This dissertation has been 1 Q-j C microfilmed exactly as received PEREIRA, Ronald Roy, 1931- VOLATILE SULFUR COMPOUNDS FROM HIGH HEAT TREATMENT OF MILK. The Ohio State University, Ph.D., 1965 Food Technology

University Microfilms, Inc., Ann Arbor, Michigan VOLATILE SULFUR COMPOUNDS FROM

HIGH HEAT TREATMENT OF MILK

DISSERTATION

Presented in Partial Fulfillment of the Requirements for the Degree Doctor of Philosophy in the Graduate School of The Ohio State University

By

Ronald Roy Pereira, B. S., M. Sc.

X X X X X -X-X-X

The Ohio State University 1965

Approved by

Adviser Dejfca^tmient of Dairy Technology ACKNOWLEDGMENTS

I wish to express my sincere appreciation to Dr. W. J. Harper,

Department of Dairy Technology, for directing the work of this

investigation and for his assistance in the preparation of this

manuscript.

My sincere appreciation is also expressed to Dr. I. A. Gould,

Chairman of the Department of Dairy Technology, for providing the

opportunity for graduate study and for his invaluable advice and

encouragement throughout my graduate program.

I am indebted to Dr. T. Kristoffersen, Department of Dairy

Technology, for his aid in the preparation of this manuscript, and

to the entire staff of the Department for their inspiration and

cooperation during my graduate program.

I wish to extend my gratitude to Dr. H. R. Conrad, Department

of Dairy Science, Wooster Experimental Station, for supplying the

radioactive milk used in this study.

My wife, Mary, deserves much of the credit for the successful

completion of my graduate program, for without her understanding and

encouragement, this task would have been impossible.

ii CONTENTS

Page

ACKNOWLEDGMENTS...... ii

TABLES ...... vi

ILLUSTRATIONS...... xii

INTRODUCTION ...... 1

REVIEW OF LITERATURE...... 3

Volatile Sulfur Compounds in Different Food Products Formed by Heat T r e a t m e n t...... 3 Vegetable Products ...... 3 Meat Products ...... 9 Milk and Milk P r o d u c t s ...... 11 Factors Affecting Heat-Release of Sulfur Compounds from Milk and Milk Products ...... 13 Mechanism of Formation of Volatile Sulfur Compounds by H e a t ...... 19 Vegetable P r o d u c t s ...... 19 Meat Products...... 2k Dairy Pr o d u c t s ...... 26 Volatile Sulfur Compounds Resulting from Irradiation of Food Products ...... 28 Meat Products...... 28 M i l k ...... 31 Light Irradiation of M i l k ...... 32 Sunlight Flavor...... 32 The Mechanism of Formation of Volatile Sulfur Compounds by Irradiation...... 3^ Gamma Irradiation ...... 3^- Light Irradiation...... 38

SCOPE OF INVESTIGATION...... kl

EXPERIMENTAL PR O C E D U R E ...... b2

iii CONTENTS (contd.)

Page

EXPERIMENTAL RESULTS ...... 65

Development of Experimental Procedures ...... 65 Corrections for Radioactivity Determinations...... 66 Colorimetric Determination of Hydrogen Sulfide ...... 72 Colorimetric Determination of Mereaptans ...... 7^ Concentration of Sulfur-35 in Milk Labelled with Different Radioisotopes...... 75 Total Radioactivity...... 75 Total Sulfur Content and Radioactivity of Milk and Milk Proteins...... 76 Free Volatile Sulfur Compounds from Unheated Sulfur-35 Labelled M i l k ...... 79 Effect of Heat Treatment on Release of Volatile Sulfur Compounds from Sulfur-35 Labelled M i l k ...... 8l The Effect of Heat Treatment on Milk Labelled with Various Chemical Forms of Sulfur-35 Sulfur Compounds...... 82 Sodium Sulfide Labelled M i l k ...... 82 Methionine Labelled M i l k ...... 8^ Sodium Sulfate Labelled M i l k ...... 811- Barium Sulfide Labelled M i l k ...... 87 Comparison of Volatile Sulfur Compounds Produced from Heat Treatment of Different Sulfur-35 Labelled Milk at 9°°C _for Two Heating Periods...... 89 Volatile Sulfur Compounds from Various Fractions of Mi l k ...... 92 Factors Affecting the Heat Release of Volatile Sulfur Compounds ...... 102 The Relationship Between Total Concentration of Hydrogen Sulfide and Radioactive Hydrogen Sulfide ...... 115 Effects of Freezing on the Thiamine Disulfide Values of Milk and Milk Products...... 321. Identification of Volatile Compounds from Heated Milk Labelled S u l f u r - 3 5 ...... 1211-

DISCUSSION ...... 125

iv CONTENTS (contd.)

Page

SUMMARY...... 131

APPENDIX ...... 135

REFERENCES...... 169

AUTOBIOGRAPHY...... l8l

v TABLES

Table Page

1. The Reproducibility and Per Cent Recovery of Total Sulfur ...... 65

2. Self Absorption of Sulfur-35 by Various Materials .... 67

3. Counting Efficiency and Figure of Merit of Radioactive Barium Sulfide in the Presence of Aqueous 3 Per Cent Mercuric Chloride and ^ Per Cent Mercuric Cyanide Solutions...... 69

b. Counting Efficiency of Radioactive Barium Sulfide in the Presence of Aqueous 8 Penitent Lead Acetate Using p-dioxane-methyl Cellosolve as Scintillating Solvent...... 70

5. The Effect of Aqueous 8 Per Cent Lead Acetate Solution on the Counting Efficiency of Sulfur-35 and Figure of Merit Value of Radioactive Barium Sulfide .... 71

6 . The Effect of Sequence of Addition of Reagent on the Development of Methylene Blue Color Intensity .... 73

7. The Optical Density Reading of Mercaptan Concentrations for Milk and Milk Products Heated at 90°C for Various Lengths of Time * ...... jb

8 . The Effect of Types of Sulfur-35 Precursors on the Radioactivity of Milk ...... 76

9. Total Sulfur and Sulfur-35 Content of Various Milks and Milk Fractions and Their Corresponding Specific Activities ...... 78

10. Free Volatile Sulfur-35 Compounds from Raw Unheated Sulfur-35 Labelled Milk ...... 80

11. Effects of Heat Treatments at 80°C and 90°C on the Formation of Volatile Sulfur Compounds from Sodium Sulfate Sulfur-35 Labelled M i l k ...... 82

vi TABLES (contd.)

Table Page

12. The Effects of High Heat Treatment at 90°C of Volatile Sulfur-35 from Milk Obtained Following Incorporation of Sulfur-35 S u l f i d e ...... 83

13. The Effect of Heat Treatment of Methionine Sulfur-35 Labelled Milk on the Formation of Volatile Sulfur-35 Compounds...... 85

1^. The Effect of Heat Treatment at 90°C on Sodium Sulfate Sulfur-35 Labelled Milk on the Release of Volatile Sulfur Compounds ...... 87

15. The Effect of Heat Treatment at 9°°C on Barium Sulfide Sulfur-35 Labelled Milk on the Release of Volatile Sulfur C o m p o u n d s ...... 88

16. The Effect of High Heat Treatment at 90°C for 30 and 60 Minutes of Different Milks Labelled with Different Sulfur-35 Radioisotopes ...... 90

17. Free Volatile Sulfur Compounds in Various Fluid Milk Products ...... 93

18. The Effect of Heat Treatment at 90°C on Barium Sulfide Sulfur-35 Labelled Skimmilk on the Release of Volatile Sulfur-35 Compounds ...... 9^

19. The Effect of Heat Treatment at 90°C on Barium Sulfide Sulfur-35 Labelled Cream on the Release of Volatile Sulfur-35 Compounds...... 96

20. The Effect of Heat Treatment at 90°C on Barium Sulfide Sulfur-35 Labelled Buttermilk on the Release of Volatile Sulfur-35 Compounds...... 98

21. The Effect of Heat Treatment at 9°0C on Barium Sulfide Sulfur-35 Labelled Acid and Rennet Whey on the Release of Volatile Sulfur-35Compounds ...... 99

vii TABLES (contd.)

Table Page

22. The Effects of High Heat Treatment at 90°C for 30 and 60 Minutes of Various Fractions of Milk Obtained from Barium Sulfide Sulfur-35Labelled Milk ...... 101

23. The Effect of pH on the Formation of Volatile Sulfur Compounds as a Result of High Heat Treatment of Sulfur-35 Labelled Barium Sulfide Mi l k ...... 103

2k. The Effect of Non-Radioactive Cystine and Cysteine Added to Sulfur-35 Barium Sulfide Labelled Milk on the Formation of Volatile Sulfur-35 Compounds After High Heat Treatment...... 106

2 5 . The Effect of p-chloromercuric Benzoate on the Formation of Volatile Sulfur-35 Compounds from Heat Treatment of Barium Sulfide Sulfur-35 Milk . . . 109

26. The Effect of Sulfur-35 Labelled Milk Saturated with Air on the Formation of Volatile Sulfur Compounds Produced by High Heat Treatment...... 112

27. The Effect of Sulfur-35 Labelled Sodium Sulfate Milk Saturated with Nitrogen on the Formation of Volatile Sulfur Compounds Produced by High Heat Treatment . . 113

28. The Concentration and Specific Activity of Hydrogen Sulfide Produced from Barium Sulfide Sulfur-35 Labelled Milk, after Heat Treatment at 90°C for Various Lengths of Time ...... 116

29. Effect of pH on the Formation of Hydrogen Sulfide and Specific Activity Value of Milk Heated to 90°C for 60 Minutes ...... 119

30. The Effect of Added Cystine and Cysteine to Milk on the Formation of Hydrogen Sulfide and Specific Activity after Heat Treatment at 90°C for 60 Minutes...... 120

viii TABLES (contd.)

Table Page

31. Total Hydrogen Sulfide and Specific Activity of Milk Saturated "with Air Prior to Heat Treatment at 90°C for 60 minutes...... 121

32. The Thiamine Disulfide Values of Milk, Skinnnilk and Cream as Determined after Storage under Freezing Conditions at -lh°C...... 123

Appendix Table

33. Information Relating to the Type of Isotope used, the Size of Dosage, Method Of Administration and the Number of Cows Involved in Obtaining Radioactive Sulfur-35 Milk ...... 137

3h. Summary of Radioactivities of Different Batches of Barium Sulfide -s35 Labelled M i l k ...... 138

35* Radioactivities of Consecutive Lots of Milk Obtained at Six Hour Intervals Following Oral Administration of Sodium Sulfide Sulfur-35 L a b e l l e d ...... 139

36. Radioactive Counts of Milk Obtained Following Intravenous Infusion of Sulfur-35 Labelled Methionine ...... lhO

37. Summary of Radioactivities of Different Batches of Sodium Sulfate Sulfur-35 Labelled Milk ...... lhl

3 8 . Total Sulfur and Sulfur-35 Content of Various Milks and Milk Fractions ...... lh2

39* Effect of Heat Treatment at 80 and 90°C on the Formation of Volatile Sulfur Compounds from Sodium Sulfate Sulfur-35 M i l k ...... 1^3

ho. The Effect of High Heat Treatment at 9°°C on the Formation of Volatile Sulfur-35 Compounds of Sulfur-35 Sodium Sulfide Labelled M i l k ...... lhh

ix TABLES (contd.)

Appendix Table Page

hi. The Effects of Heat Treatment at 90°C on Sodium Sulfate Sulfur-35 Labelled Milk on the Release of Volatile Sulfur C o m p o u n d s ...... 1^.5

k2. Effect of Heat Treatment at 90°C on Barium Sulfide Sulfur-35 Labelled Milk on the Release of Volatile Sulfur Compounds ...... 1**6

k3. Effects of Heat Treatment at 90°C on the Types and Distribution of Volatile Sulfur Compounds from Milk, Cream and Buttermilk of Sulfur-35 Sodium Sulfide Labelled Mi l k ...... 1^7

kh. Effect of Heat Treatment at 90°C on Barium Sulfide Sulfur-35 Labelled Skimmilk on the Release of Volatile Sulfur Compounds...... l W

1*5. Effect of Heat Treatment at 90°C on Barium Sulfide Labelled Cream on the Release of Volatile Sulfur Co m p o u n d s ...... 1^9

k6. Effect of Heat Treatment at 90°C on Barium Sulfide Sulfur-35 Labelled Buttermilk on the Release of Volatile Sulfur Compounds...... 15°

hj. Effect of Heat Treatment at 90°C on Barium Sulfide Labelled Acid Whey on the Release of Volatile Sulfur Co m p o u n d s ...... 151

i*8 . The Effect of Heat Treatment at 90°C on the Type and Distribution of Volatile Sulfur Compounds from Barium Sulfide Sulfur-35 Labelled Rennet Whey .... 152

i*-9- The Effect of pH on the Release of Volatile Sulfur Compounds from Barium Sulfide Sulfur-35 Milk after High Heat Treatment at $Q°C for 60 m i n u t e s ...... 153

50. The Effect of Heat Treatment at 90°C for 60 minutes in the Presence of Cystine and Cysteine on the Release of Volatile Sulfur Compounds. Milk was Labelled with Barium Sulfide S u l f u r - 3 5 ...... 15^

x TABLES (contd.)

Appendix Table Page

51. The Effect of Sulphydryl Blocking Agent, p-chloro- mercuribenzoate on the Release of Volatile Sulfur Compounds after High Heat Treatment at 90°C for 60 M i n u t e s ...... 155

52. Effect of Air on the Release of Volatile Sulfur Compounds after High Heat Treatment at 90°C for 60 minutes of Barium Sulfide Sulfur-35 Milk ...... 156

53* The Formation of Volatile Sulfur-35 Compounds from Sodium Sulfate Sulfur-35 Labelled Milk Aspirated with Nitrogen for Two Hours Prior to Heat Treatment . . 157

5k. The Relationship Between Nonradioactive and Radioactive Hydrogen Sulfide of Barium Sulfide Sulfur-35 Milk and Milk Products...... 158

55* Effect of pH on the Formation of Hydrogen Sulfide of Milk Heated to 90 C for 60 Mi n u t e s ...... 160

56. The Effect of Added Cystine and Cysteine to Milk on the Formation of Hydrogen Sulfide after Heat Treatment at 90°C for 60 M i n u t e s ...... l6l

57* The Formation of Volatile Sulfur-35 Compounds from Barium Sulfide Sulfur-35 Labelled Milk Aspirated with Air for Two Hours prior to Heat Treatment ...... 162

xi ILLUSTRATIONS

Figure Page

1. Heating Apparatus and Differential Trapping System Utilized for the Detection of Volatile Sulfur Compounds inMi l k ...... kk

2. Standard Curve for the Determination of Total Sulfur ...... 50

3. Hydrogen SulfideStandard Cu rve...... 57

k. Thiamin Disulfide StandardCu r v e ...... 63

Appendix Figure

5. Chromatogram of 2,lj-DNPH Derivatives from Sodium Sulfate Sulfur-35 Labelled Milk Heated to 90°C for 30 Minutes ...... 163

6 . Chromatogram of 2, J+-DNPH Derivatives from Sodium Sulfate Sulfur-35 Labelled Milk Heated to 90°C for 1 Hour ...... 165

7. Chromatogram of 2,U-DNPH Derivatives from Sodium Sulfate Sulfur-35 Labelled Milk Heated to 90°C for 2 Hours...... 167

xii INTRODUCTION

The chemistry of flavor components responsible for imparting characteristic food flavor is a relatively new field. Present know­ ledge of flavor chemistry is still fragmentary and obscure, because of the large number of components that may contribute to flavor,

the low concentration of compounds present and the high degree of

susceptibility of many of these compounds to chemical changes

during analysis.

In the past two decades great advances in the area of food

flavor research have been attained. Knowledge of flavor compounds

responsible for imparting characteristic flavors has been advanced

in a large variety of food products which include vegetable products, meat products and different beverage products.

In recent years, the processing of milk and milk products

has changed with the utilization of higher heat treatments for pro­

cessing. Milk is a very complex system and undergoes both physical

and chemical changes when subjected to high heat treatments. Among

the changes which are known to occur are protein destabilization,

lactose degradation, acid production, the development of browning

and changes in flavor. An important aspect in flavor alteration

is the appearance of the so-called "cooked" flavor, which has been

a major deterent to the production of fresh flavored sterile milk

products. The formation of hydrogen sulfide has been associated

1 with the development of cooked flavor for many years. However, the role of other volatile sulfur compounds in cooked flavor pro­ duction has received little attention. Recently, investigations in other areas of food flavor research have shown that volatile organic sulfur compounds such as mercaptans, sulfides and/or di­ sulfides and sulfur containing carbonyl compounds are important contributors to the flavor of a variety of cooked foods. Infor­ mation on the nature of sulfur compounds other than hydrogen sulfide and nonvolatile sulphydryl compounds in heated milk and factors associated with their formation would be useful in gain­ ing a better understanding of heat induced changes in milk and might lead to the eventual elimination of heat induced off flavors in fluid milk products. REVIEW OF LITERATURE

This review will consider the volatile sulfur compounds in food products in relationship to food flavors, the chemical nature of these compounds and the mechanism of their formation. Since much of the recent work on volatile sulfur compounds and flavor has been related to non-dairy foods, the role of sulfur compounds in cooked foods will be given major attention.

Volatile Sulfur Compounds in Different Food Products Formed by Heat Treatment

Volatile sulfur compounds have been associated with the flavor of such foods as vegetable products, meat products, and dairy products.

Vegetable Products

Cabbage

The volatile components of cabbage have been studied rather extensively (5 , 34, 38, 52, 6 0 , 7 1 , 7 9 , 131).

Garbutt and Master (52), in their investigation on the losses of volatile compounds during cooking green vegetables, described the presence of volatile sulfur from cooking cabbages as sulfide sulfur, but made no attempt to identify any of the volatile sulfur compounds.

3 Dateo et al. (3 8 ) and Hasselstrom (6 0 ) utilized a dif­ ferential trapping system to collect the volatiles removed from cooked cabbage by aspiration with nitrogen gas. The system consisted of solid lead acetate to trap hydrogen sulfide, ^ per cent mercuric cyanide to trap mercaptans, and 3 per cent mercuric chloride to trap organic sulfides. They identified the major volatile sulfur constituents of cooked cabbage as the main cause for the odor of cooked cabbage. They found dimethyl disulfide and hydrogen sulfide in dehydrated cabbage, red cabbage, saur- kraut, califlower and broccoli.

Bailey et al. (5 ) investigated the volatile sulfur com­ ponents of fresh cabbage by means of gas chromatography and mass spectrometry. Twenty sulfur compounds were reported of which five were isothiocyanates, i.e. methyl, n-butyl, butenyl, allyl, and methyl thiopropyl isocyanates; five were sulfides i.e., hydrogen, carbonyl, dimethyl, diethyl and dibutyl sulfides; nine were disulfides i.e., carbon, dimethyl, methyl, ethyl, diethyl, ethyl, propyl, dipropyl, propyl butyl, propyl allyl and diallyl disulfides. Two additional isothiocyanates and one trisulfide were tentatively identified as methyl thiomethyl and methylthio- butyl isothiocyanates and diethyl trisulfide. Isothiocyanates have also been reported in cabbage by other investigators (3^> 71,

79)• Volatile isothiocyanates of fresh cabbage were found by

Clapp et al. (3*0 to be precursors of four thioureas. The major component was identified by the use of paper chromatography and 5 countercurrent distribution as allyl thiourea and the other three as 3-methylthiopropyl thiourea, 3-butenyl thiourea and 3 -iaethylsulfinyl propyl thiourea.

Simpson and Ealliday (131) reported allyl isocyanates to be the major precursor to the formation of hydrogen sulfide during cook­ ing of vegetables.

Tubers and roots

The volatile sulfur compounds from potatoes and rutabaga have been studied by many investigators (57>6li-,79*89,128,135). The volatile sulfur compounds formed by heating potatoes were examined by gas chromatography and were found to be hydrogen sulfide, methyl mercaptan, ethyl mercaptan and dimethyl sulfide (57,128). Gumbmann and Burr (57) reported that hydrogen sulfide was produced in rel­ atively large quantities (200 to 500 ppb) over extended periods of cooking of either fresh or dehydrated potatoes. They found small amounts of methyl ethyl disulfide and methyl isopropyl disulfide from the volatiles of cooked potatoes. The volatile sulfur compounds found by Self et al (128), listed in decreasing concentrations, were hydrogen sulfide, dimethyl sulfide, methyl mercaptan and ethyl mercaptan. The formation of these compounds was directly related to the amount of heat treatment given the product.

