,fluorometric DETERMINATION OF SULFHYDRYL PLUS DISULFIDE AS AN INDEX TO

Transcription

1 ,fluorometric DETERMINATION OF SULFHYDRYL PLUS DISULFIDE AS AN INDEX TO HEAT TREA 'IMENT OF HALF-AND-HALF/ by Visit,,s iri bunri t / Thesis submitted to the Graduate Faculty of the Virginia Polytechnic Institute and State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE in Department of Food Science and Technology APPROVED: Warren K. Stone, Chairman William F. Collins \..7 Peter R. Rony October, 1974 Blacksburg, Virginia. J

2 ACKNOWLEDGMENTS I wish to express sincere appreciation to for his helpful suggestions, and assistance extended throughout my graduate study. I also want to express my gratitude to and for their patience in checking the thesis, and for his timely advice in relating to my study. A note of thanks is also due to, and for their help in running the laboratory and consumer preference tests. I also wish to express deep appreciation to my wife,, for her patience in checking and typing during preparation of the manuscript and the thesis. 11

3 TABLE OF CONTENT.3 A CKN OWLEDGMEN T.3 LIST OF FIGURES LIST OF TABLES t t t I t I I t t t I I t I t t I t t I t I t I t I t t t t t t t t t t I t I I f t f t t t t I I I t t t t t t I t t t t t t t t t I I t t I t t t t t I t I t t t t t t t t I Page ii v vii INTRODUCTION,,,,,,,.,,,,,,,,,,,,, 1 REVIEW OF LITERATURE,.,..,,,,,,,,, 4 Kinetics of Thermal Inactivation t t t t I I t t t t t t t I I t t t t I t t Protein Nomenclature Protein Denaturation I I t t t t t t t t t t I t I t t t t I I t t t I t t t I t I I I t Fluorometric Determination t I t I t I t I t I I I I I t I t I t t t t t t I t t t MA TERIAIS AND PROCEDURES t I I t t I t t t t t t t I I t I t t t I I t t t I I t I I I t t t t Equipment,.,,.,,,,,,,,,,,,,,,,,,, 23 Reagen ts,,,,.,,,,,,,,,,,,,,,,,,,, 2J Preparation of Half-and-Half 26 Preparation of Acid Whey Fraction 27 Fluorescence of FViA 29 Standard Curve Determination 31 Whey Protein Nitrogen Determination 39 RESUL'IS AND DISCUSSION 42 Sulfhydryl and Disulfide Groups 42 Whey Protein Nitrogen 46 Flavor Analysis 52 SUYiMARY AND CONCLUSIONS 56 iii

4 iv REFERENCES VITA I I I I t t I e 8 I I I I I e I f I e I t t e I t I I I e I I I I I I f I I t I I t I I I I t I I t I I I Page 60 64

5 LIST OF FIGURES Figure Page 1. Comparison of pasteurization curve and Q. burnetii (Q-fever) z curves 5 2. Milk protein nomenclature 11 J. Denaturation of the individual whey proteins in milk heated at various temperatures for JO min (Larson and Rolleri, 1955) The structure of the principal amino acids containing -SH or -SS- groups in milk proteins Tentative structure of fluorescein mercuric acetate (FMA) (Karush et al., 1964) Semimicro-Kjeldahl digestion and distillation apparatus Triple glass-distilled water apparatus Filtration of precipitated milk proteins... JO 9. Fluorescence of various fluorescein mercuric acetate concentration in 1M NaOH solution Percent decrease in fluorescence of fluorescein mercuric acetate in 1M NaOH in the dark at 25 c Standard curve for fluorometric determination of -SH+ -SS- groups... )8 12. Effect of various heat treatments of half-andhalf on concentration of -SH + -SS- in acid v

6 vi Figure Page wheys as detennined by fluorometric measurement... 4J 1J, Percent decrease in -SH + -SS- of acid whey by various heat treatments of half-and-half,, , Percent decrease in whey protein nitrogen of acid whey by various heat treatments of half-and-half Comparison between decreases in -SH + -SSand whey protein nitrogen from 65,6 0 to 79.4 C for JO min holding time... 49

7 LIST OF TABLES Table Page 1, Fat and total solids in half-andhalf and acid whey filtrates,, , Effect of concentration on fluorescence of fluorescein mercuric acetate in 1M NaOH Effect of time on fluorescence of fluorescein mercuric acetate in 1M NaOH Relationship between standard concentrations of -SH + -ss- and percent quenching by fluorometric measurement Effect of heat treatments of half-and-half on changes in concentration of -SH + -SSand whey protein nitrogen in acid whey , Percentages of -SH + -SS-, whey protein nitrogen, and whey proteins remaining in acid whey from half-and-half and milk after processing at various temperatures Relative consumer preference for half-andhalf processed by various heat treatments vii

8 INTRODUCTION The introduction of rapid-heating, high-temperature processes with very short holding times has resulted in the establishment by the Food and Drug Administration (FDA) of minimum pasteurization standards for fluid dairy foods that vary from 62.8 c for JO minutes to 100.0"C for 0.01 second, In addition, all fluid dairy foods processed by ultra-high temperature treatments that were formally designated as "sterile", now are required to be labeled as "ultra-pasteurized". The new FDA regulations require that these products be thermally processed at or above 1J7.8 c for at least 2 seconds, either after or before packaging, so as to produce a product which has.an extended shelf-life under refrigerated conditions (Federal Register, Oct. 10, 1973). Half-and-half is of considerable economic importance to the dairy industry. Annual consumption in the United States is 11.J billion pounds of milk equivalent or 9.1% of the total milk production (Pan- American Coffee Bureau, 1960). This product (half-and-half) is defined as the food consisting of a mixture of milk and cream which contains not less than 10.5~; but no more than 18.0}6 milk fat. It may be pasteurized at 65.6 c for JO minutes or by other time-temperature processes that are equivalent in microbial destruction (THRUST, 197J). Half-and-half may also be ultra-pasteurized by semi-logarithmic extrapolation of the existing time-temperature standards mentioned above. As a result of the establishment of the new time-temperature 1

