.8 6.7 4.6 3.3 6.5 Fat(%) 1.03 0.37 1.52 0.48 .34 .74 Titrable acidity(%) .10 0.07 .19 0.39 .28 .44 pH 5.88 5.2 6.4 4.57 4.40 4.81 Glass and Hedrick 1977 Table 2 2 Estimated U S T otal L iquid W hey and L iquid C ottage C heese W hey Whey 2004 2005 2006 2007 Total whey: Total cheese production 8,873,150 9,149,322 9,524,567 9,700,499 Calculated fluid whey 79,858,350 82,343,898 85,721,103 87,304,491 Calculated total solids 5,190,792 5,352,353 5,571,871 5,674,791 Total dry whey production 1,034,898 1,040,692 1,109,616 1,133,861 Cottage cheese whey: Total Cottage cheese 958,586 1,253,262 1,237,056 1,242,222 Calculated fluid whey 1, 063,55 1 7,519,572 7,422,336 7,453,332 Calculated total solids 1,072,553 4,887,722 482,451 484,466 USDA 2007

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34 Table 2 3 Lipid Oxidation Products and Related Odor Compounds Identified in Whey Precursor Compounds Odor property Linoleic acid Hexanal Cut grass Heptanal Grassy (Z) 4 Heptenal Sweet (Z) 2 nonenal Hay/rosey (E) 2 nonenal Hay (E,Z) 2,4 Decadienal Green (E,E) 2,4 Decadienal Baked Linolenic acid (E,Z) 2,6 nonadienal Cucumber (E,E) 2,4 Nonadienal Rancid (E,E) 2,4 nonadienal Frier oil Oleic acid Nonanal Soapy Octanal Green Table 2 4 Flavor compounds Originating from the A mino acids with their Odor Properties Precursor Compounds Odor property Methionine Methional Boiled potato Dimethyl trisulfide Cooked cabbage Dimethyl disulfide Cooked vegetable Proline 2 Acetyl 1 pyrroline Roasted nut Phenylalanine Phenylacetaldehyde Floral Tryptophan Skatole Animal like Serine Pyrazine Roasted nut Ethylpyrazine Roasted nut Methylpyrazine Cooked meat Threonine 2,5 dimethyl pyrazine Cooked potato 2,6 dimethyl pyrazine Cooked potato Trimethylpyrazine Roasted 2,3,5 trimethylpyrazine Roasted Leucine Methylbutanoic acid Cheesy

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35 Table 2 5 Predominant Fatty Acids Identified in Whey Compounds Aroma Acetic acid Vinegar like Propanoic acid Rancid Butanoic acid Cheesy 3 Methylbutanoic acid Sweaty Pentanoic acid Pungent Hexanoic acid Rancid Heptanoic acid Cheesy Octanoic acid Animal like Decanoic acid Soapy Dodecanoic acid Soapy Table 2 6. Major Ketones Identified in Whey and their Flavor Characteristics Ketones Flavor Characteristics 2,3 Butanedione (Diacetyl) Buttery 2 octanone Floral 1 octen 3 one Mushroom 2 nonanone Green 2 undecanone Floral 2 tridecanone Fruity green Acetophenone Musty

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36 Table 2 7. Major Sulfur Compounds Identified in Whey and their Flavor Characteristics Compounds Flavor Methional Cooked potato Dimethyldisulfide Garlic Dimethyltrisulfide Cooked cabbage Table 2 8. Formation of Aldehydes via amino acid degradation Amino Acid Aldehydes Formed Flavor Characteristics Methionine Methional Boiled potato Phenylalanine Phenylacetaldehyde Floral Leucine, Isoleucine and valine Methyl butanal Methyl propanal Nutty Table 2 9. Formation of Aldehydes via Lipid Oxidation Fatty acid Aldehydes formed Flavor Oleic acid 2 undecenal; decanal, 2 decenal, nonanal, octanal o ily, fatty Linoleic acid 3 Nonenal, 2,4decadienal,hexanal oily, fatty tallowy Linolenic acid 2,4 Heptadienal, 3 Hexenal, 3Hexenal oily, fatty tallowy

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37 Figure 2 1. Whey Solids Production

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38 Figure 2 2. Lipolysis

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39 Figure 2 3. Formation of Flavor compounds by Lipid Oxidation Degradation Products

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40 Figure 2 4. Maillard Reaction : Flavor Compound Formation Pathways and Products

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41 Fatty ac ids keto acids Methyl ketones Figure 2 5. Formation of Methylketones Figure 2 6. Formation of Predominant Sulfur Compounds in Whey Oxidation decarboxylation Strecker degradation