The most frequently reported sulfur compounds in fresh turnips and rutabaga were 2-phenyl-ethyl isocyanate and 3 -butenyl thiocyanate

(6^,79j89,98 ,135)• Hing (6 !+) reported that volatile sulfa compounds were responsible for the characteristic odor of both freshly cooked and canned rutabaga. He found hydrogen sulfide and also tenta­ tively identified methyl mercaptan, methyl sulfide and methyl

disulfide in the volatiles of freshly cooked rutabaga by chemical reactions of the mercuric salts and the retention time data on two chromatographic columns. He reported that more hydrogen

sulfide was found in freshly cooked rutabaga than in canned ruta­ baga. The concentration of hydrogen sulfide was estimated to be

2.07 mg/Kg of freshly cooked rutabagaj 0.^6 mg/Kg from drained weight of canned rutabaga and 0.77 mg/liter of brine from canned

rutabaga, after eight hours of boiling.

Onions and garlic

The volatile sulfur compounds from onions and garlic have been extensively studied (l,23,2k,28,31*77,81,10^,127,129,

13^.1^5)• Early investigators (129,81) reported that allyl-propyl

disulfide was a principal odoriferous volatile component of

onions. Recent investigation, using the modern techniques of

gas liquid chromatography, infrared analysis and mass spec­

trometry, have not been able to confirm the presence of this

compound (23,2k,10*1,13*0 •

Challenger and Greenwood (30) demonstrated the presence

of n-propyl mercaptan in raw onions, and characterized it by its

mercury, lead and silver salts. The thiol was removed from the

freshly chopped bulbs in a stream of sterile air and absorbed in

It per cent mercuric cyanide solution. On the other hand, Stahl (13*0 found in the gaseous emanation of cooked onion an abundance of propyl mercaptan, a small amount of hydrogen sulfide and traces of sulfur dioxide, dipropyl disulfide and B-hydroxy propanethiol.

The author notes that dipropyl disulfide has a definite "sweet" odor typical of disulfides, and when simply combined with n-propyl mercaptan, a credible onion odor is produced,

A number of investigators (23,24,104,127,129) using gas chromatography, infrared and mass spectrometry were able to find a variety of sulfur compounds in the volatiles of cooked onion.

Carson and Wong (24) were able to isolate and identify important sulfur flavor components, including methyl disulfide, methyl trisulfide, methyl-n-propyl disulfide, methyl-n-propyl trisulfide, n-propyl disulfide, and n-propyl trisulfide.

Self et al. (127) investigated the volatile fractions formed by boiling onions for one-half hour. He reported a large quantity of hydrogen sulfide, a large amount of methyl mercaptan, a medium amount of ethyl mercaptan, a medium amount of dimethyl sulfide and a very large amount of n-propanethiol.

Garlic owes its pungent odor to sulfur containing oils.

Kirchner (77) and Wertheim (145) reported that garlic oil consists chiefly of allyl sulfide (CgHjj^S. Later Semmler (129) reported that garlic had no allyl sulfide but found instead CgHio^ aud allyl propyl disulfide and also a trisulfide (CgH^^-Sg and a tetrasulfide (CgH^^S^. Cavallito je±—al. (3,28) alcohol extracted

(al^^cin) f*om ground garlic cloves and found it to be very unstable at room temperature. Alkaline hydrolysis of allicin

gave sulfur dioxide and allyl disulfide as products. Coffee

There are many reports in the literature demonstrating the presence of sulfur containing compounds in brewed coffee, with early investigators reporting the presence of hydrogen sulfide methyl mercaptan, furfuryl mercaptan and dimethyl sulfide (67,68,91

97,126,152).

Hughes and Smith (67,68 ) found the concentration of hydrogen sulfide in coffee.to be very small, only 2 to 3 Ppm. The hydrogen sulfide gradually disappeared on standing but reappeared upon reheating. However, Segall and Procter (126) found no hydrogen sulfide in standard coffee brew, but detected sulfur dioxide at a concentration of 27 ppb. The mercaptan content of freshly brewed coffee was determined to be 56.7 ppb. These authors suggest that there was a close relationship between the mercaptans content of coffee brew with flavor and flavor changes.

Ziatkio and Sivetz (152) studied the volatiles from coffee brew using gas chromatography and mass spectrometry. Coffee aroma essence was collected from commercial percolator vent gases which were separated and analyzed. More than thirty volatile components of roasted coffee, which contributed to the flavor and aroma, were isolated. The sulfur volatiles included dimethyl sulfide, carbon disulfide, methyl mercaptan and thiophene at a concentration of

1, 0.2, 0.1 and 0.1 per cent by weight, respectively.

More recently, Merritt et al. (97) examined volatile frac­ tions from ground roasted coffee. The volatiles were collected by distillation under high vacuum at room temperature into a receiver at liquid nitrogen temperature. Prior to examination by mass spectrometry, the distillate was further fractionated.

Subsequent analysis revealed the presence of more than 20 com­ pounds. The sulfur compounds represented if.3 per cent of the total concentration of volatiles and included carbon disulfide, 0.2 per cent; dimethyl sulfide, 0.7 per cent; methyl ethyl sulfide, 0.2 per cent; dimethyl disulfide, 3*2 per cent; and methyl-ethyl and ethyl disulfide trace. In contrast to the finding of earlier investigators for brewed coffee, this study indicated that no hydrogen sulfide or mercaptans were present in ground coffee.

Meat Products

Beef and pork

The volatile sulfur compounds from heat treatment of meat products have been investigated extensively (2 1 ,36,6 5 ,8*f,8 5 #92,

107,151).

Hornstein et al. (6 5 ) found hydrogen sulfide by boiling a lyophylized powder obtained from the water soluble extract of beef. His results confirmed the earlier results of Kramlick and

Pearson (Qk) and Crocker (3 6 ) that hydrogen sulfide came primarily from those extracts of meat which were water soluble.

Kramlick and Pearson (8 5 ) identified the volatile com­ ponents of cooked beef by paper, gas chromatography, and other chemical methods. The results revealed that the volatiles were composed of carbon dioxide, methyl mercaptan, acetone and acetaldehyde. In addition, methyl sulfide and water were 10 tentatively identified. A beef-like flavor was produced on bubbling the mixed volatiles from cooked beef through distilled water.

Yueh and Strong (151) attempted to identify the volatile components responsible for the characteristic odor of cooked beef.

Fresh lean beef was freed of fat, passed through a meat grinder, and refluxed with an equal weight of water for three hours. The filtrate of the broth was concentrated and found to contain the characteristic odor of cooked beef. Using chemical precipitation methods and gas chromatography techniques, a wide variety of volatiles were found including 6-8 mg of hydrogen sulfide per

Kg of fresh lean beef. Dimethyl sulfide was tentatively iden­ tified as well.

The volatile sulfur and hematin compounds of strained beef was studied by Luh and his co-workers (92). Strained beef con­ taining 18 per cent T.S. was canned by aseptic and conventional retorting processes. The aseptic product was sterilized by a

H.T.S.T. process, 3 sec. at 300°F in a swept surface heat ex­ changer. The retorted sample was sterilized 42 minutes at 250°F.

Retorted beef was higher in amino acid and soluble nitrogen than

the aseptic sample. The retorted product also received a more

favorable flavor rating and had 209 ug/100 g of hydrogen sulfide, whereas, the aseptic beef had 63 ug/100 g of hydrogen sulfide.

Methyl sulfide was not detected in either product.

Brennan and Barnhard (21) examined the head space con­

stituents of canned beef, by gas chromatography using flame 11 ionization detector. Some 13 volatile components were isolated and separated and 5 major constituents, hydrogen sulfide, methylthiol, ethanethiol, propanethiol, and butanethiol were tentatively identified by retention volume constants on three stationary liquid phases varying in polarity. Propanethiol and butanethiol had not previously been reported. The effects of storage on volatile components were investigated and results re­ vealed that the composition of head space constituents remained constant over a five-months storage period.

Ockerman et al. (107) examined the volatile consti­ tuents from dried cured hams using selective trapping methods, gas chromatography and infrared analysis. A wide variety of com­ pounds including aldehydes, ketones, esters and organic acids, ammonia and methylamine were detected, as well as hydrogen sulfide and trace amounts of organic disulfides and/or monosulfides.

Milk and Milk Products

Milk

The production of hydrogen sulfide by high heat treatment of milk has been recognized for many years and has been the sub­ ject of numerous investigations (11,43,54,70,90,99,109,114,150).

Factors associated with hydrogen sulfide formation by heat have beenreviewed by Gould and Sommer (54) and Patton (110).

The presence of methyl sulfide has been reported in raw milk (114,150). Patton-et aL, (114) identified methyl sulfide

in raw milk by applying gas chromatography and mass spectroscopy 12 analysis to the exhaust gases from raw whole milk stored in an air- agitated, 1,000 gallon cold wall tank. They further reported that

the threshold value for methyl sulfide was approximately 12 ppb

in distilled water at the 50 per cent level. At a concentration

slightly above this value, the compound exhibited a milk-like

flavor. These results have been confirmed by Wynn et al. (150).

Dimethyl sulfide has been reported by Jennings et al. (70)

in feed flavored milk, by Morgan and Pereira (99) in volatiles of

grass and c o m silage and Patel et al. (109) in sterile concen­

trated milk.

While this study was in progress, Bingham and Swanson (ll)

examined the volatiles of sterilized concentrated milk from whole

milk which had been forewarmed to 1^5, 165 and l85°F for 30

minutes and 265°F for 15 seconds. Gas chromatographic analysis

revealed the presence of at least 16 volatile components. Tenta­

tively identified sulfur volatiles included methyl mercaptan,

dimethyl sulfide and dimethyl disulfide.

Butter

Day et al. (U2,lf3) isolated dimethyl sulfide from bulk

butter cultures, cultured cream, cultured cream butter, the head

space of butter churns and the head space of butter samples. Pro­

cedures used for isolation of this volatile component involved

low-temperature distillation of the product or trapping of head

space vapours. The resulting highly volatile fraction was trans­

ferred from liquid nitrogen cold traps and gas chromatogrammed. 13

Far ultraviolet spectrum analysis was also utilized for identi­ fication purposes. These investigators reported that the average flavor threshold of dimethyl sulfide in butter oil was 24 ppb, but the panel preferred a concentration of 40 ppb in the oil, whereas, it was found that 10-20 ppb of dimethyl sulfide was the optimum addition to salted sweet cream butter. At concentrations of 100 ppb of dimethyl sulfide, the flavor was described as slightly feedy, whereas, 200 ppb was called definitely feedy.

Furthermore, it was observed that dimethyl sulfide had the ca­ pacity to smooth out harsh flavors of diacetyl and acids asso­ ciated with cultured flavor. Sweet cream butter to which 30 ppm of acetic acid, 500 ppm of lactic acid, 2.5 ppm of diacetyl, and

40 ppb of dimethyl sulfide had been added exhibited a smooth de­ sirable flavor in comparison with a sample without added dimethyl sulfide.

T.indav eh al- (90) tentatively identified the presence of hydrogen sulfide, methyl mercaptan, dimethyl and 2-mercap- toethanol from butter culture volatiles using gas chromatography.

Hydrogen sulfide also was tentatively identified to be present in cultured butter, by these investigators.

Factors Affecting Heat-Release of Sulfur Compounds from Milk and Milk Products

Many investigations have been made concerning the factors which may affect the release of sulfur compounds from milk and milk products upon heating. (17,44,54,55,63,86,88,102,103,118,140,

149,153). Townley and Gould (1^0) demonstrated a relationship between the amount of hydrogen sulfide liberated and the degree of heat treatment given to milk and milk products. They found that the liberation of hydrogen sulfide from momentarily heated milk occured at a specific temperature which they designated as the critical temperature. Results obtained from the critical temperature studies upon heating various products momentarily were as follows: whole milk, 76-78°C; skimmilk, 78-82°C; milk serum, 76-78°C; buttermilk and 30 per cent cream, 66-68°C. Accord­

ing to Gould and Sommer (5*0, prolonging the exposure time de­

creased the critical temperature. Furthermore, Townley and Gould

(llfO) showed that heating whole milk, 20 per cent cream, butter­ milk, skimmilk and whey at 90°C momentarily produced more hydrogen

sulfide than at their respective critical temperatures.

Lea (88) reported hydrogen sulfide being produced when

heating fresh milk at 60 and 70°C and reconstituted milk at 1*0 and

6o°C for various lengths of time. The concentration of hydrogen

sulfide in both milks increased when the heating time was increased

from 30 to 60 minutes. The liberation of hydrogen sulfide from

reconstituted milk occurs at a lower temperature than normal milk

which was attributed to a change in protein structures during

the drying process.

Boyd and Gould (1 7 ) investigated the liberation of volatile

and non-volatile sulphydryl compounds from heated milk and milk

products under different experimental conditions. In their study,

the volatile sulfur compounds were measured by the methylene blue method, and the non-volatile sulphydryls were determined by the thiamine disulfide procedure. Their results revealed that milk momentarily heated at 90°C yielded the highest TDS values (ex­ pressed as mg/l of cysteine hydrochloride) 21.0 for milk, 1 7 -1* for whey, 22.8 for 35 per cent cream, and 22.9 for buttermilk.

Hydrogen sulfide liberation began essentially instantaneously after the production of thiamine disulfide-reducing compounds.

Products heated to 90°C for 30 minutes, cooled, and aspirated with nitrogen to remove hydrogen sulfide yielded the following amounts of hydrogen sulfide expressed as gamma per literj milk ^35> cream

53 5 > buttermilk 596 and whey 2 9 3 .

Boyd and Gould (17) further reported that the original

TDS values as determined when the product was heated to 90°C mo­ mentarily, varied directly with the quantity of hydrogen sulfide that was subsequently liberated, i.e., higher for cream and butter­ milk than for whey. For all products, the decrease in TDS values resulting from the liberation of hydrogen sulfide was within the range of from 20 to 22 per cent. The results obtained for milk revealed, in general, a decrease of 1.0 mg per liter in TDS value for each 0.1 mg hydrogen sulfide removed from the sample, or a molar ratio of 2:1. Finally, the study also revealed that low- temperature preheat treatments markedly decreased the volatile and non-volatile -SH content of milk or condensed milk subse­ quently heated to 90°C for 30 minutes.

Dill et a l . ( W O examined the production of sulfur compounds in skimmilk heated by direct steam injection over a temperature and time range of 190°F to 300°F and 2 to 150 seconds. Their results showed that the heat activation of sulphydryl groups, as measured by titration with silver nitrate, followed a direct re­ lationship with temperatures in the sample heated for 2 seconds.

However, samples heated for 150 seconds and 20 seconds, exhibited maximum sulphydryl groups formation at 220°F and 260°F temperature respectively. Higher temperatures resulted in a decrease in -SH groups concentration. The decrease in titration values above the critical treatment was caused by the volatilization of sulfur com­ pounds. It was found by gravimetric analysis that a loss of as much as 8 per cent total sulfur occured in skimmilk heated to

300°F for 15 seconds,

Gould and Keeney (55) investigated the formation of active

-SH compounds of 40 per cent cream by the thiamine disulfide method, and found that the greatest concentrations of active -SH compounds produced in creams heated to 190°F, 180°F, and 170°F were after 5, 10 and 20 minutes respectively. The maxium concen­

tration was achieved by heating cream to 190°F for 5 minutes.

When the times required to heat cream to 190°F were 15 seconds,

30 seconds, and 60 seconds, the quantities of active -SH com­ pounds produced were directly and markedly increased. Further­ more, they reported a direct relationship between the fat levels

of cream and active -SH group production when creams of 20 per

cent, 30 per cent, 40 per cent and 50 per cent fat were flash heated. Flash-heating to 190°F produced three times the quantity

of active -SH compounds in 50 per cent cream as that produced in 20 per cent cream, and twice that formed in 30 per cent cream.

However, as the time exposure at 190°F was extended to 5 minutes or more, the values became more nearly the same. They reported also that the temperature of separation of milk was related in­ versely to the concentration of heat-revealed -SH compounds in

the resulting cream. The concentration of active -SH compounds

in 40 per cent cream heated to 190°F for 5 minutes were equi­ valent to 22.7, 20.5 and 19.6 mg per liter cysteine HCL when the

separation temperatures were 55°F, 100°F, and 135°F, respectively.

Kristoffersen et al. (86) studied some factors which

affect the production of hydrogen sulfide and active-sulphydryl

groups when milk is heated to high temperatures. Their results

revealed that milk from individual cows and from mixed-herd

sources varied in heat produced (90°-30 min) hydrogen sulfide

concentration from 324 to 552 micrograms per liter, and from 170

to 618 micrograms per liter, respectively. The concentrations of

heat-produced hydrogen sulfide fluctuated widely for milk which

was stored at 4°C, 15°C, and 37°C for extended periods of time.

The fluctuations followed a rather definite time pattern, with the

magnitude of the changes being related directly to the temperature

of storage and to the source of milk. These variations in heat-

produced hydrogen sulfide were accompanied by similar and simul­

taneous variation in TDS reducing substances. Changes in bacterial

numbers did not appear to account for the fluctuations in heat-

produced sulphydryl compounds. On the basis of their results the

authors considered milk to be in a "dynamic state". Furthermore, 18

Kristoffersen et al. (8 6 ) reported that low temperature preheat treatment of milk decreased the amount of active sulphydryl groups produced from heat treatment of milk. Preheating milk at 60°C for

15 minutes resulted in less heat-produced hydrogen sulfide and TDS reducing substances than did preheat treatments at either 55°C,

65°G, or 70°C.

Many researchers have shown that the liberation of hydrogen sulfide from heat-treated milk and milk products is affected by the pH (17,63,102,103,118,1^0,1^9).

Evidences have been presented by Townley and Gould (1^0) that the production of hydrogen sulfide from skimmilk whey heated momentarily to 90°C was maximum at pH 9*0* Likewise, Rettger (ll8 ) pointed out that the addition of alkali to milk facilitated the release of volatile sulfides when the milk was heated, whereas, the addition of acid retarded the release of volatile sulfides. Boyd and Gould (17) have shown that acidification of milk following heat treatment enhanced the production of hydrogen sulfide by

7 fold when compared to milk which was not acidified following the same heat treatment. Studies by Negoumy (102,103), Higgins et al.

(6 3 ) and Wormel (1^9 ) found that casein sol adjusted to alkaline pH by different alkali metal which was next heated to 90“95°C, followed by addition of IN HC1 had a direct bearing on the amount of hydrogen sulfide being released. Maximum heat release of hydrogen sulfide was reported by all investigators at alkaline pH*s.

Townley and Gould (1^0) have demonstrated that cystine added to milk at a rate of O .25 and 0.5 grams per liter retarded hydrogen 19 sulfide production when the milk was heated, whereas, cysteine added at the same rates increased sulfide production by 27 and 67 per cent, respectively, when the milk was heated. Cysteine was considered to be catalytic in respect to the increased hydrogen sulfide produc­ tion, since no hydrogen sulfide was formed from heating cysteine alone.

Mechanism of Formation of Volatile Sulfur Compounds by Heat

Vegetable Products

Various postulations have been advanced for the formation of volatile sulfur compounds in heated vegetable products. It appears that most of these compounds arise from the degradation of certain sulfur containing precursors such as amino acids, gluco- sides and other salts (5,2 2,24,25,2 6,2 7,2 8,2 9,31,32,100,101,106,108,

124,125,137,139,Ite,149)* Dimethyl sulfide, one of the major volatile constituents of cooked cabbage, has been shown to be derived from L-S-methyl- cysteine sulfoxide. Synge and Wood (139); Morris and Thompson

(lOO), and Virtanen and Matikkala (142) were able to isolate this compound from plants of the Brassica family. From garlic, Cavallito et al. (28) and Stoll and Seebech (137) isolated the compound

S-allyl-cysteine sulfoxide, which would release diallyl disulfide upon degradation by heat.

Ostermayer and Tarbell (108) postulated a mechanism for 20 the degradation of L-S-methylcysteine sulfoxide (I) by acid hy­ drolysis, which would yield the following compounds: (Equation 1)

4CH - S - CH CH(NH ) COOH +• 2H„0 ------> 6 \ 2 2 2 0 L-S-methylcysteine sulfoxide

(I)

0 t (CH3)2S2 + CH3S-SCH3 + 4NH3 4 4GH3C00H .... (eq. 1)

0

dimethyl- methanethio- disulfide sulfonate

(II) (III) (IV) (V)

These investigators indicated that methanethiosulfonate

(III) and disulfide (II) probably arose from the cleavage of thio- sulfinate (CH3SOSCH3), which in turn may have been formed from methanesulfenic acid (CH3S0H). According to Schwimmer and Weston

(124) and Schwimmer et al. (125), sulfenic acid is unstable and, therefore, may react in several ways to form other volatile sulfur compounds. Challenger (32) showed that sulfenic acid would yield methyl mercaptan and sulfinic acid in the presence of acid.