9 2 standards, an increasing need exists for a reliable, less complicated test than microbial destruction that will give an index to intensity of heat treatments of various thermal processes. The test should lend itself to routine quality control operations, The activation of sulfhydryl and disulfide (-SH+ -ss-) groups in half-and-half is the most important change accompanying heat treatment, As a result of this activation, the whey proteins, which contain most of these groups, are rendered quantitatively precipitable with the caseins at ph 4.6. Therefore, the accurate measurement of changes in the concentration of these groups in acid whey from halfand-half products processed by different heat treatments should accurately reflect the intensity of the heat treatment, Preliminary work indicated that these groups could be measured in acid whey in parts per billion concentrations by fluorometric analysis. This test has several advantages over microbiological methods for measuring heat treatment equivalents of different processes, It is easier to perform, can be automated for quality control, and the results give information on physio-chemical changes that affect quality. For example, activation of the -SH groups in the whey proteins causes cooked flavor, affects the coagulating properties, and serves as antioxygenic agents. There is no published information on the fluorometric detedllina tion of sulfhydryl and disulfide groups in the acid whey fraction of half-and-half processed by different heat treatments. In commercial operations, however, different thermal processes are used that vary over a wide range of heat treatments. Separation of the heat

10 3 activated -SH + -SS- groups and subsequent fluorometric measurement could assist processors in evaluating these processes and indicate their effects on quality and shelf-life. Accordingly, the objectives of this study are : (1) to develop a procedure for separating the undenatured whey proteins from the heat denatured whey proteins in halfand-half; (2) to develop a technique for fluorometric measurement of the -SH + -SS- groups in acid whey; (3) to determine the relationship between concentrations of -SH+ -SS- and total whey protein nitrogen in half-and-half products processed by different heat treatments; and (4) to correlate changes in -SH+ -SS- with heat treatments of halfand-half from 65.6 c to 93.3 c in increments of 2.75 c for JO min.

11 REVIEW OF LITERATURE Kinetics of Thermal Inactivation Heat is the most important treatment received during the processing of milk products, The principal objectives of heat processing ares 1. Pasteurization or sterilization to meet public health requirements, 2. Destruction of enzymes to improve keeping quality, ), Alteration of certain constituents to impart definite properties which serve to identify specific products, Basically, there are two heat processing methods in use in the dairy industry, the non-flow or vat method and the continuous flow processes (Jenness and Patton, 1959). In most instances, the heat treatment equivalents of these processes are determined by thermal inactivation of microorganisms in non-flow systems, For example, the time-temperature requirements for pasteurization currently are based upon the thermal destruction of Coxiella burnetii. All other pa.steuriza tion equivalents are calculated from this determination, Comparisons of the pasteurization and z curve for this organism are shown in Figure 1 (Enright et al,, 1957). The heat treatment equivalents of different procesges are based on the theory that, when the destruction of vegetative cells at a given temperature in a non-flow system is determined at various time intervals and plotted on semi-log pa.per, a straight line survivor curve 4

12 5 10,000 1,000 C. BURNET! I (MINIMUM TIME). Cf) 0 Z' 0 0 ~ 100 z w ~ -I- 10 ~ " " \ \\~PASTEURIZATION \ LINE \ \ \o 'o 'Q Fig, 1 - Comparison 0 pasteurization curve and.. burnetii (Q- ever) z curves,

13 6 is obtained (u.s. Department of Health, Education, and Welfare, 1973). The time required to cause a 90-fe (one log cycle) decrease in number on this curve is defined as the D-value for this organism. D-values may be calculated by the following fonnula: u D(n) = log 10 a - log 10 b where: U = Holding time a.= Initial viable count b = Final viable count n = Exposure temperature. The thennal death time ('IDT) of an organism is the time required to kill it at a given temperature in a Imown environment. Stumbo (1948) reported that in non-flow systems the change in 'IDT is also a first order reaction with respect to temperature. Hence, knowing the TuT of an organism at several different temperatures will make possible the construction of a 'IDT curve. If 'IDT's are plotted on semi-log paper against the arithmetic scale time intervals, the resulting 'IDT curve characterizes the thermal resistance of the organisms under the specific conditions of the heat treatment. The symbol z is used to express the slope of the TDT curve and represents the degrees Fahrenheit required to reduce the TDT tenfold. If one TDT value and the z value are known, a TDT curve can be constructed, and from this curve, the TDT at any other temperature may be detennined, Such curves are used to establish the thermal processing time necessary to accomplish

14 7 the extent of bacterial destruction desired and to determine the heat equivalents of various processes. A phantom TDT curve may be fabricated by plotting the D-values with their corresponding temperatures on semi-log paper. The slope of this straight line curve is the z value for the organism. This curve is defined as a phantom TDT or z curve because the exact position of the line on the graph has not been experimentally evaluated (u.s. Department of.health, Education, and Welfare, 197J). The z value from the phantom TDT curve is used to determine minimum time and temperature in a specific menstrum, and according to Kaufman and Andrews (1954), two items of information are needed: (1) how many bacteria can be tolerated, and (2) how many bacteria are present. For example, assuming an initial bacterial concentration of 10,000/ml and CJ;6 destruction or /ml, then the minimum safe processing time (MSPT) is calculated as follows: where: a = 10,000 b =.0001 D(n) = 8 min z n = Processing temperature, F MSPT = 8 x (4 + 4) = 64 min.

