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The Use of Cyclodextrins to Remove Volatile Compounds From

Selected Protein Products

Dissertation

Presented in Partial Fulfillment of the Requirements for the

Degree of Doctor of Philosophy in the Graduate School of

The Ohio State University

By

Min-Hsiu Yang, M.S.

******

The Ohio State University

1996

Dissertation committee; Approvedi

Dr. W. James Harper Dr. Michael E. Mangino " Advisor i/ Dr. Valente B. Alvarez Food Science and Nutrition \Πaduate Program UMI Number: 9631010

UMI Microform 9631010 Copyright 1996, by UMI Company. All rights reserved.

This microform edition is protected against unauthorized copying under Title 17, United States Code.

UMI 300 North Zeeb Road Ann Arbor, MI 48103 To my parents

u ACKNOWLEDGMENTS

I wish to express my deep appreciation to my parents and family; without their

continuous support, unconditional love and faith, it would have been impossible for me to

complete my ph.D. degree abroad. I am thankful for the opportunity, the financial support

and determination allowing me to work on my master and ph.D. in the United States.

I would hke to thank Dr. James Harper, my advisor, for his profound guidance and

contribution to my dissertation. His dedication, fulfillment and enthusiasm to food

research and education are attritributes I certainly admire. A great many thanks also go to

my former advisor. Dr. Denise King for her supervision during my master study and first

two years of my Ph.D. study. Her continues concern on my accomplishment is

appreciated. I would like to extend my gratefulness to Dr. Mangino and Dr. Alvarez for

their serving on my committee and valuable suggestion for this dissertation.

I thank my colleagues for their understanding my need to meet deadlines by

relinquishing to me the use of the conq)uter in our laboratory when the need arose. I

appreciate Wen-Lin’s sharing of her research, which had useful information for my use.

I appreciate the department stafi^ Carol, Tracey, Ingrin, Ed, and Mac for their support and assistance. I also would like to give my thanks to our former secretary, Judy, for her encouragement and fiiendship. I would like to express my gratitute to Dr.Glaucial

Moria Pastore for her inspiration at the early stage of my Ph.D. research.

iii I treasure the support and fiiendship from the numerous people in Columbus, ohio and Tuscaloosa, Alabama, who I have not named. I do thank God for allowing them a part in my life during the past six years.

IV m m m m ixm Rm x ■ ««««sus» ■ ■m

# 5 ■ • nm m m m m sênm t • A

# ■ B#pa»a* 3R * a * » •

- cr. James Harper ,

■*aw«se9 • aft»w«cr. oenise King

• ittataKfi9(65SS-t,'*aa

' aïE Columbus, Ohio ^Tuscaloosa, Alabama VITA

January 3, 1965 ...... Born in Taina, Taiwan

1988 ...... B.S., National Chung-Hsing University, Taichung, Taiwan

1989 ...... Research assistant. Animal Science, National Taiwan University, Taipei, Taiwan

1990-1993...... Graduate research associate. Human Nutrition and Food Management, The Ohio State University, Columbus, Ohio

1993 ...... M.S., Human Nutrition and Food Management, Hie Ohio State University, Columbus, Ohio

1993-Present ...... Graduate research associate. Food Science and Technology The Ohio State University Columbus, Ohio

VI PUBLICATION

1. Yang, M.H., Min, D.B, and King, D.L. 1992 Measurement of Upoxygenase activity by headsapce oxygen Analysis. Institute of Food Technologists Annual Meeting, New Orleans (Abstract).

2. King, D.L. and Yang, M.H. 1992 Inhibition of lipoxygenase and peroxidase by phenolic antioxidants. Institute of Food Technologists Annual Meeting, New Orleans (Abstract).

3. King, D.L. and Yang, M.H. 1993 Inhibition of lipoxygenase and peroxidase by extract of herbs. Institute of Food Technologists Annual Meeting, Chicago (Abstract).

4. Yang, M.H. and King, D.L. 1994 Effect of beta-cyclodextrin on volatile flavor compounds in soy milk. Institute of Food Technologists Annual Meeting, Atlanta (Abstract).

5. Yang, M.H. and Haiper, W. J. 1995 Effect of beta-cyclodextrin on volatile flavor compounds in whey protein concentrate. Institute of Food Technologists Annual Meeting, Anaheim, California (Abstract).

FIELDS OF STUDY

Major Field: Food Science and Nutrition

vu TABLE OF CONTENTS

DEDICATION...... ü ACKNOWLEDGMENT...... iii VTTA...... vi LIST OF TABLES...... xi LIST OF FIGURES...... xiii

CHAPTER I INTRODUCTION...... 1

CHAPTER H LITERATURE VIEW...... 3 2.1. Flavor chemistry of soy m ilk ...... 3 2.1.1. Flavor profile and origin of soy protein products...... 3 2.1.2. Analytical technology of soy flavor ...... 7 2.1.3. Control and removal of oflF-flavor Jfrom soy protein ...... 8 2.1.3.1. Lipoxygenase inactivation ...... 8 2.1.3.2. Removal of undesirable volatile components and their precursors 9 2.2. Flavor chemistry of whey protein concentrate ...... 10 2.2.1. General chemistiy of dairy flavor ...... 11 2.2.2. Origins of WPG flavor...... 13 2.2.2.1. Lipolyzed or rancid flavor in WPG...... 13 2.2.2.1.1. Origins of firee fetty acids in dairy products ...... 17 2.2.2.1.2. Control of lipolyzed flavors...... 18 2.2.2.2. Origins of oxidized flavor in milk and W PG ...... 18 2.2.2.3. Maillard reaction in milk and WPG ...... 22 2.2.2.3.I. Control of Maillard reaction...... 23 2.2.2.4. Other reaction responsible for off-flavor in W PG ...... 24 2.2.2.5. Analysis of whey protein concentrate flavor ...... 24 2.2.6. Evaluation for flavor quality of milk and dairy products in the industry...... 29 2.2.7. Solid phase microextraction (SPME)...... 31 2.2.8. Variation of volatile compounds obtained with different types of methods...... 32 2.3. Interaction of flavor and proteins...... 33 2.3.1. Effect of structure of volatile compounds...... 33 2.3.2. Effect of protein type...... 34 2.3.3. Effect of temperature...... 35 2.3.4. Effect of pH value ...... 35 viii 2.3.5. EflFect of ion concentration...... 36 2.3.6. Effect of other components...... 36 2.4. Interaction between free fatty acid and protein ...... 36 2.4.1. Fatty acid structure and binding to proteins ...... 36 2.4.2. Protein structure and binding to fatty acids ...... 37 2.4.3. Effect of pH, temperature, ion strength and other factors...... 37 2.5. Chemistry and application of cyclodextrins...... 38 2.5.1. Structure and properties of cyclodextrins...... 39 2.5.2. Application of cyclodextrins to foods ...... 39 2.5.3. Insoluble cyclodextrins ...... 42 2.5.4. Removal of narigin and limonin from citrus juice...... 44

CHAPTER m PROCEDURES...... 45 3.1. Materials...... 45 3.1.1. Soy protein...... 45 3.1.2. Whey protein concentrates...... 45 3.1.3. Cyclodextrins ...... 45 3.1.4. Chemicals and general supplies...... 46 3.1.5. Solid phase microextraction...... 46 3.2. Sample preparation...... 46 3.2.1. Preparation of protein solution...... 46 3.2.1.1. Preparation of soy m ilk ...... 46 3.2.1.2. Preparation of whey protein concentrate...... 47 3.2.2. Preparation of standard volatile conq)ound solution...... 47 3.2.2. l.Preparation of standard volatile solution for soy protein flavor analysis .. 47 3.2.2.2. Preparation of standard volatile solution for whey protein flavor analysis48 3.3. Gas chromatographic analysis of volatile compounds ...... 48 3.3.1. Analysis of soy milk flavor ...... 48 3.3.1.1. Dynamic headspace analysis of volatile compounds...... 48 3.3.1.2. Isolation and identification of volatile from soy milk with gas chromatography/mass spectrometry...... 49 3.3.2. Analysis of whey protein flavor...... 49 3.3.2.1. Solid phase microextraction (SPME)...... 49 3.3.2.1.1. Calculation and statistical analysis ...... 50 3.3.2.2. Gas chromatography/ mass spectrometry analysis of WPC...... 50 3.3.2.3. Gas chromatography analysis of standard fatty acid solution ...... 45 3.4. Use of cyclodextrins to remove volatile compounds from protein and standard chemical solution...... 51 3.4.1. Treatment of protein with P-cyclodextrin...... 51 3.4.1.1. Treatment of soy milk with P-cyclodextrin...... 51 3.4.1.2. Treatment of WPC with p-cyclodextrin...... 51 3.4.1.2.1. Effect of concentration o f P-CD on the removal of volatiles from WPC...... 52 3.4.2.Treatment of standard solution with p-cyclodextrin...... 52 ix 3.4.2.1. Treatment of standard volatile compounds of soy milk ...... 52 3.4.2.2. Treatment of standard volatile compounds of WPC...... 52 3.4.3. Treatment of WPC with various types of cyclodextrin ...... 53 3.4.3.1. WPC solution preparation ...... 53 3.4.3.2. Measurement of residual P-cyclodextrin in W PC ...... 53

CHAPTER IV RESULTS AND DISCUSSION...... 54 4.1. Soy milk investigation ...... 54 4.1.1. Identification of headspace volatiles in soy milk ...... 54 4.1.2. Removal of volatiles of soy milk by P-cyclodextrin ...... 57 4.1.2.1. EflFect of cyclodextrin on standard solution in model system ...... 57 4.1.2.2. EflFect of P-cyclodextrin on volatiles in soy milk ...... 60 4.1.2.3. EflFect of temperatiue on the removal of volatiles with P-cyclodextrin ... 62 4.2. Whey protein investigation ...... 66 4.2.1.Development of SPME methods for headspace analysis...... 66 4.2.1.1. EflFect of temperature and stirring on SPME of standard solution...... 67 4.2.1.2. EflFect of time on volatile compounds of WPC extraction by SPME ...... 70 4.2.1.3. Variation of SPME fibers ...... 72 4.2.2. Volatile compounds of WPC...... 74 4.3. Removal of volatiles in WPC with cyclodextrin ...... 78 4.3.1. EflFect of cyclodextrin on standard solution in a model system ...... 78 4.3.2. EflFect of various types of cyclodextrins on the removal of volatiles fi’om a standard solution...... 80 4.3.3. EflFect of cyclodextrin on WPC volatile compounds...... 83 4.3.3.1. Use of soluble P-cyclodextrin ...... 84 4.3.3.1.1. EflFect of 5% of P-cyclodextrin on WPC volatiles ...... 84 4.3.3.1.2. EflFect of the concentration of P-cyclodextrin on the reduction of fatty acids in WPC ...... 86 4.3.3.2. EflFect of various types of cyclodextrins on WPC volatiles ...... 89 4.3.3.3. EflFect of contact time with P-cyclodextrin polymer on the removal of volatiles fi'om WPC ...... 92 4.3.3.4. Measurement of residual P-cyclodextrin in W PC ...... 95

CHAPTER V CONCLUSION...... 100 5. l.Soy milk investigation ...... 100 5.2.Whey protein concentrate investigation...... 101

REFERENCES...... 103

APPENDICES:Additional Tables ...... 111

X LIST OF TABLES

TABLE PAGE

1. Volatile compounds associated with various odors in soy products...... 5 2. Flavor profile and responsible compounds of milk oflF-flavor...... 12 3. Flavor profiles of free fatty acids in water and emulsion...... 15 4. Flavor thresholds of volatile fatty acids ...... 16 5. Possible origin to some aldehydes obtained from the oxidation of four unsaturated fatty acids ...... 21 6. Flavor compounds identified from WPC...... 27 7. ADSA milk flavor and scoring guide ...... 30 8. Application of cyclodextrins in foods and analytical chemistry ...... 41 9. Characteristics of insoluble cyclodextrins used in this study ...... 43 10. Compounds identified from soy milk with DHA/GC/MS ...... 56 11. Reduction of flavor compounds with the addition of P-cyclodextrin after incubation at 100 °C for 5 minutes ...... 58 12. Reduction of volatile compounds in headspace with the addition of P-cyclodextrin after incubation at 30°C for 5 minutes ...... 63 13. Peak area of fatty acids extracted by SPME at different operational condition...... 68 14. Peak area of volatile compounds extracted by SPME at 40°C for 1 hour, 2 hours, 3 hours or 4 hours...... 71 15. Average, standard deviation, and C.V. of peak area extracted with two fibers ...... 73 16. Selected flavor compounds identified by GC/MS from WPC and comparison with other methods...... 75 17. Reduction of fatty acids and benzoic acid with addition of P-cyclodextrin 79 18. Effect of various cyclodextrins on fatty acids in model system...... 81 19. Reduction of volatile compounds in headspace of WPC with addition of P- cyclodextrin ...... 85 20. Effect of concentration of P-cyclodextrin on the reduction of volatile compounds in WPC...... 87 21. Peak area and reduction of fatty acids in WPC after treatment of cyclodextrins ...... 90 22. Effect of contact time between P-cyclodextrin polymer on peak area of extracted volatile compounds...... 93

XI 23. Peak area and reduction of fatty acids after addition of various cyclodextrin in model system...... 112 24. Peak area and reduction of volatile compounds in WPC after addition of P- cyclodextrin at various concentration ...... 113 25. Peak area and reduction of fetty acids in WPC after treatment of various cyclodextrins ...... 114 26. Peak area and reduction of volatile compounds after treatment of P-cyclodextrin polymer for various contact time ...... 115

xn LIST OF FIGURES

FIGURE PAGE

1. Mechanism of lipid oxidation...... 20 2. Chemical formula and molecular shape of P-cyclodextrin...... 40 3. GC/MS profiles of headspace volatile compounds mix containing no cyclodextrin (A) and volatile compounds mix containing cyclodextrin (B) after incubation at 100°C for 5 minutes ...... 59 4. GC/MS profiles of headspace volatile compounds ftom soy milk containing no cyclodextrin (A) and soy milk containing cyclodextrin (B)...... 61 5. GC/MS profiles of headspace volatile conqpounds ftom volatile compound mix containing no cyclodextrin (A) and volatile compound mix containing cyclodextrin (B) after incubation at 30°C for 5 minutes ...... 64 6. Conq)arison of the reduction of volatiles in aqueous solution with P-cyclodextrin with incubation at 30“C and 100 °C ...... 65 7. Effect of extraction time, tenq)erature, and stirring on the efficiency of SPME on fatty acids ...... 69 8. Reduction of fatty acids with addition of various cyclodextrins ...... 82 9. Reduction of fetty acids with P-cyclodextrin at various concentrations 88 10. Reduction of fatty acids with addition of cyclodextrin in WPC ...... 91 11. Reduction of fetty acids with addition of P-cyclodextrin polymer ...... 94 12. Relationship of cyclodextrin concentration and absorbance ...... 96

xm CHAPTER I

INTRODUCTION

Due to a large production of soy protein and whey protein concentrate (WPC) and

broadening in the utilization of these proteins in the U.S., a great amount of attention has

been directed to break the limitation in utilization caused by their undesirable off-flavors.

As known, beany and green flavor predominately cause a low acceptability of soy

products. Brothiness, diacetyl, bitterness, and volatile acidity of WPC is a problem of its

incorporation in plain-flavor formulated foods.

A number of studies have been made on the flavor of soy protein and whey

protein, which has lead to knowledge of origin of these volatiles and profoundly contribute to the development of treatment to eliminate ofT-flavor from these proteins. Volatile and non-volatile compounds result from lipid oxidation, lipolysis. Maillard reaction and other reactions, may be formed during the processing and storage.

Con^ared to methods previously used for flavor studies, solid phase microextraction (SPEM) is advantageous for its simplicity and ability to extract a wide range of volatile confounds. In this study, SPME with gas chromatography was evaluated for the analysis of WPC flavor. potentially to trap flavor molecules into its central cavities. In addition, success in using cyclodextrin to remove ofF-flavor of hydrolyzed soybean protein, and fish protein (Suzuki, et al., 1981) indicates that cyclodextrin may reduce odor by inclusion of responsible compounds.

The specific objectives for this study include:

1. Identify volatile compounds of soy protein and whey protein concentrate.

2. Develop methods using solid phase microextraction to measure volatiles of whey

protein.

3. Evaluate effect of cyclodextrin on volatiles in soy milk, WPC, as well as in model

systems.

4. Determine which cyclodextrin provides the most effective reduction of volatiles in WPC

and easy removal of cyclodextrin from WPC.

5. Determine operational factors including concentration of cyclodextrin, contact time, and

temperature to reach optima reduction of WPC volatile compounds. CHAPTER n

LITERATURE REVIEW

The use of all types of food proteins as food ingredients is limited to some degree

because of undesirable flavors that are characteristic of all major protem types, especially

soy protein and whey protem products. Understanding the nature of the flavor

compounds associated with these products and finding methods of improving the flavor of

food proteins would be beneficial in extending their use.

For the purpose of this study, attention has been directed to two types of food

proteins; (a) soy proteins and (b) whey proteins.

2.1. Flavor chemistry of soy milk

From a commercial viewpoint, elimination of the “beany” flavor of soybean milk,

flours, concentrates and isolates would expand the market potential of soy products as

food ingredients.