(Equation 2)

2 RSOH ► RSH + RS02H ...... (eq. 2)

sulfenic mercap- sulfinic acid tan acid

Challenger (32) further suggested that the formation of hydrogen sul­ fide from cooking vegetables could be due to the degradation of sul­ fenic acid (RCH2S0H) which in turn could arise from 21 the hydrolytic decomposition of disulfides. (Equation 3)

RCH S CHR + H O -- » RCH SH + RCH„SOH' 2 2 2 2 2 2 alkyl sulfenic disulfide acid

RCH2SO H > RCHO 4 H S . •. . . . (eq. 3)

sulfenic acid

The formation of trace amounts of hydrogen sulfide and mer-

captans during the cooking of vegetables has been suggested by

Synge and Wood (139) to arise from the decomposition of L-S-methyl

cysteine sulfoxide. According to Challenger (32) sulfoxides may

yield mercaptans and carbonyl compounds on degradation by acids.

(Equation 4)

RCH2SOCH2R ---> RCHO + RCHgSH . . . . (eq. 4)

alkyl alkyl sulfoxide mercaptan

Dimethyl B-propiothetin (I), a sulfonium compound first

isolated from red alga by Challenger and Simpson (29) may be one

possible precursor of dimethyl sulfide. The degradation of this

compound would yield dimethyl sulfide (II), acrylic acid (III),

and H®. (Equation 5)

(CH3)2S(CH2)2COOH --- > (CH3)2S2 4 CH2= CHCOOH 4 H® . . (eq. 5)

dimethyl dimethyl acrylic B-propiothetin disulfide acid

(I) (II) (HI) 22

McRorie et al. (100) were able to isolate from cabbage methylmethionine sulfonium salt which they suggested could possibly be a precursor for dimethyl B-propiothetin and methio­ nine.

On the other hand, Challenger and Hayward (31) isolated methylmethionine sulfonium salt from extracts of asparagus. The degradation of this compound produced homoserine and dimethyl

sulfide according to the following manner:

(ch3)2s(ch2)2ch(nh2)cooh — * (ch3)2s + ch2(oh)ch2ch(nh2)cooh

Dimethyl disulfide was produced by heating a solution of methionine (26,106). Obata and Mizutana (106) reported that heating methionine in the presence of plant material known to

contain pectin increased the yield of the sulfide. Casey et al.

(25) also reported similar results; however, in addition to

dimethyl sulfide, dimethyl disulfide and methyl mercaptan were

found to be present. The authors postulated that dimethyl di­

sulfide was formed from the cleavage of -S-CH3 of methionine and

by transmethylation of the methyl radical from pectin.

The mode of formation of polysulfides has been postulated

by Carson and Wong (24) and Westlake et al. (149) to arise

through the condensation of hydrogen sulfide of free sulfur with

disulfides to yield polysulfides. Bailey et al. (5) suggested

that the formation of sulfides and disulfides in cooked cabbage

were formed from sulfonium salts and sulfoxides, respectively, and he theorized that these sulfides and disulfides could possibly act as precursors in the formation of trisulfides.

Bailey et al. (5) have also proposed that the presence of carbonyl sulfide, hydrogen sulfide, and carbon disulfide in volatile components of fresh cabbage may be accounted for by the hydrolysis of isothiocyanates as follows:

RNCS + H20 ---^ RNH2 ■* COS

COS + h2o ----^ h2s + co2

2RNCS + 2H20 --- ^ 2RNH2 + C02 -f- CS2

These investigators (5), further suggested that the reac­ tion between hydrogen sulfide with unchanged isothiocyanates may result in the formation of carbon disulfide.

RNCS + H2S ------* RNHCSSH * RNH2 -f CS2

The most recent work on the mechanism of formation of low volatile compounds produced in cooked foods had been presented by Casey et al. (27). These investigators presented evidence to show that the formation of some low boiling volatiles arise from the Strecker degradation of free amino acids, with various types of sugars acting as the oxidant. They reported that the relative rates of production of volatile derivatives were dependent on the type of sugar usedj e.g., with glucose, methionine breaks down much more slowly than valine, whereas, with fructose, the opposite is true. They also reported the rate of production of volatiles was not related to their boiling point. Utilizing model systems consisting of heating an amino acid and a sugar to 110°C for 30 minutes, it was revealed that the amino acid methionine was the precursor for methyl mercaptan, dimethyl sulfide, dimethyl di­ sulfide and hydrogen sulfide. Hydrogen sulfide was also formed when either cystine or cysteine were used as precursors. Ethyl mercaptan was isolated and identified when ethionine was heated.

Meat Products

The flavor precursors in meat products have generally been considered to be water-soluble (7,9,36,65,66,84).

Crocker (36) suggested that the odor of cooked meat is due to a variety of chemical substances, presumably produced by deamination or decarboxylation of amino acids simultaneously with some breakdown of the sulfur bearing amino acid, cysteine, to yield hydrogen sulfide and propionic acid. The findings of this study were that cooked beef flavor is quite complicated chemically and consists more of odor than of taste.

Bouthilet (13,14,15,16) studied the origin of chicken flavor and suggested that it is due to a compound associated with the meat fibre rather than fat. This flavor precursor could be extracted from the fibres by steeping or by extraction with water and was demonstrated to have properties similar to glutathione.

Samples of pure glutathione gave an odor similar to chicken meat when dissolved in water and heated to 60°C, More recently, Pippen et al. (117), Peterson (116) and Kazeniac (73) demonstrated that flavor precursors in raw chicken meat can be extracted with cold

water.

Mecchi et al. (95) found volatile sulfur compounds from

cooked chicken arose primarily from muscle proteins with cystine

and/or cysteine acting as the precursors. About 90 per cent of

the hydrogen sulfide produced by heating chicken muscle came from muscle protein. Non-protein sulfur compounds found in muscle

were methionine, taurine and glutathione. Of these, only gluta­

thione produced hydrogen sulfide upon heating and accounted for

10 per cent of the hydrogen sulfide being produced. These authors

(95) also demonstrated that the rate of hydrogen sulfide produced

from heated chicken muscle could be predicted from its cystine

content.

The effect of alkaline condition on the decomposition of

proteins was studied by Robbins and Fioriti (119)* Several pro­

teins when heated to 100°C for 1 hour with an aqueous suspension

of Cd(0H)2 at pH 9*5 released hydrogen sulfide quantitatively

from cystine and cysteine. Free cystine and reduced glutathione

behaved similarly as protein bound with cystine or cysteine. How­

ever, methionine and gelatin, which contain methionine but not

cysteine or cystine, did not produce hydrogen sulfide. Sodium

hydroxide at a concentration of 1 N when substituted for Cd(0H)2>

and added to methionine and gelatin did not cause the release of

hydrogen sulfide upon heating these materials.

Robbins and Fioriti (119) have postulated a mechanism for 26 the formation of hydrogen sulfide from cystine in alkaline con­ ditions.

H-I-A-CH--S-S-CH -i-H + OH ► H-fc-CH-S-S-CH0-C-H f HO \I 2 2 I 1 l 2 | 2 II- C - CH- S - S - CH - CH » H-i-CH=S-S-CH0-^H » 2 ( | 2 | H-C-CII=S-S-CtI -£ll * H-C-C-S + H-C-CH -S I ------2 T I 2 J i H-C-C-S + Ho0 ---- > H-C-C=0 + H0S I 2 2

The authors suggested that cadium ion probably functions by trapping hydrogen sulfide and preventing it from reacting further with unreacted starting material or other products of the reaction. However, they further suggested that the possibility that a direct electrophilic attack occurred on the disulfide bond leading to a product capable of quantitatively releasing hydrogen sulfide. Accordingly, this reaction would proceed as follows:

H-i-CH2-S-S-CH2-C-H OH" H-

h- h-<|:-c=o -v h2s

Dairy Broducts

Milk proteins, such as albumins and globulins, are easily denatured by heat at temperatures above 60°C. This denaturation is accompanied by exposure of certain thiol groups and the libera­ tion of hydrogen sulfide in the denatured products (17,32,43,56,

62,65,69,72,78,81,99,114,120,130,140).

Townley and Gould (140) suggested that these groups came from 2 sources; namely, the milk serum and material associated with the fat globule membrane. Josephson and Doan (72) concluded that hydrogen sulfide came from one or more of the proteins, with the lactalbumin and fat globule membrane being the most likely sources. Greenstein (56) showed that serum proteins (albumin and globulin) and not casein were the precursors of sulphydryl group formation during heating of milk. Boyd and Gould (17) pre­ sented evidence to show that hydrogen sulfide formation was due to the cleavage of heat released sulphydryl groups, Hutton and Patton

(69) proposed a similar mechanism for hydrogen sulfide formation in heated milk.

The B-lactoglubulin fraction of serum protein is the major source of -SH groups in milk (^3,62,65,81,130). Hutton and

Patton (69) reported that by reason of the quantity present in milk, fat globulin membrane protein is a very minor source of heat released -SH groups.

Methyl sulfide in milk appears to have its origin in the feed of cows (^3,99,11^,130). Morgan and Pereira (99) have shown that methyl sulfide is a volatile constituent of grass and corn silage, and Shipe et al. (130) have presented evidence that methyl sulfide can easily be transmitted to milk via the lungs, as well as the rumen of cows. According to Day et al. (^3), two possible precursors present in plant materials may account for the presence of dimethyl sulfide in milk. These are dimethyl-^-propiothetin and methyl methioninesulphonium salt which have been isolated from algae and asparagus, respectively, by Challenger et al. (32)»

Kiribuchi and Yamanishi (78) have shown that the sulfonium salt is 28 converted to dimethyl sulfide and homoserine when green tea is heated. Methyl sulfone was isolated from cattle blood by Rusicka et al. (1 20) and could conceivably contribute to the formation of volatile sulfur compounds in dairy products. These investigators suggested that the normal concentration of dimethyl sulfide in milk may arise as a metabolic product from methionine, whereas the excess may come from particular feeds.

Volatile Sulfur Compounds Resulting from Irradiation of Food Products

The possibility of utilizing high-energy beta or gamma-ray sources for the preservation of food products has aroused consider­ able interest in recent years. Using energy from these materials, sterile products with excellent keeping qualities have been obtained in the absence of refrigeration, but off-flavors and colors have been reported as undesirable by-products of this pro­ cess (10,18,19). Since radiation energy releases volatile sulfur compounds from foods, the nature and mechanism of their formation will be discussed.

Meat Products

A variety of volatile sulfur compounds have been found in irradiated beef products, which included inorganic sulfides, mer- captans, organic sulfides and disulfides and sulfur containing carbonyl compounds (6,8,75109,HI* 122,123,124,133,134,147>

148). Stahl (13*0 was able to separate by means of gas chro­ matography irradiated meat volatiles from low temperature dis­

tillation traps and to subsequently identify the individual

components with mass spectrometry. At least 38 different vola­

tile components from raw beef were isolated after beta radiation

dosage of 2 and 4 megarep. The results revealed that non­

irradiated meat vapours contained hydrogen sulfide, methyl mer­

captan and ethyl mercaptan in concentrations of 4.3, 0.15 and

0.28 micromoles per kilogram of beef, respectively. After 2

megarep of irradiation the types and concentrations of the dif­

ferent volatile sulfur compounds in micromoles/Kg. of beef were:

hydrogen sulfide 23, methyl mercaptan 2 ,5, ethyl mercaptan 1 .1 ,

propyl mercaptan 1.5 and butyl mercaptan 0.10. After a dosage

of 4 megarep, the concentration of the various sulfur volatiles

in micromoles/Kg. were: hydrogen sulfide 39*6, methyl mercaptan

12.0, ethyl mercaptan 0 .082, propyl mercaptan 0 .59, butyl mer­

captan 0.11 and pentyl mercaptan 0.045. No sulfides or disul­

fides were found in the volatiles of non-irradiated beef and only

trace amounts of diethyl disulfide were detected in beef irra­

diated with a dosage of 2 megarep. However, a variety of sulfides

and disulfides were detected in the vapours of beef after a dos­

age of 4 megarep of irradiation. These were in micromoles/Kg. of

beef, dimethyl sulfide 1 .8 , methyl ethyl sulfide 0 .18, methyl

isopropyl sulfide 0 .15, diisopropyl sulfide 0 .053, dimethyl di­

sulfide 0.11, diethyl disulfide 0.42, ethyl isopropyl disulfide 30

0.10 and diisopropyl disulfide 0.19 micromoles.

Sliwinski and Dotty (133), using a sensitive colorimetric technique, were able to determine quantitatively the concentrations of methyl mercaptan produced under various experimental conditions.

The results indicated that the formation of methyl mercaptan from gamma irradiated beef was directly related to the radiation dosage,

the temperature of the meat during radiation and the length of

time the meat was exposed to irradiation. These investigators also reported the presence of methyl mercaptan in the volatile components

of non-irradiated meat. This is in agreement with the results of

Stahl (134).

The volatile components produced by concurrent radiation

and distillation at 5 megareps of raw ground beef was studied by

Wick et al. (147) and related to the characteristic unpleasant odor

of beef preserved by radiation. The condensate obtained from vola­

tiles of irradiated beef was found to possess a typical odor of

irradiated beef. Utilizing paper chromatography to examine 2,4-DNPH

derivatives of the distillate, and using solvent extraction and gas

chromatography techniques, melting points of semicarbazone deri­

vatives and refractive index numbers, these investigators presented

evidence to prove that 3-methylthio-propionaldehyde (methional)

was a major component of a mixture of at least 12 substances detected

of which some were sulfur containing,but not identified. Methional

had been postulated previously by Witting and Batzer (148) who

suggested that this compound, as well as 3-(methylthio)-Buty-

naldehyde, might be present in irradiated beef. However, the 31 current investigation gave no evidence for the presence of methional.

The proposal of Witting and Batzer (1^8) that methional was odor­ less was not supported by Wick et al. (75,109,119,122,L23,12l(-,ll*7).

In connection with methional, Patton and Barnes (113) suggest that the flavor and aroma of cooked meats, soups and boiled vegetables frequently suggest the presence of methional. According to him, the widespread distribution of methionine in foodstuff, and the plausibility of its conversion to methional by the Strecker degradation (111) during the cooking process lend supporting evidence to the occurrence of methional in certain foods. Another instance in which the odor of methional is strongly manifest is the various peptone and meat infusion media used for bacteriological determinations.

Milk

Sterilization of milk by irradiation has been demonstrated by Brasch and Huber (19), and Dunn et al. (^5); however, off-flavor and odors as a result of this treatment have resulted in unpalat­ able products (10,18,51).

Day et__al. (3 9 ) examined the radiation-induced flavor and odor problem of skimmilk, the effects of gamma radiation at two £ 6 dosages, 2 x 10° and 5 x 10 rep, on skimmilk and certain of its fractions were studied. The principal radiation-induced off-flavor appeared to arise from a group of potent, disagreeable smelling sulfur compounds. Further, they claimed that these compounds orig­ inated primarily from the milk proteins (casein). Day et al . (*J-0) 32 isolated and identified methyl mercaptan, methyl sulfide, and methyl disulfide by gas chromatography and mass spectrometry as the volatile sulfur components that were present in irradiated sodium caseinate sol. Methyl sulfide was identified as the sulfur compound which was considered to significantly impart off flavor.

Light Irradiation of Milk

Sunlight Flavor

A comprehensive review of activated flavor has been presented by Stull (138). Various investigators have associated sunlight de­ gradation of protein with the release of sulfur compounds causing sunlight flavor of milk (Ul,1^7,U8 ,^9 ,7^,112,115,121,122,llt3 ,l^).

Patton and Josephson (115) duplicated sunlight flavor by ex­ posing diluted aqueous solutions containing methionine to sunlight.

They also showed that the addition of mg methionine per quart of skimmilk, greatly enhanced the production of this flavor. The sulfur- containing amino acids, such as cysteine or cystine when added to skimmilk at a rate of 20 mg per quart did not exhibit any activated flavor after exposure to sunlight. The authors concluded that sun­ light flavor results from the photolysis of methionine.

Patton (1-12) presented evidence that methional was the prin­ cipal compound responsible for the formation of sunlight flavor.

Methionine and riboflavin in the presence of sunlight were considered by the author to be significant for the formation of sunlight flavor.

Also, in milk, the proteins, especially casein, are indicated as the primary source of methional because of the high concentration of meth­ ionine in casein. 33

Day et al. (hi) investigated the role of methional as a flavor compound. Methional had a strong odor and flavor and a flavor threshold value of 16 ppb.

Samuelsson and Harper (122) investigated the degradation of methionine by light under various conditions. The mixtures, that were exposed to light for irradiation for 7 hours, consisted of 200 mg of methionine, 100 ml H2O, 0.26 mg lacto-flavin, and 5 uc of methionine. The pH of the mixtures was adjusted by lactic acid. The samples were irradiated while either oxygen or nitrogen was bubbled through the mixture and the volatiles entrapped in various trapping reagents, consisting of calcium chloride, lead acetate, 4 per cent mercuric cyanide, 3 per cent mercuric chloride and 0.2 per cent 2,4-dinitrophenylhydrazine solutions. The results revealed that with increased exposure to light, the methionine concentration decreased due to degradation. Further, light irradiation caused the formation of i^S, sulfides and disulfides, mercaptans, to C3 aldehydes, as well as methional. Increased concentrations of mercaptans, sulfides and disulfides were formed by light exposure as the pH was reduced from 6.8 to 4.8. The authors concluded that methional probably plays an important role in the development of sunlight flavor, but it must be assumed that substances such as mercaptans and sulfides, also contribute to the flavor.

Samuelsson (121) exposed methionine sulfur-35 labelled milk to light in the presence of oxygen. The volatiles produced were f" fractionated and the products obtained supported the theory that cabbage-like sunlight flavor is caused by the presence of mercap­ tans, sulfides and disulfide substances. The author suggested that these substances can be caused by the oxidative breakdown of methionine. This study further revealed that in comparison to the whey proteins, the casein portion was degradated less, if at all.

Also, lowering of pH caused the formation of increased quantities of flavor compounds which is in agreement with the evidence pre­ sented by Samuelsson and Harper (122) for free methionine.

The Mechanism of Formation of Volatile Sulfur Compounds by Irradiation

Gamma Irradiation

The formation of volatile sulfur compounds by irradiation has been investigated by many investigators (6,37*6l,93>l3*0»

Batzer and Dotty (6) demonstrated that the undesirable odor formed by gamma irradiation of beef arose, from some water

soluble compounds of the meat. Marbach and Dotty (93) showed that hydrogen sulfide was released in smaller quantities from ground beef with high fat content (20 per cent) than from beef containing less fat (10 per cent) at the same radiation dosage. The authors

(93) demonstrated that irradiated meat had a considerable reduc­ tion in the glutathione content when compared to non-irradiated

samples. They suggested that hydrogen sulfide arose from the degra­

dation of glutathione. This is consistent with that reported by

Dale and Davis (37) who demonstrated previously that hydrogen sul­

fide is released from solutions of glutathione and cysteine by 35 irradiation. Furthermore, the formation of hydrogen sulfide from cysteine is independent of the presence of oxygen. Stahl (134) also suggested that volatile organic sulfur compounds released by irradiation of beef was from the decomposition of amino acids, and that the variety of sulfur compounds found were formed by recom­ bination of fragments resulting from the degradation products of amino acids in the proteins of meat fiber.

Hedin et al. (61) demonstrated that irradiation odor of beef was shown to arise chiefly from a water soluble, non-dialy- zable fraction. This fraction was a mixture of at least two elec-

trophoretically separable proteins. They further suggested that the odor was associated with sulphydryl or closely related compounds since cysteine and methionine were not detected in the proteins after irradi­ ation and since the odor was eliminated by sulphydryl blocking agents such as Sodium p-chloromercuribenzoate, n-maleimide and mer­

curic acetate.

In order to elucidate the mechanism of formation of volatile

organic sulfur compounds, the degradation of amino acids and the product of decomposition were isolated and examined by several inves­

tigators (39,40,46,82,83,94,148).

Kolousek et al. (82) and Kopoldova et al (83) investigated

the irradiation degradation products of radioactive methionine (I).