15 8 This point (64 min at (n) F) can now be plotted on the fabricated TDT curve and a line with a slope of 10 F drawn through it, This line represents the true TDT or z curve for this organism under the conditions assumed and from this TDT curve various time-temperature combinations can be selected which will be equivalent to reduce the population from 10,000/ml to 0,0001/ml, Recent thermal destruction studies have been conducted using Coxiella burnetii (Q-fever) as the test organism (Harper, 1970), The data in Figure 1 show the relationship between the milk pasteurization curve and the 'IDT or z curve for. burnetii, In the low temperature range, 140 F to 145 F, the z curve is to the right of the pasteurization curve, On the basis of these findings and a confidence limit of 97.7%, the temperature employed for the vat pasteurization of milk was raised to 145 F and a temperature of 150 F was suggested for higher fat products, such as, half-and-half (u.s. Department of Health, Education, and Welfare, 1973). The present temperature-time standard of pasteurization of milk is to heat the milk to at least 145 F for at least JO minutes or 161 F for 15 seconds (u.s, Department of Health, Education, and Welfare, 1965), In both cases, all pathogenic microorganisms in the vegetative state are destroyed and most of the physical and chemical properties, such as, color and flavor remain practically unchanged, With the trend toward increased product shelf-life and greater output of milk, the Public Health Service has established minimum pasteurization standards for processes with shorter holding times and

16 9 higher holding temperatures than are now being used. These processes have been given the general label of ultra-high temperature (UHT) pasteurization. Read et al. (1968) reported that five alternate timetemperature combinations for milk and milk products were selected as standards for UHT pasteurization in plate heat exchange systems. These standards are as follows: 191 F for 1.0 second 194 F for 0.5 second 201 F for 0.1 second 204 F for 0.05 second 212 F for 0.01 second. These minimum time-temperature standards for UHT pasteurization were calculated by semi-logarithmic extrapolation of the existing timetemperature combinations for ice cream mix pasteurization (155 F for 30 minutes or 175 F for 25 seconds). In connection with commercial application, however, processors may exceed these standards for either time or temperature, or both, thereby increasing the need for a rapid test for determining the relative intensities of various heat treatmen ts. During the thermal processing of milk products, the higher heat treatments affect the heat-sensitive proteins. The principal effect is denaturation which increases with the intensity of heat treatments and is used as an index to the heat treatment of dried milk products (American Dry Milk Institute, 1965).

17 10 Protein Nomenclature In discussing milk protein denaturation, it is necessary to distinguish among the various proteins, because they differ in composition and properties, The committee on milk protein nomenclature (Rose et al,, 1970) reviewed changes and additions to the nomenclature of the proteins of cow's milk, They classified these proteins into two distinct fractions as shown in Figure 21 (a) the caseins, which coagulate by acidifying milk to a ph value near 4,6 at 20 c; and (b) the whey or milk-serum proteins, which remain after the caseins have been removed, The caseins exist in milk primarily in the form of rathe~ large micelles (40 - JOO mµ in diameter) containing calcium, inorganic phosphate, magnesium, and citrate, They are not considered to be heat denaturable like the whey proteins and other milk proteins, The caseins generally exist in solution as random coils and have little ordered structure that can be disrupted by heat (Jenness and Patton, 19.59; Herskovits, 1966; McKenzie, 1967), The caseins are divided into alpha, beta, gamma and kappa caseins. s Alpha -casein is the largest casein component, It constitutes s 45-55% of skim milk protein and has a molecular weight of about 23,000 (Rose et al., 1970), Beta-casein has been demonstrated to exist in three genetically determined forms (Kiddy et al., 1966) and has a molecular weight of about 25,000, There has been increased interest in this component

18 MILK PROTEINS CASE I NS ( /o) WHEY PROTEINS (20.5 /o) K as {3 y MW. 19,000 23,000 24,100 30,650 a-lactolbumin 14,437,8-Lacfoglobulin 36,000 Proteose - Peptone 4, I ,000 lmmunog lobulins 170,000-1, 000, 000 Fig. 2 - Milk protein nomenclature

19 12 over the past five years. Gamma-casein has a molecular weight of about 30,650 which is found to be dependent on ph, buffer ion, and temperature. Kappa-casein exists as a colloidal caseinate complex in raw skim milk with alpha -casein and beta-casein. It represents 8-15% of the s skim milk proteins and is a carbohydrate-containing protein with a monomer molecular weight of around 19,000 (Woychik et al., 1966). McKenzie (1967) and Sawyer (1969) reported that the surface of the caseinate micelle is rich in kappa-casein and that it is probably available for interaction with beta-lactoglobulin. This phenomenon was also reported by Sabarwal and Ganguli (1972). They indicated that skim milk heat stability is dependent on the relative amount of kappacasein and beta-lactoglobulin present in the milk, Prior to 1934, it was generally considered that the whey proteins of milk consisted of two definite substances, a lactalbumin and a lactoglobulin fraction, and comparatively insignificant amounts of several other substances of the nature of proteins (Webb and Johnson, 1965). Jenness and Patton (1959) pointed out that whey protein consists essentially of alpha-lactalbumin and beta-lactoglobulin which represents 11.5% of the total milk proteins. Rose et al. (1970) reported that whey proteins can be divided into four groups: alpha-lactalbumin, beta-lactoglobulin, immunoglobulins and a proteose-peptone fraction, Beta-lactoglobulin has a molecular weight of about 36,000 and represents 7%to 12% of the total skim milk protein and 40% to 60% of the

20 13 whey proteins. This protein is of great interest in protein chemistry, primarily because it belongs to a family of simply prepared, crystalline proteins. In milk, however, beta-lactoglobulin is the principal protein having a free sulfhydryl group in the form of a cysteine residue. From the amino-acid analysis of beta-lactoglobulin, it is known that for each 36,000 molecular weight dimer, it contains two -SH groups and four -SS- linkages (McKenzie, 1971). The interactions of beta-lactoglobulin with kappa-casein, when milk is heated, determine characteristics of milk which are of considerable importance to the dairy industry. For example, the heat stability of skim milk is dependent on the relative amount of kappa-casein and beta-lactoglobulin in the milk. Alpha-lactalbumin is second in concentration to beta-lactoglobulin and has a molecular weight of approximately 14,440. It represents 2% to 5% of total skim milk proteins and contains no free sulfhydryl groups. However, alpha-lactalbumin has a rather high content of cystine and is, therefore, a good source of -SS- groups. The immunoglobulins are present in milk in low concentration and represent 1% to 2% of the total skim milk proteins. Although these proteins contain small amounts of cysteine (-SH), they have a rather high content of cystine. The term "immunoglobulin" is general and applies to a heterogeneous family of large molecular weight proteins that share common physic-chemical characteristics and antigenic determinantes (Rose et al., 1970). Rowland (1938) observed that if milk is heated to 96 c (205 F)