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42 CHAPTER 3 MATERIALS AND METHOD S Liquid Cottage Cheese Whey Liquid cottage cheese whey was obtained from Publix, Lakeland, Florida. Whey was collected in 1 gallon High Density Polyethylene (HDPE) containers, transported to the Food Science & Human Nutrition Dept., University of Florida, Gainesville, Florida and stored at 200C. The pH of the whey was 4.5 Equipments Solid Phase Microextraction (SPME) The SPME device for manual sampling consisted of a holder assembly and replaceable fiber coated with bonded 2cm 50/30 m Divinylbenzene/ Carboxen/ Polydimethylsiloxane (DVB/CAR/ PDMS) were purchased from Supelco. According to the manufacturers instruction, the fiber was conditioned by heating in the injection port of the gas chromatograph at 240C for 60 minutes. A 40 -mL amber glass vial containing 10 ml of sample sealed with PTFE silicone septa was placed in a water bath at 400C for 30 min for sample/headspace equilibration. The SPME fiber was exposed to the headspace for 40 min for the adsorption of the volatile compounds. Upon volatile extra c tion, the SPME fiber was withdrawn and introduced into the injection port of the GC where the analytes are thermally desorbed and entered the GC column for separation and analysis Gas Chromatography-Flame Ionization Detector (GC -O) Volatiles from liquid cottage cheese whey were separated using an HP 5890 gas chromatograph (Hewlett Packard, Palo Alto, Ca, USA) equipped with a flame ionization detector, a sniffing port and splitless injector. The SPME fiber was injected into a capillary column DB 5 (30

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43 temperature was programmed with an initial temperature of 40C and ramped to 240C at 8/min with a final hold time of 5 minutes. Samples were injected using splitless mode with an i njector port temperature of 240C and the detector at 250C. The effluent was split 1:2 between a Flame Ionization Detector(FID) and a sniff port (Datu, Geneva, NY) Whey Treatments Fresh Whey (FW) The fresh liquid cottage cheese whey to be used as control was divided into 10 ml aliquots and was frozen at 200C until analysis Frozen whey (W) was thawed at 50C for 15 hours before treatments. Temperature Fluctuated Whey (TFW ): FW at 50 C was heated to 600 C for 4 hours by placing it in a hot water ba th at a temperature of 600C. Whey was cooled from 600 C to 220C in 15 minutes in another clear 16 ounze wide mouth glass jar immersed in ice water. Whey was held in a water bath of 220C for 4 hours. Whey was cooled from 220C C to 50 C in another clear glas s jar immersed in ice water. The whey was subjected to this initial heating (600C), cooling (220C) and cooling (50C) to produce TFW. The TFW was aliquoted into 10 ml using 40 ml Pyrex glass test tubes and sealed using phenolic screw caps. The aliquoted tes t tubes were frozen at 200C until analysis. Low Temperature Whey (LTW): FW at 50 C was transferred to a glass jar (16 ounze, clear straight sided, round with 89 400 White Metal Plastisol Lined Cap). The whey in glass jar was stored at 50C for 14 days to produce LTW. The LTW whey was aliquoted into 10 ml using 40 ml Pyrex glass test tubes and sealed using phenolic screw caps. The aliquoted test tubes were frozen at 200C until analysis.

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44 Foamed Whey (FOW): The FW was transferred to a glass jar (1 6 ounze, c lear straight sided, round with 89400 white metal plastisol lined cap). Foam was generated by incorporating air for 30 minutes in the whey using a glass jar (1 6 -ounze, clear straight sided, round with 89 400 White Metal Plastisol Lined Cap) and a device t hat was connected to an air cylinder (Breathing Air Grade D size 300 gas cylinder, Air Gas, Gainesville, FL). The foam was allowed to settle every 10 minutes for 30 minutes in order to prevent overflow. At the end of the treatment, the foamed whey was aliquoted into 10 ml using 40 ml Pyrex glass test tubes and sealed using phenolic screw caps. The aliquoted test tubes were frozen at 20 0C Volatile Flavor Compound Analysis Optimization of the Headspace -SPME Method: Extraction Temperature Experiments were performed on the control whey samples at temperature ranging from 25C to 40C to determine the efficiency of the extraction temperature. 40 C was chosen as the extraction temperature of the volatiles. Sample/headspace equilibration time After sample temperature optimization, the exposure time was studied in control sample. A 40 -mL amber glass vial containing 10 ml of sample sealed with PTFE -silicone septa was placed in a water bath at 400C for 30 min before fiber exposure produced better peaks when chromatographed. Fiber exposure time A 40 mL amber glass vial containing 10 ml of sample sealed with PTFE -silicone septa was placed in a water bath at 400C for 30 min and fiber -exposing time of 40 minutes produced better peaks when chromatogra phed. Panelist Three Panelists (2 females and 1 male) were recruited based on their interest and availability. Each panelist analyzed the sample in triplicate accounting for 9 sniffs per sample.