2.1.1 Flavor profile and origin of soy protein products

Kabrener et. al (1971) used a sensory panel to characterize soy flour, concentrates and isolates. They attributed beany, com meal, musty, toasted, bitter, chalky, and astringent odors and flavors as common characteristics of these products. oxidation was proposed (Kabrener, et al., 1974). More recently, MacLoed and Ames

(1988) summarized 334 volatile components comprising 18 aliphatic hydrocarbons, 3

alicyclic hydrocarbons, 14 terpenoids, 31alphatic alcohols, 31 aliphatic aldehydes, 32

aliphatic ketones, 4 alicyclic ketones, 10 aliphatic carboxylic acids, 10 lactones, 13

aliphatic esters, 1 alicyclic ester, 6 aliphatic esters, 7 aliphatic amines, 1 aliphatic nitrile, 5

chlorine-containing compounds, 67 benzenoids, 5 pyrroles, 1 pyridine, 19 pyrazines, 3

thiazoles, and 5 sulfur heterocyclic compounds, identified from soybeans, flours,

concentrates, isolates and textured soy protein. The description of odors associated with

volatile compounds are listed on Table 1. They are formed from lipid oxidation and

degradation, derivation of sugars or amino acids, and thermal decomposition of phenolic

acids, thiamin and carotenoids. It is believed that lipoxygenase-mediated conversion of

polyunsaturated fatty acids to aldehydes and alcohols is a major contributor to off-flavors in soy products (Sessa, 1979).

Lipid oxidation, including autoxidation and enzymatic oxidation, coverts unsaturated fatty acids to hydroperoxides. Lipoxygenase is one of the major enzymes responsible for the deterioration of soybeans (Kinsella and Damodaran, 1980). The enzyme catalyzes the oxidation of polyunsaturated fatty acids and esters containing a Table 1 Volatile compounds associated with various odors in soy products

Odors Associated volatile compounds

Green, grassy, beany pentan-l-ol Hexan-l-ol Heptan-l-ol 3 -Methylbutan-1 -ol Oct-l-en-3-ol Pentanal Hexanal Heptanal Cis-& trans-pent-2-enal Cis-hex-3-enal Trans-hex-2-enal Hept-2-enal Hexa-trans-2, trans-4-dienal Nona-2,4-dienal Nona-trans-2,cis-6-dienal Deca-2,4-dienal Undecan-2-one Butenone Hydrogenated Cis- and trans-non-6-enal Cis-non-7-enal Octa-trans-2, trans-6-dienal Nona-2,6-dienal Deca-2,7-dienal Higher alcohols Lactones Straw-like Nona-2,6-dienal Cooked soybean, repulsive 4-vinylphenol Burned Maillard reaction products Sulfurons Hydrogen sulfide Methanethiol Cooked cabbage/ vegetable Dimethyl sulfide Maillard reaction products Roasted 4-Ethyl-2-mthylthiazole Cis-& trans-3, 5-dimethyl-1,3- dithin Thialdine Alkylprazines Deep-fried Decan-trans-2, trans-4-dienal Table 1 Volatile compounds associated with various odors in soy products (continued)

Odors Associated volatile compounds

Buttery Butanedione Pentane-2, 3-dione Oily/fatty, tallow-like, putty Hexanals Heptanal Octanal Cis- & trans-hept-2-enal Cis- & trans-oct-2-enal Non-2-enal Trans-non-3-enal Trans-dec-2-enal Trans-undec-2-enal Hepta, -trans-2, trans-4-dienal Nona-trans-2, trans-6-dienal Deca-2, 4-dienal Oct-3-en-2-one Musty, moldy, earthy Oct-l-en-3-ol Geosmin Acetophenone Mushroom Oct- l-en-3-ol Oct-l-en-3-one Oxidized, cardboard-like,oily. Higher alka-2,4-dienals paint-like (e.g. C7,C8,C9, CIO)

Fishy Aliphatic animes Cis. Hept-4-enal Deca-trans-2, cis 4,cis-7-trienal

Potato Penta, 2,4-dienal

Cucumber Trans-non-2-enal Cis-nou-3-cnal Trans-non-6-enal Nona-trans-2, trans-6-dienal Celery BovoUde Catty 4-Mecapto-4-methylpentan-2-one Fusel Long-chain alcohol Hay-like, bamyard-like, bran like Isophorone cis-cis 1,4-pentadiene system, forming conjugated hydroperoxides (Eskin and Grossman,

1977). The ratio of 9 to 13 hydroperoxides depends on the enzyme source and reaction

conditions. The secondary products resulting from hydroperoxides breakdown, such as

aldehydes, alcohols and ketones, lead to undesirable taste and odors in soybean (Gardner,

1989).

2.1.2. Analytical technology of soy flavor

Since 1960, an improvement in fractionation by using gas chromatography and

identification by using micro infrared spectrometry with mass spectrometry and NMR has

brought remarkable progress in soy flavor research, which allowed more compounds

contributing to objectionable flavor of soybeans or soy oil to be identified (Chang, 1979).

A systematic characterization of the carbonyl compounds in reverted soybean oil was

developed at Rutgers State University. A total of 71 volatile compounds were identified

from reverted soybean oil (Smouse and Chang, 1969).

In an early study, by identifying the 2,4-dinitrophenylhydrazones of carbonyl

compounds with chromatography and spectral analysis, n-hexanal, acetaldehyde 2-

heptenal, and acetone were reported as major volatile carbonyl compounds steam-distilled

from full-fat soybean flasks (Sessa et al., 1969)

In 1970, Wilkens and Lin (1970) conducted gas chromatographic and mass spectral analyses of soy milk volatiles and classified them as aldehydes, acetals, esters, sulfur compounds, hydrocarbons, aromatic compounds, ketones, alcohols and others. Hexanal was the major volatile compound. Most recently, dynamic headspace sampling/gas chromatography with mass

spectrometry (DHS/GC/MS) was used to analyze soy milk flavor (Yu, 1993 and Ha et al.,

1992). This method only isolates and examines the volatile compounds which exist in the

vapor state above the food material. The method is appreciated for its solventless

extraction and less time-consuming procedure (Lee, 1993). Thirty-eight compounds

including hydrocarbons, alcohols, aldehydes, ketones, ester, benzene, furanoids, sulfur

compounds, and chloride compound, were identified with DHS/GS/MS in soy milk (Yu,

1993).

2.1.3. Control and removal of off-flavors from soy protein

A means of improvement for soy flavor have been discussed in review of MacLeod

and Ames (1988) and Kinsella and Damodaran (1980). More than one hundred methods

have been patented for improvement of the bitterness and beany flavor in soy protein

products.

The most important of these include: lipoxygenase inactivation and removal of undesirable volatile compounds and their precursors.

2.1.3.1. Lipoxygenase inactivation

A rapid inactivation of lipoxygenase is required to eliminate formation of off-flavor of soy protein products. Heating soybeans or a combination with an increased pH have been used commonly to inactivate lipoxygenase activity during soaking or processing.

However, heat inactivation results in protein dénaturation and generates a cooked or toasted flavor, which may reduce the hmctional properties, or result in undesirable flavor

(Kinsella and Damodaran, 1980).

A preparation of soy milk by grinding the unsoaked beans in water at 100°C for 10

minutes inactivated lipoxygenase and yielded a good flavored product (Kinsella and

Damodaran, 1980). Alkaline/acid inactivation and chemical inactivation have been

developed in many combinations. NaOH, NH 4OH, NazCOs, HCl, NH4CO3 , and CaCk

aqueous solution have been used to soak soybeans and inactivate lipoxygenase. In

addition, hydrogen peroxide caused irreversible inactivation of lipoxygenase by attacking

its non-heme iron. Cysteine also significantly reduced the ofi^flavor of soy protein, which

may be due to the interaction with the active site of lipoxygeanse, or with linoleic acid

peroxide, or the generation o f hydrogen peroxide.

The treatment combining heat and soaking in aqueous ethanol (SOg beans/200ml)

inhibited effectively lipoxygenase. However, a resoaking of soybeans previously e?q)osed

to 30% alcohol resulted in regeneration of most of the %oxygenase activity with a lower nitrogen solubility index (NSl) of soy protein (Kinsella and Damodaran, 1980).

2.1.3.2. Removal of undesirable volatile conq)onents and their precursors

These methods have included treatments involving heat, solvents, pH adjustment, enzymes, others methods and various combination of these methods (MacLeod and Ames,

1988).

A heat treatment at atmospheric pressure or elevated pressure and toasting are used commonly for deflavoring of soy protein (McLeod and Ames, 1988). Infi'ared 10

radiation (IR), high frequency electric heating, high frequency irradiation at low

temperature and microwave irradiation may be used to improve soy flavor. Processes,

using alkali treatment, often in addition to acidification, as well as heating will efrectively

remove off-flavors from soy protein.

To avoid denaturalization of protein and generation of nutty flavor caused by

heating treatment, solvent, such as methanol, pentane, hexane, ethanol, or propanal has

been used to extract oil from soybeans to improve soy flavor (MacLeod and Ames, 1988).

Use of a mixture of enzymes incorporating carbohydrates, proteases, lipases, oxidase and

pectinases was proposed to break the bond between volatile compounds and soy protein

and oxidize aldehydes. However, the addition of protease may result in some loss of

protein functionality.

How and Morr (1982) found that the use of activated charcoal and ion-exchange

resins removed significant amount of phenolic conq)ounds from soy isolates, but bitter and

astringent flavor remained comparable with those of untreated products. The flavor

improvement may be due to reduction of conq)ounds other than phenoUcs. Chemical

methods including the use o f sulfites to react with carbonyl compounds, cheating agents

such as polyphosphate or citric acid to conq)lex trace metals have been employed to

improve flavor of soy products (MacLeod and Ames, 1988).

2.2 Flavor chemistry of whey protein concentrate .

Whey proteins, from both acid and sweet whey sources, have been used increasingly as food ingredients during the past 15 years. Flavors associated with whey 11

carry through into concentrated whey protein products and are considered undesirable by

the food industiy (Morr and Ha, 1991).

2.2.1. General chemistry of dairy flavor

A great number of studies or reviews have been reported for the flavor analysis of

milk (Adda, 1986; Badings and Neeter, 1980; Esldn, 1986; Shipe, 1980), heated milk

(Calvo and Hoz, 1992; Shibamoto et al., 1988) and dairy products (Badings and Neeter,

1980; Manning and Nuisten, 1985; Badings ,1991; Forss, 1979; Hammond; 1989). Since

whey protein concentrate is a by-product of cheese, which is made from milk, an

understanding of milk flavor will help to understand WPG flavor. A summary done by

Shipe et al. (1978) illustrated nomenclature, standard, and bibliography of off-flavors of

milk based on the research done from 1950 to 1976. According to the causes of dairy

flavors, they have been stated as heated flavor, lipolyzed flavor, microbial flavor, light

induced flavor, oxidized flavor, transmitted flavor and miscellaneous flavors, which included those flavors that either cannot be attributed to a specific cause or specifically defined in sensory terms. Table 2 summarizes the sensory description, causing factors, and compounds contributing to these flavors. These flavors varies with cows, surroundings, feed, the means of processing, the use of chemical, enzymatic and microbial manipulation, and the storage (Shipe, 1980; Manning and Nursten, 1985). Table 2 Flavor profile and responsible compounds of milk off-flavor

Terms used Flavor profile Causes and predominating factors Compounds involved

Heated Flavors sulfurous, heated abnormally high temperature processing lactones, maltol caramelized, scorched methyl ketones vanillin, benzaldehyde Light-induced flavor burnt, oxidized wavelength and intensity of sunli^t, exposure time, carbonyl compounds translucence of the container, methional, sulfides, levels of ascorbic acid and riboflavin mercaptanSjdisulfides Lipolyzed flavors goaty, soapy ,butyric excessive agitation, homogenization, free fatty acids bitter separation or clarification, and heat shock C4,C6,C8,C10,C12 Microbial Flavors acid, malty, and fimity Streptococcus lactis. acetic acid stale, bamy, unclean, Pseudomonas fragi, Bacillus propionic acid foreign, rancid aldehyde, alcohol Oxidized flavors oxidized, cardboard,cappy a reaction between oxygen and lipids, hydrocarbons, acids, metallic, tallowy, oily, accelerated by copper ions, pro-antioxidants, alcohols, aldehydes, and fishy and light, and inhibited by antioxidants. and ketones. Transmitted flavors feed, weed, cowy, and transfered via cow’s feed, surroundings in milk, bamy flavor via the respiratory and/or digestive system and blood system Miscellaneous flavors absorbed, astringent, absorbed from the environment, amino acids bitter, chalky, chemical, associated with high process temperature, iodine compounds flat, and foreign flavors, caused by proteolysis, and improper procedures chlorine compounds lack freshness contamination from cleaners, and sanitizers.

From Shipe et al., 1987

N> 13

2.2.2. Origins of WPC flavor

Sweetness, saltiness, bitterness, brothiness, astringency, non-volatile acidity,

volatile acid, and diacetyl were used to describe whey flavors (McGugan et al., 1979). A

review by Morr and Ha (1991) addressed that Mallard reaction, lipid oxidation and

riboflavin decomposition predominately contribute off-flavor of WPC. However, the

chemical mechanism of Maillard reaction and lipid oxidation occurring in WPC is still

waiting to be investigated.

2.2.2.1 Lipolyzed or rancid flavor in WPC

Lipolyzed flavor is used to describe the flavor produced by lipase catalyzed

hydrolysis of milk fat triglycerides. Al-Shabibi et al. (1964) reported that of 16 fatty acids

added milk, only hexanoic, octanoic, decanoic, and dodecanoic acids were associated with

rancidity of milk. Later, an observation by Scanlan et al. (1965) indicated that even

numbered fatty acids from butyric acid to dodecanoic acid contributed equally to the rancid flavor, whereas the fatty acids above dodecanoic acid contributed little. It is believed that no single fatty acids but a mixture of free fatty acids predominately contribute lipolyzed flavor in milk.

The content of free fatty acids in fresh milk is lower than thresholds, therefore the significance of their contribution to normal milk is not clear. However, the excessive concentration of free fatty acids contributes lipolyzed flavor in milk and other bland flavored dairy products. On the other hand, a fairly large amount of free short chain fatty acids, such as C4, C6 and C8 contributes characteristic flavor of cheese (Jeon, 1994). 14

Table 3 describe the characteristic flavor of free fatty acid from C2 to C12 (Shibamoto et

al., 1980).

The thresholds of free fatty acid in various media are summarized in Table 4

(Shibamoto et al, 1980)

Thresholds of fetty acids vary with the type of media and pH of media. The conclusion

made by Shipe indicated that fatty acids chain length decreases the threshold values in water but increases in oil. In other words, the flavor threshold decreases as the afSnity for the dispensing media decreases (Shipe, 1980). But an observation conducted by Brennand et. al(1989) reported that thresholds for n-chain fatty acids generally increased from butanoic (C4) to octanoic (CIO) acid, which may been attributed at least in part to both increasing vapor pressure of fatty acids as well as decreasing water solubilities. However, the thresholds decreased again for the C9 and CIO fatty acids. Fatty acids with larger molecular weight than C12 exhibit high thresholds, presumably because vapor pressures are relatively high. 15

Table 3 Flavor profiles of free fatty acids in water and emulsion

Fatty acids Flavor description In water In Emulsion

Acetic acid Sour not-oily Butyric acid buttery smooth, mild Hexanoic acid chemical heavy Octanoic acid dry, herb fishy, oily, thick Decanoic acid milky, creamy milky Dodecanoic acid waxy, slightly creamy buttery,oily

From Shibamoto, 1980 16

Table 4 Flavor thresholds of volatile fatty acids.

Fatty Acids Thresholds (ppm) Water Oil milk Literature

Acetic (2:0) 8.0 0.079 B 54.0 - C Butyric (4:0) 6.8 0.6 25.000 A;C 1.5 0.400 B Hexanoic (6:0) 5.4 2.5 14.000 A;C 0.2 0.630 B Octanoic (8:0) 5.8 350 - A 0.8 0.126 B Heptanoic (7:0) 3.0 D 0.28 (pH 2) E Decanoic (10:0) 3.5 200 7.000 A;C 0.7 0.063 B 2.2 (pH 3.2) E 8.7 (pH 4.5) 11.3 (pH 6.0) Nonanoic (11:0) 3.0 D 2.4 (pH 2.0) E Dodecanoic (12:0) 700 8.500 A 0.5 0.010 B Tetradecanoic (14:0) 10.0 D 5,000 C Hexadecanoic (16:0) 10.0 D 10,000 C

A. Patton, 1964 B. Shibamoto, 1980 C. Kinsella, 1969 D. Cherkinski, 1978 E. Brennand et al., 1989 17

At normal pH (6.6), milk would exhibit some buffer action, tending to sequester

the acids and raise their thresholds above those with water (Patton, 1964). This did not

agree with the results obtained by Shibamoto (1980) that thresholds of fatty acids in milk

were generally lower than those in water. As the pH of the system is lowered, the

threshold value for the fatty acid decreases (Brennand et al., 1989). To insure that the

fatty acids were protonated and thus would exhibit maximum volatility, a pH 2.0 solution

was adapted to determine thresholds of fatty acids occurring in milk fat.