21 14 for 30 min, about 80;'& of the whey proteins are changed so that they are precipitated by acidification to ph 4.6. He considered that this 80}~ represented beta-lactoglobulin and alpha-lactalbumin and that the remaining 20% was a separate protein to which he applied the name proteose-peptone, Rose et al, (1970) defined the proteose-peptone fraction as that portion of the protein system not precipitated by heating at 90 c to 100 c (194 F F) for twenty minutes and subsequent acidification to ph 4.7. They estimated that this fraction represented 2% to &/o of total skim milk proteins and 20% of the whey protein fraction, Recent reports have indicated that the proteose-peptone fraction contains -SH+ -SS- groups (Senter et al,, 1972). The detailed composition of this fraction, however, is unknown, Protein Denaturation When milk is subjected to a heat treatment, changes may occur that affect its physio-chemical properties (Pofahl and Vakaleris, 1968), Neurath et al, (1944) defined denaturation as "any non-proteolytic modification of the unique structure of a native protein giving rise to definite changes in chemical, physical, or biological properties," An important manifestion of protein denaturation involves an unfolding or at least an alteration of the folded structure of a protein molecule without breaking its covalent peptide bonds. Accordingly, protein denaturation can contribute to cooked flavor and texture of foods containing proteins (Jenness and Patton, 1959).

22 15 Larson and Rolleri (1955) showed the effect of heat treatments on individual whey proteins in milk (Fig, 3), The denaturation curves obtained for each of the whey proteins show that the immunoglobulins are rendered acid precipitable by the least heat treatment, followed in order by beta-lactoglobulin and alpha-lactalbumin, The proteosepeptone fraction is the most resistant to heat denaturation and is not denatured at all by the heat treatments applied, An important manifestation of protein denaturation is the increased reactivity of specific groups resulting from structural modification of the protein molecule, The activation of -SH or -SS- groups (Fig, 4) is one of the most readily detectable of the chemical changes accompanying denaturation (Putman, 1953). The heat activated sulfhydryl groups contribute to cooked flavor, affect firmness, and account for antioxygenic properties of milk products (Jenness and Patton, 1959; Webb et al,, 1974), Jenness, 1954 also reported that denaturation of beta-lactoglobulin as measured by sulfhydryl (-SH) activity follows first-order kinetics and has an activation energy of about 80,000 calories per mole, By boiling raw milk in a water bath for JO min, Senter et al,, (1972) reported that the heat-denatured proteins were removed, The remaining undenaturable protein fraction contained approximately 33% of the total -SH + -SS- groups. Reports in the literature, however, are conflicting as to the exact nature of these heat-induced changes, Crowe et al, (1948), Boyd and Gould (1957), Pofahl and Vakaleris (1968), Vakaleris and Pofahl (1968a), and Senter et al, (1972) reported increases after heating in

23 16 -II) E Ol -~ ::::!.,;;.! _J ' z w I- 0 a: Q ;; _ ::l 0::: w en o w 0::: ::l I- ct 2 w 0 z ::> MIN. HEAT TREATMENT ( C) Fig, J - Denaturation of the individual whey proteins in milk heated at various temperatures for JO min (Larson and Rolleri, 1955).

24 17 CYSTEINE CYST I NE COOH I NH 2 -C-H I CH 2 I SH COOH I NH 2 -C-H I CH 2 I s I s I CH2 I NH2-C-H I COOH Fig. 4 - The structure of the principal amino acids containing -SH or -SS- groups in milk proteins.

25 18 -SH with corresponding decreases in -SS-. Other workers have reported decreases in -SH under similar conditions (Larson and Rolleri, 1955; Yoshino et al., 1962; and Sasago et al., 1963). These conflicts may be due to differences in techniques used and to the sensitivities of the methods employed. The published methods for the quantitative estimation of -SH + -SS- groups in whey proteins are as follows: 1. Harland and Ashworth (1945) adapted the use of thiamine disulfide to the determination of sulfhydryl groups in milk in a study of the effects of various heat treatments. 2. Crowe et al. (1948) reported on a modification of Chapman and Mc- Farland's ferricyanide procedure for evaluating sulfhydryl groups in milk. 3. Larson and Jenness (1950a) determined protein sulfhydryl groups with iodine and 0-iodosobenzoate by an amperometric titration. 4. Larson and Jenness (1950b) measured the sulfhydryl groups of milk by an iodimetric titration. 5. Sasago et al. (1963) determined the -SH + -SS- groups in milk by the para-chloromercuribenzoatedithizone method. Fluorometric Determination Recent advances in analytical techniques provide more sensitivity than in the previously published methods for the measurement of -SH and -SS- groups. An example is the fluorometric determination of -SH + -SS- groups. Measurements by this technique are approximately 1,000 times more sensitive than colorimetric and spectrophotometric tech-