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45 Odors that were detected atleast 4 times were considered arom a active. A sliding scale with 4 pointers (none, slight, moderate and strong) was used to indicate the aroma intensity. Panelist responses were captured using chromperfect software (Justice Innovations, Denville, NJ). The odor descriptor was recorded for e ach sample Training of Panelist for GC -O As this study did not require panelists to identify finer differences between the odors, the panelists were trained only to identify the presence of odor, record the odor intensity and describe the odor in few words. Training of the panelist began by familiarizing them with the scale and the system in a short time period. During this period the panelists performed the GC O analysis of the FW twice where identification and the intensity of the odor eluted odor wa s recorded using a 4 point sliding scale as described above. Panelists were not instructed about the expected intensity of the odors and each panelist used the intensity category, which in his / her opinion fitted best the intensity of the odor perception caused by the eluted odorant. Test data were recorded only after panelists demonstrated an ability to replicate analyses. After the successful test, the trained panelists carried out sniffing the samples and recorded the perceived intensities of the odor. The readings of the panelists were disregarded if their odor description did not match the retention time or linear retention index (LRI) values did not match with the published odor properties Identification of Odorants Identification of aroma active co mponents was achieved by comparing linear retention indices and aroma descriptors with those from a database or published values. The n alkane (C8 C20) series was analyzed under the same GC conditions in DB 5 column, in order to calculate LRI. A scatter pl ot or calibration curve of retention time along with retention indices of alkane standards were graphed on Excel. Retention indices for the standards were obtained by

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46 multiplying the carbon number by a factor 100. A polynomial was obtained from a regressio n. This equation was then used to calculate linear retention indices for individual components in fresh and treated liquid cottage cheese whey Statistical Analysis Statistical differences between samples were determined by using a two -way analysis of vari ance (ANOVA) test, at the 95% confidence level, as it allows for the comparisons of more than two samples. The advantage of using a two -way ANOVA is that any error due to the panelists can be distinguished.

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47 CHAPTER 4 RESULTS AND DISCUSSI ON Gas chromatography olfactometry (GC/O) is a useful collection of techniques that combine olfactometry or the use of human detectors to access odor activity with the separation of volatiles. In this study, the whey volatiles were separated using highresolut ion capillary gas chromatography. The use of a non-polar chromatographic phase is advantageous because nonpolar compounds tend to remain on a non-polar column longer and thus would be better separated. Useful information about the volatiles that provide any aroma sensation can be obtained by the selectivity and sensitivity of the human nose combined with the resolving power of the GC. This is particularly useful for those potent aroma compounds, that have extremely low thresholds, which are detected at th e sniffing port, but are below the detection limits of flame ionization detector. An example of this can be noted in Figure 4 1. It can be seen that butanoic acid (rancid) had no FID peak in the region of 5 6 minutes. The analytical concentration of butano ic acid was below the detection levels of the FID detector, yet the concentrations exceeded the aroma threshold to be aroma active. Since individuals do not have the same sensitivity to all the aroma compounds and since there are differences between indivi duals in assessing the aroma eluting from the sniffing port, a compound was considered aroma active only if it was detected at least 4 times in the 9 sniff runs. The effects of hypersensitivities and anosmias are minimized in this study by using 3 panelist s. The panelists were not instructed about the expected intensity of the odors and were given free choice to describe the odor. Each panelist used the intensity category which in his / her opinion fitted best the intensity of the odor perception caused by the eluted odorant. Table 4 1 shows the descriptors used by each panelist during free -choice profiling of the odors contributing to the off -flavors in the fresh and treated liquid cottage cheese whey