A variety of sulfur-35 containing compounds were isolated and iden­

tified, these included methyl mercaptan (III), methionine sulfoxide

(IV), methyl-ethyl sulfide (II), methionine sulfone (V), sulfenic

acid (VI) and homocysteric acid (VII). The following equations 36 were postulated by the authors as a possible mechanism for the for­ mation of these compounds.

eH3-S-(CH2)2CHNH2-COOH * CH3-S-CH2-CH3

(I) (II)

CH3-S(CH2)2CHNH2-COOH >CH3-CH2-CHNH2-COOH * CH3-SH

(I) (III)

CH3-S-(CH2)2CHNH2COOH >CH2*S‘CH3 (I) OCH-jCHNH COOH 1 2 (IV)

CH *S*CHo 0 2 1. 3 OCH2CHNH2COOH ■> CHo-S-CH0 2 H 3 OCH2CHNH2COOH

(IV) (V)

0

CH2-S-CH3

OCH2CHNH2COOH - ► c h 3c h 2-s o 2h

(V) (VI) 0

CH CH -SO H. ->CH -S=0 3 2 2 2 \ OH

(VI) (VII)

35 According to Martin et al. (94), when S -D-L methionine or

S35-glutathione was added to ground meat and the mixture irradiated, 37 hydrogen sulfide and methyl mercaptan as volatile sulfur-35 com­ pounds were found. Furthermore, they indicated that most of the methyl mercaptan appeared to be formed directly from free meth­ ionine. This observation was also reported by other investigators

(46,82,83), However, some mercaptans were produced during the

irradiation of the glutathione sample (94), perhaps as a result of

an indirect process or the products of a secondary reaction. Hydro­ gen sulfide was found in both cases after irradiation, however, the

observed isotope dilution indicated that most of the hydrogen sul­

fide apparently originated from other sulfur-containing precursors

in meat. Duran and Tappel (46) reported similar results when methionine was irradiated. They postulated that methyl mercaptan was formed by the cleavage of S-methyl group of methionine. However,

the relationship between the yield of hydrogen sulfide and the

dosage of irradiation suggested that hydrogen sulfide was produced

mainly by a secondary reaction.

Day et al. (39,40) suggested that sulfur compounds, such as

mercaptans and organic sulfides, originated from milk proteins

during irradiation although the specific amino acids involved were

not known--they postulated the formation of the identified com­

pounds arising from the production of free radicals involving air,

amino acids and water. The radicals could combine in various ways

to yield compounds identified in this study as shown in the follo­ wing scheme: 38

COMPOUND INTERMEDIATE ORIGIN

Methyl mercaptan CH3S* + *H Methionine + H2O

or

CH • + .SH Many sources + cysteine Methyl disulfide CH3S* + CH^S • Methionine + H20

or

Oxidation of methyl mercaptans

Methyl sulfide CH^ S’’ + CH^ Methionine 4 many sources

Light Irradiation

The mechanism of formation of volatile sulfur compounds by light irradiation has been investigated by (59,105,111,121,122).

According to Patton (111), the principal chemical consti- tuent in sunlight irradiated milk which”produces sunlight flavor is methional. He postulated that methional is formed from meth­ ionine by the Strecker degradation in the presence of lactoflavin and oxygen in the following manner:

CH3-S-(CH2)2 -CHNH2— co2h ------>

Methionine

ch3s(ch2)2cho + nh3 t co2

Methional

Besides methional, Samuelsson and Samuelsson et al. (121,122) have presented data to show that hydrogen sulfide, mercaptans, sulfides

and disulfides are also formed in light irradiated milk. According to them, methional is probably formed by the Stracker degradation of methionine. However the following mechanism for the forma­ tion of hydrogen sulfide, mercaptans, sulfides and disulfides was proposed:

CH -S-CH -CH -CHNH -COOH ^ 2 Methionine 1 2 3 4 5 6

1. Hydrogen sulfide is formed on cleavage between 1 - 2 and 2-3. When H® is present in the system hydrogen sulfide is formed. The.amount of hydrogen sulfide increases with decreasing pH.

2. Sulfides are probably due to a cleavage between 3 - 4 and/or 4-5. This would lead to the formation of methyl sulfide and methyl-ethyl sulfide.

3. Mercaptans are formed by a cleavage between 2 - 3 but may also be formed by a cleavage between 4 - 5 and 1-2. Oxyda- tion of mercaptans can give disulfides. The degradation of meth­ ional could also lead to the formation of mercaptans.

Obata et al. (105) reported that when beer was exposed to sunlight, an unpleasant flavor occured which was due to the forma­ tion of volatile mercaptans.Furthermore, the investigators re­ ported that humulones and lupolones of hops were related to the for­ mation of "sunlight" flavor. When humulone and cysteine were ex­ posed to sunlight under CO^ atmosphere, "sunlight" flavor deve­

loped and 3-methyl-2-butenyl mercaptan was identified as the com­ pound which imparted the off flavor. 40

Recently, Harper and Brown (59) demonstrated the formation of hydrogen sulfide, mercaptans, organic sulfides, and sulfur-35 containing carbonyl compounds when solutions of sulfur-35 cystine, as well as, radioactive milk labelled with sulfur-35 cystine were exposed to fluorescent light and sunlight, respectively. They reported that the formation of these volatile sulfur compounds re­ sulted from the degradation of radioactive cystine by light. SCOPE OF INVESTIGATION

The major aim of this study was to identify the types of volatile sulfur compounds that are formed as a result of heat treat­ ment of milk and to investigate factors related to the heat induced formation of different kinds of volatile sulfur compounds in fluid milk products.

The aspects which were investigated included—

1. The influence of different types of sulfur-35 radio­ isotope precursors on the distribution of volatile sulfur com­ pounds in raw and heated milk.

2. The effects of time of heating milk upon the types and distribution of the various volatile sulfur compounds.

3. The relationship of different fluid milk products to

the distribution of heat-produced volatile sulfur compounds at a

given temperature for different varying lengths of time.

4. The influence of pH, cystine, cysteine, sulphydryl blocking agents and oxygen level on the relative concentration of different types of volatile sulfur compounds in heated milk.

5. Identification of the nature of sulfur-35 compounds

formed by the high heat treatment of milk.

41 EXPERIMENTAL PROCEDURE

Source of Milk

The milk utilized throughout this study, unless otherwise specified, was radioactive milk, sulfur-35 labelled. Four types of sulfur-35 isotopes were employed. These were, sodium sulfate

(NagS^o^), sodium sulfide (NagS^)^ methionine

CHNHgCOOH, and barium sulfide (BaS^). The radioactive materials were introduced into the cows either intravenously, intraruminally or orally by placing the isotope material in a gelatin capsule, then wrapping the capsule in a small quantity of feed and forcing this ball of material down the esophagus of the animal. Milk was removed from the cows at intervals varying from four to twelve hours, depending on the particular study. Milk from each milking was analyzed for radioactivity and the lots with the greatest activities were either utilized singly or combined for heat studies. The milk was preserved by freezing at minus ll<-0C. Prior to heating, the frozen milk was thawed out in running tap water at 20°C.

Heating Procedure

Milk was heated in a totally closed system to prevent loss of

volatile compounds formed during the heating process (Figure l). A

quantity of milk (200 - 300 ml) was placed in a 250 or 500 ml pyrex

test tube, suspended in a rheostat-controlled water bath, which was

H2 43 maintained at a temperature of 91-92°C. The time to bring the tem­ perature of the milk to 90°C varied from three to five minutes.

Prior to heating, half a ml of a 10 per cent (w/v) Dow Corning anti­ foam agent (Silicone Product) was added to the- milk. After the addition of antifoam solution, nitrogen gas was turned on for one minute to check the trapping system for leaks and to facilitate mixing of antifoam material in milk. A 12 mm O.D. fritted glass cylinder extending to 5 ram from the bottom of the heating vessel was used for dispersing the nitrogen gas.

Since the milk was heated in a totally closed system, it was necessary to provide slight pressure to avoid back siphoning of milk and trapping reagents during the heating experiment. Sufficient nitrogen pressure was applied to maintain the milk at a constant level in the gas tube,but with no nitrogen gas being introduced into the system. Fig. 1. Heating Apparatus and differential trapping system utilized for the detection of volatile sulfur compounds in milk. n /

J f TE SOS 1 V/ // //■ u fr i « 'A' 3ZD - n2

8^ \ \ Samling \y vy \ J \ 3 \J tube \i/ \i/ v V Agitator 2,4-DNPH HgCI2 Hg(CN)2 CaCL Pb(OAc). Thermometers

Hedting tube

•p- Heating unit VJI 46

Immediately after the conclusion of heat treatment, the heat­ ing vessel was removed and the milk sample was cooled in an ice water bath to 24°C, which required 2 to 3 minutes. During cooling, care was taken to maintain the proper gas pressure in the system so as to prevent back siphoning of milk and trapping reagents. Following cooling, the samples were aspirated with high purity nitrogen gas at a rate of 200 cc per minute for 2 hours to remove any volatile com­ ponents that were formed.

Fractionation of Volatile Sulfur Compounds

The volatile sulfur components formed by heating were sepa­ rated into hydrogen sulfide, mercaptans, sulfudes and/or disulfides and 2 ,4-dinitrophenylhydrazine derivatives by a modification of the

absorption train of Dateo et al. (38) (Figure 1).

The vapors from the heating flask were passed through various traps connected in series. The water cooled condenser and

the two calcium chloride drying tubes served to remove moisture

from the gas mixture. Next, the gas mixture passed through a tube

containing 0.4 gm of finely powdered crystalline lead acetate,

Pb(C2H202)2*3H20, which removed hydrogen sulfide as insoluble lead

sulfide (PbS). The emerging gas from the lead acetate tube next

flowed through two tubes containing 10 ml of aqueous 4 per cent

(w/v) mercuric cyanide solution,(Hg(CN)2)» to trap mercaptans as

mercuric salts. From the mercuric cyanide tubes, the gas mixture

flowed through three tubes each containing 10 ml of aqueous 3 per

cent (w/v) mercuric chloride solution, which served to trap dialkyl sulfides and dialkyl disulfides, as addition complexes of mercuric chloride. Finally, the gas mixture was swept through three tubes containing 0.1 per cent 2,4-dinitrophenylhydrazine solution in

2N hydrochloric acid. The 2,4-dinitrophenylhydrazine reagent is specific for carbonyl compounds including, sulfur-containing carbonyl compounds.

Determination of Total Sulfur in Milk and Milk Fractions

Total sulfur in milk, casein and whey proteins, were esti­ mated by a modification of the procedure of Klipp and Barney (8 0 ).

The modified method involved the oxidation of dried samples in a

Parr oxygen bomb model 1901 (2).

Milk samples were dried by freeze drying. The casein samples were prepared by acidification of milk to pH 4.6 with 10 per cent acetic acid. The precipitated casein was redispers.ed with sodium hydroxide and subsequently reprecipitated. Thi6 procedure was repeated three times. Following reprecipitation the casein was washed twice with distilled water. The casein was dried at 35°C for 48 hours. The whey protein fraction was centrifuged at high speed to remove all residual casein. The clear whey fraction was boiled for

10 minutes and the precipitated proteins were removed by centrifuga­ tion. The precipitate was washed twice with distilled water prior to

drying in 35°C for 48 hours.

Attempts to use trichloroacetic acid to precipitate the whey

proteins were unsuccessful because the precipitates were extremely

corrosive to the chamber of the bomb, due to residual trichioacetic

acid in the whey protein samples. 48

Both the casein and whey fractions were ground into a fine powder before combustion in the oxygen bomb. The modified proce­ dure was as follows: Half a gram of freeze dried milk was placed in a metal combustion capsule, and twenty milliliters of 3 per cent

H2O2 were then placed in the bomb. The apparatus was a.ssembled according to instructions (2). A 10 cm fuse was utilized for the purpose of igniting the sample. Following this, the bomb was charged with 25 atmospheres of purified oxygen. The bomb was then placed in a water bath and next wired to an electrically operated ignition box and ignited. The bomb was allowed to remain in the water bath for at least 15 minutes following ignition. After this period, the excess gases in the bomb were released slowly and at an even rate so that the pressure within the bomb was reduced to atmos­ pheric in not less than one minute. The bomb was then opened and all parts of its interior, including the combustion capsule, valve passages and electrodes were washed with a fine jet of deionized water. The washings were transferred to a 100 ml beaker and made alkaline to phenolphthalein with diluted 10 per cent ammonium hydroxide.

After neutralization, the solution was boiled to remove excess moisture and hydrogen peroxide. Boiling was terminated when approximately 15 ml of solution remained in the beaker. The aqueous solution was passed through a Dowex 50-H resin column 1.5 cm in diameter and 15 cm long followed by an equal volume of deionized water. The effluent was adjusted to pH 4 with diluted ammonium hy­ droxide and/or diluted hydrochloric acid. The total effluent was 49 not in excess of 40 ml. Next, to a 100 ml volumetric flask was added 10 ml of 0.1 M sodium acetate buffer, 50 ml of 95 per cent

ethyl alcohol, the collected effluent and sufficient deionized water

to bring the total to 100 ml. The mixture was transferred to a 250 ml flask containing 0.3 gm of barium chloranilate. The flask was

stoppered and shaken on a mechanical shaker for 15 minutes. The

excess and BaSO^ were removed by filtering through Whatman

42 filter paper, and the filtrate (chloranilic acid) was read im

a Bausch and Lomb colorimeter at a wave length of 530 millimicrons

against a reagent blank. The amount of sulfur in the sample was

obtained by referring to a standard curve (Figure 2). The standard

curve was obtained by triplicate analysis of five different known

concentrations of sulfuric acid and analyzing for sulfur content as

previously described.

Measurement of Radioactive Sulfur Compounds

The quantity of radioactive sulfur compounds was determined

with either a gas flow Geiger type detector or by means of liquid

scintillation counting.

The gas flow equipment consisted of a Nuclear-Chicago Model

181A Decade Scaler, Model D-47 Gas Flow Counter, Model C-110A Auto­

matic Sample Changer and a Model C-111B Printing Timer. The D-47

Gas Flow Counter was equipped with a "Micromi" window. The gas mix­

ture used consisted of 99.05 per cent helium and 0.95 per cent iso­

butane at a flow rate of 50 cc per minute. Initially the activities Fig. 2. Standard curve for the determination of total sulfur OPTICAL DENSITY 0-2 0-4 0-3 0-5 01 0-6 OA SLU CONCENTRATION SULFUR TOTAL 0

AT PR MILLION PER PARTS 100

200

300

400

500 1 5 52 of milk, milk fractions and volatile sulfur compounds from each trap were determined by removing 0.2 and 0.5 ml aliquots and placing them in precleaned 30 mm O.D. by 2 mm round stainless steel planchets

(33). The samples were dried slowly under an infrared lamp by ro­ tating the sample with a motorized turntable at 30 rpm. Each sample was counted three times with a total of 2,000 counts being registered for each count. The average of the three determinations was taken aa the true count after correction for background was made. The mean standard deviation was calculated to be 0.3 ie. 0.1 counts per minute.

Background was determined by counting a blank planchet at regular intervals during the counting of radioactive samples.

After the acquisition of a Tri-Carb Liquid Scintillation

Spectrometer Model 314X, radioactivities of the volatile sulfur compounds in mercuric cyanide, mercuric chloride and lead acetate were determined by counting aliquots from each trapping solution.

Since no method was available for counting sulfur-35 in aqueous

salt solution the method utilized was experimentally developed from

the basic procedure of Bruno and Christain (21) for the determination

of carbon-14 in aqueous bicarbonate solution. The scintillating

solution utilized in this study consisted of the following:

p-dioxane ------833.0 ml

Ethylene glycol monomethyl ether "cellosolve" ---- 167.0 ml

2,5-diphenyloxazole (PPO) 10.0 gm

l,4-bis-2-(5-phenyloxazolyl)-benzene (POPOP) 0.5 gm

Naphthalene --- 50.0 gm 53

Counting temperature was 5°C and the counting vials were poly­ ethylene (25 ml) with polyethylene screw caps.

The determination of radioactivities of mercaptans in the mercuric cyanide traps, consisted of mixing 3 ml of trapping solu­ tion with 17 ml of scintillating solution. The determination of alkyl sulfides and dialkyl disulfides in the mercuric chloride traps consisted of combining 2 ml trapping with 18 ml of scintil­ lating reagent.

The determination of radioactive hydrogen sulfide consisted of dissolving 0 .^ gm of the solid lead acetate containing trapped radioactive lead sulfide in 5 ml of distilled water. Because lead sulfide is insoluble, it settled to the bottom of the counting vial when mixed with the previously described scintillating solvent.

Consequently, it was necessary to further modify the procedure for counting insoluble sulfur-35 lead acetate. The procedure adopted was a modification of the method of Gordon and Wolfe (5 3 ) using thixotropic gel (Cab-o-sil) powders for the determination of radio­ activity in emulsions and solids in aqueous suspension. The pro­ cedure consisted of mixing 2 ml of the lead acetate solution with

18 ml of a k- per cent by weight of Cab-o-sil in the Dioxane-methyl cellosolve scintillating solution. Upon shaking this mixture vigor­ ously, a thick homogenous gel resulted with the fine particles of

PbS dispersed throughout the gel.

The determination of the radioactivity of the material in the

2,k-dinitrophenylhydrazine traps was not possible with liquid scintil­ lation techniques because of color quenching by this reagent and the 54

low levels of sulfur-35 present. Consequently, the planchet method utilizing the gas flow counter described previously was utilized

throughout the study.

The radioactivities of milk, cream, whey and skimmilk were determined with the scintillation spectrometer by mixing thoroughly

0.5 ml of material with 19.5 ml of a 4 per cent by weight of Cab-o-

sil in Dioxane-cellosolve scintillating solution, and counting as

described previously.

Calculation of specific activity

The specific activity values reported in this investigation

are defined as the net counts per minute per unit weight of sulfur.

These values were obtained by two methods. In one instance, the

specific activity values were determined by obtaining the ratio be­

tween the net counts per minute of one gram of milk or milk product,

and the total sulfur content as determined by the oxygen bomb method

in parts per million for one gram of the corresponding product. The

second approach to determine specific values was to take the ratio

of the net counts per minute of radioactive sulfur in the hydrogen

sulfide from one liter of milk or milk product and the total amount

of hydrogen sulfide for one liter of milk.

'Quantitative Measurement of Hydrogen Sulfide

Hydrogen sulfide concentration was determined by a modifica­

tion of the methylene blue method of Marbach and Dotty (93). In this

method, the liberated hydrogen sulfide was collected as the black

precipitate of lead sulfide in the crystalline lead acetate trap. After the aspiration of the heated milk sample was completed and

the hydrogen sulfide trapped as lead sulfide in the lead acetate

trap, the trap was disconnected and the contents placed in a 20 hy

150 mm pyrex tube. Five milliliters of distilled water were used to

rinse out the trap and the contents collected in the test tube. The

excess lead acetate was dissolved by shaking the mixture gently with

the black insoluble lead sulfide dispersed in it as finely divided

particles. Methylene blue development was accomplished by adding

to this mixture, 1 ml of 0.1 normal sodium hydroxide, 1 ml of

N-N dimethylphenylenediamine reagent and 0.2 ml of Reisser’s

solution in succession. The test tube was stoppered tightly, in­

verted and the contents of the tube shaken for 10 minutes on a

mechanical shaker to mix the reagents. After 10 minutes, 10 ml

of distilled water was added to the mixture, the mixture shaken

again and allowed to stand at room temperature for 30 minutes for

maximum color development. Color intensity m s determined on a

Model 31^ Bausch and Lomb Spectrophotometer against a reagent blank,

using a wave length of 665 millimicrons. The amount of hydrogen

sulfide m s determined by referring the readings to the standard

curve, Figure 3*

Preparation of Reagents

1. N-N dimethylphenylenediamine solution: Dissolve 5 grams

of N-N dimethylphenylenediamine reagent in one liter of concentrated

hydrochloric acid, the solution should have an absorption of 0.03 or

less at 500 millimicrons. When protected from light, the solution is stable indefinitely.

2, Reisser solution: Dissolve 67.6 grams of ferric chloride hexahydrate in distilled water, dilute to 500 ml and mix this solution with 500 ml of nitric acid solution, containing 72 ml of boiled concentrated nitric (specific gravity 1.42). This solution is also stable indefinitely when protected from light.

Preparation of standard curve

The standard curve was prepared by a modification of the

Marbach and Dotty (93) procedure. A large crystal of ^ 2 3 * 9 ^ 0

(about 3 grams) was washed with distilled water until just the core

(about 0.1 gm) remained. This piece of washed Na2S*9H20 was next dissolved in distilled water and made up to 1 liter. The sodium sulfate solution was standardized with standard iodine solution using 0.2 per cent starch solution as indicator. The iodine solution in turn was standardized with standard sodium thiosulfate solution

(132). Different aliquots of the standardized solution were analyzed and optical density was determined at 665 millimicrons against a reagent blank. The standard curve was obtained.by plot­ ting known concentrations of sulfide from a range of 28 to 168 micrograms against the corresponding optical densities at a wave

length of 665 millimicrons (Figure 3)•

Determination of Mercaptans

The determination of mercaptans in the mercuric cyanide traps was carried out according to the colorimetric technique of Figure 3. Hydrogen sulfide standard curve OPTICAL DENSITY - 0 UFR CONCENTRAT' SULFUR G&1CR06RA&S 4 lit ®4 I© © 58 59

Tsugo and Matsuoke (14-1). The procedure involved the reaction of

4-. 5 ml of trapping mercuric cyanide solution vith 0.5 ml of dimethyl- p-phenylenediamine hydrochloride (0.6 gm per 100 ml of 6 N HCl) and

0.5 ml of 0.02 M ferric chloride. The mixture was mixed and shaken vigorously and then incubated at 30°C for thirty minutes. A red

color developed in the presence of mercaptans. The color inten­

sity was determined with a Bausch and Lomb colorimeter at a wave

length of 530 millimicrons.