26 19 niques, and are capable of almost indefinite extension. In many instances it is possible to determine less than nanogram (.001 µg) quantities (Turner, 1966). The fluorometric detennination of -SH + -SS- groups was first reported by Karush et al. (1964). They demonstrated the sensitivity of the quenching reaction by spraying an alkaline solution of fluorescein mercuric acetate (FMA - Fig. 5) on paper chromatograms with disulfidecontaining spots and measured the quenching reaction in an alkaline solution of disulfide with a fluorometer. The quenching reaction in solution has provided a very sensitive quantitative assay for disulfide groups in proteins and peptides. The theory of the fluorometric technique involves an interaction of the fluorescent molecule in FMA with -SH and -SS- molecules in a highly alkaline solvent solution (1M NaOH). This causes the fonnation of a covalent linkage between the mercury and sulfur atoms that is responsible for the quenching reaction. They also reported that this reaction is approximately a one-to-one reaction between FMA and disulfide. This fact indicates that only one mercury atom per FMA molecule is utilized for combination with sulfhydryl. This is sufficient to quench its fluorescence assay. However, measurements are made in extremely dilute solutions ( µmole/ml), and special precautions must be followed in connection with purity of solvents and cleanliness of glassware. Additional development of Karush's work has provided a very sensitive quantitative assay for disulfide groups in milk proteins and

27 HO 0 0 (CH 3 COO)Hg Hg (OOCCH3) COOH Fig. 5 - Tentative structure of fluorescein mercuric acetate (Fr.IA) (Karush et al., 1964) I\) 0

28 21 peptides. Vakaleris and Pofahl (1968b) adapted Karush's method to the study of -SH + -SS- groups in the milk protein system and applied it to the study of the effects of heat on these groups. Pofahl and Vakaleris (1968) also studied the effect of heat on the -SH + -SS- groups by a spectrofluorometric method. Senter et al. (1972) used a fluorometric procedure to determine -SH + -SS- groups in rennet whey from milk processed by turbulent-flow ultra-high temperature processes. They reported the presence of these groups in the proteose-peptone fraction, Heat treatments in excess of those employed for pasteurization also have an effect on the nitrogen distribution. The principal change, which becomes evident with increasing heat treatment, is a decrease in whey protein nitrogen. These changes in whey protein nitrogen are used as an index to heat treatment by the dry milk industry. The American Dry Milk Institute (1965) modified and adapted Kuramoto's method as a standard procedure for determining heat treatment classification of dry milk products. The following classes were established: Class High heat Low heat Medium heat Whey Protein Nitrogen Not more than 1.5 mgs/gm Not less than 6.o mgs/gm 1.51 to 5,99 mgs/gm. It has been acknowledged that heat activation of -SH + -SS- and changes in whey protein nitrogen distribution in milk can be separately used as an index to heat treatment. However, there are no publications that

29 22 correlate the relationship between -SH+ -SS- and whey protein nitrogen.

30 MATERIALS AND PROCEDURES Equipment 1. Turner Model 111 Fluorometer, Serial No. B-2298, G. K. Turner Associates, Palo Alto, California. 2. Corning Model 12 Research ph Meter, Serial No. A 16399, Scientific Instrument, Corning Glass Works, Medfield, Massachusetts. J. Semimicro-Kjeldahl Digestion and Distillation Apparatus (Fig. 6). 4. Babcock Cream-test Apparatus. 5. Mojonnier Apparatus, Model No. D-1589, Mojonnier Bros. Co., Chicago, Illinois. 6. Two-stage homogenizer, Model A, cap. 200 gal/hr, press. JOOO psi, Union Steam Pump Co., Battle Creek, Michigan.?. Mettler Type B-6 Semimicro-balance. 8. Triple Glass-distilled Water Apparatus (Fig.?). 9. Kimble 10-ml volumetric Flasks for light-sensitive materials, Transmission of glass is ~ at JOO mµ, 1% at 400 mµ, and 4% at 500 mµ. 10. Laboratory helically coiled tube heat exchange system. 11. Recording Instrument, Taylor Instrument Companies. 12. Delaval Separator-clarifier No. 240, The Delaval Separator Co., Poughkeepsie, Chicago, Illinois. Reagents 1. L-cysteine (Free base), M.W , Nutritional Biochemicals 23

31 r.. I., '~ Fig. 6 - Semimicro-Kjeldahl digestion and distillation apparatus. ~

32 25 Fig. 7 - Triple glass-distilled water apparatus.

33 26 Corp., Cleveland, Ohio. 2. Glutathione (Oxidized), M.W , Nutritional Biochemicals Corp., Cleveland, Ohio. 3. Fluorescein Mercuric Acetate, Nutritional Biochemicals Corp., Cleveland, Ohio. 4. Sodium Hydroxide pellets, Chemically pure reagent. 5. Hydrochloric Acid (Sp gr 1.19), Chemically pure reagent. 6. 7x Cleaning Glassware, Linbro Chemical Co., Inc., New Haven, Connecticut.?. Selenized Hengar Granules, Hengar Co., Philadelphia, Pennsylvania. 8. Sulfuric Acid (Sp gr 1.84), Chemically pure reagent. 9. Boric Acid: Z'b solution. 10. Indicator: Two parts.2% alcoholic methyl red solution and 1 part.2% alcoholic methylene blue solution. 11. Petroleum Ether, B.P. 45-6o 0 c. 12. Ethyl Alcohol, Chemically pure reagent. 13, Ammonium Hydroxide (28% NH 3 ), Strong ammonia solution. 14. Anhydrous Ethyl Ether, Chemically pure reagent. Preparation of Half-and-Half The homogenized half-and-half products for this study were prepared from raw milk obtained from the university herd. The raw milk was separated into skim milk and heavy cream. The fat content of the cream was determined by the Babcock procedure (American Public Health