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48 Volatile compounds responsible for off -flavors of liqu id cottage cheese whey were tentatively identified using gas chromatography-olfactometry (GC O). The tentative identifications of the volatile compounds contributing to off -flavors were based on their odor properties and calculated LRI values on a DB 5 col umn. The aroma intensities of the volatile compounds were normalized for each panelist to minimize differences between panelist uses of the intensity scale. Aroma intensities detected on the DB 5 column were normalized based on the butanoic acid, which had the highest peak height among all the volatiles (Tabel 4 2). The normalized intensities (NI) of the volatile compounds were classified as low, moderate and high. Normalized intensities from 1 to 3.9 were considered as low intensity compounds. NI from 4 to 7.9 was considered as moderate intensity compounds and NI from 8 to 10 were considered as high intensity compounds. Volatile Aroma Compounds contributing to the Off Flavors in Fresh whey (FW) or Control A total of 1 2 volatile off -flavor compounds were det ected in FW (Table 4 2) The volatile off-flavor compounds mainly included fatty acids, aldehydes, ketones, sulfur and nitrogen containing compounds. Based on their high intensities, volatile compounds such as butanoic acid (rancid), methyl butanoic acid (c heesy), 3 -nonenal (fatty), 2 6 nonadienal (waxy), methional (potato), dimethyltrisulfide (cabbage) are thought to be the major compounds contributing to the off-flavor of FW. Fatty Acids Butanoic acid was the major straight chain fatty acid contributing t o the rancid aroma in FW. It had the highest intensity (NI 10) among all the compounds detected in FW. Butanoic acid with rancid odor was previously detected in liquid whey, dry whey and in a variety of cheeses (Karagul and others 2003; Mahajan and others 2004). Rancidity was found to be released by

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49 short chain fatty acids and was observed at about 35 mg/kg. Most free fatty acids including butanoic acid are derived from lipolysis of milk fat. Although proteolysis and lactose fermentation can contribute fre e fatty acids. Methylbutanoic acid was found to be another important fatty acid in FW and it contributed to the cheesy aroma. Methylbutanoic acid was previously detected in liquid cheddar whey. It is a key flavor component in Swiss cheese. It can be generated by the hydrolyis of milk fat. It is also attributed to the metabolism of amino acid, leucine. Short chain fatty acids have low thresholds but contribute largely to flavor notes than long chain fatty acids, which have high flavor thresholds. No lo ng chain fatty acids were identified in this study. Sulfur Containing Compounds Methional (boiled potato) and dimethyltrisulfide (cabbage) were detected at moderate intensities in FW. These compounds have previously been detected in liquid whey, dry whey and in a variety of cheeses. Methional is said to have a low threshold and could be smelled strongly at a concentration of 0.5 ppm (Kamadia and others 2006). This compound is generated by amino acid (methionine) catabolism involving a decarboxylation to a mines, followed by oxidation via Strecker degradation to aldehydes (Bendall 2001; Aston and Dulley 1982; Da Silva and others 1993; Fu and others 2002; Boscaini and others 2003). It has been reported as a compound formed by the reaction between methionine and riboflavin in skim milk in the presence of light. It further degrades to several noxious and putrid compounds including dimethyl sulfide and dimethyl disulfide. Dimethyl tris ulfide (DMTS) was detected at moderate intensity in FW. DMTS was previously identified in liquid whey, dry whey and a variety of cheeses. A recent study confirmed the role of dimethyl trisulfide for the cabbage off -flavor in whey protein isolate. DMTS lev els were 1.94 0.26 ppb in whey protein isolate with cabbage flavor and 3.25 0.61 ppb

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50 in whey protein isolate without cabbage flavor (Wright and others 2006). Cabbagy defects were correlated with total volatile sulfur in a study on the volatiles of milk tre ated at high temperature for 3 or 90 seconds at 1400C and stored at ambient temperature (Jaddou and others 1978). DMTS is formed from the degradation of methionine. Methionine can degrade to form dimethyl sulfide and methanethiol; and methanethiol can be further oxidized to form dimethyl disulfide and dimethyl trisulfide. Aldehydes and Ketones Unsaturated aldehydes such as nonenal (fat) and 2,6 nonadienal (waxy) were detected at high and moderate intensities respectively. Saturated aldehyde, nonanal (soa py), was detected at moderate intensities and pentanone (fruity) was detected at low intensity in FW. These compounds were prominent in liquid and dry whey. Flavors of aldehyde are said to be very potent that even a threshold of less than 1 ppm produces a strong odor (Mills and Solms 1986). Unsaturated aldehydes, including nonenal, are collectively associated to the cardboardy off flavor in liquid cheddar whey (Karagul and others 2003). Unsaturated aldehydes are formed from the autooxidation of unsaturated fatty acids (Wadodkar and others 2002; Scanlan and others 1968; Rerkrai and others 1987; Moio and others 1993) Nonanal (soapy) has been reported to be the result of the autooxidation of unsaturated fatty acids (c18: 1 and c18: 2) and also as a result of s pontaneous decomposition of hydroperoxides promoted by heat (Grosh 1993). Pentanone was the only ketone identified at low intensity (NI 3.9). It exhibited a fruity odor. Although fruity notes were detected in fermented whey and whey produced from fermented milk, the odor was associated to 3 -heptanone and pentanol respectively. Other Compounds Butanol (sweet) was identified as a moderate intensity compound in FW. It has been previously detected at high intensity in liquid cheddar cheese whey. Its intensity varied with