Separation of Carbonyl Compounds

The volatile components in the 2,4-dinitrophenylhydrazine traps were separated by two dimensional thin layer chromatography

using the method of Anet (l).

The thin layer plates were prepared on 7 7/8 hy 7 7/8 inches

glass plates. The plates were coated with a slurry of Silica gel G

at a concentration of 20 gm in 100 ml of distilled water, at a thick­

ness of 0.008 inch. The coated plates were air dried for 15 minutes

at room temperature before placing in a hot air oven at 110°C for

one hour for the purpose of activating the plates.

The solvent systems used were: Solvent I. benzine-ethyl

acetate-petroleum ether 8:1:1 (v/v) and Solvent II. toluene-ethyl

acetate 1:1 (v/v).

The 2,4-dinitrophenylhydrazine derivatives were extracted

from the 2,4-dinitrophenylhydrazine traps with ether. After extrac­

tion, the ether solution was dried with sodium sulfate and then evap­

orated to dryness. The solid 2,4-dinitrophenylhydrazine derivatives were dissolved in a small quantity of ether before application on the cooled activated thin layer plates. Five to ten lambda of material were spotted at a point 1.5 by 1.5 cm from a corner of the plate. To insure a uniform solvent front traveling up the plate, the developing chamber was saturated with Solvent I by lining its walls with filter paper. The plates were then placed in the devel­ oping chamber containing Solvent I to a depth of 0.5 cm. The sol­ vent mixture was permitted to travel up the plate to a distance of

15 cm from the origin. The plates were removed and air dried to eliminate all traces of the Solvent I. The dried plates were turned around 90° and placed in Solvent II. Again, the solvent mixture was permitted to travel 15 cm up the plate from the point of origin.

The plates were removed and again air dried. The spots were devel­ oped by spraying the dried plates with a solution of 2 per cent sodium hydroxide in 95 per cent ethyl alcohol. Different colored

spots ranging from light yellow to deep purple would appear after the plates were allowed to stand at room temperature for a few minutes.

The size, color and relative position of the spots were recorded by

placing a tracing paper over the developed plates and drawing appro­

priate outlines around the various spots.

Determination of Mon-Volatile Sulphydryl Groups ( -SH) by the Thiamine Disulfide (TPS) Method

For the determination of sulphydryl compounds in heated milk

and milk products, a modification of Earland and Ashworth’s thiamine

disulfide method was used (58). 61

Milk samples for the thiamine disulfide determination were obtained from the heating vessel by means of a 6 mm glass siphon tube which extended to within 3 centimeters of the bottom of the heating vessel. The outlet of the siphon tube was fitted with a piece of tygon tubing, 3 cm in length and closed off by means of a

Fischer screw clamp. An aliquot of milk was removed from the heat­ ing vessel by means of nitrogen pressure after the milk sample had been heat-exposed and cooled to 24°C.

The TDS reducing materials were measured as follows: Two ml of milk was placed in a 16 by 150 mm pyrex test tube, and 1 ml of thiamine disulfide reagent was added. The contents of the tube were agitated and four drops of isobutyl alcohol were added. The samples were allowed to stand at room temperature for a period of two hours, after which 2 ml of 10 per cent trichloroacetic acid were added to precipitate the proteins. The tubes were centrifuged at 2,500 rpm for five minutes and the aqueous layer was filtered through a small wad of cotton into a second test tube. One ml of the filtered sample was made to a volume of 50 ml with distilled water. Two ml of this diluted filtrate was used for the estimation of the amount of thiamine present.

The two ml of diluted filtrate was placed in a 20 by 150 mm pyrex test tube, and then 3 ml of distilled water and 15 ml of iso­ butyl alcohol were added. Next, two ml of potassium ferriccyanide reagent was added and the mixture immediately agitated by a stream of air introduced through a fine bore glass tubing. The air aspir­ ation was continued for two minutes, after which the mixture was 62 allowed to stand for two minutes. The aqueous layer which separated was siphoned off, and approximately 2 grams of anhydrous sodium sul­ fate was added. The tubes were centrifuged for 2 minutes and the alcohol was decanted into matched curvettes and the fluorescence was determined by a Model 12 Coleman Photofluometer previously standard­ ized by a standard quinine sulfate solution. The readings were referred to a standard curve, Figure 4.

The standard curve was prepared from a standard thiamine hydrochloric solution containing 1 microgram of thiamine per milli­ liter. Aliquots of the standard thiamine solution containing from

0.1 to 1.0 micrograms were used for the preparation of the standard curve, for the thiamine disulfide determination Figure 4.

Preparation of reagents and standard solutions for the TPS determination

1. Thiamine disulfide solution: prepared by dissolving ^t-O mg. of thiamine disulfide reagent (prepared according to the method by

Harland and Ashworth (58 ) from 15 ml of a solution containing 2 ml of 0.1 N hydrochloric acid).

2. Potassium ferriccyanide solution: This oxidizing agent was prepared as recommended by AOAC (4). One gram of potassium ferric­

cyanide was made to 100 ml with distilled water. Two ml of this solu­ tion was diluted to 50 ml with 25 per cent sodium hydroxide.

3 . Quinine sulfate standardized solution: This solution was

prepared by diluting one volume of a stock solution (dissolve 10 mg of

quinine sulfate in 1 liter of 0.1 N sulfuric acid. This solution

fluoresced to about the same degree as the thiochrome from one micro­

gram of thiamine. Figure 4. Thiamin disulfide standard curve. 6 k

M m o

0 © • ©

THIAMIN HYDROCHLORIDE

CONCENTRATION, MICR06RAMS EXPERIMENTAL RESULTS

Development of Experimental Procedures

Methods utilized in this investigation, which were developed experimentally, included the determination of total sulfur, evaluation of counting procedure for determining radioactivity and the quantita­ tive estimation of hydrogen sulfide and mercaptans. This section pre­ sents results concerning the suitability of the experimentally developed procedures.

An evaluation was made to determine the precision and accuracy of the method for determining total sulfur. Varying concentrations of sulfur in the form of cysteine hydrochloride, were added to the combustion cap­ sule containing 0.5 gram of freeze dried milk. The freeze dried sample of milk was analyzed for total sulfur content and was used as a control.

The reproducibility of the method applied to eighteen replicate samples and recovery of added cysteine hydrochloride are shown in Table 1.

Table 1. The reproducibility and per cent recovery of total sulfur.

Concentration Cysteine No. of sulfur, ppm Standard Per cent HC1 ppm samples recovered deviation recovery added range average range average

Control 4 774-925 860 ±63 75.5-83.8 81.8

300 10 226.5-251.5 246.2 ± 80 75.5-83.8 81.8

400 8 294.0-334.0 311.7 ±138 73.5-83.5 77.9

65 The standard error in estimating total sulfur content ranged from

+7.0 per cent for milk to + 4 per cent for milk with 400 ppm added sulfur.

The average per cent recovery for 300 and 400 ppm added cysteine were 81.86 per cent, and 77.9 per cent respectively. All total sulfur values pre­ sented herein were corrected for 100 per cent recovery using the mean recovery value of 79.8 per cent.

Corrections for Radioactivity Determinations

Gas flow counter

Corrections of radioactive counts were necessary for loss in half-

life, self absorption and background. When the gas flow counter was uti­

lized, self absorption corrections were made for the loss of radioactivity.

Since doubling the concentration of radioactivity had essentially no effect

on self absorption, all analysis was conducted with a constant concentra­

tion of radioactive material. Self absorption was determined for 4 per

cent mercuric cyanide, 3 per cent mercuric chloride, 0.1 per cent 2,4-

dinitrophylhydrazine solution, 8 per cent lead acetate, milk and milk pro­

ducts, respectively. The correction values for self absorption were cal­

culated in terms of a factor by which the estimated net count per minute

of the sample must be multiplied to provide the actual net count per minute.

Data are presented in Table 2 for correction of self absorption of sulfur-

35 for milk, casein, whey protein, 4 per cent mercuric cyanide, 3 per cent

mercuric chloride, 0.1 per cent 2,4-dinitrophenylhydrazine and 8 per cent

lead acetate. Self absorption correction factors varied widely with the

different materials, ranging from l',05 to 3.11. The data revealed that

aqueous lead acetate and casein produced the greatest amount of self 67 absorption of radioactivity requiring a correction factor of 3.10 and

3.11, respectively. These were followed in order of decreasing self absorption by fluid milk, 3 per cent mercuric chloride, 4 per cent mercuric cyanide whey proteins and 0.1 per cent 2,4-dinitrophenylhy- drazine, and the corresponding self-absorption factors for these materials were 2.64, 1.79, 1.66, 1.54 and 1.05, respectively.

Table 2. Self absorption of sulfur-35 by various materials.

Correction Sample NCPM Self absorption Standard factor derivative mean

Sodium sulfate - ---- 1592 0

Sodium sulfate - milk 603 2.37 t 0.18

Sodium sulfate - casein (0.05 gm) 496 3.11 * 0.20

Sodium sulfate - whey protein (0.1 gm) 1030 1.54 + 0.20 mm

Sodium sulfate - 4 % Hg (CN)2 956 1.66 ♦ 0.18

Sodium sulfate - 3 7. HgCl2 885 1.79 V 0.29

Sodium sulfate - 0.1 7. 2,4-DNP 1515 1.05 + 0.06

Sodium sulfate - 8 % Pb (CH3C02)2 513 3.10 0.19

j NCPM in control Correction factor s NCPM in sample Based on average of 4 trials.

Liquid Scintillation Spectrometer '

The evaluation of counting efficiency involved the addition of a constant amount of sulfur-35 material, and different concentrations in volumes from 1 to 6 ml of aqueous solution of mercuric cyanide, mercuric 68

chloride and lead acetate trapping solution added to individual poly­

ethylene vials. The solutions were brought to a final volume of 20 ml

with a liquid scintillation solution. The vials were capped, gently

shaken and each counted for 10 minutes at 5°C. The counting efficiency

for varying concentrations of trapping solution was calculated by dividing

the net counts per minute (NCPM) obtained for each sample by the net

counts per minute for the control which contained no added salt solution.

The computation of experimental data was by the figure of merit system

adapted from that proposed by Kinard (76) for evaluating solvent systems

for tritiated water. The figure of merit value was obtained by multiply­

ing the per cent of total aqueous solution in the counting vial by the

corresponding per cent counting efficiency. The results for counting

efficiency and figure of merit values for mercuric cyanide and mercuric

chloride are presented in Table 3.

In both cases, there was a decrease in efficiency with an in­

crease in the volume of reagents used. This ranged from 80.9-2.7 per cent

and 58.1-1.1 per cent for mercuric cyanide and mercuric chloride, respec­

tively. The largest figure of merit values were obtained with 2 ml of

reagent in both cases. However, in the counting of mercuric cyanide,

there was no statistical differences between the use of 2 and 3 ml of

reagents.

Difficulties were encountered in counting hydrogen sulfide in the

form of lead sulfide. Table 4 presents results of counting radioactive barium sulfide in various amounts of 8 per cent lead acetate solution.

It was noted that the counts in successive determinination of the same Table 3* Counting Efficiency and Figure of Merit of Radioactive Barium Sulfide in the presence of Aqueous 3% Mercuric Chloride and 4% Mercuric Cyanide Solutions

Aqueous Labelled Scintil­ Mercuric Chloride Mercuric Cyanide Salt Barium lating % ncpm % Figure ncpm % Figure Solution Sulfide Solvent Aqueous x 10-3 Efficiency of x 10-3 Efficiency of ml ml ml Solution Merit Merit

0 0.1 19.9 0 .5 . 76.9 100 50 266.4 100 50

1 0.1 18.9 5.5 44.7 58.1 319 215.3 80.8 444.4

2 0.1 17.9 10.5 33.2 43.1 452.5 152.2 57.1 599.5

3 0.1 16.9 15.5 16.1 20.9 323.9 94.6 35.5 550.2

4 0.1 15.9 20.5 4.0 5.2 106.6 45.2 16.9 346.4

5 0.1 14.9 25.5 1.1 1.4 35.7 19.2 7.2 183.6

6 0.1 13.9 30.5 0.9 1.1 33.5 7.3 2.7 82.3 70 vial decreased gradually with time corresponding to the formation of lightly colored yellow precipitates. It was observed that the rate of de­ crease of counts with time was directly proportional to the volume of lead acetate used.

Table 4. Counting efficiency of radioactive barium sulfide in the presence of aqueous 8 per cent lead acetate using p-dioxane-methyl cello- solve as scintillating solvent.

8 per cent labelled p-dioxane- Net counts per minute for 4 consecutive lead barium methyl determinations after (minutes) acetate sulfide cellusolve 0 60 120 180 ml. solution

0 0.1 19.9 43998 43890 43990 43875

0.1 0.1 19.8 38287 37260 36881 36861

0.5 0.1 19.4 37218 36686 34833 31751

1.0 0.1 18.9 32569 27533 25959 25108

1.5 0.1 18.4 27276 21913 21311 20225

2.0 0.1 17.9 25523 20143 18806 17961

The thixotropic gel counting technique of Funt (50) was used to overcome this problem. The determination of counts in the 4 per cent thixotropic gel proved to be successful with little deviation in counts between successive determinations. Color quenching occurred only after 72 hours. The data in Table 5 for counting radioactive barium sulfide in the presence of 8 per cent lead acetate and 4 per cent thixotropic gel re­ vealed that counting efficiencies ranging from 56.9 per cent for 0.5 ml 71 to 9.4 per cent for 6 ml of lead acetate solution. The vial which con­ tained 2 ml of 8 per cent lead acetate solution had the highest figure of merit value 476,7,

Table 5, The effect of aqueous 8 per cent lead acetate solution on the counting efficiency of sulfur-35 and figure of merit value of radioactive barium sulfide.

8 per cent 4 per cent per cent NCPM per cent figure of lead thixotropic salt X10"3 efficiency merit in. acetate gel solution sol- ml. ml.

0 19.9 0.5 86.4 100.0 50.0

0.5 19.4 3.0 49.2 56.9 170.7

1.0 18.9 5.5 48.5 54.5 299.7

1.5 18.4 8.0 39.4 45.6 364.8

2.0 17.9 10.5 39.2 45.4 476.7

2.5 17.4 13.0 23.1 26.7 347.1

3.0 16.9 15.0 20.0 23.1 346.5

3,5 16.4 18.0 15.4 17.8 320.4

4.0 15.9 20.5 11.3 13.0 266.5

4.5 15.4 23.0 8.0 9.4 216.2

Based on the figure of merit value, the determination of radio­ activities in the 3 per cent mercuric chloride and 8 per cent lead ace­ tate traps involved the use of 2 ml of each salt solution, and 3 ml of

4 per cent mercuric cyanide solution. Counts obtained from counting various aliquots of trapping solutions, by the liquid scintillation 72

method, were corrected for counting efficiencies which involved multi­ plying counts per minute by the following correction factors: mercuric

cyanide 2.81, mercuric chloride 2.32 and lead acetate 2.32.

Colorimetric determination of hydrogen sulfide

The procedure used in this investigation was determined experi­ mentally, Direct application of the method of Dotty et al. (93) was found

to be unsatisfactory. Because of this, a modification was developed for

the determination of lead sulfide in the lead acetate trap. The modified method has been presented in the procedure section.

During the modification it was observed that the sequence of

addition of reagents affected the development of methylene blue color.

The following sequences were used:

Tube 1. 2 ml of sodium sulfide was added to 0.4 gram solid lead acetate followed by the addition of 3 ml of water and 1.0 ml 0.1 N sodium hydroxide.

Tube 2. 2 ml ofsodium sulfide was added to a solution made up of 0.4 gm lead acetate and 3 ml water, this was followed by 1 ml 0.1 N sodium hydroxide.

Tube 3. 2 ml of sodium sulfide was added to a solution made up of 0.4 gm lead acetate, 3 ml water and 1 ml of 0,1 N sodium hydroxide.

To each solution was added 1 ml amine and 0.5 ml Ressier solution.

Each mixture was then shaken vigorously and allowed to stand at room temp­

erature for 10 minutes. After which 10 ml of water was added to the reac­

tion mixture, shaken again, and allowed to stand at room temperature for a

further thirty minutes for color development. The results are presented

in Table 6 . 73

Table 6 . The effect of sequence of addition of reagent on the development of methylene blue color intensity.

Tube No. Optical density at 665 mu Trial 1 2 3 Average

1 0.475 0.475 0.468 0.473

2 0.420 0.415 0.420 0.418

3 0.495 0.495 0.500 0.495

The results indicated good reproducibility for all three tubes re­ gardless of the method used in the addition of the various reagents. In tube one the O.D. ranged from 0.468 to 0.475 with an average of 0.473.

For tube 2, the O.D. ranged from 0.415 to 0.420 with an average of 0.418.

Lastly, in tube 3 when sodium sulfide was added to a solution of lead ace­ tate and water, the O.D. ranged from 0.495 to 0.500 with an average of

0.495. These results indicated that the greatest color development was found in tube 3 and the lowest in tube 2. Because-of this, it was decided that color development for the subsequent determination of hydrogen sulfide followed the steps taken for tube 3.

During the determination of hydrogen sulfide by the colorimetric technique, it was noticed that a greenish blue color developed when the concentration of sulfur was low, when 1.5 ml of amine solution and 0.5 ml of Ressier solution were used for methylene blue color development. Conse­ quently, a study was conducted to ascertain the effect of quantity of added reagents on the corresponding color development. Experimentally it was 74 determined by varying the ratio of amine and Ressier reagent that 1.0 ml of amine solution and 0.2 ml of Ressier solution provided optical density values at 665 mu which were from 0.1 to 0.5 higher than the original combination of 1.5 ml amine and 0.5 ml reagent regardless of concentration. In addition, a typical methylene blue color was observed with the modified reagent concentration even with the lowest concentration of sodium sulfide solution. Therefore, it was decided to use 1 ml of amine solution and 0.2 ml Ressier solution for the development of methy­ lene blue color as described previously.

Colorimetric Determination of Mercaptans

The colorimetric method for mercaptans was applied to the mercuric cyanide trapping solution obtained from heating various milk and milk pro­ ducts at 90°C for varying lengths of time. The optical density values for milk, cream, skimmilk, and buttermilk heated and unheated are shown in

Table 7.

Table 7. The optical density reading of mercaptan concentrations for milk and milk products heated at 90°C for various lengths of time.

Product Optical density reading at 590 mu for product heated at 90°C for different ______timeg (minutes)______0 30 60 120

MLlk 0.005 0.015 0.015 0.015

Cream 0.015 0.012 0.015 0.015

Skimmilk 0.010 0.005 0.005 0.005

Buttermilk 0.005 0.015 0.005 0.005 75

The results revealed that the optical density values were too low to provide accurate estimate of mercaptan concentration. The values showed no consistent trend in respect to optical density values with in­ creased time of heating with any products. Because of this, the data was considered unreliable. Therefore, this method could not be utilized in this investigation.

Concentration of Sulfur-35 In Milk labelled with Different Radioisotopes

Total Radioactivity

Sulfur-35 was employed throughout this investigation to obtain radioactive milk to determine the effect of heat on the formation of volatile sulfur compounds. Different chemical forms of sulfur-35 isotopes were utilized and included methionine, sodium sulfide, sodium sulfate and barium sulfide. Only single trials were made with sulfur-35 methionine and sodium sulfide because of high cost. The milks were obtained at k, $,

8 or 12 hour intervals after incorporation of isotopes into the cow. All the different isotopes were administered into the rumen of the cow except for methionine which was incorporated into the cow by intravenous infusion.

Information concerning the type of isotope used, the size of dosage, the method of administration of the isotope and the specific cows used to obtain each lot of radioactive milk are shown in Appendix Table 33*

The time required for the milk to reach maximum activity and the net count per minute for milks using different chemical forms of sulfur-35 are summarized in Table 8 .

The rate of incorporation of sulfur-35 into the milk varied with the chemical forms of sulfur-35 utilized. Maximum counts were obtained at 76

Table 8 . The effect of types of sulfur-35 precursors on the radio­ activity of milk

Time required to reach Net Counts per Minute Type of milk maximum count per milliliter of milk Range Average

Sodium sulfide-s35 6 — 3,783

Methionine-S^5 12 -- 11,531

Sodium sulfate-S^5 24- 1380-2262 1,821

Barium sulfide-s35 2k 8714-16922 12,032

Appendix Tables 34,35,36, and 37

6 , 12 and 24 hours for sodium sulfide, methionine, sodium sulfate and barium

sulfide sulfur-35 labelled milk, respectively. Since there was only one

lot each of methionine and sodium sulfide milk, no range in activities

were observed, whereas, in sodium sulfate milk, the maximum counts for the

different lots of milk ranged from 1380 to 2262 counts per minute per milli­

liter of milk with an average of 1821 counts per minute. The different lots

of barium sulfide milk had maximum counts ranging from 871^ to 16,922 counts

per minute with 12,032 counts per minute as an average.