2.2.2.1.1 Origins of free fatty acids in dairy products

Lipolysis is catalyzed by indigenous milk enzymes or a bacterial lipase. P-type

esterases including glycerol tricarboxyl esterase, aliphatic esterases, diesterases, and

lipases (EC 3.1.1.3.), are the predominate enzymes which responsible for hpolysis m

bovine milk. Lipoprotein lipase (EC 3.1.1.3.4) plays an important role in moving lipids from blood to the mammarygland and its presence in milk may be due to leakage from the tissue (Esldn, 1990). Lipase and esterase produced by Pesudomonas fragi also are involved in dairy lipolysis. However, the presence of fat globule membrane inhibits hpolysis by protecting the Upoysis subtract -triglycerides from hpase. Mechanical agitation, foaming , and repeated abrupt temperature changes results in disruption of fat- glohule membrane and enhance hpolysis - so caUed induced hpolysis. On the other hand, milk may become hpolyzed even without any physical treatment - so caUed spontaneous hpolysis. The transfer of cofactor protein or of hpoproteins containing this protein from blood to milk was proposed to activate hpase (Olivercrona, 1980). 18

2.2.2.1.2. Control of lipolyzed flavor

Fleming (1980) discussed the effect of temperature on lipolysis, It is evident that a

rapid milk cooling retards the development of FFA and rewarming to temperature above

20-25°C followed by re-cooling of the milk may enhance lipolytic activity. In addition, the

effect of mechanical agitation on lipolysis depends not only on the previous temperature

histoiy of milk, but also on the temperature during agitation, the nature of the mechanical

treatment, and the characteristics of the milk, some of which are not yet fully understood.

Lipases are inhibited by the presence of proteins and chemicals. Lipase action is inhibited by some of other protein components of plasma lipoproteins, the major protein from egg yolk lipoproteins, hemoglobin and myoglobin, some albumin preparations, and a glycoprotein isolated from arota (Fleming, 1980). The inhibition caused by these other proteins can often be relieved in a competitive manner by lipase cofactors (Olivercrone,

1980). It was reported that a number of chemicals and proteins, such as aureomycin, penicillin, streptomycin, terramycin, a, (3, and v casein, as well as P- lactoglobulin have inhibitory effect on lipase by forming complexes between them and lipase (Shipe, 1980).

2.2.2.2 Origins of oxidized flavor in milk and WPC

In fluid milk, the oxidized flavor arises primarily from the oxidation of the fatty acids in the phospholipids of the fat globule membrane. As shown in Figure 1 (Hamilton,

1989), the mechanism of lipid oxidation is comprised of three steps: initiation, propagation, and termination. The formation of free lipid radicals (R’) is initiated by a catalyst such as copper ions. The propagation step results in formation of peroxyl radicals 19

(ROO’) and lipid radicals (R’). The oxidation reaction may be terminated when two radicals are combined to quench free radicals. Hydroperoxides formed in the initial step are flavorless and unstable and break down to produce flavor compounds, such as alcohols, aldehydes, and ketones.

Some of the aldehydes that can be formed from the different fatty acids in milk and dairy products are illustrated in Table 5 (Shipe, 1980). The secondary reaction such as isomerization, e.g. non-3 enal to non-2 enal., potentially generate flavorful products.

Oxidation of fat depends on variability of fresh raw milk, the concentration of ascorbic acid, the storage temperature, and milk processing. 20

Initiation RH------>R’ +H’

Propagation R'+Oz------>ROz’

ROz’+RH-—- - > ROzH+R’

Termination R’+ R ’------> R-R

ROz’+ R’ > ROzR

Where RH=polyimsaturated fatty acid,

R’, RO’, ROz’= lipid free radicals.

Figure 1 Mechanism of lipid oxidation (From Hamilton, 1989) 21

Table 5 Possible origin to some aldehydes obtained from the oxidation of four unsaturated fatty acids.

Fatty acid Hydroxide position at Aldehyde obtained

Oleic C ll Octanal C8 2-undecenal C9 2-decenal CIO Nonanal Linoleic C13 Hexanal C9 2,4-decadienal Cll 2-octenal Linolenic C16 propanal C14 2-pentenal C12 2,4-heptadienal C13 3-hexanal C ll 2,5-octadienal C9 2,4,7-decatrienal Arachidonoic CIS hexanal C13 2-octenal C12 3-nonenal C ll 2,4-decadienal CIO 2,5-undecadienal C7 2,5, 8-tridecatrienal

From Shipe, 1980 22

2.2.2.3. Maillard reaction in milk and WPC

The Maillard reaction, nonenzymatic browning, generally requires the presence of

an amino-bearing compound, usually a protein, a reducing sugar, and some water

(Whistler and Daniel, 1985). Lactose and casein are the two principal reactants in the

browning of milk products. Although the reaction could occur at low temperature, even

at refrigerated temperature, the Maillard reaction is usually associated with those areas of

the food that have been dehydrated by heat (Whitfield, 1992).

The principle reaction steps involved in the Maillard reaction were described by

Hodge in 1953 (Whitfield, 1992). The initial products of Maillard reaction, Amadori

products and Heyns products, do not contribute to flavor. However, a dehydration and

deamination by thermal processing results in rearrangement and degradation of these

compounds and generates flavor compounds, such as furfiirals and furan derivatives. The interaction of Maillard products and Strecker degradation products, amino ketones, leads the production of some heterocyclic compounds, such as pyrazines, oxazoles, thiophenes, and heterocyclic compounds with more than one sulfur atoms. The Strecker degradation of methionine is another source of sulfur-containing intermediates.

Whitfield (1992) reviewed volatile compounds formed from interactions between

(1) Maillard products and carbonyl compounds, (2) amino acids and carbonyl compounds,

(3) amino acids and derivatives of fatty acids, and (4) Maillard reaction products, triglycerides and phospholipids, which indicated the presence of lipid may accelerate the 23

Maillard reaction by providing free radicals and contribute to formation of some

alkylpyrazines, thiazoles, furans and pyrroles.

Destruction of essential amino acids, particularly lysine and probably histidine, has

been shown to occur during the storage and browning of nonfat dry milk of high (7.6%)

moisture content. Obviously, the destructiveness of amino acids occurs in Maillard

reaction, before or after the development of colored pigments, as well as the Strecker

degradation results in a loss of nutritive value of dairy products (Whistler and Daniel,

1985) The Maillard reaction occurred during storage of WPC also results in the decrease

functionality of WPC, such as low solubility and a lower pH value (Kehrberg and Johnson,

1975).

2.2.2.3.1 Control of Maillard reaction

An observation for storage stability of dried sweet cheese whey indicated that the degree of browning of stored WPC generally increased with increasing storage temperature, moisture content, and length of storage time (Kehrberg and Johnson, 1975).

An elevated pH values favored the rate of brown pigment formation. Browning is controlled in dairy foods by limiting heat treatment, moisture content, and time and temperature of storage. In milk products, active sulfhydryl groups serve as natural inhibitor in retarding heat-induced browning. Sodium bisulfite, sulfur dioxide, and formaldehyde also retard browning in milk system and amino acids-sugar system.

Removal of lactose and addition of antioxidants also inhibit the non-enzymatic browning of non-fat dry milk (Morr and Ha, 1991). 24

2.2.2A. Other reactions responsible for oflF-flavor in WPC

Riboflavin and ascorbic acid are considered as the primary and secondary factors

responsible for light-induced “oxidized “ flavor in fluid milk (Morr and Ha, 1991). Light

induced flavor associated with a degradation of riboflavin and cystine caused by

photoxidation was observed in skim milk, whey, and serum protein Abactions but not in

casein and fi'ee amino acid j&actions, which indicates that serum proteins are primary

source of light induced flavors, with riboflavin acting as a sensitizer (Sattar and deMan,

1975). When riboflavin and methionine were exposed to light, methional was formed

(Esldn, 1990). The formation of free radicals in dry milk protein by mechanical energy was associated with gluey-flavor of protein, which indicated the possible involvement of free radicals in the formation of glueyness in casein (Hansen et al., 1970).

Heat treatment also contribute to formation of ofiT-flavors compounds in milk.

Besides products of oxidation and Maillard reaction, benzaldehyde, diacetyl and acetophenone were found in heated milk (Scanlan et al., 1968). These compounds were also found in WPC (Lee, 1993 and Mills, 1993).

2.2.2.S Analysis of whey protein concentrate flavor

A large number of studies have been done on the analysis of milk, heated milk and dry milk, which included solvent extraction/steam stillation (Ferretti and Flanagan, 1972), a headspace concentration system (Wellnitz-Ruen et al., 1982; Christensen and

Reineccius, 1992; Badings and Jong; 1984), and solid phase extraction (Coulibaly and

Jeon, 1992). Super critical extraction was used for the analysis of cheese flavor (Roudot 25

et al., 1993). Sequentially, flavor compounds were separated and identified with gas-

liquid chromatography/ mass spectrometry or gas chromatography/mass spectrometry

(Ferretti and Flanagan; 1972; Wellnitz-Ruen, 1982). However, only a few of studies have

been done on the flavor of whey protein concentrate (Morr and Ha, 1991).

In 1971, spray-dried whey protein extract was obtained through continuous

distillation and extraction with dichloromethane by Ferretti and Flanagan (1972).

Products identified couq)rised seven alkylpyrazines, three furans, two pyrroles, a-methyl-r-

butyrolactone, isobutyramide, N-methyl-2-pyrrolidinone, 3-hydroxy-2-butanone,

benzaldehyde, phenol, benzyl alcohol, maltol, dimethylsulfone, propionic, butyric and

benzoic acid. Besides dimethylsulfone, which accounted for more than 50% of the steam

distillate, propionic acid, butyric acid and alkylpyrazines were the major components.

An observation of flavor compounds of whey powder subjected to accelerated

browning was done by Ferretti and Flanagan (1971a) with dichloromethane/ vacuum

distillation extraction and GLC/MS. Fifty-five compounds including pyrrols, furans, fatty

acids, and lactones were found. Ferretti and Flangagan (1971b) also identified flavor

compounds of commercial spray-dried whey. Alkylpyrazines, furans, pyrroles, lactones, benzaldehyde, phenol, benzyl alcohol, maltol, dimethylsulfone, and fatty acids were found

and identified. A vacuum distillation and dietyl ether was used to isolate volatile compounds from WPC by Mills (1993). Conq)ounds identified include saturated and unsaturated aldehyde, methyl ketones, alcohols, alkyl pyrazines and saturated fatty acids. 26

Recently, dynamic headspace sampling with GC/MS was used to analyze flavor of

WPC by Laye (1994) and Lee (1993). Acetonitrile/water extraction provided a large range of volatile compounds from WPC, which included free fatty acids and other compounds (Li, 1995). Flavor compounds of WPC identified in these references are list in

Table 6. 27

Table 6 Flavor compounds identified from WPC.

Alchols Furans Ethanol 2-propyl furan 2-Propanol 2-pentyl furan 1-Pentanol 2-furanamethanol l-Octen-3-ol 2-acetyl furan 1-Hexanol furfury alcohol Ketones 2-propionylfbran 2-propanone 2-pentanone Chlorinated hydrocarbons 2.3-butanedione trichlorofluoro methane 2-octanone 1,1,1 ,-trichloro ethane 2-decanone dichloro methane 3,5-octadien-s-one tiichloro methane acetophenone bromodichloro methane heptan-2-one tetrachloroethene isocyano-methane Aldehydes hexanal Free fatty acids propanal, 2-methyl- acetic acid heptanal hexanoic acid octanal octanoic acid nonanal benzoic acid 2.4-heptadienal butyric acid 2-nonenal propionic acid 3-methyl butanal pentanal Pyrazines 2 methyl pyrazine Esters 2.5 dimethyl pyrazine acetic acid methyl ester 2.6 dimethyl pyrazine acetic acid ethyl ester 2,3 dimethyl pyrazine acetic acid pentyl ester 2, 3,5 thrimethyl pyrazine ethyl acetate tetramethyl pyrazine alkylpyrazine Aliphatic hydrocarbons 2-methyl-5 (6)- ethyl pyrazine pentane 2-methyl-5 (6)-vinyl pyrazine heptane octane Pyrroles decane 2-formypyrrole undecane n-methyl-2-formypyrrole n-methyl-2-pyrrolidinone 28

TABLE 6 Flavor coirpounds identified fi'om WPC (continued)

Aromatic hydrocarbons Lactones benzene a-methyl-r-butyrolactone benzene, dichloro methyl benzene Amide ethyl benzene isobutramide 1,4-dimethyl benzene 1,3-dimethyl benzene Others 1,2-dimethyl benzene diacetyl limonene C3 alkyl benzene acetophenone dichloro benzene benzaldehyde toluene benzyl alcohol Sulfur containing compounds dimethyl disulfide dimethyl sulfone maltol

From Laye 1994; Lee, 1993; Mills, 1993; Mills,1986; Ferretti and Flangan, 1971 29

2.2.6. Evaluation for flavor quality of milk and dairy products in the industry

Sensory evaluation and acid degree value are commonly used to evaluate flavor in the dairy industry.

American Dairy Science Association (ADSA) developed scorecards for sensory evaluation of a number of dairy products, including milk. A scoring guideline of milk flavor defects was given in Table 7 (Shipe, 1980). The total score is used to rate the overall flavor quality on a 10 point scale. A score of 10 is given to a sample that has a

“typical fresh” taste and is free of undesirable flavors. Samples lacking in flavor or having off-flavors are scored lower. It is recommended that the classification of undesirable flavors based on their causes will be useful to locate the origins of off-flavors for quality control.

Acid degree value (ADV) have been used to evaluate the hydrolytic rancidity of milk. However, the extraction for ADV analysis is more effective on long chain fatty acids than short chain fatty acids. Since long chain fatty acids are less responsible for the rancidity of milk and dairy products, ADV is not an accurate indicator of milk rancidity

(Duncan and Christen, 1990). 30

Table 7 ADSA milk flavor and scoring guide

Flavor intensity Flavor Slight Definite Pronounced

Astringent 8 7 5 Bamy 5 3 1 Bitter 5 3 1 Cooked 9 8 6 Cowy 6 4 1 Feed 9 8 5 Fermented/fruit 5 3 1 Flat 9 8 7 Foreign 5 3 1 Garlic/onion 5 3 1 High acid 3 1 Lacks freshness 8 7 6 Malty 5 3 1 Metallic 5 3 1 Oxidized 6 4 1 Rancid 4 1 *,.2L Salty 8 6 4 Unclean 3 1 ...S. a unsalable. From Shipe, 1980 31

2.2.7. Solid phase microextraction (SPME)

The extraction of organic compounds from a sample matrix usually is composed of

purge and trap or headspace methods for concentrating volatiles, and liquid-liquid

extraction, solid phase extraction, or supercritical fluid extraction for semivolatiles and

nonvolatiles (Supleco, 1995). These methods have various disadvantages, including cost

and excessive preparation time.

Solid phase microextraction (SPME), a sovlentless extraction process, was

introduced recently. It can be used to extract volatile and nonvolatile compounds in both

liquid and gaseous samples by exposing the fiber to the matrix (Supleco, 1995). The

analytes adsorbed will be released thermally and analyzed, when SPME fiber is inserted into the gas chromatograph injector port. A SPME unit consists of a length silica fiber coated with a high temperature phase, such as polyacrylate, or polydimethylsiloxane. The range of extraction depends on the polarity of SPME fiber. It is recommended that polydimethylsiloxane is suitable for the extraction of volatile and mid- to nonpolar semivolatiles and polyacrylate coating fiber is for polar semivolatile compounds.

Polydimethylsiloxane has been used for extraction of volatile flavor components from saffron, dill seed, and separmint, with limited preparation (Wooley and Mani, 1994).

Polyacrylate fiber provided a rapid and easy adsorption of polar semivolatiles, such as phenols, fi'om water (Shirely, 1995). Yang and Peppard (1994) used headspace sampling

SPEM and immersion SPEM to monitor 25 common flavor componenets in spiked water, ground coffee, fhiit juice beverage, and butter-flavored vegetable oil. The sensitivity of 32

immersion SPEM was comparable, or liigher than that of conventional solvent extraction

(dichloromethane) for most esters (e.g., ethyl isolvalerate,cis-methyl cinnamate, trans­

methyl cinnamate), terpenoids (e.g., linalool, a-terpineol,(3-terpinol) and y-decalactone in

fruit juice beverage. The weak affinity of the 100pm polydimethylsiloxane phase for fatty

acids in the beverage was considered as an advantage, because it reduced the potential for

interference with the flavor compounds.

2.2.8. Variation of volatile compounds obtained with different types of methods

The variations of two dynamic headspace sampling systems were compared by

Imhof and Bosset (1991). A chemical mix containing 11 compounds was used to evlauted

the reproducibility of Takmar LSC 2000 and Rektorik MWS-1 systems. Takmar LSC

2000 system generally achieved better repeatability with coefficients of variation below

16%, whereas coefficients of vaiiation obtained from the Rektorik MWS-1 system were as

high as 50% for several compounds. Ha et al. (1992) reported that the realtive standard

deviation (%RSD) of hexanal, 2-hexenal, 1-heptanol, and l-octen-3-ol obtained from

soaked soy beans with DHS/GC/MS were 17.0, 30.4, 33.1, and 24.5%, respectively. The

coefficients of variation of five major headspace volatile compounds from whey protein

concentrate, including dimethyl disulfide, hexanal, dimethyl trisulfide, nonanal, and benzothiazole were 4.8%, 4.3%, 16.9%, 6.2%, and 14.3%, respectively.