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51 whey produced at different time periods at different starter culture blends (Karagul and others 2003). An unknown compound exhibited sour note at moderate intensity in FW. Sour odor is associated with acetic acid in liquid cheddar cheese whey. Gallordo and others, 2005, suggested that the probiotic, yogurt and cheese cultures influence the intensity of the acid or sour odor at different levels in fermented whey and whey produced from fermented milk. Sotolon (spicy) was detected at moderate intensity in FW. This compound was previously identified in liquid cheddar cheese whey, sweet whey powder and low fat cheeses. Sotolon, a lactone, is thought to be the result of Maillard reactions The precursors for lactones are triglycerides containing a hydr oxyl acid moiety (Parlimnet and others 1966). Effect of Low Temperature Treatment on the Off -Flavor Compounds of Whey The objective of this experiment was to determine if there were changes in the odor intensities of volatile off -flavor compounds in fresh whey occured as a result of storage at refrigeration temperature (<50C) for 14 days. Figure 4.2 shows the comparison of the intensity of the volatile aroma compounds contributing to the off -flavors in FW and LTW. Acidic Compounds No significant change in the odor intensies of butanoic acid (rancid) was observed in the whey subjected to low temperature for 14 days (LTW). Butanoic acid (rancid) was detected at high intensities in both FW and LTW. The level of butanoic acid has been shown to increase in its c oncentration from 3 to 5 mg/kg when stored at 200C between 20 and 50 days Storage at a temperature of 50C for 14 days did not have an effect on the odor intensity of the butanoic acid Methylbutanoic acid ( cheesy) had high odor intensity in FW but was appar ently lost in LTW. Methylbutanoic acid is a key flavor component in Swiss cheese ( Preininger and others 1996). It was attributed to the metabolism of amino acid leucine (Munoz and others 2003; Ortigosa and others 2001). Q uantity of methylbutyric acids produ ced was dependent on PAB strains in Swiss

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52 cheeses. The largest part of methylbutyric acids (52 75%) was produced during the cold storage (Thierry and others 2004) However, in the case of this experiment the cheesy aroma was not detected after the FW was t reated at low temperature (50 C) for 14 days. Aldehydes and Ketones: Low temperature treatment did not have an effect on the intensities of nonanal (soapy) and 2,6 nonadienal (waxy) and pentanone (fruity). However, 3-nonenal (fatty) was reduced by approxim ately (60%) after low temperature treatment. Similar results were published by Contarini and others, 1997 who observed a decrease in aldehyde content in heat treated milk stored at 4 C. Pentanal (malt) was generated in LTW with a moderate intensity (NI 4. 46). Straight chain aldehydes such as pentanal, heptanal, were thought to be formed as a result of the -oxidation of unsaturated fatty acids (Molimard and Spinnler 1996). Octenal (grassy) odor was generated in the LTW. Alkenals including octenal are secon dary reaction products of fat oxidation (Mills 1993) Sulfur and Nitrogen Containing Compounds No significant changes in the intensities of the methional (boiled potato) and dimethyltrisulfide (cabbage) were observed after the low temperature treatment. Ac etyl pyrroline (roasted), however, was generated in LTW. It had moderate odor intensity (NI 5.32) in LTW. Acetyl pyroline was previously detected in the liquid cheddar cheese whey, sweet whey powder and whey protein isolate. It is noted for its popcorn lik e flavor. It has been reported as a thermally induced aroma active compound. Proline is the most important precursor of 2 acetyl 1 pyrroline. The reaction between the amino acids proline or ornithine and the sugar -degradation product (2 -oxopropanal) generates 2 acetyl 1 -pyrroline ( Schieberle and others 1993 ). Also, 2 acetyl 1 -pyrroline is thought to be formed by the interaction of pyruvaldehyde with 1 -pyrroline (Bendall 2001; Karagul and others 2001). 2-Acetyl 1 -pyrroline has very low odor thresholds and

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53 is often cited as a compound that directly contribute to roast or smoky flavors (Da Silva and others 1993; Karagul and others 2003). Other Compounds Butanol (sweet), unknown (sour) and sotolon (spicy) notes that were detected at moderate intensities in FW but were not detectable in the LTW. Butanol (sweet) odor was found to increase in its concentration when stored at room temperature (220C) for 5 months. But butanol (sweet) could not be perceived after FW was subjected to refrigeration temperature (<50C) for 14 days. Butanol (sweet) was postulated to be a non-enzymatic browning product (Jeon and others 1978). Stark and Fross et al suggested that alkohols were formed by the oxidation of unsaturated fatty acids from the primary alkoxy radicals resulting f rom the decomposition of lipid hydroperoxides. Effect of Temperature Fluctuations on the OffFlavor Compounds of Whey The objective of this experiment was to determine if there are changes in the odor intensities of volatile off -flavor compounds in fresh whey occurred as a result of temperature fluctuation. Figure 4 3 shows the comparison of the intensities of the volatile aroma compounds contributing to the off -flavors in FW and TFW Acids Temperature fluctuations of whey did not have a significant effec t on the odor intensities of the butanoic acid (rancid). The level of the butanoic acid is said to increase after heat treatment of non -fat dry milk (Karagul and others 2001). In this study, the exposure of FW to subsequent heat and cold during temperature fluctuations did not have an effect on the intensity of butanoic acid. However, methyl butyric acid (cheesy) was apparently lost in TFW. These two compounds have been earlier detected in liquid and dry whey and in a variety of cheeses.