Total sulfur content and radioactivity of milk and milk proteins

The total sulfur, sulfur-35 and specific activity content of milk,

casein and whey protein were investigated for milk obtained from cows

treated with sulfur-35 sodium sulfate, methionine and barium sulfide. The

specific activity is the ratio of concentration of sulfur-35 to total 77 sulfur content in counts per minute per microgram of sulfur. The corresponding radioactivities and specific activities results are presented in Table 9*

The total sulfur content for milk, casein and whey protein of all three types of sulfur-35 labelled milk were similar. For the different milks, the total sulfur content ranged from 3^50 to ^370 ppm; the three caseins, 7060 to 8020 ppm and the three whey proteins, 10520 to 10800 ppm.

Generally, the radioactivity per gram of material was high­ est for the whey proteins,, except for sodium sulfate milk in which the casein fraction had slightly higher activity than the whey pro­ tein fraction. The specific activity varied among the same fraction from milk labelled with different chemical forms of sulfur-35 and also varied among the components in a given lot of milk. The results could be best evaluated in terms of relative specific activity in which milk was used as the unit of comparison. Based on relative specific activity values, selective labelling was apparent.

Methionine labelled milk showed the greatest selective labelling into milk proteins because this milk had the highest ratio of specific activity values for casein and whey protein sulfur-35 when compared to the specific activity of sulfur-35 of methionine milk. The relative specific activity values for casein and whey protein were 3.^6 and

3.82, respectively. The second highest selective labelling into milk proteins of sulfur-35 material was observed in the barium sulfide milk.

The barium sulfide labelled milk had relative specific values of 2.5^ and 2.55 for the casein and whey proteins, respectively. The 78

Table 9* Total sulfur and sulfur-35 content of various milks and milk fractions and their corresponding specific activities

Type of Wet counts sulfur-35 Total per minute labelled sulfur per gm dry Specific activity milk ppm weight NCPM/ sulfur relative

Sodium sulfate

Milk 3,9^0 9,179 2.5 1.00

Casein 7,360 18 > 1 6 2.4 0.91

Whey proteins 10,800 17 >59 1.6 0.62

Methionine

Milk ^,370 34,250 7-9 1.00 Casein 8,020 219,750 27.4 3.46

Whey proteins 10,960 331,557 30.3 3.82

Barium sulfide

Milk 3 >50 70,512 20.4 1.00

Casein 7,060 366,223 51.9 2.54

Whey proteins 10,520 543,222 51.6 2.55

Appendix Table 38 79 variations in relative specific activities suggested wide differences in the specific activity of the non-protein fractions of milk. The specific activity of non-protein sulfur content in milk labelled with different chemical forms of sulfur-35 could not be determined directly.

The per cent of sulfur in the non-protein fractions of milk were similar, being 1.7 per cent, 0.9 per cent and 1.5 per cent for sodium sulfate, methionine and barium sulfide milk, respectively.

The specific activity of non-protein sulfur-35 was greater than pro­ tein sulfur for sodium sulfate labeled milk; whereas for sodium sulfide and methionine sulfur-35 labeled milk,the opposite was found.

Free Volatile Sulfur Compounds from Unheated Sulfur-35 Labeled Milk

Free volatile sulfur compounds were determined by aspirating raw milk with nitrogen gas at 200 cc per minute for 2 hours at room temper­ ature. The nitrogen gas was passed through differential trapping

systems to separate hydrogen sulfide, mercaptans, organic sulfides and

2,4-dinitrophenylhydrazine (2,4-DNPH) derivatives.

Kesults are presented in Table 10 for the free volatile sul­

fur compounds of raw unheated sulfur-35 milks labeled by sodium sul­

fate, sodium sulfide and barium sulfide, respectively. All the milks

examined contained free volatile mercaptans, sulfides and/or disulfides

in varying concentrations. Sulfur-35 containing carbonyl compounds were

found only in those milks labeled with sodium sulfate, sodium sulfide

and one lot of barium sulfide milk. No radioactive hydrogen sulfide

was detected in the volatiles of either sodium sulfate or sodium sulfide

milk. The data indicated that the milks obtained from different cows 8 0

Table 10. Free volatile sulfur-35 compounds from raw unheated sulfur-35 labelled milk

Net counts per minute per liter of milk Sample Type of Cow Organic 2,4-BNEH Hydrogen isotope no. Mercaptans sulfides derivatives sulfide

1 Sodium Ht82 160 213 320 0 sulfate

2 Sodium 1502 18 195 khk 0 sulfide 1^06

3 Barium 1555 230 333 108 123 sulfide

h Barium 1507 117 *<•33 0 0 sulfide

5 Barium 1577 233 1000 0 133 sulfide

Appendix Tables kO, hi and h2 81 labeled with barium sulfide varied in the amount and type of free vol­ atile sulfur compounds present. The sulfur-35 concentration in mercap­ tans of all three samples of barium sulfide milk from different cows were similar. However, the sulfur-35 concentration of sulfides and/or disulfides varied from 333 to 1,000 counts per minute and the sulfur-35 containing carbonyl compounds varied from 0 to 108 counts per minute.

The concentration of hydrogen sulfide was quite similar for milk from cows number 1555 and 1577* tut none was detected from cow number 1507.

The concentration of free volatile sulfur compounds was extremely low.

Based on the specific activity of the sulfur in the milk, the concen­ tration of the free sulfur compounds ranged from 1 to 40 parts per billion.

Effect of Heat Treatment on Release of Volatile Sulfur Compounds from Sulfur-35 Labeled Milk

Preliminary investigations were conducted to determine if dif­ ferent types and quantities of volatile sulfur compounds were formed by high heat treatment of milk. In these initial trials, milk from a cow treated with radioactive sodium sulfate was heated to 8o°C and 90°C for

10, 30 and 60 minutes. Data for 10 minutes of heating are in Table 11.

Although no precipitates were observed in the chemical traps, low concen­ trations of radioactivities were found in all four types of reagents. At

80°C, no volatile sulfides were detected after 10 minutes of heating, whereas, appreciable amounts of sulfides were found in the mercuric chloride traps after 90°C of heating. Similarly, the concentration of mercaptans and sulfur-35 containing 2,4-dinitrophenylhydrazine deriva­ tives were much higher at 90°C than 8o°C. Because of the low level of radioactivity found in the trapping reagents after heat treatment at 8o°C, all subsequent heat trials were conducted at 90°C. 82

Table 11. Effects of heat treatments at 8o°C and 90°C on the formation of volatile sulfur compounds from sodium sulfate sulfur-35 labelled milk

Treatment Net counts per minute per liter of lilk ' Temperature Time organic 2,4-DMPH hydrogen °C Minutes mercaptans sulfides derivatives sulfide a CO H O o 53 0 27 present ON i O o —1 613 786 i4o6 present

Appendix table 39

Sulfur-35 content was not quantitated.

The effect of heat treatment on milk labelled with various chemical forms of sulfur-35 compounds on the formation of volatile sulfur compounds

Investigations relating to the formation of volatile sulfur com­

pounds as a result of high heat treatment involved the utilization of

milk labelled with different chemical forms of sulfur-35 radioisotopes.

The different radioisotopes used in obtaining radioactive milk included

sulfur-35, sodium sulfide, methionine, sodium sulfate and barium sulfide.

Due to high cost of both sulfur-35 sodium sulfide and methionine, only

limited investigations were made using these isotopes.

Sodium sulfide labelled milk

Sodium sulfide milk obtained following incorporation of the

radioactive compound into the rumen, was used in initial experiments.

Milk was withdrawn at 6 hour intervals and those lots of milk with the

highest activities were pooled and used for heat studies. Milk was

heated at 90°C for 30, 60, and 120 minutes. The volatile sulfur compounds from heat treatment of sodium sul­ fide milk are presented in Table 12. The data are presented in terms of the change in net counts per minute from the unheated control.

The limited data showed that hydrogen sulfide, mercaptans and organic sulfides were formed by heat treatment. At all three times of heating, heat released hydrogen sulfide was produced,in highest concen­ trations, followed by mercaptans and then organic sulfides.

Table 12. The effects of high heat treatment at 90°C of volatile sulfur-35 from milk obtained following incorporation of sulfur-35 sodium sulfide

Treatment Change in net counts per minute per liter■ of milk Temperature Time organic 2,4-DNPH hydrogen °C Minutes mercaptans sulfides derivatives sulfide

90 30 + 234 - 49 - 444 4- 487

90 6o + 88 * 64 - 324 + 285

90 120 - 18 -195 - 3 + 1^5

Appendix table 40. For raw milk ncpm were 18, 195> 444 and 0 for mercaptans, organic sulfides, 2,4-DNPH derivatives and hydrogen sulfide, respectively.

In the case of the organic sulfides, only heating at 90°C for

60 minutes caused the formation of any of these compounds. The concen­

tration of 2,4-DNPH derivatives were lower in the heated milk than in the

raw milk for all three times of heating. Maximum heat released hydrogen

sulfide and mercaptans occurred after 30 minutes of heating, with definite

decreases in the concentrations of both of these compounds with heating

periods longer than 30 minutes. After heating for 2 hours at 90°C, the Qk concentrations of volatile mercaptans, organic sulfides and 2,k-DNPH derivatives were all less than were present in the unheated raw milk.

Methionine labelled milk

Methionine sulfur-35 milk was obtained following intravenous infusion of the isotope into the cow. Milk was withdrawn at 6 hour intervals, and those lots of milk with the highest activities were used for heat studies. Heating times were 30 , 60 and 120 minutes at temper­ atures of 90 and 95°C.

The results for the formation of volatile sulfur compounds from heating sulfur-35 methionine labelled milk are presented in Table 13•

Eadioactive hydrogen sulfide was the only heat released volatile sulfur compound present in milk under all conditions of heat treatment. At both heating temperatures, maximum formation of radio­ active hydrogen sulfide was found in the milk heated for 30 minutes.

In milk heated at 90°C, heating longer than 30 minutes caused only slight decrease in the hydrogen sulfide, whereas at 95°C the concentration of hydrogen sulfide was much lower after 120 minutes of heating than after

30 or 60 minutes of heating. At 90°C, mercaptans and 2,k-DNPH derivatives were found only after 30 minutes of heating. At 95°C, mercaptans and 2,4-DNPH derivatives were present in maximum concentra­ tions after 30 minutes of heating. No organic sulfides were observed in milks heated at either 90 or 95°C.

Sodium sulfate labelled milk

Sulfur-35 sodium sulfate was incorporated into the cow through the rumen. Milk was collected at 8 hour intervals, and those lots of milk with the highest activities were pooled and used for heat trials. Table 13. The effect of heat treatment of methionine sulfur-35 labelled milk on the formation of volatile sulfur-35 compounds.

Heat Treatment Net counts per minute per liter of milk Temperature Time Organic 2,4-DNPH Hydrogen Minutes Mercaptans Sulfides Derivatives Sulfide

90 30 35 0 221 254

90 60 0 0 0 206

90 120 0 0 0 228

95 30 101 0 196 255

95 60 158 0 37 219

95 120 0 0 120 134

CD VJ1 86

Milk was heated at 90°C for 10, 30, 6o and 120 minutes. As shown previously, the highest specific activity in this milk was associated with the non protein sulfur. The results of this study are presented

in Table l4.

Heat release of all four types of volatile sulfur compounds was

evidenced by the increase in radioactivity in all four types of trapping

reagents. In contrast to earlier trials with milk labelled using other

chemical forms of sulfur, heat released 2,if-DNPH derivatives were

observed for all 4 heating times used in this study. Variations were

observed in the concentrations of heat released volatile sulfur com­

pounds in milks heated for different periods of time and variation

in radioactivity in various traps was noted between trials. After 10 minutes of heating, 2,4-DNPH derivatives and mercaptans generally were

formed in the highest concentrations; after 30 minutes of heating, mercaptans were formed in the highest concentrations; after 60 minutes

of heating, hydrogen sulfide was formed in the highest concentrations,

and after 120 minutes of heating, organic sulfides and 2,4-DNPH deriva­

tives were released in the highest concentrations.

The greatest concentration of mercaptans were formed after

heating the milk for 30 minutes at 90°C. On the other hand, the concen­

tration of radioactive organic sulfides, hydrogen sulfide and 2,4-DNPH

derivatives was highest after heating milk at 90°C for 120 minutes. 87

Table lb. The effect of heat treatment at 90°C on sodium sulfate sulfur-35 labelled milk on the release of volatile sulfur compounds

Treatment Change in net counts per minute per liter of milka Temperature Time organic 2, if-DNPH hydrogen °C Minutes mercaptans sulfides derivatives sulfide

90 10 4- 213 f 19b + 296 + 195

90 30 ¥ kk9 f 137 + 22 * 2b8

90 60 + 133 + 27 + 1^3 *■ l6l

90 120 + 99 t J+56 + b02 ■i 308

Appendix table 1+-1. aFor raw milk ncpm were l6o, 213, 320 and 0, for mercaptans, organic sulfides, 2,1)—DNPH derivatives and hydrogen sulfide, respectively.

Barium sulfide labelled milk

Milk, from cows which had been dosed with sulfur-35 barium

sulfide, was collected at the regular morning and evening milkings

following the administration of the radioactive sulfur. The milk ob­

tained 2b hours after incorporation of the isotope was used. Since the

barium sulfide was utilized somewhat slowly in the rumen of the cow, it

was possible to obtain milk with much higher radioactivity than when

other isotopes were used. The average count of the milk used was

9800 counts per minute. The milk was analyzed for volatile sulfur com­

pounds and thiamine disulfide reducing substances after 30, 60 and 120

minutes of heating. The results of the effect of varying heating times

on the volatile sulfur compounds and total sulfydryl groups are presented

in Table 15* Table 15. The effect of heat treatment at 90°C on barium sulfide sulfur-35 labelled milk on the release of volatile sulfur compounds

Change in net counts per minute (TDS) Treatment per liter of milka mg. of cysteine Temperature Time organic 2,i|-DHPH hydrogen hydrochloride °C minutes mercaptans sulfides derivatives sulfide per liter of milk

90 30 * IO87 - 378 - 18 + 3012 18.6

90 60 * 1382 - 305 - 85 + 10,672 16,6

90 120 + l6kk - 285 + k6 1 8112 20.0

Appendix Table k2

^ o r raw milk ncpm were 203, 525, 108 and 95 for mercaptans, organic sulfides, 2,4-DNPH derivatives and hydrogen sulfide, respectively. 89

Heat treatment resulted in the release of hydrogen sulfide and mercaptans, but no organic sulfides and very small amounts of 2,4-DHPH derivatives -were formed at any of the times used in these studies. At all heating times, the concentration of hydrogen sulfide was at least three times greater than that of the mercaptans. Maximum concentration of sulfur-35 hydrogen sulfide was found after 60 minutes of heating, whereas the maximum concentration of sulfur-35 mercaptans was present after 120 minutes of heating. The mercaptans showed the greatest increase during the first 30 minutes of heating, followed by a slow increase in concentration with prolonged heating times. The greatest increase in hydrogen sulfide occurred between 30 and 60 minutes of heating.

Comparison of volatile sulfur compounds produced from heat treatment of different sulfur-39 labelled milk at 90°C for two heating periods

A comparison of the volatile sulfur compounds formed after high heat treatment at 90°C for 30 and 60 minutes, respectively, for methionine, sodium sulfide, sodium sulfate and barium sulfide sulfur-35 labelled milk are presented in Table 16. The values pre­ sented were obtained by dividing the counts per minute of each class of volatile compound by the net counts per minute of hydrogen sulfide, of the same milk.

The ratios of concentration of sulfur-35 in the mercaptans, organic sulfides and 2,i»—DHPH derivatives in relation to the concen­ tration of sulfur-35 hydrogen sulfide provide a means of determining whether the type of sulfur used in obtaining the radioactive milk had any effect on the nature of the sulfur-35 volatile compounds released by heat treatment. Table 16 . The effect of high heat treatment at 90°C for 30 and 60 minutes of different milks labelled with different sulfur-35 isotopes

Heating Ratio between counts per minute of each sulfur-35 Type of time volatile and counts per minute of sulfur-35 sulfur-35 at 90° hydrogen sulfide isotope minutes organic 2,4-DNPH hydrogen mercaptans sulfides derivatives sulfide

Methionine 30 0.14 0 O.87 1.00

Sodium sulfide 30 0.52 0.29 0 1.00

Barium sulfide 30 0.42 0.02 0.01 1.00

Sodium sulfate 30 2.45 l.4o 1.38 1.00

Methionine 6o 0 0 0 1.00

Sodium sulfide 6o 0.37 0.91 0.42 1.00

Barium sulfide 60 0.42 0.02 0 1.00

Sodium sulfate 6o 1.83 1.50 2.87 1.00 91

The ratios obtained showed that for the milks heated for 30 minutes, hydrogen sulfide was present in higher concentrations than any other volatile sulfur compound when methionine, sodium sulfide or barium sulfide provided the source of the radioactive sulfur. Essen­ tially no radioactive organic sulfides were formed in these milks by heating, and only methionine labelled milk contributed significantly to heat released 2.}k ~DNPH derivatives. These milks were similar in respect to the fact that the specific activity of the sulfur was higher in the protein than in the non-protein sulfur fraction. The results with milk labelled with sodium sulfate were quite different.

Furthermore, this milk had a higher specific activity in the non-protein

sulfur fraction than in the protein sulfur. Heating this milk resulted

in concentration of radioactive mercaptans, organic sulfides and

2,k -DNPH derivatives all being higher than the concentration of radio­ active hydrogen sulfide.

Similar results were obtained for the milk heated for 60 minutes, except that no radioactive volatiles other than hydrogen sul­ fide were found in milk labelled with methionine.

These results revealed that the chemical form of the sulfur used to obtain sulfur-35 labelled milk does have a definite influence

on the type of radioactive volatile sulfur compounds formed by heat

treatment. The results suggested further that organic sulfides and

2,J+-DHPH derivatives are formed from different sulfur precursors in

the milk than mercaptans and hydrogen sulfide. 92

Volatile sulfur compounds from various fractions of milk

To further understand the formation of volatile sulfur com­

pounds as a result of high heat treatment of milk, experiments were

conducted to investigate the formation of volatile sulfur compounds

from skimmilk, cream, buttermilk and whey fractions of milk.

Preliminary investigations on the formation of volatile sulfur

compounds from skimmilk, buttermilk and cream were made with sodium

sulfide sulfur-35 labelled milk (Appendix Table ^3). A single trial

indicated inconsistency in the concentration of volatiles formed from

the different products, which probably was because of the low level of

radioactive sulfur-35 ia the milk. Since the results for the formation

of volatiles from these fluid products appeared to be unreliable, sub­

sequent trials were then conducted with barium sulfide sulfur-35

labelled milk with higher activity.

Barium sulfide milk was fractionated into skimmilk, cream,

buttermilk, acid whey and rennet whey. Each product was aspirated for

two hours at room temperature to determine the free radioactive volatile

sulfur compounds present in these products. The results are presented

in Table 1 7 . The data revealed the presence of varying concentrations

of free radioactive sulfur compounds in the form of mercaptans, organic

sulfides, 2,4-DNPH derivatives and hydrogen sulfide, in the different

milk products. Except for buttermilk, the concentration of free mer­

captans and organic sulfides was higher for all products than for the

raw milk. The concentration of mercaptans was two and nine times

higher in skimmilk and acid whey than in milk. The organic sulfide

content of skimmilk, cream and rennet whey were about four to five times

higher than for milk. However, the concentration of free radioactive 93

2,4-DNPH derivatives were generally similar for the different milk products. The concentration of free hydrogen sulfide from buttermilk and acid whey was significantly higher than those found in unheated milk.

Table 17. Free volatile sulfur compounds in various fluid milk products

Net counts per minute per liter of product Product organic 2,4-DNPH hydrogen mercaptans sulfides derivatives sulfide

Milk 203 525 108 95

Skimmilk 560 2155 198 83

Cream 324 2823 342 132

Buttermilk 0 287 4o4 218

Acid whey 1116 581 331 164

Rennet whey 409 1986 — 70

Appendix tables 42, 44, 45, 46, 47, and 48.

Results obtained from heating various fluid milk products obtained from barium sulfide sulfur-35 labelled milk appeared to be consistent and provided a means of following the effect of heat on the formation of volatile sulfur compounds from different milk "products.