The variations of SPME for 17 phenols from water were reported by Shirey

(1995). The range was from 4.6% (2-chlorophenol) to 50.3% (2,4-Dinitrophenol) at the concentration of 5ppb to 200ppb. The variations of flavor compounds including ethyl 33

acetate, ethyl butyrate, limonene, ethyl hexanoate, cis-3-hexenyl acetate, cis-3-hexanol,

benzaldehyde, linalool, diethyl succinate, neral, 2-methylbutyric acid, y-hexalactone, 1-

carvone, geranial, anethole, hexanoic acid, phenylethyl alcohol, P-ionone, cinnamic

aldehyde, triacetin, y-decalactone, heliotropin, triethyl citrate, ethylvanillin, and vanillin

were reported and ranged from 1.4% to 17.8%.

2.3. Interaction of flavors and proteins

Some volatile compounds are known to interact with proteins, with the strength of

the bonds between volatiles and proteins varying as a function of both the protein and the

volatile compound. The effects of structure of volatiles and proteins, temperature, pH,

and components added on flavor-protein interaction have been investigated (Gremil, 1974;

Mills and Solms, 1984; O’neill and Kinsella, 1987; Franzen and Kinsella, 1974).

Understanding the binding of volatile compounds to proteins is important to the

development of any method to remove volatile compounds from food proteins.

2.3.1 Effect of structure of volatile compounds

Gremli (1974) investigated the interaction of compounds with soy protein by

adding various alcohols, aldehydes, and ketons into aqueous soy protein solution. The

result indicated that alcohols did not interact with soy protein, while aldehydes strongly

react with soy protein. A certain of percentage of aldehydes was still retained by the

protein in a high vacuum transfer system, which means that the interaction of aldehyde with soy protein appears to have reversible and irreversible features. None of the ketones 34

were retained during the high vacuum transfer, therefore, the interaction between ketones

and soy protein must be reversible.

An investigation of interaction of flavor confounds with whey proteins was

conducted by Mills and Solms (1984). The binding of aldehydes and methyl ketones

increased with increase in molecular chain length.

2.3.2 Eflfect of protein type

The interaction between 2-nonanone and soy protein was observed. On an

equivalent weight basis soy protein, P-conglycinin and glycinin had approximately 5,2 and

3 primary binding sites per 100,000 daltons and affinity constants (K) of 570,3050, and

540 m‘*, respectively (O’neill and Kinsella, 1987). The highest affinity constant of (3-

conglycinin showed that P-conglycinin was responsible for most of flavor binding by soy protein in aqueous solution. P-Conglycinin also had relatively strong binding with alphabetic carbonyls compared to the affinity constant of 2440 and 820 m ' for P- lactoglobuhn and bovine serum albumin.

The conq)osition o f protein may also affect the interaction. Casein has less cystine and lysine but more aromatic amino acids, methionine, arginine, and histidine than whey protein. Among the twenty common amino acids studied, lysine adsorbed the greatest amount of carbonyl compounds (Kim and Min, 1989). Therefore, casein may have relatively weak binding with flavor conq)ounds because of its low lysine content. Whey protein concentrate consist of a variety of proteins: P-lactoglobulin, a-lactalbumin. 35

immunoglulins, and bovine serum albumin, which are heat sensitive and more susceptible

to dénaturation during processing than casein (Hasen and Heinis, 1992). Compared to

casein, whey protein had greater binding with d-limonene and benzaldehyde, which may be

related to dénaturation of the whey protein during processing.

Franzen and Kinsella (1974) examined the binding of volatile compounds with

various proteins including lactabumin, bovine serum albumin, leaf protein concentrate,

single cell protein (SCP), textured vegetable protein (TVP), soy protein isolate, and soy

protein concentrate. Although the headspace volatiles were significantly decreased in an

aqueous protein system, the protein showed no consistent or predictable effects with

regard to the magnitude of flavor binding, and no one flavor compound was preferentially

bound by all of the proteins.

2.3.3 Effect of temperature

Heating causes some milk proteins to denature. As the amount of heat increased

fi-om pasteurization to UHT temperatures, the milk proteins unfold to a greater extent,

which creates more reactive sites on the protein to complex with flavor compounds, such

as vanilla (Hansen, 1989).

2.3.4. Effect of pH value

pH value also affected the binding of heptanal and nonanone on whey protein. At pH 6.89, the binding of heptanal was greater than the that of nonanone, whereas at pH

4.66, the reverse occurred. Heptanal had greater binding on whey protein at pH 6.89 than that at pH 4.66, but reduced binding occurred with nonanone (Mills and Solms, 1984). 36

2.3.5. Effect of ion concentration

It was suggested that the presence of added calcium ions, during the co­

precipitation of casein and whey protein by heat, improved the flavor stability of casein by

reducing the browning reaction (Buchanan, et al., 1965). Higher calcium levels developed

less color on heating, but there was little evidence of any effect of calcium content on the

flavor stability (Ramshaw and Dunstone, 1969).

2.3.6. Effect of other components

Salt and lactose content had little effect on the binding of heptanal on whey

protein. However, the binding of heptanal and nonanone decreases as the residual lipid

content was reduced (Mills and Solms, 1984). The addition of water decreased headspace

volatiles by decreasing the sur&ce area and solubility the flavor with the protein (Franzen

and Kisella, 1974).

2.4. Interaction between free fatty acid and protein

Since ffee fatty acids are one of major contributors of off-flavor in dairy products

and were evaluated for the flavor quality of WPC in this study. Investigation of the interaction of ffee fftty acids and proteins is reviewed.

2.4.1. Fatty acid structure and binding to protein

Spector et al. (1969a and 1969b) investigated the binding between long chain fatty acids and various proteins with radioactive labeled on C14 of fatty acids. Plamitate and pahnitoleate had stronger binding with bovine serum albumin (ESA) than oleate, linoleate, stearate, myristate, or laurate. Similarly, palmitate still exhibited tighter bonding with 37

beta-lactoglobulin than stearate, oleate, and laurate. Apparently, changes in the length or

de^ee of unsaturation doesn’t afTect the strength of association with BSA. But

substitution of carboxyl group of fatty acids (such as methyl palmitate) resulted in a

decrease of binding of palmitate to BSA.

2.4.2.EfTect of protein structure and binding to fatty acids

Plamitate was bound more tightly by bovine serum albumin (BSA) than human and

rat serum albumins (HSA and RSA). However, oleate and laurate were bound more

tightly to HSA than to BSA. A number of proteins including ribonuclease, fibrinogen, a-

amylase, trypsin, a-chymotrypsin, lysozyme, streptococcal protease, and subtilisin only

bound less than 2% of the amount that was bound by the same quantity (weight) of BSA.

P-Lactoglobuhn took up about 20% as much palmitate as BSA (Spector et al., 1969a).

Another study showed that more 14C-l-plamitate fi'om the media containing beta-

lactoglobulin (BLG) was taken by Ehrlich ascites tumor cells in vitro than fiom those

containing bovine albumin, which indicated plamitate was bound less firmly to BLG than

BSA (Spector et al., 1969b).

2.4.3. Effect of pH, temperature, ion strength and other factors

pH, temperature, and ion strength also affect the binding of fatty acids with B LG ,

HSA and BSA. The afSnity was increased when pH was raised fiom 5.6 to 8.7 and decreased as the ionic strength of medium was raised. Palmitate binding was decreased in the presence of6 M urea and when protein either was exposed to elevated tenq)erature or 38

was acetylated prior to incubation (Spector, et al., 1969b). Pamitate was bound more

tightly by both human and bovine albumin at 23°C than at 37°C.

2.5. Chemistry and application of cyclodextrins

Cyclodextrins have been shown a promising effect on the removal of odor,

protection of susceptible materials and other applications by complexing with guest

molecules. A general introduction of their structures and uses is given.

2.5.1. Structure and properties of cyclodextrins

Cyclodextrins are a group of cyclic nonreducing oligosaccharides, built with a - 1,4

links from six, seven, or eight glucopyranose ring, respectively known as alpha, beta, and

gamma cyclodextrins. Cyclodextrins are produced by the action of cyclodextrin

transglycosidase (CTG) on prehydrolyzed starch (Szejtli, 1988).

All the glucosyl-o-bridges point into the center of the molecule, producing a donut

shape. There are ffee hydroxyl groups in each successive glucose unit on the rim of there

donut shape molecules- the C6 primary hydroxyls on the narrower side and the C2 and C3

secondary hydroxyls occupying the wider side (Figure 2). These two hydrophilic planes confer hydrophilicity upon the molecules. Therefore, cyclodextrins are water soluble. On the other hand, in the cavity, there are three bands, one on the top of the other; two bands of C-H groups and, in between, a band of glycosidic oxygens, forming a cooperative array of binding sites and creating a relatively nonpolar lipophilic microenvironment. The characteristic of intramolecular hydrophobicity and extramolecular hydrophilicity allows 39 the entrapping of a guest compounds and provides water-solubility of the inclusion complex (Pagington, 1987; American Maize Products Co., 1993).

2.5.2. Application of cyclodextrins to foods

A number of reviews regarding applications of cyclodextrin in foods, cosmetics, toiletries, and pharmaceuticals are available (Allegre, 1994; Pagington, 1985; Nagatomo,

1985; and Szejtli, 1984). Hashimoto (1984) and Nagai (1981) reported the research and application of cyclodextrins done in Japan. Industrial applications of cyclodextrin in foods are listed on Table 8 , 40

CHjOH

OH HO CH,OH

CH:OH

Apolar cavity Secondary hydroxyl rim

Primary hydroxyl rim

Figure 2 Chemical formuJar and molecular shape of P-cycldoextrin. (From Pagington, 1986). Table 8 The application of cyclodextrins in foods and analytical chemistry.

Functions Examples (Reference)

In foods Protection of active ingredients against oxidation, light Protect flavorings added in snack food (A) induced reaction, heat, and loss by volatility or sublimation Protect flavorings during thermal processing (A) Protect spices in powder or paste form (B)

Elimination (or reduction) of undesired tastes and microbiological Reduce bitter taste of orange juice (C) contaiminations, tiber/other undesired components, Reduce cholesterol content of egg yolk (D) hygroscopicity Decaffeination (E)

Improvement of solubility of insoluble materials Enhance solubility of vitamins A, D, E, and K (F) Improve solubility of colors in soft drink (A)

Technological advantages Powder tea, yeast drink, and natural fruits (B) Improve émulsification of whipping creams (B)

In analytical chemistry As a mobile phase in thin-layer chromatography, HPLC and GC

A. Pagington, 1987 B. Hashimoto, 1988 C. Shaw and Wilson, 1985 D. Smith et al. 1995 E. Yu, 1980 F. Pitha, 1981 42 2 .5.3.Insoluble cyclodextrins

The name cyclodextrin derivatives donates compounds which contain only one or

two cyclodextrin rings: those containing three or more units are termed cyclodextrin polymers, as their molecular weights exceed 3000 (Szejtli, 1982).

The solubility of cyclodextrin derivatives depends on the groups substituting hydroxyl groups. Méthylation and alkylation will increase the solubility of cyclodextrins, which could be use to carry out a specific application in foods and pharmaceuticals

(Szejtli, 1988). Acylation and polymerization will impart insolubility to P-cyclodextrins.

These materials may be easily used as process aids to remove specific components from a mixture of materials with centrifugation or filtration. The characteristics of insoluble cyclodextrins used in this study are listed in Table 9 (American Maize Products Co.,

1995).

Cyclodextrin polymers are produced by the reaction of cyclodextrin and some hi or polyfunctional compounds, such as aldehydes, ketones, allyl halides, isocyanates, epoxides

(e.g. epichlorohydrin, ethyleneglycol diepoxypropyl ether) etc. (Szejtli, 1988). These compounds can couple with the hydroxyl groups of cyclodextrin. Up to a certain molecular weight the polymers are water soluble; crosslinking and the increase of molecular weight lead to the development of a swelling, yet insoluble structure. 43

Table 9 Characteristics of insoluble cycldoextrins used in this study.

Moisture Residual Empirical pH Average Ash (%) ffee beta- formula (Filtrated - Degree of CD 1% solid substitution solution) (’HNMR) Acetylated 0 .0 % <50ppm C84H 112 O56 6.39 20.5 <0 .1 beta-CD % Acetylated 0 .6 % <50ppm C72H96O48 6 .6 15.8 <0 .1 Alpha-CD % Beta 90% N.D. (C42H70O35 .C3 N.D. N.D. N.D. Epichloroh HsO) y ydrin Co­ polymer N.D. means not reported (From American Maize Products Co., 1995) 44

2.5.4. Removal of naringm and limonin from citrus fruits

The use of cyclodedxtrin to remove bitter compounds is an excellent example for establishing the procedure to remove undesirable flavor from soy protein and whey protein concentrate.

Konno et al. (1982) reported that the addition of (3- cyclodextrin resulted in signifrcant reduction of bitter taste in naringin or limonin solution and Citrus Natsudaidai juice based on sensoiy evaluation. An occurrence of increased solubility of naringin and a

NMR shifts of P- cyclodextrin after an addition of cyclodextrin indicated the formation of naringin-cyclodextrin complex.

Polymer of P-cyclodextrins gave more effective reduction of bitterness in citrus fruit juice than monomer of beta cyclodextrin. A column procedure for removing bitterness from citrus juice and grape fruit juice with polymer of P-cyclodextiin has been developed by Shaw et al. (1983 and 1985). Reduction of limonin and naringin concentration ranged from 30-59% and from 33-48%, which made juice significantly preferred in flavor tests over their controls. A generation of beta cyclodextrin polymer with 2% sodium hydroxide was carried out without loss in debittering capacity after 30 times (Shaw and Busling, 1986). CHAPTER m

PROCEDURES

3.1 Materials

3.1.1 Soy protein

Vinton 81 soybeans were provided by Steyer Seeds, Inc. (TiflSn, OH).

3.1.2 Whey Protein concentrates

Based on Li’s (1994) finding that 60% protein whey protein concentrate contained

the largest quantity of volatile components out of six different WPCs studied, this WPC

was selected for use in this study. The WPC (60% protein) was provided by Calpro

ingredient conçany (Corona, California). The product was produced from the whey of

Cheddar, mozzarella and Monterey Jack, which was pre-treated with acidification and

without diafiltration or any post treatment. The content of fat and lactose were 4 to 5%

and 25%, respectively.

3.1.3. Cyclodextrins

P-cyclodextiin, acetylated «-cyclodextrin, acetylated P-cyclodextrin, P- cyclodextiin polymer were supplied by American Maize-products company (Hammond,

Indiana).

45 46

3.1.4 Chemicals and general supplies

Acetic acid, propionic acid, butanoic acid, pentanoic acid, hexanoic acid, heptanoic

acid, octanoic acid, nonanoic acid, decanoic acid, benzoic acid, phenyl benzoate, 1-

hepatanol, 1-hexanol, l-octen-3-ol, nonanal, pyrrole, benzaldehyde, biphenyl, 2-

furanmethanol, maltol, hexanal and l-octen-3-ol were purchased from Aldrich Chemical

Co. (Metuchen, NJ). Tetradecanoic and hexadecanoic acid, pentane, propanal, pentanal,

2-hexanal, 1-pentanol, 2-heptanal were obtained from Sigma Chemical Co. (St. Louis,

MS). Furan-2-carboxaldehyde, methyl sulfone, decanal were obtained from Fluka Co.

(Ronkonkoma, NY). HPLC grade of acetonitrile and water were purchased from Fisher

(Pittsburgh, PA).

3.1.5 Solid Phase Microextraction

Polyacrylate coating solid phase microextraction fiber assembly (85 pm fiber), serum bottles, teflon coated rubber septa and aluminum caps were obtained from Supelco,

Inc. (Bellefonte,PA).

3.2 Sample preparation

3.2.1 Preparation of protein solutions

3.2.1.1. Preparation of soy milk

Four hundred grams of soybeans were soaked in water at room temperature for 16 hours. Soaked soybeans and water, weighing 4000g, were ground with a commercial grinder. Soy milk was automatically separated from residue during the grinding 47

processing with a grinder capable to retain the residue in the grinder. The soy milk was

used immediately.

3.2.1.2 Preparation of whey protein concentrate

A 12% WPC aqueous solution was made with double distilled water. Ten ml of

WPC solution and a stirring bar was placed in a 25ml glass serum bottle and sealed air

tight with a teflon-coated-rubber septum and an aluminum cap. All samples were sealed

and stirred magnetically at room temperature for 10 minutes. The microextraction was

conducted after 7 hours of equilibration at 5°C.

3.2.2. Preparation of standard volatile compound solutions

Solutions of volatile compounds of soy milk and whey protein concentrate were

prepared.

3.2.2.1 Preparation of standard volatile solutions for soy protein flavor analysis

One hundred pi of each flavor compound, hexanal, l-octen-3-ol, trans-2-hexanal,

n-pentenal, 2-heptenal, pentan-l-ol, pentanal, heptanal, ethanol, and propanal were

dissolved and diluted with distilled water to 250ml. One ml of solution was diluted to

100ml with distilled water. Ten ml of diluted solution was pipetted into a 25 ml serum

bottle. The serum bottles were immediately sealed with a teflon-coated rubber septum and

aluminum cap. Then serum bottles were placed into a shaking water bath at 100°C or

30°C for 5 minutes, then cooled by placing them immediately into ice bath. All serum bottles were refrigerated at 5°C for 2 hours before analysis of headspace. Although 48

addition of internal standard is suggested to calibrate concentration of analytes, internal

standard was not used in this study to avoid the complexion between cyclodextrins and

internal standard.