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54 Sulfur Containing C ompounds Odor intensitiy of methional (boiled potato) did not change significantly in TFW. It was surprising to note that the dimethyltrisulfide (cabbage), which was detected at a moderate intensity in FW, was not be perceived in the TFW. A study by Vaz quez and others, 2006b reported that the thermal processing of milk generated sulfur off -flavors with high odor activity values. In the same study, higher levels of sulfur compounds were found in heat treated milk than other nonheat treated milk. It has b een reported that the sulfydryl groups of whey proteins, lactoglobulin, are exposed by heat and liberate sulfur compounds including dimethyltrisulfide which are responsible for the cooked flavor of heated milk (Christensen and Reineccius 1992; Pat rick and Swaisgood 1976; Simon and Hansen 2001). In contrast to the above published studies, the whey subjected to temperature fluctuations did not exhibit a cabbage odor. In addition, Burbank and others 2007 noted that the sulfur compounds including dimet hyl trisulfide, at lower concentrations exhibited agreeable cheddar cheese flavor notes instead of the characteristic cabbage or odor. Aldehydes and Ketones Among the aldehydes detected in the FW, intensities of 3 nonenal (fat) and 2, 6 nonadienal (waxy) were lost after the FW was temperature fluctuated. However, nonanal (soapy) and Pentanone (fruity) was intensified after temperature fluctuation. Straight c hain aldehydes are reported to be common oxidation products in milk. They can result from the auto -oxidation of unsaturated fatty acids and spontaneous decomposition of hydroperoxides promoted by heat. Concentration of the nonanal was the highest among the other aldehydes when the heat treated milk was subjected to 600C (Vazquez -Landaverde and others 2006a). Similar trend was observed in the present study, where the odor intensity of nonanal (soapy) was significantly higher in TFW, in which the whey had be en subjected to 600C. On the

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55 other hand, Contarini and others 1997, observed a decrease in aldehyde content in UHT milk stored at room temperature. Similarly, 3 -nonenal (fat) and 2,6 nonadienal (waxy) were lost in this study when the FW was temperature fluctuated. Intensity of fruity odor (Pentanone) was found significantly higher in the TFW. Nitrogen Containing Compounds Dimethyl pyrazine (roasted) and 2 Isobutyl 3 -methoxypyrazine (musty), which were not detected in FW were detected in TFW. Dimethyl pyra zine (roasted) was detected at high intensity. It was previously detected at moderate intensities in dry whey. 2 Isobutyl 3 methoxypyrazine (musty) was detected at high intensity in both liquid and dry whey. These compounds are also responsible for the nutty aroma in Parmesan cheese (Qian and Reineccius 2002). These compounds are the products of Maillard reaction (Friedman and others 1996; Alasalvar and others 2003). These compounds are formed during concentration, spray drying or other processes involved in the manufacturing of whey powder. Effect of Foaming on the OffFlavor Compounds of Whey The objective of this experiment was to determine if there are changes in the odor intensities of volatile off -flavor compounds in fresh whey occurred as a result of foaming. Figure 4 3 shows the comparison of the intensities of the volatile aroma compounds contributing to the off-flavors in FW and TFW Figure 4 4 shows the comparison of the intensity of the volatile aroma compounds contributing to the off -flavors in FW and FOW Acids No significant change in the odor intensies of butanoic acid (rancid) was observed in the whey subjected to foam. Butanoic acid (rancid) was detected at high intensities in both FW and FOW. However methylbutanoic acid ( cheesy), which had high odor intensity in FW was