34 27 Association, 1972). The weight of skim milk required to standardize the cream to 10.5"fo milk fat was calculated and added to the cream. The percentage fat in the standardized half-and-half was also checked by the Rose-Gottlieb ether extraction procedure (American Public Health Association, 1972). The half-and-half was warmed to 6o 0 c, homogenized at 2,500 psi, rapidly cooled to 4.4 c in a laboratory helically coiled tube heat exchanger, and stored at 3.3 c, until processed. The homogenized half-and-half was heated to the desired temperature in the fifty gallon vat equipped as required by the U.S. Department of Health, Education, and Welfare (1973) for processing of Grade "A" dairy products. (This vat was equipped with an air-space heater, inlet and outlet valves, and an indicating and a recording thermometer.) The product was agitated during the entire heat treatment process, immediately cooled to 3.3 c in a laboratory helically coiled tube heat exchanger, and packaged in steril containers. Percent fat and total solids of the final product were determined by the Rose- Gottlie b ether extraction procedure (American Public Health Association, 1972). The homogenized half-and-half contained percent fat and 19.3 ± 0.1 percent total solids (Table 1). Preparation of Acid Whey Fraction Acid whey was prepared from the half-and-half products by a slight modification of the procedure of Swanson and Sanderson (1967) for separating whey proteins in skim milk at ph 4.6. The sample was warm-

35 Table 1 - Fat and total solids in half-and-half and acid whey filtrates. Half-and-half Acid whey filtrate Heat t5eatment Total Fata Total solidsa Fata solidsa ( c) (~~) (%) (%) (~;) 6ob ob ob ob Meand 6.36 ± C I\) ()) Meand 6.11 ± ,9c o o C ,2C Meand 5.88 ± 0.06 a Average of duplicate tests. bpreheat temperature, no holding time. 0 Processing time 30 min. dmean average ± standard. deviation.

36 29 ed to 4 c and the ph adjusted to 4.7 by slow addition of 1M HCl. It was then warmed to 30 c in a water bath. The caseins, fat and heat denatured proteins coagulated and formed a gel. The gel was broken by gentle stirring and the ph adjusted to 4.6 with 1M HCl. The mixture was then filtered through Whatman No. 40 paper and the filtrate collected as acid whey (Fig. 8). The mean percentage of fat and total solids was 0.02 ± 0.02 and 6.12 ± 0,06, respectively. The clear appearance and low fat content of the acid whey were necessary to make accurate fluorometric analysis. There were no significant changes in percentages of fat in acid whey from half-and-half processed by the different heat treatment (Table 1). However, the percentages of total solids in acid whey from half-and-half processed in the range of 65.6 c to 71.1 c for JO min showed a significant decrease {P <.05), and additional significant decreases (P <.05) were observed for processes between 73.9 c to 93.3 c. The mean average percentage of total solids in acid whey from unpasteurized half-and-half was 6,36 ± 0,07. Fluorescence of FMA The optimum FMA concentration which would give the amount of fluorescence suitable for these measurements was determined by testing a series of standard FMA solutions. A 100 µm stock solution of FMA was prepared by weighing 8.5 mg into a 100-ml volumetric flask and diluting to volume with 0.1M NaOH. From this stock solution, aliquots were diluted with 0.1M NaOH to give a series of working solutions

37 30 Fig. 8 - Filtration of precipitated milk proteins. Precipitate contains caseins, denatured whey proteins, and fat. Filtrate (acid whey) contains undenatured whey proteins, lactose and soluble salts.

38 )1 having the following concentration of FMA: 10,0 µm, 5.0 µm, 1,0 µm, and 0,5 µm, From each working solution, 1 ml was transferred to a 10- ml ruby-colored flask and diluted to volume with 1M NaOH, As shown in Table 2 and Figure 9, the 2 µm and 5 µm FMA working solutions gave readings in the range of 20% to 60% transmission which is the highest accuracy range for the fluorometer. Since the 5 µm solution was suitable for the quenching reaction, all further work was done with a 5,0 µm FMA working solution, This solution was prepared daily due to possible deterioration by exposure to light, After determining the optimum FMA concentration it was necessary to select the best reaction time to insure the maximum percent decrease in fluorescence, Karush et al, (1964) reported that the FMA undergoes slow degradation in 1M NaOH and that it was greatly accelerated by exposure to room light, The effect of time on F'MA in 1M NaOH stored in the dark was investigated by preparing a sample containing 1 ml of 5 µm FMA working solution and 9 ml of 1M NaOH, As shown in Table J and Figure 10, the percentage decreases of fluorescence leveled off at lj,1% in 40 minutes, Vakaleris and Pofahl (1968b) reported similar results in their studies, Therefore, all further work was done using a reaction time of one hour to insure the maximum percent decrease in fluorescence, Standard Curve Determination In order to measure the concentration of -SH + -SS- groups in acid whey, a standard curve was prepared from a stock solution of ox-

39 32 Table 2 - Effect of concentration on fluorescence of fluorescein mercuric acetate in 1M NaOH. FMA Cone.a Fluorescence reading (%) Sample (µm) A B Mean , ,0 63, a Concentration of working solution

40 33 w (...) 2 LU (...) CJ) L J o:: 0 ::::> _J lj._,,,,_ ' LU (...) u:: ltj a.., ~ ~ ~ 120 ~ ~,, 0..~,_~---~-'-~-'-~-'-~..L..-~L---1~---l CONCENTRATION OF FLUORESCE~N MERCURIC ACETATE (µ.m) Fig. 9 - Fluorescence of various fluorescein mercuric acetate concentration in 1M NaOH solution.

41 34 Table 3 - Effect of time on fluorescence of fluorescein mercuric acetate in 1M NaOH. Fluorescence reading Time A B Mean Decrease in fluorescence (min) (%) (%) (1~) (%) , , , , , ,

42 w 12 u z w II u en LiJ 10 c::: 0 :::> _J lj.. z 9 8 l1j 7 en <( l.ij IZ u Lu I- 4 z Lu u a::: w Q. 3 2 Oo-~~._~~~_._~~--'~~~-"-~~~'--~~-J TIME IN MIN. Fig Percent decrease in fluorescence of fluorescein mercuric acetate in 1M NaOH in the dark at 25 c.