The results for the formation of volatile sulfur compounds from heat treatment of skimmilk obtained from milk labelled with sulfur-35 barium sulfide are shown in Table 18. Table 18. The effect of heat treatment at 90°C on barium sulfide sulfur-35 labelled skim­ milk on the formation of volatile sulfur-35 compounds

Change in net count per minute (t d s ) Treatment per liter of milka Mg. of cysteine Temperature Time organic 2,4-DNPH hydrogen hydrochloride °C minutes mercaptans sulfides derivatives sulfide per liter of skimmilk

90 30 180 - 380 + 76 + 6919 10.3

90 60 + 560 - 225 - 88 4 7202 l4.0

90 120 + 52 4 387 - 4l + 6709 13.6

Appendix Table 44

^ o r raw skimmilk ncpm were 560, 2155, 198 and 83 for mercaptans, organic sulfides, 2,4-DNPH derivatives and hydrogen sulfide, respectively. 95

Heating of skimmilk for various periods of time demonstrated that hydrogen sulfide was formed in the highest concentrations among all the radioactive volatile sulfur compounds detected. The ratio of heat released hydrogen sulfide to heat released mercaptans was 30-100 to 1 as compared to 3-5 to 1 previously found for whole milk. In con­ trast to earlier findings with milk, heat released radioactive organic sulfides and 2,4-DNPH derivatives were found after 30 and 120 minutes of heating, respectively. The maximum concentration of mercaptans and hydrogen sulfides were formed after 60 minutes of heating.

Maximum thiamine disulfide reducing materials were obtained from skimmilk following heat treatment at 90°C for 6o minutes. This corresponded to the maximum concentration of sulfur-35 in hydrogen sulfide and mercaptans.

The results for heating cream are presented in Table 19. After

30 minutes of heating, radioactive organic sulfides were present in highest concentration in the cream, followed by hydrogen sulfide and the mercaptans. After 6o and 120 minutes of heating, the concentra­ tion of hydrogen sulfide was slightly higher than the organic sulfides.

No heat release of 2,4-DNPH derivatives were noted at any of the times of heating. The concentration of both hydrogen sulfide and organic sulfides increased greatest during the first 30 minutes of heating, reached a maximum after 60 minutes of heating, and decreased slightly between 60 and 120 minutes of heating. A similar trend was observed for the total sulphydryl content, whereas the volatile mercaptans in­ creased steadily over the entire two hour heating period. The thiamine Table 19• The effect of heat treatment at 90°C on barium sulfide sulfur-35 labelled cream on the formation of volatile sulfur-35 compounds

(TDS) Change! in net counts per minute mg. of cysteine Treatment per liter of creama hydrochloride Temperature Time organic 2,4-DNPH hydrogen per liter °C minutes mercaptans sulfides derivatives sulfide of cream

90 30 + 281 + 4514 - 8 f 3536 14.3

90 6o + 724 + 5542 + 8 + 6602 14.4

90 120 +1320 + 4317 - 88 4 4927 13.6

Appendix Table 45 a „ For raw cream, ncpm were 2791* 2623, 342 and 13b for mercaptans, organic sulfides, 2,4-DNPH derivatives and hydrogen sulfide, respectively. 97

disulfide reducing materials formed from heating cream were relatively

constant throughout the whole heating period.

The results for high heat treatment of buttermilk are presented

in Table 20. Heat treatment of buttermilk resulted in the formation

of all types of volatile sulfur compounds. Hydrogen sulfide was pro­

duced in the highest concentration, followed by the organic sulfides,

mercaptans and then by the 2,14—DNPH derivatives. The maximum concen­

tration of hydrogen sulfide was found after 6o and 120 minutes of

heating, whereas the highest concentration of heat released organic

sulfides were observed after 30 minutes of heating. Only a small

concentration of mercaptans were present in heated buttermilk and the

concentration was generally independent of the time of heating.

The total sulphydryl groups were highest after 30 minutes of

heating and decreased markedly between 30 and 60 minutes of heating.

The decrease in the concentration of total sulphydryl groups was

paralleled by a large drop in the organic sulfides and an increase in

hydrogen sulfide. The decrease in counts per minute in organic sul­

fides was approximately the same as the increase in counts per minute

in hydrogen sulfide.

The results for the formation of volatile sulfur compounds and thiamine disulfide reducing materials from the high heat treatment of acid and rennet whey are presented in Table 21. The data reveals that

only hydrogen sulfide is formed in significant concentrations from the

heat treatment of either acid or rennet whey. The heat released hydrogen

sulfide was 28, 15 and 5 times greater in rennet than acid whey after

30, 60 and 120 minutes of heating, respectively. The greatest increase Table 20. The effect of heat treatment at 90°C on barium sulfide sulfur-35 labelled butter­ milk on the formation of volatile sulfur-35 compounds

(TDS) Change in net counts per minute mg. of cysteine Treatment per liter of buttermilka hydrochloride Temperature Time organic 2,4-DNPH hydrogen per liter °C minutes mercaptans sulfides derivatives sulfide of buttermilk

90 30 + 388 i 1342 4 57 + 3849 10.1

90 60 t 388 + 383 + 190 4 5246 5.1

90 120 t 135 4 289 + 186 + 5390 i 4.5

Appendix Table 46

^or raw buttermilk, ncpm were 0, 2, 87, 4o4 and 218 for mercaptans, organic sulfides, 2,4-DKPH derivatives and hydrogen sulfide, respectively. Table 21. The effect of heat treatment at 90°C on barium sulfide sulfur-35 labelled acid and rennet whey on the formation of volatile sulfur-35 compounds

(TDS) Change in net counts per minute Mg. of cysteine Treatment per liter of wheya hydrochloride Temperature Time organic 2,4-DNPH hydrogen per liter °C minutes mercaptans sulfides derivatives sulfide of whey acid rennet acid rennet acid rennet acid rennet acid rennet

90 30 + 65 -229 - 53 -156 * 83 — *125 +3268 2.31 11.50

90 60 -295 -229 -159 -509 -145 - ♦207 *3285 2.98 13.70

90 120 +286 + 13 - 54 - 6 l -248 — +785 *4035 4.10 15.40

Appendix Tables 47 and 48

^ o r raw acid whey, ncpm were 1116, 581, 331 and 164, for raw rennet whey, ncpm were 409, 1986, 0 and 70 for mercaptans, organic sulfides, 2,4-DNPH derivatives and hydrogen sulfide, respectively. 100 in hydrogen sulfide occurred between 0 and 30 minutes for rennet whey and between 60 and 120 minutes for acid whey. Small amounts of mer­ captans were released from both wheys after 120 minutes of heating.

The thiamine disulfide reducing substances were 5, V a n d 3*8 times greater in rennet than acid whey after 30, 60 and 120 minutes of heat­ ing. Maximum sulphydryl groups and radioactive volatile sulfur com­ pounds were produced in both wheys after 120 minutes of heating. The concentration of thiamine disulfide reducing materials formed from rennet whey were about four fold greater than those which were formed from acid whey after 30, 60 and 120 minutes of heating.

A comparison of the volatile sulfur compounds formed after high heat treatment at 90°C for 30 and 60 minutes, respectively, for milk, skimmilk, cream, buttermilk, acid whey and rennet whey obtained from barium sulfide sulfur-35 labelled milk are presented in Table 22.

The results revealed that with the exception of cream, hydrogen sulfide is produced in the highest concentration of the volatile sulfur compounds from the high heat treatment of various fluid milk products. The major compound released from heating cream was organic sulfides. After 30 minutes of heating the major organic volatile component was mercaptans in milk and acid whey, organic sulfides in cream and buttermilk and 2,1*-DNFH derivatives in acid whey.

Buttermilk was the only product which released all four types of volatile sulfur compounds upon heat treatment for both 30 and 60 minutes. Based on these results, it would appear that a major portion of the precursors of heat released organic sulfides and hydrogen sulfide follow the fat phase of milk and the precursor of 2,k-DNPH derivatives follows the serum phase of the milk. 101.

Table 22. The effects of high heat treatment at 90°C for 30 and 60 minutes of various fractions of milk obtained from barium sulfide sulfur-35 labelled milk

Ratio between counts per minute of each Milk Heating sulfur-35 volatile and net counts per fraction time at minute of sulfur-35 hydrogen sulfide 90°C organic 2,4-DNPH hydrogen minutes mercaptans sulfides derivatives sulfide

Milk 30 0.36 0 0 1.00

Skimmilk 30 0.02 0 0.01 1.00

Cream 30 0.08 1*28 0 1.00

Buttermilk 30 0.10 0.35 0.01 1.00 Acid whey 30 0.50 0 0.66 1.00

Rennet whey 30 0 0 — 1.00

Milk 6o 0.12 0 0 1.00

Skimmilk 60 0.08 0 0 1.00

Cream 60 o.ll 0.87 0 1.00

Buttermilk 6o 0.07 0.07 0.03 1.00

Acid whey 6o 0 0 0 1.00

Rennet whey 6o 0 0 0 1.00 102

Factors Affecting the Heat Release of Volatile Sulfur Compounds

To better understand the mode for the formation of volatile sulfur compounds from high heat treatment of milk, factors which may enhance or retard the formation of these volatiles were investigated.

Factors included were pH, cysteine and cystine, sulphydryl blocking agent, air and nitrogen.

Thus far, the formation and release of volatile sulfur com­ pounds from high heat treatment of milk, as well as other milk frac­ tions, have been studied at the normal pH of milk. Since previous investigators (63, 102, 103, 119> 1^0, IU9 ) have shown that pH is an important factor in heat release of hydrogen sulfide, study was con­ ducted to determine the effect of pH on the heat formation of other volatile sulfur compounds.

The milk utilized was barium sulfide sulfur-35 labelled milk, orally administered. The activity of the milk utilized was 9^00 counts per minute per milliliter of milk. One lot of milk (pH 6 .6 ) served as a control. Other lots were adjusted to pH 7-7 and 9*7 respectively, using 25 per cent sodium hydroxide. The heating procedure consisted of heating milk at the desired pH to 90°C for 60 minutes. The results are presented in Table 2 3 .

In general, increasing the pH appeared to affect the release of

sulfur-35 mercaptans and organic sulfides, as well as hydrogen sulfide.

The concentration of mercaptans formed from heating milk at pH 7*7 was Table 2 3 . The effect of pH on the formation of volatile sulfur compounds as a result of high heat treatment of sulfur-35 labelled barium sulfide milk

(TDS) Changes in 3net counts per minute mg. of cysteine Treatment per liter of milka hydrochloride Temperature Time organic hydrogen per liter °C minutes pH mercaptans sulfides sulfide of milk

90 60 6.7 + 99 - 191 + 8193 18.6

90 60 7*7 +713 + 873 +13,503 15.5

90 60 9.7 t 57 - ikQ 4^9,087 27.8

Appendix Table ^9

aFor raw milk, the ncpm were 203, 525 and 95 for mercaptans, organic sulfides and hydrogen sulfide, respectively. 104 increased by approximately four fold over that of normal milk, whereas, about the same concentration of sulfur-35 mercaptan was formed at pH 6.7 and 9*7* The formation of volatile organic sulfides at pH 7*7 revealed that these compounds were increased by approximately nine fold

over that of normal milk. However, there was no heat release of

organic sulfides in milk which was adjusted to pH 9 .7 .

An increase in the pH of milk from 6 .7 to 7*7 or 9*7 was

followed by a general increase in the formation of hydrogen sulfide, which is in agreement with the results reported by other investiga­

tors. At pH 7 »7 j "the amount of radioactive hydrogen sulfide increased by approximately 1.6 fold over that of normal milk and at pH 9-7j the

increase was approximately sixfold.

The amount of heat released non-volatile sulphydryl groups as

determined by the thiamine disulfide procedure revealed that the pH of milk at 7*7 appeared to lower the availability of non-volatile -SH

groups, so that the TDS values were lower than that obtained for

heated milk. On the other hand, the TDS values of milk adjusted to

pH 9*7 increased by approximately 50 per cent over that of normal milk.

Cystine and cysteine

Since Townley and Gould (1^0) demonstrated that cystine added

to milk retarded hydrogen sulfide production when the milk was heated

and that the addition of cysteine increased the formation of hydrogen

sulfide, a study was initiated to investigate the effects of these two

sulfur containing amino acids on the formation of organic volatile

sulfur compounds other than hydrogen sulfide. 105

The milk in this experiment was barium sulfide sulfur-35

labelled milk. The sulfur amino acids used were nonradioactive and were added at a rate of 0.5 grams per liter of milk prior to heating.

The heating conditions were as those described above. The results

for this experiment are presented in Table 2k.

The results revealed that when cystine was added to milk, the

free radioactive volatile sulfur pattern was altered. The concentra­

tion of mercaptans decreased slightly, the organic sulfides disappeared and radioactive hydrogen sulfide level doubled. When cysteine was added to milk, the free mercaptans, sulfides and/or disulfides were

less than that observed for milk which contained no cysteine. How­ ever, cysteine promoted the release of free sulfur-35 hydrogen sulfide from unheated milk containing 0.5 gm of cysteine per liter almost equivalent to that produced by heating normal milk at 90°fl for 60 minutes. The increase in sulfur-35 hydrogen sulfide for unheated

cysteine was approximately 70 fold.

When portions of the same milk,which contains 0.5 gm per liter

of cystine, was heated to 90°C for 60 minutes the formation of sulfur-35 hydrogen sulfide was drastically retarded. The concentration of hydrogen sulfide was only 3 per cent of that present in heated milk with no added cystine. The formation of organic sulfides was approx­

imately ^ fold greater than for the control milk which received the

same heat treatment. The results thus indicated that cystine promoted

the formation of organic sulfides. However, the formation of mercaptans

was 1.6 fold that of milk which contained no cystine but receiving

identical heat treatments. Table 24. The effect of non-radioactive cystine and cysteine added to sulfur-35 barium sulfide labelled millc on the formation of volatile sulfur-35 compounds after high heat treatment.

(TDS) Heat Net Counts per Minute mg. cysteine Treatment Per Liter of Milk Hydrochloridi Temperature Time Material Organic Hydrogen per Liter C Minutes Added Mercaptans Sulfides Sulfide of Milk

Room 0 None 203 525 95 0

Room 0 DL-Cystine 139 0 166 0

Room 0 L-Cysteine HC1 68 125 5,500 258

90 60 None 302 334 8,288 18.6

90 60 DL- Cystine 497 1,308 260 12.5

90 60 L-Cysteine HC1 883 1,005 33,648 189

Appendix table 50 107

When cysteine was present in milk, and the milk heated, the mercaptan and organic sulfide concentrations were increased by

approximately 3 fold, respectively, -when compared to milk which con­

tained no added cysteine, but receiving the same heat treatment.

Consequently, it may be assumed that cysteine increased the formation

of both mercaptans and organic sulfides. The increase in the levels

or organic sulfides from the heat treated cysteine milk was slightly r — less than those of heat treated cystine milk, but the addition of

cysteine to milk promoted the formation of more mercaptans in milk

than by adding cystine to milk. The formation of radioactive hydrogen

sulfide from high heat treatment of milk with added cysteine at a

rate of 0 .? grams per liter revealed that 4 times as much sulfur-35

containing hydrogen sulfide are produced from this type of milk than

from milk which contained no added cysteine.

The thiamine disulfide reducing materials from heated milk which did not contain added cystine or cysteine had a TDS value of

18.6. The thiamine disulfide values for milk which contained cystine

and subsequently heated had a TDS value of 12.5 which showed that

cystine retarded the formation of non-volatile sulphydryl groups.

Unheated milk containing added cystine contained no non-volatile

sulphydryl groups, but unheated milk which contained added cysteine

exhibited a 258 fold increase in the TDS value over that of unheated

milk which did not contain any cysteine. Milk which contained added

cysteine and then heated to 90°C for 60 minutes had only a 10 fold

increase in the amount of non-volatile sulphydryl groups when compared

to milk which was heated under the same conditions. 108

Sulphydryl blocking agent

A study was conducted to determine -whether the inorganic sul­ fides and volatile organic sulfur compounds are formed from the active native sulphydryl groups that are available in milk prior to heat treatment or whether they are formed as a result of the degra­ dation of sulfur-sulfur and/or sulfur carbon bonds because of high heat treatment of milk. The sulphydryl blocking agent p-chloro- mercuribenzoate was utilized at a concentration of 5 micromoles per liter of milk to block all native sulphydryl groups present in unheated milk.

The investigation of this phase of the study consisted of utilizing sulfur-35 barium sulfide milk. The activity of the milk was 18,000 counts per minute per milliter of milk. The radioactive milk was divided into 4 lots. Lot 1 was aspirated with nitrogen at room temperature for 2 hours, and the volatile material trapped by the various trapping reagents. Lot 2 was heated to 90°C for 60 minutes, cooled to room temperature and aspirated with nitrogen for 2 hours.

To lot 3 p-chloromercuricbenzoate at a concentration of 5 micromoles per liter was added, and the mixture allowed to react all room temper­ ature for 1 hour. Following this period, the milk was aspirated for

2 hours with nitrogen and the sulfur volatiles trapped. Lastly, lot ^ milk also contained p-chloromercuricbenzoate at a concentration of 5 micromoles per liter, after allowing the mixture to react at room temperature for 1 hour, the milk was heated to 90°C for one hour, then cooled to room temperature and aspirated with nitrogen gas for 2 hours and the volatiles trapped. The results for this experiment are shown in Table 2 5 . Table 2 5 . The effect of p-chloromercuric benzoate on the formation of volatile sulfur-35 compounds from heat treatment of barium sulfide sulfur-35 milk

p-chloro- (TDS) Treatment mercuri- Net counts per minute rag. of cysteine Temperature Time benzoate per liter of milk hydrochloride °C minutes micromoles organic hydrogen per liter per liter mercaptans sulfides sulfide of milk

Room 0 None 137 171 80 0

Room 0 5 127 if 5 9 0 0

90 60 None 302 33^ 8288 ’ 18.6

90 60 5 b2 901 171 if.91

Appendix Table 51 110

The blocking agent affected the formation of volatile sulfur compounds in both the unheated and heated milks. The results indi­ cated that the sulphydryl blocking agent in unheated milk reduced the formation of sulfur-35 hydrogen sulfide, but did not affect the vola­ tile mercaptans, when compared to milk which did not contain any sulphydryl blocking agent. However, the three fold increase in the levels of organic sulfides in the mercuric chloride traps indicated that the formation of these volatiles were excellerated by the presence of the sulphydryl blocking agent in the raw milk.

The presence of 5 micromoles of p-chloromercuribenzoate in milk heated to 90°C for 60 minutes altered the concentration levels of vola­ tile sulfur compounds. Volatile mercaptans were detected at levels of only 14 per cent when compared to the levels found in the heated control milk. The formation of hydrogen sulfide in heated milk also was suppressed by the blocking agent. However, the results indicated that three times as much organic sulfides were formed as a consequence of heating in the presence of the sulphydryl blocking agent, when compared to heated milk with no added p-chloromercuribenzoate.

The low TDS value of k .91 detected in milk containing the sulphydryl blocking agent as compared to 18.6 for heated control milk indicated that the majority of the non-volatile sulphydryl groups were masked by the blocking reagent.

Air

The approach to this study involved using barium sulfide sulfur

35 labelled milk with an activity of 16500 counts per minute per milli­ liter of milk. The milk was divided into 2 lots. One lot was heated at Ill

90°C for 60 minutes, and the other lot was aspirated with air at room temperature for 2 hours prior to heat treatment at 90°C for 60 minutes. During the heat treatment of lot 2 milk, the positive pres­

sure was also maintained with air. The results are presented in

Table 26.

The results of aspiration of milk with air for 2 hours prior to heat treatment on the formation of volatile sulfur compounds in

comparison to milk which received no air treatment revealed that there were differences in the formation of volatile sulfur compounds.

Similarly, the levels of sulfur-35 hydrogen sulfide produced from high heat treatment of air aspirated milk was lower in comparison to milk which was not air aspirated. The amount of hydrogen sulfide produced from air aspirated milk was only 23 per cent of that for the control milk which was not aspirated with air. Milk which was aspirated with air revealed that more mercaptans were formed than organic sulfides.

The mercaptan concentration was 5 times as much in milk saturated with air when compared to milk which was not aspirated with air. Wo

organic sulfides were produced from either milk. The thiamine disul­ fide values for milk which had received air treatment prior to heat

treatment was 11.0 as compared to 18.6 for milk which did not receive

any air aspiration.