3.2.2.2. Preparation of standard volatile solution for whey protein_flavor analysis

A 1000 ppm stock solution containing acetic acid, propanoic acid, butanoic acid, hexanoic acid, heptanoic acid, octanoic acid, nonanoic acid, decanoic acid, benzoic acid, tetradecanoic acid, and hexadecanoic acid was prepared with acetonitrile. The stock solution was diluted to 80 ppm with HPLC grade water. Ten ml of the 80 ppm standard solution was pipetted into 20 ml serum bottle and sealed with a teflon-coated-rubber septum and an aluminum cap. All bottles were sealed and stirred for 10 minutes at room temperature. Microextraction was conducted after an equilibration for 24 hours at room temperature and carried out at 40 °C with stirring for 2 hours.

3.3.Gas chromatographic analysis of volatile compounds

3.3.1 Analysis o f soy milk flavor

3.3.1.1. Dynamic headspace analysis of volatile compounds

The procedure of dynamic headspace analysis followed the method of Laye et al.

(1993). The serum bottle was connected to the Model LSC 2000 purge and trap concentrator (Tekmar, Cincinnati, OH). Headspace volatile were purged 10 minutes with ultra pure helium at a flow rate of 40 ml/min. Volatdes were sequentially absorbed on a

Tenax TA trap (Tekmar), desorbed by flash heating, cryogenically focused at the liquid 49

nitrogen cooled capillary interface, and automatically injected into the GC/MS and heating

for 1 minute at 180°C.

3.3.1.2. Isolation and identification of volatiles from soy milk with gas

chromatography/Mass spectrometry

A Hewlett-Packard 5890 Series II Gas Chromatography and 5971A Mass

Selective Detector (MSD), equipped with a DB-5 fused silica capillary column (30m X

0.25 um film thickness, J & W Scientific Inc., Rancho Cordova,CA) was used to analyze

headspace volatile organic compounds using ultra high purity helium as the GC carrier gas

at 1 ml/min. The temperature was programmed from 32°C to 80°C at 2°C/min. Mass

spectral analysis was done with an ion source temperature of 180°C and ionization energy

of 70eV. Flavor compounds were identified by matching retention index (RI) with

NIST/EPA/MSDC 49K Mass Spectral Database and the retention time standards.

3.3.2. Analysis of whey protein flavor

3.3.2.1. Solid phase microextraction (SPME)

A fused silica fiber coated with 85 pm of polyacrylate was placed into the headspace of bottles containing WPC for adsorption of flavor compounds. The adsorption was conducted for 2 hour at 40°C with and without stirring. After sample adsorption, the fused silica fiber was introduced into the gas chromatography injector at

250°C for 10 minutes, where the adsorbed analytes are thermally desorbed and delivered to a capillary GC column. 50

3.3.2.1.1. Calculation and statistical analysis

The peak area of each volatile compound was recorded. Means (M), standard

deviations (SD) and coefficient of variations (C.V.=SD/M*100) of peak area were

calculated. The use of internal standards added to WPC before treatment with

cyclodextrin was not practical, because the cyclodextrins absorbed the internal standard as

well of the volatile compounds in the WPC.

Data were analyzed by ANOVA procedure of MINITAB (1994). Tukey’s method

was used for multiple comparisons.

3.3.2.2. Gas chromatography/ mass spectrometry analysis of whey protein solution

A Hewlett-Packard 1800A GC/MS system with an electronic ionization detector,

and equipped with a Hewlett-Packard INNOWax capillary column (crosslinked

polyethylene glycol, 30mX0.25mm i.d., 0.50|im film thickness) was used to analyze and

identify volatile compounds in the headspace of WPC. Ultra high purity helium was used

as the GC carrier gas at the flow rate of 1 ml/min. The temperature was programmed

from 38°C to 80°C at 2°C/min, to 160°C at 6°C/min, to 220°C at 10°C/min, and

maintained at 220°C for 3 min. Peaks were identified by comparisons of acquired mass

spectra with that in the NB S/Wiley database processed by HP ChemStation on HP Vectra

PC. Authentic standards were run to confirm compounds by matching retention time.

3.3.2.3. Gas Chromatographic analysis of standard fatty acid solution

Analysis of fatty acids in model system was carried out on a Hewlett-Packard

5890A Plus gas chromatography equipped with a flame ionization detector and a cross­ 51

linked polyethylene glycol-TPA FFAP capillary column (25mX0.32mm i.d., 0.52pm film

thickness). Nitrogen was used as the carrier gas with fiow rate of 2.8ml/min. The

temperature was programmed from 32°C to 80°C at 2°C/min, to 160°C at 6°C/min, to

220°C at 10°C/min and held for 7 minutes. Authentic compounds listed as possible flavor

compounds of WPC from previous GC-MS analysis were injected to confirm their

presence in WPC by matching retention time.

3.4 Use of CD to remove volatile compounds from protein and standard chemical solution

3.4.1 Treatment of proteins with P-cyclodextrin.

3.4.1.1. Treatment of soy milk with P-cyclodextrin

Two hundred ml of soy milk was placed in a 250ml flask and lOg of P-cyclodextrin was added. Soy milk added cyclodextrin was placed into 100°C shaking bath for 15 minutes and subsequently cooled in ice bath. Soy milk was refrigerated at 5°C overnight.

Ten ml of soy milk was pipetted into a 25 ml serum bottle. Sealed serum bottles containing soy milk were placed in a 5°C refiigerator before subjecting them to dynamic headspace analysis.

3.4.1.2 Treatment of WPC with P-cyclodextrin

Effect of P-cyclodextrin on volatiles of WPC were determined by the comparison between WPC and WPC with added cyclodextrins. Ten ml of 12 % WPC was pipetted into a 25ml serum bottle containing 0.5g of P-cyclodextrins and a IX 5/16 inch stirring 52

bar. These bottles were sealed with a teflon-coated-rubber septum and an aluminum cap

and stirred for 10 minutes at room temperature. Volatiles were analyzed with

SPME/GC/MS.

3.4.1.2.1. Effect of concentration of P-cyclodextrin on the removal of volatiles from WPC

Ten ml of 12% WPC aqueous solution was placed in a 25ml serum bottle

containing 0.05g, O.lg 0.25g or 0.5g of P-cyclodextrin and sealed. After ten minutes of

stirring and 24 hours of equilibration, headspace volatiles were extracted with SPME at

40°C for 2 hours with SPME.

3.4.2 Treatment of standard solution with P-cyclodextrin

3.4.2.1 Treatment of standard volatile compounds of soy flavor

Five tenths of a gram of cyclodextrin was added into a serum bottle containing 10

ml soy milk. The serum bottles were immediately sealed with a teflon-coated rubber

septum and an aluminum cap. Headspace volatiles were analyzed with DHS/GC/MS after

a refrigeration at 5°C for 2 hour.

3.4.2.2 Treatment of standard volatile compounds responsible for WPC flavor

After addition of P-cyclodextrin into a 25 ml serum bottle containing 10 ml standard free fatty acid solution, the solution was stirred magnetically for 10 minutes at room temperature. Microextraction was conducted at 40°C with stirring for 2 hours after an equilibration for 24 hours at room temperature. 53

3.4.3. Treatment of WPC with various types of cyclodextrins

3.4.3.1. WPC solution preparation

Ten ml of 15% WPC solution was pipetted into a serum bottle containing lOg of

P-cyclodextrin, acetylated a-cyclodextrin, acetylated P-cyclodextrin, or P-cyclodextrin

polymer and a stirring bar. The bottles were stirred magnetically for 10 minutes allowed

to stand for 24 hours at room temperature before SPME/GC analysis.

3.4.3.2. Measurement of residual P-cyclodextrin in WPC

The method used was that described by the study of Smith et al. (1995). A freshly

prepared phenolphthalein solution (0.5 ml 0.006M phenolphthalein in ethanol and 99.5ml

IM NaHCOs). The pH was adjusted to 10.5 with 5M KOH. The WPC aqueous solution was diluted fivefold with HPLC water. Then, 0.995ml phenolphthalein solution and 5ul

diluted WPC were mixed and absorbance was determined at 546 nm. A standard curve of cyclodextrin content was made based on the readings obtained from the mixture of WPC and 0.15, 0.25, 0.5, 0.75, and 1.0 % (WAV) of P cyclodextrin. The absorbance of supernatant after centrifugation was measured. The efficiency of centrifugation with

1475, 3020, 5900, or 8700g at 5°C for 10 minutes to remove residual P-cyclodextrin was evaluated. CHAPTER IV

RESULTS AND DISCUSSION

Two food proteins were utilized in these investigations; (a) soy proteins and (b)

whey proteins. For both proteins, head space volatiles were separated and identified and

the removal of volatile compounds by cyclodextrins was evaluated. The work was

initiated with soy milk and then the direction was changed to whey proteins because of a

change in the fimding for the program.

4.1. Soy milk investigations

The first part of this study was centered on the volatiles in soy milk, using dynamic

headspace san^hng and GC/MS analysis and investigation of the effect of P-cyclodextrin

on the removal ofvolatUes fi'om soy milk.

4.1.1. Identification of headspace volatiles in soy milk

Using dynamic head space analysis, the volatile compounds in soy milk were

separated by gas chromatography on an DB-5 fused silica column and identified by

matching retention index (RI) with mass spectrometry and the retention time of standards.

Although there are more than 26 peaks eluted by gas chromatography, only 14

conqrounds were identified by mass spectrometry. The retention time, means of identification, quality detected by MS of these compounds are listed in Table 10.

54 55

References to literature reporting the presence of the various volatile compounds in soy

milk is also shown in Table 10.

With the exception of three compounds, all the products identified were known

from previous investigation. However, more than forty compounds identified by Wilkens

and Lin (1970) including aldehydes, acetals, esters, benzothiazoles, ethylcyclohexene,

decane, 3-methyldecane, benzene, benzaldehyde, ketones, alcohols, l-pentyfiiran,

hexanoic acid, y-nonalactone and others were not found in this study. It was suggested

that these less volatile compounds did not exist in the headspace, particularly since the

dynamic headspace sampling was conducted right after samples were taken out from the

refrigerator. Unsurprisingly, the compounds found in this study were similar to those

found by Ha et. al. (1992) and Yu (1993) using the same method; dynamic headspace

sampling/GC/MS.

The major volatile compounds identified were pentane, hexanal, 2-methyl-2- butene, 1-hexanol, ethanol and l-octen-3-ol. Except for 2-methyl-2-butene, these compounds have also been shown as the major volatile compounds in soy milk by Wilkens and Lin (1970), Ha et al.(1992), and Yu. (1993). Pentane composed 40% of total volatile in soy milk in this study, while Yu reported that 7% to 20% of the volatile compounds were contributed by pentane in a different varieties of soy beans. The lower pentane content in this study may result from the difference of variety and preparation 56

Table 10 Compounds identified from soy milk with DHA/GC/MS

Retention time Name %‘ Means of ID^ Quality^ Ref^

1.61 Pentane 40.50 M.S. & RT 50 A,B,C 1.67 2-Butene, 2-methyl^ 5.90 MS 64 1.84 1,4-pentadiene 0.63 MS 72 A 1.94 Hydroxylamine, 0.71 MS 45 0-(2-methylpropyl/ 2.42 Heptane, 2,4-dimefiiyl^ 0.65 MS 56 2.49 2-Prop anone 0.86 MS 74 C 3.25 Ethyl acetate^ 0.31 MS 90 B,C 4.09 Ethanol 3.24 RT A,B,C 4.35 2-ethyl fiuran 0.69 MS 83 B,C 5.08 Pentanal 1.80 RT 87 A,B,C 6.02 Trichloromethane 1.14 MS 83 C 7.57 Dimethyl disulfide 1.03 MS 64 B,C 8.02 Hexanal 25.23 MS&RT 40 A,B,C 14.54 1-Pentanol^ 0.49 RT A,B,C 23.14 1-Hexanol 6.23 M S& R T 64 A,B,C 28.20 l-octen-3-ol 3.04 MS&RT 59 A,B,C

* * The average content % of three trials ^ The method used for identification: MS means mass spectrometry and RT means retention time ^ Quality of mass spectrometry References reporting the presence of volatile compounds: A, Wilkens and Lin, 1970, B, Ha et al., 1992 and C, Yu, 1993 ^ Confound was found in only one trial among three.trials 57

Hexanal comprised about 25 % of the volatiles sampled in both this study and the

study of Wilkens and Lin (1970), but there was no further information addressed about the

percentage of other compounds in Wilkens and Lin’s (1970) study. Ha and coworker

(1992) reported hexanal comprised 58% of total headspace volatile for soaked soy beans.

The results from Yu’s study indicated that the content of hexanal in soy milk ranged from

20% to 48% for different varieties of soy beans. With extremely low threshold, hexanal is

probably one of the major compounds contributing to the disagreeable aroma of soy milk

(Wilkens and Lin, 1970).

4.1.2. Removal of volatiles of soy milk by P-cyclodextrin

The efficiency of the removal of volatile compounds by P-cyclodextrin was evaluated both in a model system and for soy milk.

4.1.2.1 Effect of cyclodextrin on a standard solution in a model system

A model system was used, which was prepared by standards including hexanal, 1- octen-3-ol, trans-2-hexanal, n-pentenal, 2-heptenal, pentan-l-ol, pentanal, heptanal, ethanol, and propanal.

The effect of p-cyclodextrin on the concentration of headspace flavor compounds in an aqueous model system after incubation at 100°C for 5 min was evaluated by dynamic headspace analysis (Table 11 and Figure 3). The reduction of flavor compounds caused by the addition of cyclodextrin was calculated by the difference between soy milk added cyclodextrin and the control (without cyclodextrin). After incubation with cyclodextrin at 58

Table 11 Reduction of flavor compounds with the addition of P-cyclodextrin after

incubation at 100°C for 5 minutes

Compounds Average peak area (XI0®)’ Reduction

Without P-CD With p-CD Pentane 705.8 664.3 N.D.'' Ethanol 24.9 23.9 N.D.' Pentanal 470.1 213.4 N.D.'* Hexanal 1,123.1 304.2 77.61% 2-Hexenal 31.6 14.0 55.80% 1-Pentanol 256.0 139.6 54.52% 2-Heptenal 95.4 30.3 68.20% 1-Hexanol 1.8 0 100.00% l-Octen-3-ol 300.0 44.8 85.08% Total area 3,105.4 1588.0 67.22%

'Data based on mean of duplicate runs significant difference existed between original solution and P-cyclodextrin treated samples. (p<0.05) 59

Without cyclodextrin

1.20+07-

le + 0 7 - i

8000000 c fljs

“ I* 'I • I J ■ I T — I— f - * r - rime—> 5.0 0 10.00 15.00 20.00 25.00

kbundance 1.40+07- With cyclodextrin 6 1.20+07

10+07

8000000

6000000-

4000000-

2000000 -

0 - L_i '1 r “T— r - I T -1—t- rime—> 5 .0 0 10. 00______15.00 20.00 25.00

Figure 3 GC/MS profiles of headspace volatile compounds from volatile compound mix containing no cyclodextrin (A) and volatile compound mix containing cyclodextrin (B) after the incubation at 100°C for 5 minutes. 60

100°C for 5 minutes, all flavor compounds tested, except for pentane, ethanol and

pentanal, were significantly reduced; with a reduction of peak area ranging from 54%

to 100% (p<0.050), One-hexanol, derivative from the added volatile compounds was

removed up to 80%, as compared to the control. Hexanal, the predominant compound

responsible for beany flavor of soybeans, was decreased by 60.8%, More than 67% of

total peak area of flavor compounds were removed fi'om the model system.

4.1.2.2. Effect of (3-cyclodextrin on volatiles in soy milk

P-cyclodextrin (0.5 g) was added to 10 ml of soy milk to observe the effect of

cyclodextrin on volatiles of soy milk following incubation at 100°C for 15 minutes and refrigeration at 5°C overnight. The GC/MS profiles of headspace volatile compounds from soy milk containing cyclodextrin and no cyclodextrin are illustrated in Figure 4.

Based on the statistic analysis, there was no significant difference (P< 0.05) found in the peak area of pentane, ethanol, and total peak area. All of l-octen-3-ol and 77% of 1- hexenol were removed from the headspace, which is similar to observation made in model system that P-cyclodextrin was most effective in the reduction of l-octen-3-ol. The concentration of hexanal was reduced by 50%, which was lower than the reduction found in the model system. The weaker effect may be due to the barrier effect of other compounds existing in soy milk, which could retard the interaction between cyclodextrin and volatile compounds. The bonding between protein and flavor compounds may contribute to this barrier effect. 61

Abundance 2500000- Without cyclodextrin A

2000000 15 c

1500000- X

c 1000000- c - 3 C 1 V 5 c ~ 13 2 JL 9 500000- J

0 - ki,k„ 11 .1 . .1 ^ 1 1 1 1 1 rime—> 5.00 10.00 15.00 20.00 25.00 1

Abundance 2500000- With cyclodextrin

2000000

1500000

1 0 0 0 0 0 0 -

500000-

I ' I ' 5.00 10.00 15.00 20.00 25.00

Figure 4. GC/MS profiles of headspace volatile compounds from soy milk containing no cyclodextrin (A) and soy milk containing cyclodextrin (B). PLEASE NOTE

Page(s) not Included with original material and unavailable from author or university. Filmed as received.

UMI 63

Table 12 Reduction of volatile compounds in headspace with the addition of P- cyclodextrin after incubation at 30°C for 5 minutes.