43 J6 idized glutathione and L-cysteine that was 100 µm with respect to the -SH and to the -SS- groups. This solution was prepared by dissolving mg of oxidized glutathione and mg of L-cysteine in 100 ml of triple distilled water. From this stock solution, the following working solutions of -SH + -SS- were prepared in triple distilled water: 2 µm, J µm, 4 µm, and 5 µm. One ml of 5 µm FMA working solution was pipetted into each of six ruby-colored, 10-ml volumetric flasks. One ml of triple distilled water was added to the first flask to obtain total fluorescence, i ml or 1 ml from the above working solutions was pipetted into each of the five remaining flasks to give the following additions of -SH + -SS- to each flask:.002,.003,.o04,.005, and.006 µmole, respectively. Each flask was diluted to volume with 1M NaOH, the contents were mixed thoroughly, and held in the dark for one hour at room temperature. The fluorescence of each sample was determined with a Turner Model 111 fluorometer at an excitation wavelength of 415 to 4J6 nm and 510 nm for emission. The fluorescence readings were then converted to percent quenching by the following formula: % quenching = Total fluorescence - fluorescence reading of sample x loo. Total fluorescence For the standard curve, percent quenching and the corresponding -SH + -ss- concentration are presented in Table 4 and plotted in Figure 11. This curve shows a direct proportion between percent quenching and -SH + -SS- concentration. The slope is 10,256 and the intercept is The linear regression is:

44 37 Table 4 - Relationship between standard concentrations of -SH + -SS- and percent quenching by fluorometric measurement. Fluorescence reading -SH + -SS- A B Mean Percent (µm) (%) (~~) (%) quenching Blank 66.o 66.o Blank Blank Blank : Blank Blank Blank oo

45 J8 50 (.!) z I 40 u z w :::> CJ 30 I- Z w u 0:: 20 w 0... y = 10256x SH+-SS-(JL MOLES/ ml) Fig Standard. curve for fluorometric detennination of -SH + -SSgroups.

46 39 Cone. of -SH+ -ss- in µmoles per ml= Percent guenching + z.o, 10,256 From this standard curve, concentrations of -SH + -SS- groups in acid whey were determined. Acid whey samples were diluted 1:500 with triple distilled water for analysis of acid whey from half-and-half that was heated in the range of 6o,o 0 c to 79,4 c for JO min, For higher heat treatments, 82.2 c to 93,3 c, it was necessary to lower the dilution to 1:250 in order to bring the fluorescence reading of -SH+ -SS- within the range, Measurements of the -SH + -SS- in the diluted acid whey were conducted by the procedure that was used for measuring -SH + -SS- in the standard curve, except that 1 ml of diluted acid whey was used instead of 1 ml of the working solution of -SH + -ss-. Whey Protein Nitrogen Determination Whey protein nitrogen was determined by a modification of the AOAC (1970) method. 1. One ml of well mixed acid whey sample was transferred into a 100- ml Kjeldahl digestion flask. 2. A selenized Hengar granule and 6 ml of concentrated sulfuric acid (Sp gr 1.84) were added to the sample in the flask. 3. A blank determination was made as a check on reagents and technique by adding 1 ml of distilled water instead of the acid whey sample.

47 40 4. Percent recovery-1 of nitrogen was made by adding 1 ml of standard ammonium chloride solution instead of the acid whey sample. 5. The mixture was digested slowly at first at a low temperature, and gradually increased after white fumes (so ) appeared in the digestion flask The digestion was continued until the color became water clear. Any remaining amber color indicated that digestion was incomplete, and that further digestion was needed.?. The digestion flask and its contents were cooled to room temperature and 15 ml of distilled water were added. The contents of the digestion flasks were transferred to a 500-ml vacuum-jacketed distillation flask. Each flask was rinsed three times with 3 ml portions of distilled water. Twenty milliliters of 50% carbonatefree NaOH solution was added by allowing it to run slowly down the side of the distillation flask. 8. The distillation flask was mounted on the steam distillation unit. The distillate, containing the released ammonium nitrogen, was collected in a 150-ml beaker containing 20 ml of ;:% boric acid and 3 drops of indicator. The tip of the condenser tube was kept below the surface of the acid. Distillation was continued until 100 ml of distillate was collected in the beaker. Then the beaker and 1 calculations of percent recovery of nitrogen were by the following formula: % Recovery nitrogen ~ Net ml of HCl x Nonnality x 14 x 100 Milligrams of nitrogen added '

48 41 its contents were lowered from the condenser and distillation was continued for one minute, The end of the condenser tube was rinsed with distilled water into the receiving beaker, 9. The distillates were titrated with standard HCl solution to the first permanent tinge of lavender color, Calculations of percentage of nitrogen were by the following formula: % Nitrogen = Net ml of HCl x Normality x 0,014 x 100, Wt, of sample (1 e) 2 2Based on 1 ml of acid whey = 1 g,

49 RESULTS AND DISCUSSION SulfhJ'.:dz:Yl and Disulfide Groups The effect of various heat treatment temperatures of half-andhalf on the concentration of -SH + -SS- in acid whey are shown in Figure 12. As the heat treatment temperatures were increased from 65.6 c through 82.2 c, concentrations of -SH + -ss- in the acid whey progressively decreased. The linear regression curve for µmoles of -SH + -SS- per ml of whey (Y) versus processing temperatures (X) is: There were no additional decreases in the concentration of -SH + -SSby heat treatments in the temperature range of 82.2 c to 93.3 c (Table 5). The percentage decrease in the -SH + -SS- content of acid whey from half-and-half is shown in Figure 1J. The linear regression curve for percent decrease of -SH + -ss- per ml of whey (Y) versus the processing temperatures (X) is: For every 2.75 c increase in temperature, the decrease in -SH+ -SSis 11.09%. The fluorometric measurements of -SH + -SS- in acid whey from )Obtained from a least squares computer program. It is valid only from 65.6 c to 82.2 c for JO min processing time. 42