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56 apparently lost in FOW. Methyl butyric acid is a key flavor component in Swiss cheese(Preininger and others 1996). It was attributed to the metabolism of amino acid leucine (Munoz and others 2003; Ortigosa and others 2001) Thierry and others 2004, suggested that the quantity of methylbutyric acids produced in swiss cheeses was highly dependent on Propionic acid bacterium (PAB) strain. The largest part of methylbutyric acids (52 75%) was produced during the cold storage, during which no significant PAB growth or lactic acid consumption could be detected. Aldehydes and Ketones Among the aldehydes, no significant change in the odor intensities of nonanal (soapy) and 2,6 nonadienal (waxy) was observed between the FW and FOW. Pentanone (fruity) odor was lost in the FOW. 3 nonenal (fat), which was detected at high intensity in FW, was not detected in the FOW. Pereda and others 2007, suggested that milk fat globule membrane (MFGM) is affected in Ultra High Pressure Homogenized ( UHPH) milk, and as a result the fatty acids are more exposed, and furthermore, at 300 MPa, the higher temperature achieved can also promote hydroperoxide formation. Whereas in milk treated with high hydrostatic pressure (100 800 MPa for 30 min), the fat gl obules are not disrupted ( Ye and others 2004). Vazquez Landaverde and others 2006a, explained that under high pressure, oxygen becomes more soluble and hence it could increase the formation of hydroperoxides, leading to more aldehyde formation. Since previous studies have explained an increase in the aldehyde concentration upon fat globule disruption and oxygen reaction under high pressure, It may have been expected that foaming the whey for 30 minutes in whey may impact the aldehyde levels and the odor intensities were expected to be high for the aldehydes in FOW. In contrary, there was no significant change in the odor intensities of the aldehydes nonanal (soapy) and 2,6 nonadienal (waxy). Surprisingly the fatty odor produced by 3 nonenal was lost in the FOW. It may be assumed that the nonanal may

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57 have oxidized to its corresponding acid or reduced to alcohol, which resulted in the loss of soapy flavor. Sulfur and Nitrogen Containing Compounds lactoglobulin, are exposed by heat to form sulfur compounds (Christensen and Reineccius 1992; Patrick and Swaisgood, 1976; Simon and Hansen, 2001). In this study, no significant change was observed in the odo r intensities of methional and DMTS between FW and FOW. 2 -methoxy3, 6 dimethylpyrazine (roast) and 2Isobutyl 3 -methoxypyrazine (musty) were generated with high intensities in FOW. Both these compounds were detected at high intensities in FOW. The product ion of pyrazines from the reaction of carbohydrates and amine compounds has been studied extensively over the past several years. Hodge and others 1972 proposed that amino acids and carbohydrates were important precursors for pyrazines formed during the nonenzymatic browning reaction. Ferretti and others 1970 reported that the pyrazines were from the reaction of lactose with casein. Shibamoto and others 1977 further explained the formation of pyrazine compounds as the reaction between sugars and amines, whi amino carbonyl amino carbonyl intermediates produces pyrazine compounds. Pyrazines were also obtained as a result of lipid autoxidation (Rizzi and 1972).

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58 Figure 4 1. Partial FID (top) and Aromagram (bottom) overlay of fresh liquid cottage cheese whey analyzed on DB 5 column Butanoic acid FID response 2,6 nonadienal (cucumber) Retention Time (min)

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59 Table 4 1. Descriptors used by each panelist during free -choice profiling of the odors contributing to the off -flavors in the fresh and treated liquid cottage cheese whey Odor number Whey Retention Time Odor Descriptors Panelist 1 Panelist 2 Panelist 3 1 FW 2.75 Sweet Caramel Sugary 2 FW, LTW 3.35 Fruity Apple like Some fruit 3 LTW 3.84 Malt Malt Malt 4 FW 4.21 Sour Acidic Vinegar 5 FW, LTW, TFW, FOW 5.36 Rancid Cheese Rancid Cream Vomit 6 TFW,FOW 5.86 Baked Charred Roasty 7 FW 6.05 Cheesy Swiss cheese Cheese 8 FW, LTW, TFW, FOW 6.64 Cooked potato Boiled potato Boiled potato 9 LTW 7.09 Roasted nut Roasted peanut Nutty 10 FW, LTW,FOW 7.7 Boiled cabbage Cooked vegetable Cabbage or lettuce like 11 LTW 9.49 Green Grassy Grass 12 FOW 9.5 Earthy Mildew Dusty 13 FW 9.99 Cumin Spice Curry 14 FW, LTW 10.24 Fatty Oily Frier oil 15 FW, LTW, TFW, FOW 10.35 Soapy Detergent Soapy 16 FW, LTW, FOW 10.91 Cucumber Cucumber melon 17 TFW,FOW 11.4 Wet wood Musty, dusty like Wet sand

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60 Table 4 2. Aroma intensities based on the normalized peak heights of samples analyzed using DB 5 column. Descriptors are listed in ascending order of LRI Compound Number LRI Compound Normalized Aroma Intensities 1 660 Butanol 4.24 2 709 Petanone 6.9 3 762 Unknown 4.9 4 829 Butanoic acid 10 5 873 Methyl butyric acid 9.94 6 903 Methional 7.95 7 928 Acetyl pyrroline 5.32 8 963 Dimethyltrisulfide 7.94 9 1093 Sotolon 4.9 10 1107 3 nonenal 9.4 11 1113 Nonanal 4.51 12 1145 2,6, Nonadienal 7.94

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61 Figure 4 2. Aroma intensities for the FW and LT W on DB 5 column. Intensities were normalized peak height based on butanoic acid, which had the highest peak height. Peak number s correspond to those listed in Table 4 3.