Compounds Average Peak Area (X 10®) ‘ Reduction

Without p-CD With P-CD Pentane 702.1 584.6 N.D.^ Pentanal 892.0 545,0 N.D.^ Hexanal 1,361.4 353.3 60.78% 2-Hexenal 49.1 31.6 35.66% 1-Pentanol 476.1 330.9 30.49% 2-Heptenal 107.9 49.6 54.08% 1-Hexanol 1.4 0 100.00% l-Octen-3-ol 299.5 58.4 80.50% Total area 4,088.6 2,279.7 44.24%

‘mean of duplicate runs ^’^No significant difference existed between duplicated or triplicate samples of cyclodextrin added soy milk and controls (p<0.05). 64

PUaundance Without cyclodextrin

2.5e+07 T3C JS

2e+07

1.5e+07- g 0 Î O rn C 1 B c le+07 3 K 0 î c. u 1 5000000 i X (N

T I T " '' rime— >______5.00 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

Abundance 3e+07 With cyclodextrin g

2.5e+07-

2e+07

1 . 5e+07 -

le+07

5000000 -

1 0 J L 1 L . 1 i 1 1 rime—> 5 .0 0 10.00 15.00 20.00 25.00 30.00 35.00 40.00 45.00

Figure 5 GC/MS profiles of headspace volatile compounds from volatile compound mix containing no cyclodextrin (A) and volatile compound mix containing cyclodextrin (B) after the incubation at 30°C for 5 minutes. 65

Total area '/////////////////A

1-octen-3ol 'mmsssmsmm!.

1-Hexanol ’m m m m m m sm

2-Heptenal ■ 30C E3 lOOC

1-pentanol

2-Hexenal

Hexanal w m m s s m s s m

T ------1-- 1...... I 1" 1----P— I------1— — ! 0 20 40 60 80 100 120

Reduction (%)

Figure 6 Comparison of the reduction of volatiles in aqueous solution with P- cyclodextrin with incubation at 30°C and 100°C. 66

Since insoluble P-cyclodextrin polymer still provided reduction of volatile

compounds, the higher solubility of P-cyclodextrin at 100°C may not contribute to greater

reduction of some volatile compounds at 100°C. The temperature may affect the

complexion between P-cyclodextrins and volatile compounds. A study by Wilson and

coworkers (1989) indicated that cyclodextrin polymer cross-linked with epichlorohydrin

supplied by American Maize company removed less naringin and limonin from grapefruit

juice at 5°C than at ambient temperature. However, limonin was removed from navel

orange juice at 5°C as well as at ambient temperature (26°C). An increase of temperature

of egg yolk to 50°C was employed to increase cyclodextrin solubility during cholesterol

removal (Smith et al, 1995). It seems that the effect of an increase of temperature on the

reduction may vary with the guest compounds included and the food systems utilized.

4.2. Whey Protein Investigation

After results showed that P-cyclodextrin could remove volatile compounds from

soy milk, attention was directed toward whey protein concentrates. A 60% whey protein

concentrate, found to contain a high level of volatile compounds by Li (1995) was used in

this investigation. Major attention has been given to volatile fatty acids and benzoic acid,

which were found to be the major volatile compounds in the 60% WPC (Li, 1995).

4.2.1. Development of SPME methods for head space analysis:

Because of lack of accessibility to dynamic head space equipment, this investigation evaluated a new method, solid phase micro-extraction (SPME), for the 67

investigation of head space volatiles. Investigation was needed to determine the

conditions best suited for the use of this method for determination of volatile compounds

in whey proteins. Preliminaiy investigation showed that a polyaciylate fiber was more

effective that a polydimethylsiloxane fiber, so that the polyacrylate fiber was used for all

fiuther studies.

Since no essential volatiles were extracted by SPME at room temperature for 10

minute, which had been used previously with incorporation of salt and stirring for the

analysis of orange juice (Supelco, 1994), it was necessary to evaluate the efficiency of

longer time, higher tenqperature and stirring for extraction.

4.2.1.1. Effect of terrqrerature and stirring on SPME of standard solution

The incorporation of heat, stirring and longer extraction time were considered to

improve the extraction of volatile fi’om the whey protein concentrate. Trials were run first

with a model system. Ten ml of lOOppm standard solution was extracted either at room

temperature or 40°C for 10 minutes or 30 minutes with or without stirring.

Generally, an increase of temperature, stirring and operation time enhanced the

amount of volatiles extracted (Table 13 and Figure 7). The C2, C3, C4, benzoic, C12,

C14, and C16 fetty acids were affected less dramatically by these factors, which may be

due to their high vapor pressure. To receive sufficient amount of fatty acids for analysis, heating to 40°C, stirring and longer operation time were incorporated. 68

Table 13 Peak area of fatty acids extracted by SPME at different operational condition.

Rm temp. 10 Rm temp. 30 Rm.temp. 40°C 30min. 40°C 30min. min. min. stirring 30 stirring (xIO*) (x lO ^ min. ( X 10^) (xlO*) (x lO ^ C2 12.3 16.1 16.9 9.8 20.6 C3 46.2 66.8 72.5 66.8 82.0 C4 97.2 174.5 211.8 1,576.3 246.5 C6 253.0 635.3 1,079.4 1,249.3 2,123.8 C7 271.1 714.2 1,325.4 1,948.9 3,364.3 C8 305.5 807.8 1,591.4 2,862.4 4,799.6 CIO 1,998.3 504.5 11,788.7 2,610.9 5,012.6 Benzoic 0.0 0.0 57.7 191.6 287.8 C12 5.2 36.9 127.9 247.6 729.3 Phenyl 474.6 1,132.8 2,170.1 4,259.4 6,869.2 benzoate C14 1.1 3.6 26.4 19.5 178.2 C16 0.6 119.4 10.3 3.5 48.5 Data based on a single run 69

7000000 -

6000000 -

5000000 -

- 1 4000000 - t-2 1-3 ^4 - * - 5

100000 --

03 06 08 Benzoic phenyl benzoate 02 04 07 O10 012 014 016

compounds

1 .Room temperature for 10 minutes 2.Room temperature for 30 minutes 3 Room temperature for 30 minutes with stirring 4.40°C for 30 minutes 5.40°C for 30 minutes with stirring

Figure 7 Effect of extraction time, temperature, and stirring on the efficiency of SPME on fatty acids. 70

4.2.1.2. Effect of time on volatile compounds of WPC extraction by SPME

To determine operation time to obtain desirable amount of volatiles, ten ml of 12%

WPC was extracted with SPME at 40°C for 1 hour, 2 hours, 3 hours, or 4 hours with stirring to determine appropriate time to process SPME for WPC. The results are presented in Table 14.

The result showed that 2 hours was sufficient to extract most compounds, although 4 hour extraction resulted in a remarkable increase in the concentration of C8,

C9, CIO, C12, C14, C16 fatty acids, and benzoic acid. Two-hour extraction also was sufficient for other volatile compounds, such as pentanol, hexanol, l-octen-3-ol, heptanol and methyl sulfone. However, to allow more than ten samples be analyzed in a day, extraction of 2 hours was chosen for subsequent trials. 71

Table 14 Peak area of volatile compounds extracted by SPME at 40°C for 1 hour, 2 hours, 3 hours or 4 hours.

Compounds Peak Area 1 hour 2 hours 3 hours 4 hours

Pentanol 7273 7773 7270 7471 Hexanol 2653 3558 3202 3621 Nonanal 5601 7734 6945 8966 l-octen-3-ol 1548 2193 1771 2248 Heptanol 2645 3535 3291 3820 C2 1925 1197 1149 1394 C3 3435 6048 5434 6206 C4 2446 4323 3479 4512 2-Furamethanol 658 918 1129 657 C5 2514 3174 3399 4898 C6 47642 79672 60129 76388 Methyl sulfone 4541 4480 4570 4505 C7 19770 21558 15908 22513 Maltol 3958 5214 6263 9345 Biphenyl 6024 7406 6359 7555 C8 29422 53469 45494 74191 C9 20575 20648 14700 32169 CIO 37781 69361 54146 157527 Benzoic 127366 364271 376121 952382 C12 48891 95384 92977 127364 Phenyl benzoate 23474 0 43439 11580 C14 9756 10801 4121 48346 C16 4399 6261 3694 15821

Result based on a single run 72

4.2.1.3. Variation of SPME fibers

Because of the requirement of 2 hour for each extraction, two SPME fibers were

used alternatively every hour, which allowed to complete extraction of more than 10

samples in a day. However, it was suspected that there was a significant variation

between two SPME fibers. This study was conducted to evaluate the variation of two

SPME fibers from a same lot. The headspace volatiles above ten ml of 100 ppm fatty acid

standard solution was extracted with SPME at 40°C for 2 hours with a stirring.

Average, standard deviation, and coefficient of variation (C.V.) of peak area were

listed in Table 15. Fiber 2 has a relatively lower C.V. for C5, C6,C7,C8,C9, CIO, benzoic

acid and C12 than fiber 1, but a higher C.V. of C14 and C16. The C.V. of peak area of

each fatty acid ranged from 15% to 110%. Except for C14 and C l6, comapared to the

C.V. with dynamic headspace sampling method and SPME reported by other researchers,

the C.V. are still satisfactory (below 20%). C14 and C16 have relatively higher C.V.s,

due to their low solubilty and high vapor pressure. However, C14 and C16 fatty acids were not concerned because they are less responsible for off-flavor compared to other fatty acids. Since there is variation among fibers, it is suggested that the same fiber should be used to eliminate the problem. Alternatively, two fibers which had been tested and shown to have a C.V. less than 20 % could been used for an experiment. Table 15 Average, standard deviation, and C.V. of peak area extracted with two fibers

Fiber 1 Fiber 2 Fiber land Fiber 2 Average Stdev C.V. Average Stdev C.V. Average Stdev C.V.

C2 5276 887 16.81 5206 908 17.44 5025 929 18.49 C3 22093 2987 13.52 22569 3351 14.85 21267 3835 18.03 C4 44772 5852 13.07 46527 5383 11.57 43791 6783 15.49 C5 100735 15028 14.92 108037 11103 10.28 99640 16951 17.01 C6 219702 33929 15.44 232944 19069 8.19 215326 37354 17.34 C7 366875 58637 15.98 383226 22825 5.96 355905 62869 17.66 C8 474535 77266 16.28 481354 16375 3.40 453419 79381 17.51 C9 573113 91521 15.97 567347 9653 1.70 541991 91725 16.92 CIO 466433 69724 14.95 450809 7768 1.72 435917 72870 16.72 C12 83281 10752 12.91 82741 5263 6.36 78417 13984 17.83 C14 5508 1314 23.85 2460 1884 76.62 3931 2026 51.53 C16 0 0 0 1037 521 50.27 709 783 110.47 Benzoic 14073 3304 23.48 13108 1033 7.88 13426 2102 15.66

Values of average, standard deviation, and C.V. based on triplicate runs 74

4.2.2. Volatile compounds of WPC

Volatiles in the headspace of 10ml of a 12% solution of WPC was extracted by

SPME at 40°C for 2 hours. The analytes were sequentially introduced into an INNOWax

column. The results are shown in Table 16. More than sixty compounds were found in

WPC with the anafysis o f SPME and GC/MS, which provided more conq)ounds and a

wider range of flavor profile conq>ared to distillation method (Mills, 1993; Ferrtti and

Flanagan, 1971), dynamic headspace analysis (DHA) (Lee, 1993; Laye, 1994) and

acetonitrile/water extraction (Li, 1995) It should be noted that the same WPC reported by

Li (1995) was used in this investigation.

Since the WPC sample used in this study was as same as that used in Li’s study, a

similar composition of volatiles was found in these two studies. Both acetonitrile/water

extraction and SPME could efficiently extract fatty acids fi'om WPC. But compared to 29

confounds found with a acetonitrile/water extraction, SPME seems have higher capacity of extraction with sixty compounds reported. Dynamic headspace sampling analysis is not as effective as SPME and solvent extraction of fatty acids. 75

Table 16 Selected flavor compounds identified by GC/MS fi'om WPC and comparison with

other methods.

Compounds Peak Area% Means of I.D*. Distillation^ DHA^ Solyent'’

Alcohols 1-Pentanol 1.85 MS,R V' V 3-Octen-2-one 0.88 MS l-Octen-3-ol 0.75 MS,R V 1-Hexanol 0.70 MS,R V 1-Heptanol 0.92 MS,R 1-Octanol 1.45 MS l-Octyn-3-ol 0.17 MS 1-Nonanol 0.13 MS Ketones 2-Propanone, 1-OH 0.21 MS ^l 3,5 Octadien-2-one 0.20 MS V 3-Octen-2-one 0.88 MS 2-Tridcanone 0.12 MS Cyclohexanone 0.08 MS 4-Amino-1,2,4 triazol 0.19 MS -5-one Aldehydes Octanal 0.24 MS V Propanal, 2-methyl 0.04 MS V Hexanal 0.04 MS V V Nonanal 0.35 MS,R V V Octanal 0.24 MS V 2-Undecenal 0.17 MS 2-ButenaI,2-methyl 0.31 MS Fatty acids Acetic acid 1.93 MS,R V V Propanoic acid 0.23 MS,R V V Butanoic acid 0.44 MS.R V V Pentanoic acid 0.47 MS,R Heptanoic acid 2.36 MS.R V Hexanoic acid 13.63 MS,R V V Octanoic acid 5.65 MS,R V V Nonanoic acid 1.93 MS,R V Decanoic acid 5.65 MS,R V Aromatic hydrocarbons Benzoic acid 42.72 MS,R V V Phenyl benzoate 0.64 MS,R V V Benzaldehyde 0.27 MS,R V V 76

Table 16 Selected flavor compounds identified by GC/MS from WPC and comparison with other methods, (continued)

Compounds Peak Area% Means of I.D*. Distillation^ DHA^ Solvent"

Sulfur containing comoound Dimethyl sulfone 0.06 MS,R V Furans 2-Furancarbox 0.41 MS,R aldehyde 2,5-DiOH furan 0.01 MS 2-Furanmethanol 0.29 MS,R V V 2-Furancarbox aldehyde. 0.09 MS 5-methyl 2(3H>Furanone, 0.21 MS 5 butyldihydro 2,5-Dimethyl-4-OH-3(2H) 0.18 MS 2(3H>Furanone, 0.39 MS di hydro-5-pentyl

Pyrroles IH-pyrrole 0.01 MS,R Maltol 1.76 MS,R V V

Pvrans 2H-Pyran-2-one, 0.15 MS tetrahydro-6-penty

2H-Pyran-2-one, 0.16 MS tetrahydro-3,6,-dimethyl Amines

6-Undecylamine 0.16 MS 2-Heptanimes,5-CH3 0.11 MS Biohenvl 3.83 MS,R V

1. Means of identification (MS means GC/MS; R means retention time). 2.Mills, 1993 and Ferrtti & Flanagan, 1971. 3. Dynamic headspace analysis, Laye, 1994 and Lee, 1993. 4. Acetonitrile/water extraction, Li, 1995. 5.”V“ means presence of compounds 77

Due to the relatively high polarity of the polyacrylate fiber, both polar and

nonpolar compounds were adsorbed. A number of alcohols, ketones, aldehydes, fatty

acids, esters, sulfur containing compounds, furans, pyrazines, pyrrols, and amines were

identified by GC/MS in this study.

Fatty acids from acetic acid to decanoic acid and benzoic acid comprised of 50%

of total flavor compounds in WPC extracted with SPME (Table 17). Particularly, benzoic

acid contributed 40% of the total peak area of the volatile compounds. A significant

increase of benzoic acid content during the manufacture cheeses and cultured dairy

products may be attributed to action of lactic acid bacteria, which could produce benzoic

acid from hippuric acid (Richardson and Gray, 1981). It is also suspected that benzoic

acid, phenyl benzoate and benzaldehyde are derived from benzoyl peroxide, a bleaching

agent used for the manufacture of Mozzarella cheese.

Although, there was no dodecanoic, tetradecanoic acid, and hexadecanoic acid

detected on the GC/MS profile, they were eluted with a longer running time, as shown in

at the later experiments using GC alone. These fatty acids may be produced by enzymatic

lipolysis from triglycerides or from microorganisms formed during cheese making.

Derivatives of lipid oxidation such as aldehyde, ketones, and alcohols have been reported

responsible for stale ofF-flavor in WPC (Laye, 1994), these compounds comprised 10% of

total flavor peak area of WPC. Dimethylslfone may be derived from dimethyl sulfide, which was found in WPC by Laye (1994). Other compounds, furans, pyrrols, pyrans, fliranmethol, and maltol could been formed via Maillard reaction (Lee, 1993). 78

4.3. Removal of volatiles in WPC with cyclodextrin

To understand the effect of cyclodextrins on the removal of volatiles from WPC

(12 % WPC solution) and a model system containing fatty acids and benzoic acid were

used to observe the change of volailes in the headspace after addition of cyclodextrin.

4.3.1 .Effect of cyclodextrin on standard solution in a model system

Ten ml of a standard solution containing 80ppm each of acetic, propanoic,

butanoic, hexanoic, heptanoic, octanoic, nonanoic, decanoic, tetradecanoic, hexadecanoic

acid and benzoic acid was prepared for SPME analysis. Addition of 0.5g of cyclodextrin

was followed by an equlibration at room temperature for 18 hours. To avoid protein

dénaturation, room temperature incubation was used instead of 100°C in this study.

SPME was conducted at 40°C for 2 hours.