50 4) E 0 ' en 1.6 Lt.I _J 0 c- ~ I en en I + J: en I y = x TEMP. C, 30 MIN. HOLD Fig, 12 - Effect of various heat treatments of half-and-half on concentration of -SH + -SS- in acid wheys as determined by fluorometric measurement,

51 Table 5 - Effect of heat treatments of half-and-half on changes in concentration of -SH + -SS- and whey protein nitrogen in acid whey. Hean decrease in whey Mean -SH + -SS- Mean decrease Mean whey protein Heat treatment in wheya in -SH + -SS-a protein nitrogena nitrogena (OC) (µmoles/ml) (%) (mg/ml) (%) 60.ob 2.20 ± ± ± ± ± ± ± ± ± ± c 1.53 ± ± ± ± ± o ± c 0.95 ± ± J ± o ± ± ± 3.48 o.44 ± ± C o.46 ± o ± ± ± o ± ± ± ± J.3C o.42 ± o ± 3.89 aaverage of triplicate determinations + standard deviation bttomogenization temperature, no holding time CHolding time 30 min t=

52 45 I CJ) 100 en I :c en I z 60 L J (/) ~ LU r~ 40 (.) t1j 0 t- z 20 w (.) cc L J a_ 0 y=4.031x TEMP. C, 30 MIN. HOLD 0 Fig Percent decrease in -SH + -SS- of acid whey by various heat treatments of half-and-half.

53 46 half-and-half as a function of various heat treatments are recorded in Table 3, The data are expressed as the average of triplicate determination + standard deviation, The results show that there are no substantial variations in the -SH + -SS- content of acid whey from unpasteurized half-and-half, Similar results were reported by Senter et al, (1972) for raw milk, They found that the mean -SH + -SS- concentration in rennet whey was 2,89 ± 0,26 µmoles/ml and that 32% was located in the proteose-peptone fraction and undenaturable proteins, In acid whey from half-and-half that was processed between 82,2 c and 93,3 c, the average concentration of -SH + -SS- was 0,44 ± 0,04 µmoles/ml, These data show that 807~ of the -SH + -SS- groups of acid whey reside in the denaturable whey proteins and 20fo in the proteosepeptone and undenaturable whey proteins, The difference in percentages of -SH + -SS- in the proteose-peptone and undenaturable whey proteins in acid whey and in rennet whey are primarily due to the method of separation (acid vs, rennet) and to the different kind of products (half-and-half vs, milk), Whey Protein Nitrogen Effects of various heat treatments of half-and-half on the percent decrease in whey protein nitrogen (WPN) are shown in Figure 14, As processing temperatures were increased from 65,6 c to 79.4 C for JO min, the concentration of whey protein nitrogen in the acid whey progressively decreased, The linear regression curve for percent decrease of whey protein nitrogen (Y) versus processing temperatures (X)

54 47 z CL ~ ;.> z 80 ljj (/) <{ 60 0 L!J a:: (.) t J 0 40!- Z' w (.) a:: 20 L!J a. y=3.231x '---'-~~--L-~~-L~~--1..~~--1~~.J TEMP. C, 30 MIN. HOLD Fig Percent decrease in whey protein nitrogen of acid whey by various heat treatments of half-and-half,

55 is: Y = 3.231x There were no significant changes in whey protein nitrogen by heat treatments in the temperature range of?9.4 c to 93.3 c. A comparison between percentage decrease in -SH + -SS- and whey protein nitrogen is shown in Figure 15. The results show that these two methods are very closely related, as indicated by the correlation coefficient of In addition, these results show that the fluorometric measurement of -SH + -SS- in acid whey from half-and-half is a reliable index to heat treatments in the range of 65.6 c to 82,2 c for 30 min processing times. The fluorometric measurement of the -SH + -SS- reflected a wider range of heat treatment of 65.6 c to 82.2 c as compared to 65,6 c to 79.4 c for whey protein nitrogen by the semimicro-kjeldahl method (Table 5), Therefore, it appears that the fluorometric measurement of -SH+ -SS- is a more sensitive test than the semimicro-kjeldahl as an index to heat treatments of half-and-half, Table 6 shows a comparison of the amounts of -SH + -SS-, whey protein nitrogen, and whey protein remaining in acid whey from halfand-half and milk processed by various heat treatments, Percentage calculations for milk are based on 100% remaining after heat treatment of 4o 0 c for 30 min, and for half-and-half, the percentages are based 4obtained from the least squares computer program, only from 65,6 c to 79,4 c for 30 min processing time, It is valid

56 49 100, ~ ~ 2! Q_ ~ ;;..> z 80 L.J CJ) 60 <{ l J ~ u L J Cl 40 t- y = 0.829x r=o. 99 Lu u ~ 20 Lu a.. 0'--~~--'~~~--'"~~~--~~~_,_~~~~ PERCENT DECREASE IN -SH+ -SS- Fig Comparison between decreases in -SH + -SS- and whey protein nitrogen from 65.6 c to 79.4 c for JO min holding time.

57 50 Table 6 - Percentages of -SH + -SS-, whey protein nitrogen, and whey proteins remaining in acid whey from half-and-half and milk after processing at various temperatures, 30 min heat Half-and-half Milk treatment -SH + -ss-a Whey protein nitrogenb Whey proteinc (oc) (%) (7~) 0~) od 100.od , , ,5 50, ,0 23, ,5 40, asulfhydryl plus disulfide concentrations were determined by fluorometric measurement and percent decrp.ases calculated by the following formula: Y = 4.0J1X - 253,262. bwhey protein nitrogen concentrations were determined by semimicro- Kjeldahl and percent decreases calculated by the following formulas Y = 3,231x cpercent whey protein nitrogen was determined by micro-kjeldahl (Rowland, 1938) and calculated as whey protein (%N x 6.JB), Percentage decreases were based on 100}~ for heat treatments of 40 c with JO min holding time. dpercentage decreases were based on 10~fa for heat treatments of 6o 0 c with no holding time,