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62 Figure 4 3. Aroma i ntensities for the FW and TFW on DB 5 column. Intensities were normalized peak height based on butanoic acid, which had the highest peak height. Peak numbers correspond to those listed in T able 4 3. 6

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63 Figure 4 4. Aroma intensities for the FW and FOW on DB 5 column. Intensities were normalized peak height based on butanoic acid, which had the highest peak height. Peak numbers correspond to those listed in T able 4 3.

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64 Tab le 4 3 Aroma active peaks with tentative identification based on aroma attributes and retention characteristics for liquid cottage cheese whey samples as detected by GC O on a non polar columns Component # LRI (DB 5) Component name Descriptor 1 660 Butanol Sweet 2 709 Pentanone Fruity 3 762 Unknown Sour 4 829 Rancid Butanoic acid 5 870 Cheesy Methylbutanoic acid 6 903 Methional Boiled potato 7 963 Dimethyltrisulfide Cabbage 8 1093 Sotolon Spicy 9 1107 3 nonenal Fat 10 1113 Nonanal Soapy 11 1145 2,6 nonadienal Cucumber 12 722 Pentanal Malt 13 928 Acetyl pyrroline Roasted nut 14 1064 Octenal Green 15 885 Dimethyl pyrazine Roast 16 1175 2 Isobutyl 3 methoxypyrazine Musty 17 1066 2 methoxy 3,6 dimethyl pyrazine Musty

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65 CHAPTER 5 SUMMARY AND CONCLUSI ON GC Olfactometry was applied to study the effect of storage, temperature fluctuations and foaming on the aroma active off -flavor components. When fresh and treated whey were considered, a total of 19 aroma active off -flavor components were detected and 17 were tentatively identified. Storage, temperature fluctuations and foaming affected the number, types and relative intensities of the off -flavor compounds perceived. Sweet (butanol), sour (unkown compound), cheesy (methyl butyric aci d), spicy (sotolon) perceived in fresh whey were lost in all three treated wheys. Cabbage (dimethyl trisulfide), fatty (nonenal) and cucumber (2, 6 nonadienal) off -flavors were lost in the temperature fluctuated whey. Fatty (nonenal) off -flavor was lost in the foamed whey. Whey subjected to storage, generated the malt (pentanal), roasted nut (acetyl pyrroline), grassy (octenal) off-flavors. Musty (2 -methoxy3, 6 dimethylpyrazine) and roasty (unkown compound) flavors were generated by temperature fluctuated whey. Foamed whey generated burnt (furfuryl alcohol), roast (dimethyl pyrazine), earthy (2 -methoxy3, 6 dimethylpyrazine) and musty (2 Isobutyl 3 -methoxypyrazine) off -flavors. Temperature fluctuated whey intensified the fruity (pentanone) and soapy (nonana l) off-flavors and fatty off -flavor was reduced by the whey stored at low temperature. On the other hand, rancid (butanoic acid) and methional (cooked potato) did not vary in their intensities in any of the three treated wheys. As expected, thermally unsta ble compounds such as sotolon, nonenal, 2,6 nonadienal were not perceived after the whey was subjected to temperature fluctuations. Generation of potent odorants such as 2 -methoxy3, 6 dimethylpyrazine) and musty (2 Isobutyl 3 -methoxypyrazine) in temperatu re fluctuated whey may be result of Maillard reactions that occurred during temperature fluctuations

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73 BIOGRAPHICAL SKETCH Uma Devi Anguswamy graduated from St. Marys higher secondary school, India in 1994. She received a Bachelor of Science degree in Clinical Nutrition and Dietetics from Avinashilingam University, India in 1997. Uma Devi Anguswamy will complete her Master of Science in food science and human nutrition at the University of Florida in 2010. Her research was in the field of flavor chemistry and was conducted at the Food Science and Human N utrition Department in Gainesville, Florida, USA. Upon graduation, Uma Devi Anguswamy wish to gain hands on experience in USA before she returns to her county, India, where her knowledge and skills will be applied in a food industry.