The results are presented in Table 17. In a model system, 100% of acetic acid and

dodecanoic acid could be removed. The occurrence of total reduction on acetic acid may

be due to the extremely low concentration of acetic acid. The reduction of propanoic

acid, butanoic acid, and pentanoic acid ranged from 62% to 78%. More than 90% reduction of acids from hexanoic acid to hexadecanoic acid was achieved through the additon of P-clycldextrin. A comparison of GC profiles between standard solution containing no P-cyclodextrin and that containing P-cyclodextrin, indicates a dramatic reduction of fatty acids and benzoic acid caused by P-cyclodextrin (Table 17). 79

Table 17 Reduction of fatty acids and benzoic acid with the addition of P-cyclodextrin.

Compounds Average peak area (10^) Reduction (%)‘

Without B-CD With B-CD Acetic acid 0.02 0.00 100.0 Propanoic acid 0.85 0.32 62.1 Butanoic acid 1.77 0.61 65.5 Pentanoic acid 4.96 1.05 78.8 Hexanoic acid 16.11 1.58 90.2 Heptanoic acid 47.61 1.83 96.2 Octanoic acid 105.91 2.06 98.0 Nonanoic acid 174.69 2.43 98.6 Decanoic acid 161.15 2.24 98.6 Tetradecanoic acid 16.88 0.01 99.7 Hexadecanoic acid 2.64 0.01 99.7 Benzoic acid 8.50 0.02 99.7

Data is based on mean of triplicate runs 1. Significant difference exists between the P-CD added fatty acids and controls from acetic acid to decanoic acid and benzoic acid (P<0.05), dodecanoic acid (P=0.059), tetradecanoic acid(p=0.083), and hexadecanoic acid (p=0.115). 80

The results agreed with the observation of Schlenk and Sand (1964) that fatty

acids from hexanoic acid to decanoic acid and a- and P-cyclodextrin tended to form

crystal inclusion compounds, which enhanced the solubility of fatty acids in water.

4.3.2. Effect of various types of cyclodextrins on the removal of volatiles from a standard

solution

The addition of 0.5 g of acetylated a-cyclodextrin, acetylated P-cyclodextrin, P- cyclodextrin, and P-cyclodextrin were evaluated in this section. Samples were equlibrated for 24 hour at room temperature. Mean peak areas of coffecicient of variation are presented

In a model system, P-cyclodextrin resulted in the most significant reduction of all fatty acids selected at the concentration of SOppm, whereas acetylated P-cyclodextrin gave less effect on fatty acids (Table 18 and Figure 8). Acetylated P-cyclodextrin and P- cyclodextrin polymer do not appear to significantly affect C2, C3, and C4. However, there is no difference among the effects of P cyclodextrin, acetylated P-cyclodextrin, and

P-cyclodextrin polymer on C l2, C l4, and C16 fatty acids acids. 81

Table 18 The effect of various cyclodextrin on fatty acids in model system

Compounds Reductions (%)

P-cycldoextrin Acetylated P-cyclodextrin P-cyclodextrin polymer

C2 45.66 a 7.88 b* 11.83 ab* C3 35.17 a 9.64 b* 12.11 ab* C4 46.68 a 23.29 a* 11.77 a* C6 84.03 a 13.36 c 40.40 b C7 93.00 a 21.68 c 59.87 b C8 96.22 a 33.64 c 75.64 b C9 97.05 a 46.85 c 83.93 b CIO 96.94 a 57.72 b 88.65 a Benzoic 97.20 a 66.38 b 76.53 ab C12 94.75 a 73.34 a 80.99 a C14 96.52 a 78.81 a 78.57 a C16 95.21 a 75.96 a 82.09 a

Data based on the mean of three runs Each letter designates the same degree of significance. * means no significant difference with controls 120

100

c 0 BAcelylaled b-CD u T33 O poly met CD 01 ■ b C D

fatty acids

Figure 8 Reduction offàtty acids with addition of various cyclodextrins

8 83

At the concentration of 5% WAV, P-cyclodextrin always gave a positive reduction

on fatty acids and benzoic acid, whereas the weakest effect was found after the addition of

acetylated P-cyclodextrin. The result indicated that the use of P-cyclodextrin polymer is

the best choice among insoluble cyclodextrins, although the reduction is not as well as P-

cyclodextrin.

For this experiment, the concentration used was higher than that used in the

previous model system (100 ppm compared to 80 ppm). Also, longer equilibration was

required to later experiment for the activation of new fiber to replace a broken fiber. The

concentration of fatty acids of this experiment in headsapce was higher than that of

previous model system. In addition, a greater C.V.s of fatty acids resulted from a long period taken to finish the experiment. However, the same trends were observed in that effectiveness of P-cyclodextrin tended to increase with an increase in chain length of fatty acids.

4.3.3. Effect of cyclodextrin on WPC volatile compounds

P-cyclodextrin (soluble cyclodextrin) was evaluated in preliminary studies for its accessibility. In later studies, acetylated a-cyclodextrin, acetylated P-cyclodextrin, and P- cyclodextrin polymer (insoluble cyclodextrins) were evaluated for their easy removal from

WPC. 84

4.3.3.1 Use of soluble P-cyclodextrin

4.3.3.1.1 Effect of 5% of P-cyclodextrin on WPC volatiles

Volatiles of ten ml of 12% WPC containing 0.5 g of P-cyclodextrin were extracted

with SPME after being stored under refrigeration for 7 hours. The peak area of each

volatile compounds were compared and reduction was calculated by the decrease of

volatile after addition of cyclodextrin. Reulsts are in Table 19.

In WPC system, P-cyclodextrin had the least effect on acetic acid (55.6%),

whereas the rest of fatty acids and benzoic acid were reduced by more than 90%.

Especially, butanoic acid, pentanoic acid, heptanoic acid, octanoic acid, nonanoic acid, and

decanoic acid, which were almost completely removed from the headspace. A sensory

study by adding fatty acids into milk (Scanlan et al., 1965) indicated that the even-

numbered fatty acids from butanoic acid to dodecanoic acid contributed to the rancid

flavor in milk, whereas fatty acids above dodecanoic acid contributed little. Therefore, the

elimination of these fatty acids with an addition of P-cyclodextrin should help to improve

the flavor of WPC. Ninety five percent of benzoic acid, the major volatile compound

found in this WPC, was reduced (Table 19). Besides fatty acids, most of the aldehydes,

ketones, and alcohols were totally removed after the addition of cyclodextrin. These

volatile compounds, such as pentanol, hexanol, l-octen-3-ol, heptanol and others, are also

contribute undesirable flavor. The removal of these flavors will benefit the addition of

WPC in plain-flavor foods. 85

Table 19 Reduction of volatile compounds in headspace of WPC with addition of P-

cyclodextrin.

Compounds Average peak area (10^) * Reduction (%)

Without B-CD With B-CD 1-Pentanol 351.5 2.0 99.6 1-Hexanol 121.5 0.4 99.7 Nonanal 77.5 0.0 100.0 3-Octen-2-ol 158.2 0.0 100.0 l-Octen-3-ol 174.0 0.0 100.0 Heptanol 200.2 0.0 100.0 Acetic acid 305.0 138.5 55.6 2-Furancarboxaldehyde 65.3 8.0 87.7 Pyrrol 30.8 0.0 100.0 Benzaldehyde 50.0 1.4 97.3 Propanoic acid 41.4 3.4 91.7 1-Octanol 263.1 0.0 100.0 3,5 -Octadien-2-one 68.5 0.0 100.0 Ethanol,2- 58.5 37.4 36.0 (2-ethoxyethoxy)- Butanoic acid 76.0 0.0 100.0 1-Nonanol 27.3 0.0 100.0 2-Furanmethanol 46.3 52.9 -14.2 Pentanoic acid 107.5 0.0 100.0 Hexanoic acid 2486.0 143.4 94.2 Dimethyl sulfone 6.3 0.0 100.0 2(3H)-furanone, 5 40.8 0.0 100.0 butyldihydro- Maltol 299.1 161.9 45.9 Biphenyl 702.2 28.2 96.0 2(3H)-Furanone, 67.1 0.0 100.0 dihydro-5-pentyl Octanoic acid 1080.1 0.0 100.0 Nonanoic acid 389.9 0.0 100.0 Decanoic acid 656.8 0.0 100.0 Benzoic acid 8230.1 380.2 95.4

' Based on mean of duplicate runs. 86

4,3.3.1.2 Effect of the concentration of P-cyclodextrin on the reduction of fatty acids in

WPC

To determine the optima concentration providing desirable reduction of fatty acids,

Four concentrations of P-cyclodextrin, 0.05%, 0.1%, 0.25%, and 0.5% were tested. The

results are presented in Table 20, with peak area and coefficient of variation presented in

Appendix Table 24.

Generally, an increase in concentration of P-cyclodextrin increased the removal of

volatile compounds. The most effective reduction of pentanol, heptanol, benzaldehyde,

octenol, C4, and C6 occurred at the concentration of 5%. However, the addition of 2.5%

of cyclodextrin resulted in a same reduction as 5.0% for octen-3-ol, C3, C5, C7, maltol, biphenyl, C8, C9, CIO, benzoic acid. C l2, C l4, and phenyl benzoate. The reduction of

C16 was less reproducible at the elevated concentrations of P-cyclodextrin than others, because of its low vapor pressure and low solubility, which may result in insignificant effects between control and treated samples. 87

Table 20 Effect of concentration of P-cyclodextrin on the reduction of volatile compounds in WPC.

Compounds Reduction (%) 0.05% 0.1% 0.25% 0.5%

Penatanol 10.79 d* 28.76 c 46.42 b 66.12 a Octen-3-ol 41.14 b 63.34 ab 79.41 a 81.20 a Heptanol 42.73 d 65.85 c 80.84 b 89.91 a C2 0.00 * 6.00 * 8.80 * 25.41 * Decanal 0.00 b* 14.84 ab* 67.48 a 55.75 a Benzaldehyde 9.14 c* 29.56 b 41.35 b 58.53 a C3 0.00 c* 8.62 be* 24.08 ab 38.71 a Octenol 44.53 d 69.18 c 83.43 b 92.08 a C4 9.10 b* 14.44 b* 21.14 b* 49.72 a C5 10.01 b* 0.00 b* 28.40 ab* 55.56 a C6 23.88 d 41.83 c 64.89 b 80.58 a Methyl sulfone 16.71 ab* 7.01 b* 47.11 ab* 70.42 a C7 36.83 c 61.85 b 80.83 a 90.89 a Maltol 42.64 ab* 34.54 ab* 53.25 ab* 68.11 a Biphenyl 39.70 ab* 10.38 ab* 35.96 ab* 68.14 a SC 46.07 c 72.12 b 85.54 ab 94.21 a 9C 40.12 c 66.81 b 80.69 a 91.81 a IOC 35.39 c 65.86 b 84.42 ab 94.09 a Benzoic 29.28 c 46.82 be 67.84 ab 83.12 a C12 21.69 c* 51.04 b 77.16 a 90.53 a Phenyl benzoate 10.16 c 20.82 be 55.53 ab 62.89 a C14 22.42 c* 28.32 be* 56.95 ab 82.13 a C16 28.94 * 4.28 * 47.12 * 52.58 *

Data based on means of triplicate runs A same alphabetic letter means no significant difference between two treated samples at p=0.05. means no significant difference with controls. □ 0.50% ■ 1.00% 02.50% a 5.00%

u

o O o c . c n j CO CD c Q. 0 ) Q) 0 ) Q. b j C o

compounds

Figure 9 Reduction of fatty acids with addition of cyclodextrin at various concentration 00 00 89

4.3.3.2. Effect of various types of cyclodextrins on WPC volatiles

Among cyclodextrins tested in this study, P- cyclodextrin performed most effective

reduction of fatty acids (C2 to Cl 2) (Table 21, Figure 10 and Appendix Table 25). The

reduction of C2 to C12 caused by P-cyclodextrin polymers ranged from 50% to 100%.

The concentration of most fatty acids was lower than previous study because of the use of

smaller stir bars (1/2X 5/16 compared to 1X5/16 inch). The lower reduction of fatty acid may be associated with less vigorous agitation. However, there is no significant reduction of C3 after a treatment with cyclodextrins, which agrees with the observation that propanoic acid is compatible with a-cyclodextrin, but it has no satisfactory fitting in the larger cavities (Szejtli, 1988).

A significant reduction was found for C2,C6,C9, CIO, C12 after the addition of beta cyclodextrin polymer. A lower reduction by P-cyclodextrin polymer than P- cyclodextrin may be due to the lower concentration of P-cyclodextrin in the polymer, which was composed of 90% of water on a weight basis. Acetylated P- and acetylated a- cyclodextrins provide less effect on fatty acids. Only C9,C10, and C12 were reduced by

19%, 52% and 69%, respectively, with the treatment of acetylated beta cyclodextrin.

Although P-cyclodextrin gave the most desirable reduction of free fatty acids, its higher solubility may cause a residue of P-cyclodextrin in the WPC solution. To remain less residual P-cyclodextrin in WPC and obtain desirable removal of fatty acids, P-cyclodextrin 90

Table 21 Peak area and reduction of fatty acids in WPC after treatment of cyclodextrins

Compounds Reduction(%) Acetylated-P-CD Acetylated a-CD p-CD P-CD polymer

C2 1.03 c* 0.00 c* 87.32 a 38.26 b C3 31.24 * 0.00 * 41.67 * 0,00 * C4 14.04 b* 3.84 b 61.38 a* 19.06 b C5 10.47 b* 8.52 b* 53.20 a 19.09 b C6 21.66 b* 9.31 b* 100.00 a 80.01 a C7 3.49 b* 0.00 b* 81.91 a 39.73 ab* C8 37.99 ab* 24.03 b* 100.00 a 70.61 ab* C9 19.29 b 7.94 b* 94.19 a 51.21 a CIO 51.69 ab 41.64 b* 93.47 a 69.72 ab Benzoic acid 10.52 b* 0.00 b* 67.09 a 54.39 b* CI2 69.16 a 50.23 ab* 100.00 a 100.00 a C14 69.91 * 76.32 * 68.53 * 78.95 * C16 24.15 * 47.51 * 85.95 * 86.30 *

Average reduction is based on triplicate runs A same alphabetic letter means no significant between two treated samples. * means no significant difference from controls. c o □ A-a!phaCD u ■A-BetaCD ■O3 « SBetaCD q : B Polymer

Compounds

Figure 10 Reduction of fetty acids with addition of cyclodextrin in WPC

'•O 92

polymer is more suitable than other commercial insoluble cyclodextrins to remove free

fatty acids from WPC.

4.3.3.3. Effect of contact time on the removal of volatiles from WPC

The reduction of volatiles after the contact of 10 minutes, 2 hours and 30 hours

was determined (Table 22, Figure 11 and Appendix Table 26). This information would be

used to determine the contact time sufficiently provide optima reduction of volatiles.

Apparently, there is no significant reduction of C2, decanal, methyl sulfone, maltol.

C l4, and C l6 occurred after the treatment with beta cyclodextrin polymer. Most of

compounds, except for C8, were reduced by the max. amount after the period of 10 min.

Therefore, contact of 10 min is general sufficient to achieve optima reduction.

An application of P-cyclodextrin polymer to debitter citrus juices reported that the

capacity of the resin to remove limonin or naringin could be reached in 15 minutes, since no appreciable further decrease was noted up to 60 minutes (Shaw and Wilson, 1983). A patent using beta-cyclodextrin to remove cholesterol from egg yolk employs a contact time 30 minutes or 60 minutes at 5°C, 10°C, or 15°C, whereas another patent instructed that a stirring of oil-in water emulsion of egg yolk and polymerized cyclodextrin at temperature higher or equal to 1°C, preferably at 15°C, for some seconds to some minutes could be sufficient to permit the formation of cyclodextrin-cholesterol complexes. 93

Table 22 The effect of contact time on peak area of extracted compounds

Reduction (%)

Compounds lOmin 120 min 30 hours

Pentanol 41.53 a 48.45 a 49.98 Octen-3-ol 58.44 a 64.60 a 63.05 Heptanol 58.29 a 70.06 a 67.62 C2 17.10 * 0.00 * 38.26 Decanal 0.00 * 0.00 * 0.00 Benzaldehyde 30.64 b 30.24 a 0.00 C3 26.51 a 7.39 a* 34.38 Octanol 49.95 a 64.89 a 69.92 C4 21.48 a 13.47 a* 17.28 C5 31.86 a 23.33 a 17.95 C6 57.10 a 59.19 a 54.71 Methyl sulfone 16.37 * 3.39 * 15.49 C7 48.93 a 48.93 a 53.70 Maltol 25.02 * 66.91 * 47.91 Biphenyl 15.23 a 29.58 a 27.49 C8 58.55 b 72.69 a 68.82 C9 45.32 a 65.20 a 58.05 CIO 41.82 a 58.24 a 58.94 Benzoic acid 78.13 a 75.05 a 80.41 C12 54.36 a 46.62 a 48.45 Phenyl benzoate 43.10 a 20.55 a 55.84 CM 51.41 * 0.00 * 55.53 C16 0.00 * 0.00 * 100.00

Average reduction (%) is based on triplicate runs. A same alphabetic letter means no significant between two treated samples. * means no significant difference from controls reduction %

b o s §

î

0I “*5

1 g

I' i f§ e 2,

■ ■ ■ ■ B"t l.llllllllllllllllllllllllllll I

Oi —^ —k o M o ||i- t^6 95

4.3.3.4. Measurement of residual P-cyclodextrin in WPC

Aqueous solution of P-cyclodextrin at the concentration 0.15%, 0.25%, 0.5%,

0.75%, and 1% were made to determine standard curve. A linear relationship existed between concentration of cyclodextrin and absorbance (R^=0.991)(Figure 12). In

Vikmon’s study, the decrease of color intensity of phenolphthalein is proportional to CD concentration between the concentration of 0 and 50pg/ml, 0 and 300pg/ml, 0 and

3000pg/ml for P,y and a-cyclodextrins, respectively 96

0.35 y = 0.2908X + 0.0329 = 0.9991 ^ 0.3 --

0.25 - g I 0.2 I 0.15 -

0.05 --

0 0.2 0.40.6 0.8 1 concentration (%)

Figure 12 Relationship of cyclodextrin concentration and absorbance 97

WPC containing 5% of P-cyclodextrin were centrifuged at 1475Xg, 3020Xg, 5900Xg and

8700Xg at 5°C for 10 minutes. The absorbance at different centrifugal force and

concentration calculated are listed as following:

without 1475Xg 3020Xg 5900Xg 8700Xg centrifugation absorbance 0.144667 0.143667 0.169667 0.14 0.173333 Con.(%) 0.384 0.381 0.470 0.368 0.483

The result indicates that the samples treated with centrifugation didn’t have less

CD content than control. Since centrifugation only removes insoluble compounds, the P-

cyclodextrin is apparently retained in a soluble form in supernatant of WPC samples. An observation under microscopy indicated no any crystal form of P cyclodextrin founds in control appear in WPC samples after centrifugation, which supports that there are soluble

P-cyclodextrins existing even after the removal of soluble cyclodextrin.

Absorbance of sample treated with acetylated a-cyclodextrin, acetylated P- cyclodextrin, and P-cyclodextrin polymers also were determined. The reduced absorbance by addition of P-cyclodextrin was obtained by substrating the absorbance of whey protein concentration plus phenophthalein at pH 10.5. Solution with added polymer or acetylated a- or P-cyclodextrin had a increased color (positive absorbation). The increase in color may be due to either a shift in pH caused by addition of insoluble cyclodextrins or by light scatter by insoluble cyclodextrins. However, the pH is increased only by 0.1 to 0.2, which indicates that increase of color may not be due to the shift in pH. In addition, the degree 98

of color increase was much less after centrifugation. This suggested that the increase of

color may be caused by light scatter by insoluble cyclodextrin and insoluble cyclodextrins

was removed by centrifugation. Although centrifugation reduced insoluble cyclodextrins,

the degree of the decrease could not be quanitated.

Ultrafiltration system with a membrane (molecular weight cutoff 1000-2000) was

used to separate inclusion compounds and P-cyclodextrin monomer, naringin or limonin

(Shaw and Buslig, 1986). However, the ultrafiltration was processed in a sample model

system. It may not be suitable for real food system for the removal of other compounds

with P-cyclodextrin.

An incubation with a - amylases formed by or derived from microorganisms of the

groupAspeprgillus niger, Aspergillus oryzae, Bacillus polymyxa, Bacillus coagulans and

Flavobacterium or domestic hog pancreas amylase was used to remove cyclodextrin residue from egg yolk or egg yolk plasma after decholesterolization (Cully et al., 1990). A hydrolyzation of P-cyclodextrin with both cyclodextrin glycosyl transferase (CGTase) and an amylase or other enzymes may be an alternative used to remove P-cyclodextrins from food systems (Hedges et al., 1993). Besides the problem of residual beta-cyclodextrins, soluble complex may also remain in WPC. A study of Shaw and Busing (1986) found that addition of excess beta cyclodextrin to an aqueous naringin, limonin solution or grapefruit juice caused no change in naringin or limonin concentration after removal of undissolved monomer by filtration. Apparently, the complex does not precipitate from solution under 99

the operational condition. Since P-cyclodextrin has not been approved as food additive,

the use of P-cyclodextrin polymer or other insoluble cyclodextrins as a process aid may be

an alternative to avoid residual P-cyclodextrins.

Centrifugation at SOOOx g was suggested in a patent to remove the complex of

cholesterol and P-cyclodextrin from egg yolk. Smith et al. found that centrifugation at

8700 xg could remove all cyclodextrin residue when the molar ratio of CD; cholesterol is

4 , whereas 3.2% of CD was remained at the ratio of 4.84. Since the P-cyclodextrin

remaining in the WPC was not considered in their study, there was no further discussion

addressed. However, the efficiency of centrifugation on the removal of cyclodextrin may depend on the type of food system applied, and the molecular ratio of guest compound: cyclodextrin used. CHAPTER V

CONCLUSION

5.1. Soy milk investigation

In model system, an addition of P-cyclodextrin resulted in reduction on major

beany flavor compounds, pentanal, hexanal, 2-hexenal, 1-pentanol, 2-heptenal, 1-hexanol,

l-octen-3-ol, and total amount o f volatiles were found. There was no significant effect on

the level of pentane and ethanol.

Qne-hexanol and l-octen-3-ol were reduced by more than 80% by the addition of

cyclodextrin at both 100°C and 30“C. Hexanal, the major conqiounds responsible for the

beany flavor of soy beans, was reduced by more than 50%.

In soy milk, P-cyclodextrin was less effective in reducing flavor compounds than in

the model system. However, the concentration of pentanal, hexanal, 1-hexenol, and 1-

octen-3-ol declined significantly (37% to 100%.). Similar to the result observed in the

model system, the greatest reduction was noticed on 1-hexenol and l-octen-3-ol. Hexanal

accounted for 25% of total volatiles, which agrees with the result reported by Wilkens and Lin (1970) that hexanal comprised about 25 wt% of the volatiles of soy milk. Fifty percentage of hexanal was removed fi'om headspace after the addition of cyclodextrin.

1 0 0 101

5.2. Whey protein concentrate investigation

In this study, solid phase microextraction with a polyacrylate fiber was a

comparable methods to other methods for the analysis of WPC flavor for its extraction

ability for both polar and nonpolar compounds. However, the incorporation of heat,

stirring, and long extraction time was necessary to receive sufficient amount of volatile

extracted. More than sixty of compounds, such as fatty acids, aldehydes, ketones,

alcohols, esters, furans, pyrazines, and pyrroles were identified by GC/MS in this study.

Benzoic acid and fatty acids are major compounds of flavor compounds of the flavor

compounds extracted by SPME from aqueous solution.

In a WPC aqueous solution, the addition of cyclodextrin resulted in remarked

reduction of flavor compounds in WPC. Fifty-five percents of acetic acid was reduced, whereas other fatty acids and benzoic acid were reduced more than 90%. Butanoic acid, pentanoic acid, nonanoic acid, and decanoic acid were not found in the headspace of WPC after cyclodextin treatment. Other compounds, such as aldehydes, alcohols, benzaldehyde, pyrroles, and furans, also were significantly affected by addition of P-cyclodextrin. The results obtained from the model system also agreed with those from WPC aqueous system that the addition of P-cyclodextrin remove fatty acids and benzoic acid range from 65% to

100%. This information reveals a potential use of P-cyclodextrin to exclude off-flavor of

WPC. 102

Although P-cyclodextrin provideed superior reduction of volatiles in WPC than acetylated P-cyclodextrin, acetylated a-cyclodextrin and P-cyclodextrin polymer, the use of P-cyclodextrin polymer was suggested for its more effective reduction than other insoluble cyclodextrins and its easy removal from WPC.

It is essential to determine the appropriate concentration of cyclodextrin used and contact time between WPC and cyclodextrin to reach optima reduction of volatiles. It was found that the concentration of 2.5 g P-cyclodextrin and 10 minutes of contact time between P-cyclodextrin polymer and WPC was sufficient to obtain optima effect for tested condition in this study. REFERENCES

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Additional Tables

111 Table 23 Peak area and reduction of fatty acids after addition of various cyclodextrin in model system

Control C.V. beta red(%) C.V. A-beta red (%) C.V. polymer red (%) C.V. C2 27298 b 25.9 14834 a 45.66 30.0 14834 ab 7.88 177.3 22314 ab 11.83 74.1 C3 110124 b 22.2 71397 a 35.17 27.9 99506 be 9.64 109.9 96792 abc 12.11 75.1 C4 297262 b 17.8 158505 a 46.68 13.4 268022 23.29 128.8 262286 ab 11.77 49.7 ab C6 2040469 8.1 325843 a 84.03 4.2 1767869 13.36 63.7 1216099 b 40.40 9.8 d c C7 3713787 6.4 260177 a 93.00 2.4 2908665 21.68 54.6 1490373 b 59.87 7.2 d c C8 5527021 6.2 209018 a 96.22 1.8 3667591 33.64 39.2 1346656 b 75.64 4.4 d c C9 6225165 7.6 183870 a 97.05 1.6 3308614 46.85 27.0 1000563 b 83.93 2.8 d c CIO 4561438 13.0 139681 a 96.94 1.9 1928558 57.72 20.2 616959 a 88.65 2.3 c b benzoic 469472 c 24.7 13157 a 97.20 3.8 157862 b 66.38 31.57 110172 ab 76.53 14.1 C12 1786845 32.3 93813 a 94.75 2.4 476293 a 73.34 14.0 339596 a 80.99 2.3 b C14 674971b 43.4 23518 a 96.52 1.5 143002 a 78.81 13.8 144671 a 78.57 6.6 C16 210623 b 39.9 10091 a 95.21 3.6 50627 a 75.96 22.0 37724 a 82.09 12.4 Data based on the mean of three runs. Each letter designates the same degree of significance

S) Table 24 Peak area and reduction o volatile in WPC after addition of P-cyclodextrin at various concentration Con C.V. 0.5% C.V. Red% 1% C.V. Red% 2.5% C.V. I Red% 5% C.V. Red% Pentanol 16678 d 3.4 14856 d 11.8 10.789 11867 c 2.9 28.76 8925 b 4.5 46.42 octen-3- 5643 a 3.0 66.12 3715 c 7.5 2154 b 10.7 41.14 1343 ab 19.0 ol 63.34 753 a 10.2 79.41 666 a 90.5 81.20 heptanol 4.5 5533 d 5.8 42.73 3299 c 5.2 65.85 1850 b 3.7 80.84 975 a 7.7 89.91 C2 7058 23.9 8047 23.7 0.00 7061 21.0 6.00 6933 29.4 8.80 5538 14.6 25.41 decanal 2647 b 29.1 2643 b 26.9 0.00 2100 ab 9.2 14.84 737 a 123.1 67.48 1096 a 6.5 benzalde 4861 3.0 55.75 4423 c 9.8 9.14 3431b 8.9 29.56 2857 b 11.0 hyde c 41.35 2020 a 5.7 58.53 C3 2987 c 6.9 3199 c 1=3 0.00 2780 be 10.8 8.62 2310 ab 14.9 24.08 1861a 5.6 38.71 Octeaol 14822 e 2.7 8171 d 11.5 44.53 4533 c 1.7 69.18 2436 b 7.2 83.43 C4 i l o j a 8.1 8176 b 16.3 9.10 7630 b 7.4 14.44 7013 b 15.6 21.14 4481a 19.0 49.72 C5 10694 b 5.0 9512 b ^ 5 _ 10.01 10792 b 29.4 0.00 7537 ab 17.6 28.40 C6 4678 a 15.9 55.56 170749e 6.1 127862d 5.0 23.88 97740c 7.5 41.83 58957b 4.0 64.89 32640 a 80.58 methyls 4476 b 4.2 3733 ab 17.7 16.71 4177 b 49.6 7.01 2372 ab 3.8 47.11 ufone 1327 a 3.7 70.42 42038 d 7.5 26059 c 13.7 36.83 15662 b 14.6 61.85 7871a 4.5 80.83 90.89 6391b 9.3 3734 ab 153.5 42.64 4094 ab 32.9 34.54 2932 ab 30.5 53.25 1983 a 18.1 68.11 Bipheny 9945 10.4 6121 110.0 39.70 8763 8.0 10.38 6294 11.0 35.96 3106 10.2 1 b ab 68.14 ab ab a 95976 d 9.5 50600 c 10.8 46.07 26219 b 14.4 72.12 13524ab 4.2 85.54 9C 5404 a 94.21 42148 d 13.2 24526 c 15.0 40.12 13660 b 20.5 66.81 7842 ab 6.9 80.69 3254 a 91.81 IOC 88625 d 13.9 55453 c 11.2 35.39 29598 b 24.3 65.86 13355ab 7.6 84.42 4989 a 770757d 12.0 525102c 11.7 29.28 402069 34.8 46.82 238800 9.6 67.84 127162a 29.6 83.12 be ab C12 19168 d 12.3 14609 c 7.1 21.69 9237 b 26.0 51.04 4229 a 13.0 77.16 1738 a 90.53 phaiyl 7.2 3538 c 6.0 10.16 3122 be 9.9 20.82 1761 ab 71.3 55.53 1459 a 8.6 62.89 C14 4079 c 12.6 3174 c 46.7 22.42 2932 be 21.2 28.32 1769 ab 25.2 56.95 728 a 82.13 39.8 2147 6.3 28.94 3174 40.5 4.28 1633 20.2 Data based on triplicate runs. 47.12 1381 52.58 A same alphabetic letter means no significant difierence between two treated sample at p=0.05 Table 25 Peak area and reduction of fatty acids in WPC after treatment of various cyclodextrins

C.V. A-beta C.V. Red% A-alpha C.V. Red% beta C.V. Red% polyme C.V. Red% C2 9329 c 1.3 9299 c 5.1 1.03 9830c 4.3 0.00 1182a 18.7 87.32 5732 b 12.2 38.56 C3 1207 23.7 830 7.1 31.24 1238 38.5 0.00 704 56.0 41.67 1295 000 7094 b 11.5 6098 b 7.2 14.04 6822 5.1 3.84 2740 a 22.7 61.38 5735 b 4.1 19.06 C5 5139 c 6.1 4601 5.8 10.47 4701 2.7 8.52 2405 8.2 53.20 4158 6.3 19.09 be be a b C6 4252 b 10.1 3331 b 16.2 21.66 3856 b 10.4 9.31 Oa 0.0 100 850 a 99.9 4898 b 21.8 4727 b 39.5 3.49 5274 b 28.6 0.00 886 a 16.1 81.91 2952 ab 3641b 34.1 2258 ab 9.7 37.99 2766 b 3.4 24.03 0.00a 0.0 100 1070 ab 4079 c 5.9 3292 b 8.8 19.29 3755 be 9.6 7.94 237 a 99.1 94.19 1990 a 4964 c 39.1 2398 ab 24.95 51.69 2897 be 20.7 41.64 324 a 18.7 93.47 1503 ab 17.1 17367 b 19.9 15540 b 12.5 10.52 17582 b 5.1 0.00 5716 a 11.0 67.09 7921b 2873 b 36.7 886 a 87.2 69.16 1430 ab 13.5 50.23 0.00 a 0.0 100 0.00 a 0.0 100 C14 2466 71.26 742 98.3 69.91 584 34.3 76.32 776 62.5 68.53 519 21.1 78.95 C16 2029 109.7 1539 98.8 24.15 1065 55.6 47.51 285 173.2 85.95 278 173.2 86.30 A same alphabetic letter means no significant between two treated samples Table 26 Peak area and reduction of volatile compounds after treatment of P-cyclodextrin polymer for certain contact time, Control C.V.(%) lOmin C.V.(%) Reduction C.V.(%) 120 mm Reduction % Pentanol 16071b 10.7 9396 a 5.0 41.53 1.3 8285 a 48.45 Octen-3-ol 3099 b 9.3 1288 a 15.6 58.44 21.5 1097 a 64.6 heptanol 8941b 11.2 3729 a 4.9 58.29 13.00 2677 a 70.06 C2 5199 14.1 4308 20.2 17.1 67.4 5848 0.00 decanal 2810 a 5.6 3339 a 9.4 0.0 56.45 2839 a 0.00 benzaldehyde 5490 b 7.1 3808 a 9.5 30.64 8.82 3830 a 30.24 C3 3290 b 1.5 2418 a 10.4 26.51 36.8 3047 ab 7.39 Ocetanol 14596 b 10.2 7306 a 9.1 49.95 7.4 5125 a 64.89 C4 9003 b 8.8 7059 a 8.5 21.48 19.9 l 7791 ab 13.47 C5 9730 b 9.3 6630 a 6.3 31.86 16.8 7460 a 23.33 C6 173848 b 5.7 74583 a 3.6 57.10 14.33 70949 a 59.19 methyl 4378 a 8.4 3661 a 18.2 16.37 12.8 4229 a 3.39 Sulfone 7C 44613 b 8.3 22785 a 4.0 48.93 33.9 20657 a 53.70 Maltol 4732 a 35.3 3548 a 45.0 25.02 173.2 1566 a 66.91 Biphenyl 10478 b 8.9 8882 a 6.0 15.23 6.47 7379 a 29.58 C8 107641c 6.8 44614 b 3.0 58.55 13.19 29401 a 72.69 C9 55281b 15.3 30228 a 5.7 45.32 14.7 19238 a 65.20 CIO 117025 b 17.3 68087 a 8.5 41.82 23.3 48871 a 58.24 benzoic 837231b 15.1 183086 a 12.5 78.13 23.9 208857 a 75.05 C12 26508 b 24.1 12895 a 12.6 54.36 29.1 14151 a 46.62 phenyl 5242 b 28.5 2983 a 7.6 43.10 61.9 2370 a 20.55 C14 4147 a 22.3 2015 a 13.3 51.41 97.3 4929 a 0.00 C16 553 a 115.7 |845a 20.9 0.00 86.7 915 a 0.00 Average value based on triplicate runs A same alphabetic letter means no significant diSerence between two sanq)les at p=0.05.

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