Nitrogen

Experiments were conducted to determine the types and distribu­

tion of volatile sulfur compounds released from heat treatment of milk

which had been exhaustively aspirated with nitrogen prior to heating. Table 26. The effect of sulfur-35 labelled milk saturated with air on the formation of volatile sulfur compounds produced by high heat treatment

(TDS) Aspira­ Change in net counts per minute mg. of cysteine Treatment tion per liter of milka hydrochloride Temperature Time with organic hydrogen per liter °C minutes gas mercaptans sulfides sulfide of milk

90 6o None f 99 - 191 <■ 8193 18.6 VO CVJ D— 90 6o Air + 532 1 4 2333 11.0

Appendix Table 52

^ o r raw milk, the ncpm were 203, 525f and 95 for mercaptans, organic sulfides and hydrogen sulfide, respectively. 112 113

In contrast to all previous studies on factors affecting heat released volatile sulfur compounds, sodium sulfate sulfur-35 labelled milk was used in these trials. The milk was aspirated with nitrogen gas for two hours at room temperature to remove free volatile sulfur compounds and to saturate the milk with nitrogen prior to heating.

The control sample was aspirated an additional two hours through the trapping system to determine if any residual volatile sulfur com­ pounds remained in the milk. The other lots of milk were heated to 90°C and held for 10, 30 and 60 minutes, cooled and analyzed for volatile sulfur compounds. The results of this experiment are presented in

Table 2 7 .

Table 27 . The effect of sulfur-35 labelled sodium sulfate milk saturated with nitrogen on the formation of volatile sulfur compounds produced by high heat treatment

Treatment Net counts per minute per liter of milk Temperature Time organic 2,4-DNPH hydrogen °G minutes mercaptans sulfides derivatives sulfide

0 0 0 0 0 0

90 10 U 65 0 k26 129

90 30 U 3 533 *1-00 308

90 60 5^+6 160 k8o 133

Appendix Table 53 Ilk

The data for the control sample shoved that no radioactivity was detected in any of the four trapping reagents, indicating that all of the free volatile sulfur compounds were removed from the milk.

The effect of heat on the nitrogen treated milk was somewhat similar to that obtained for unaspirated milk in that mercaptans, organic sulfides and 2,1— DNPH derivatives were also produced in higher concentrations than hydrogen sulfide.

The distribution of volatile mercaptans following heat treat­ ments of milk at 90°C for 10, 30 and 60 minutes indicated that the highest concentration of radioactive mercaptans occurred after heat treatments for only 10 minutes, and then the concentrations of mer­ captans gradually decreased with increased holding time. Previous results on milk which was not aspirated with nitrogen prior to heat­ ing revealed that the greatest concentration of mercaptans occurred after 30 minutes of heat treatment at 90°C. Possibly, the lowering of the oxidation-reduction potential as a result of nitrogen aspira­ tion for 2 hours prior to heat treatment may have contributed to the distribution of volatiles in the various traps.

It was observed that no organic sulfides were formed after

10 minutes of heat treatment, whereas, at this heat treatment the greatest quantity of mercaptans occurred. After 30 minutes of heat treatment, the presence of organic sulfides were detected; this level dropped off as the heating was continued at 90°C for 6o minutes; and this was followed by an increase in the concentration of mercaptans.

The concentration of volatile 2,1— DNPH derivatives were relatively constant after 10 and 30 minutes of heat treatment respectively, but increased slightly as the holding time at 90°C was

increased to 60 minutes.

The concentration of hydrogen sulfide after 10 and 60 minutes

of heating were quite similar, but lower than that detected after 30 minutes of heating.

The Relationship Between Total Concentration of Hydrogen Sulfide and Radioactive Hydrogen Sulfide

Further information on the mechanism of heat released volatile

sulfur compounds could be obtained by determining the ratio of radio­

active sulfur-35 to total sulfur for the volatile components released

from high heat treatment of milk. Only hydrogen sulfide was present

in high enough concentration to determine the concentration of total

sulfur quantitatively.

The specific radioactivity of hydrogen sulfide was calculated

by dividing the counts per minute of radioactive hydrogen sulfide in

the lead sulfide trap by the .total sulfur content determined colori-

metrically to give counts per minute per microgram of hydrogen sulfide.

The technique was applied to the hydrogen sulfide produced from the

heat treatment of various fluid milk products, to provide more infor­

mation on the effect of different factors on H2S formation.

The total concentration and specific radioactivity of hydrogen

sulfide produced from barium sulfide, sulfur-35 labelled milk and milk

products after heat treatment at 90°C for various lengths of time are

shown in Table 28. The results revealed that although radioactive

hydrogen sulfide was recovered from unheated milk, the concentration Table 2 8 . concentration and specific activity of hydrogen sulfide produced from barium sulfide sulfur-35 labelled milk, after heat treatment at 90°C for various lengths of time

Time of Heating 90°C in Minutes

______Raw______;______30______60______120______Concen- Concen- Concen- Concen- Product tration Specific tration Specific tration Specific tration Specific H„S activity H^S activity HoS activity H„S activity ug/1 cpm/ug ug/1 cpm/ug ug/1 cpm/ug ug/1 cpm/ug

Milk 0 — 336 9.56 395 24.84 512 16.18 Skimmilk 0 — 368 17.19 377 17.13 540 12.78 Cream 0 — 430 7.22 587 10.07 674 7.28 Buttermilk 0 — 554 7.20 1,017 5.44 1,078 5.29 Acid whey 0 — 131 1.07 242 1.22 111 9.57 Rennet whey 0 — 490 6.81 567 5.91 650 6.31

Appendix table 5^

j- 1 t-1 c \ 117 of hydrogen sulfide was too low to be determined colorimetrically and therefore, the specific activity could not be calculated.

For heated products, total hydrogen sulfide concentration followed similar trends to those previously reported for radioactive hydrogen sulfide in respect to the effect of heat treatment. However, the specific activity of the hydrogen sulfide varied between different products heated for the same period of heating time and for a given product at different heating periods.

The concentration of hydrogen sulfide produced after heat treatment at 90°C for 30 minutes for all products ranged from 131 to

55** ug per liter. Acid whey had the lowest concentration of hydrogen sulfide and buttermilk the greatest, whereas, skimmilk and milk had quite similar concentration. Contrary to previous investigations, cream and rennet whey also had similar concentrations of hydrogen sulfide. The specific activity calculated for products obtained under the same heating period varied between 1.07 for acid whey to 17*19 for skimmilk. Milk had a value of 9*56, and cream buttermilk and rennet whey had values of 7 *22, 7.20 and 6 .8 , respectively.

After 60 minutes of heating the concentration of hydrogen sulfide ranged between 2^2 ug per liter for acid whey and 1017 ug per liter for buttermilk. Again, milk and skimmilk had similar levels of hydrogen sulfide, 380 ug per liter, and cream and rennet whey having approximately the same concentration of 660 ug per liter, respectively.

These observations were also extended to heat treatments of 90°C for

120 minutes. The specific activities for products heated to 90°C for

60 minutes and 120 minutes range from a high of 24.8^ for milk to a 118

low of 1.22 for acid whey for 60 minutes and l6.8 for milk to 5.29 for buttermilk for 120 minutes of heating. Buttermilk and rennet whey had very similar specific activity values for both 60 and 120 minutes of

heating, whereas, there were marked differences in specific activity

values for the rest of the products heated at 90°C for 60 and 120

minutes.

The specific activity values for rennet whey and buttermilk

and cream had rather similar values for all heating periods. Acid

whey had similar values after 30 and 60 minutes. This trend was also

observed for skimmilk. However, after 120 minutes of heating, the

specific activity value for acid whey increased by 8 fold but those

for skimmilk decreased. The specific activity of milk had a maximum

value after 60 minutes of heat treatment.

These differences in specific activities for the different

milk products at the various heating periods suggest that the heat

released radioactive hydrogen sulfide does not come from the same

precursor in different products heated for a given time or an

individual product throughout a prolonged heat treatment.

The total hydrogen sulfide content and specific activity data

produced by heating milk at different pH's are shown in Table 2 9 . The

results revealed that total hydrogen sulfide content increased by 3

fold when the pH was raised from 6.7 to 7*7 and an additional 7 fold

from 7*7 to 7*9* However, the increase in hydrogen sulfide concentra­

tion did not follow a first order reaction, and the kinetics of this

reaction suggested the involvement of more than one component. This 119 observation was supported by the decrease in specific activity of hydrogen sulfide as the pH was increased from 6.7 to 7*7 'and finally to 9 .7 .

Table 2 9 . Effect of pH on the formation of hydrogen sulfide and specific activity value of milk heated to 90°C for 60 minutes

HgS microgram Specific pH per liter activity of milk of milk cpm/ug.

6.7 395 20.98

7.7 1V75 9.21

9.7 9033 5 . ^

Appendix Table 55

The effect of added cystine and cysteine to milk on the forma­

tion of total sulfur and specific activity are shown in Table 30. The

results revealed that hydrogen sulfide in unheated milk was detected

colorimetrically only in the milk with added cysteine. For this milk

radioactive hydrogen sulfide was released and the specific activity

was approximately one half of that obtained with milk heated at 90°C

for 60 minutes.

In the cystine treated milk, the total hydrogen sulfide pro­

duction was depressed by k fold of that found in the control milk,

whereas, the specific activity was decreased 10 fold. This indicated

that the majority of hydrogen sulfide in cystine treated milk was coming 120 from cystine and not a radioactive sulfur component of the milk system.

Table 30. The effect of added cystine and cysteine to milk on the formation of hydrogen sulfide and specific activity after heat treatment at 90°C for 60 minutes

Added compound ILjS micrograms Specific 0.5 gm/liter Treatment per liter activity of milk of milk cpm/ug

None unheated 0 0

Cystine unheated 0 0

Cysteine unheated 615 8.9^

None heated 395 20.9^

Cystine heated 111 2.3^

Cysteine heated 8173 h.ll

Appendix Table 56

In the cysteine treated heated milk, there was a 20 fold increase in the formation of total hydrogen sulfide with a corres­ ponding 5 fold decrease in specific activity. Therefore, only 1600 ug per liter of hydrogen sulfide of milk was sulfur-35 origin, and

6500 ug per liter of hydrogen sulfide were of non-sulfur-35 origin.

This suggested that even though cysteine appeared to contribute directly to hydrogen sulfide production, there was an activation of heat released hydrogen sulfide as evidenced by the ^ fold increase in radioactive sulfur-35 released hydrogen sulfide. 121

The effect of air aspiration on the formation of total hydrogen sulfide and the corresponding specific activity are illus­ trated in Table 31* '-Che amount of total hydrogen sulfide was essentially the same for both normal and aerated milk. However, the specific activity of the hydrogen sulfide from the aerated milk was only about one third of that in the normal milk, thus suggesting that aeration altered the precursor of the heat released hydrogen sulfide.

Table 31. Total hydrogen sulfide and specific activity of milk saturated with air prior to heat treatment at 90°C for 60 minutes

Treatment Hydrogen sulfide Specific with air microgram per activity liter of milk cpm/ug

None 395 20.98

Air 335 7.1

Appendix Table 57

Effects of Freezing on the Thiamine Disulfide Values of Milk and Milk Products

This phase of study was conducted to ascertain the effect of freezing of milk and milk products on the formation of thiamine disulfide reducing materials after high heat treatments. Milk or other milk products utilized in this investigation were preserved by freezing at -lk°C. It had been demonstrated by Kristofferseir 122 et al. (8 6 ) that the IDS values of milk fluctuated after incubation at 37°C up to *f8 hours. Because of this, the effect of storage at

-lU°C on the TDS values was studied.

The milk was obtained from the university dairy. The milk was nonradioactive and consisted of mixed herd milk. The milk was divided into two lots. One lot was warmed to 32°C and separated by a DeLaval Model 100 laboratory separator. The skimmilk and cream portions were collected. Two hundred milliliters each of milk, skimmilk and cream were placed in individual pint bottles and placed in the freezer for storage. The thiamine disulfide values of the individual samples were determined after heat treatment at 90°C for 30 minutes at three to four days intervals for three weeks dura­ tion. Milk samples were withdrawn for TDS determination after cooling the heated milk to 2k°C.

The results of this study are presented in Table 32. Each value in this table is the average of three TDS determinations for the same sample of milk. The results indicated'that regardless of the product involved, the TDS values varied from one determination to the next after storage. The results suggested that freezing apparently did not affect the milk system because the quantity of heat activated sulphydryl groups varied in samples of milk products held for times at -l4°C. Table 32. The thiamine disulfide values of milk, skimmilk and cream as determined after storage under freezing conditions at -l4°C

TDS values of cysteine hydrochloride per liter of product

Product Days after storage in freezer 0 3 6 10 13 17 20

Milk 14.5 13.4 16.5 14.8 16.2 13.6 14.8

Skimmilk 12.5 13.6 14.5 11.0 13.9 11.9 11.6

Cream 11.6 16.3 16.2 13.9 15.1 13.0 14.5 12^

Identification of Volatile Compounds from Heated Milk Labelled Sulfur-35

Attempts to identify individual mercaptans and organic sul­ fides in the mercuric cyanide and mercuric chloride traps by regen­ eration techniques were unsuccessful because of the very low concen­ trations of volatile sulfur compounds present in these traps.

Limited attempts to apply gas chromatography for identification of individual sulfur compounds in the trapping reagents were not successful.

The volatile components in the 2,4-DNPH traps were investi­ gated by means of thin lay chromatography. The chromatograms

(Appendix Figures 5> 6 and 7)"were obtained by directly superimposing a piece of tracing paper over the developed plate and drawing the outlines of the various spots. Some twenty components were separated.

The color of the individual compounds ranged from light yellow to deep purple. Attempts to determine which of these spots was radio­ active, using radioautographic techniques, failed to reveal any radioactivity even with exposure times up to 27 days.

Differences were observed in the chromatographic patterns obtained from the 2,if-DNPH traps of milk heated to 90°C and 95 °C for various length of times. The results are difficult to interpret, but alterations in chromatograms were obtained in respect to the time and temperature to which the milk was heated. DISCUSSION

This investigation has revealed the utility of using radioactive sulfur as a tool for detecting the presence of trace amounts of volatile sulfur compounds in both unheated and heated milks. Previously other in­ vestigators (141) have shown that the success of selective trapping vola­ tile sulfur compounds was dependent upon the formation of visible precipi­ tate in the trapping reagents. The results of this study, in which no visible precipitates were formed in any of the trapping reagents, have shown that very low levels of the different types of volatile sulfur com­ pounds can be detected by using sulfur-35 as a tracer. In addition, the use of radioactive sulfur provides several other advantages, which included:

(a) selective labelling of sulfur components of milk, (b) means of eval­ uation of precursors of sulfur-35 volatile compounds through determination of specific activities, (c) the ability to follow volatile sulfur compounds without interference from other classes of volatile chemical compounds formed by the same heat treatment.

Methyl sulfide has been reported by several investigators as a normally occuring compound in raw milk (114,150), Although individual sulfur compounds could not be detected by the method used in this study, radioactive organic sulfides were found in raw milk. Furthermore, methyl sulfide could be expected to be a major constituent of the organic sulfides.

In addition to organic sulfides, the presence of other volatile sulfur com­ pounds in raw milk has been shown by this study to include mercaptans,

125 126

2,4-DNPH derivatives and hydrogen sulfide. Although present in ppb concentrations, these compounds would be expected to be important in the flavor of raw milk. Of significance is the finding that the con­ centrations of the free volatile sulfur compounds in milk could be influenced by a variety of factors other than heat treatment. The significant increases in free organic sulfides in raw skimmilk, cream and rennet whey, the increase in hydrogen sulfide, organic sulfides and mercaptans by addition of cysteine, the increase in mercaptans in acid whey are illustrations of the factors found in this study that alter the free volatile sulfur content of the raw milk products. The reasons for such variation are not readily apparent. One possible explanation might lie in the absorption of volatile sulfur precursor compounds on to the fat phase of milk, followed by release of sulfur components following some processing technique that "exposed" the volatile

groups. An observation made during this study that free fat from sul­

fur labelled milk caused spurious counts in a gas flow counter might

support this hypothesis.

Limited data suggest that the chemical form of sulfur-35 may

influence the type of free volatile sulfur compounds found in the milk.

Barium sulfide milk appeared to have higher concentrations of mercap­

tans, organic sulfides and hydrogen sulfide than milks labelled with

other forms of sulfur-35• Milk labelled with sodium sulfate and

sodium sulfide contained more free 2,k-DNPH derivatives than milk

labelled with other sulfur-35 precursors. Variations in the concen­

trations of free volatile sulfur compounds were also observed in milk

obtained from different cows using the same type of radioisotope.

Several investigators have suggested that methyl sulfide in raw 127 mill; comes directly from the feed (43,99,114,130). The fact that the in­

troduction of inorganic radioactive sulfur-35 salts into rumen of cows resulted in free volatile radioactive sulfides and mercaptans in milk,

suggests that rumen fermentation also may have contributed to the presence of free volatile sulfur compounds in raw milk. This would explain the very high free radioactive organic sulfide content in one of the three different milks labelled with sulfur-35 barium sulfide.

Based on the assumption that the specific activity of sulfur-35 compounds in raw milk would be the same as that of the specific activity of the milk, the concentration of free volatile sulfur compounds in milk would range from 1 to 4.0 ppb, with values in separated cream increasing to 10 ppb for organic sulfides. The flavor threshold values for volatile

sulfur compound in milk reported by Patton and Barnes (112) are 0.05,0.021,

0.012 and 0.002 ppm for methional, methyl disulfide, methyl sulfide and methyl mercaptan, respectively.

The differences in specific activities of sulfur-35 in the various milks, caseins and whey proteins revealed that the chemical form of sulfur-

35 used to obtain radioactive milk affected the distribution of sulfur-35

isotope in the milk system. The selective labelling of protein was appar­

ent for milks labelled with methionine, sodium sulfide and barium sulfide, whereas the use of sodium sulfate resulted in selective labelling of the non-protein sulfur fractions. With methionine labelled milk, no radio­ activity could be found in the amino acids cystine or cysteine (35). Whey proteins and casein had the same specific activity when barium sulfide was utilized as the radioactive sulfur precursor. This finding suggests that the sulfur amino acids in these proteins are labelled equally. This 128 assumption has been confirmed by Conrad (35) who has isolated methionine

% and cysteine form the same barium sulfide milk used in this study and

found the specific activity of the two amino acids to be identical.

The selectivity of labelling apparently had an effect on the

volatile sulfur compounds formed by heating milk, as evidenced by the

variation found in the radioactive volatile sulfur compounds isolated

from heated milk. In milks in which the specific activity of the protein

was high in respect to the non-protein sulfur, the highest radioactivity

in the heat released volatiles was found in the hydrogen sulfide. When

the specific activity of the non-protein fraction was high, mercaptans,

organic sulfides and 2,4-DNPH derivatives exhibited higher content of

sulfur-35 than did hydrogen sulfide. Only relatively low levels of radio­

active hydrogen sulfide were found upon heating milk labelled with methio­

nine. The differences in relative amounts of radioactive volatile sulfur

compounds formed upon heating milk labelled with different chemical forms

of sulfur-35 compounds, suggests that multiple precursors of heat released volatile sulfur compounds exist in milk. This viewpoint is substantiated

further by the difference iiv.the type and concentration of radioactive volatile sulfur compounds obtained by heating different fluid milk products

obtained from milk labelled with a single chemical form of sulfur-35, as well as by variations in the specific activity of heat released hydrogen

sulfide. The cyclic appearance of volatile organic sulfur-35 compounds with various times of heating that was observed in some trials with sodium

sulfate labelled milk also suggested that multiple precursors for heat- released sulfur compounds do exist in the milk system.

Factors such as pH, reducing agents and oxidizing agents, aeration 129 and sulfydryl blocking agents that have been known to influence the heat release of hydrogen sulfide-also affected the heat release of volatile organic sulfur compounds,, Increasing the pH of milk has been known to make the milk more heat labile, with increased production of hydrogen sul­ fide, These studies, while confirming the increased production of hydrogen sulfide, showed that the mechanism of heat released hydrogen sulfide at alkaline pH levels is not the same as at the normal pH of milk, since the specific activity of heat-released hydrogen sulfide decreased with in­ creased pH0

The addition of non-radioactive cysteine to milk caused a marked increase in the concentration of hydrogen sulfide, mercaptans and organic sulfides, released during heating. The decreased specific activity of hydrogen sulfide in the heated milk suggests that degradation of the added cysteine may have contributed to the formation of volatile sulfur compounds.

However, the increase in the radioactive volatile sulfur compounds revealed that the addition of cysteine-catalysed degradation of sulfur compounds in milk, Conversly, the addition of non-radioactive cystine to milk resulted in the inhibition of the formation of radioactive hydrogen sulfide and promotion of the formtion of radioactive organic sulfides upon heating.

The sulphydryl blocking agent p-chloromercuribenzoate was effective in essentially eliminating the isolation of radioactive hydrogen sulfide and mercaptans from heated milk. This finding provides further evidence for the interrelationship between total sulphydryl groups and hydrogen sulfide reported previously by other investigators (17,86), However, no definite relationship was apparant between the thiamine disulfide reducing sub­ stances and the radioactive volatile mercaptans found in the different 130 heated milk. The fact that organic sulfides were found in heated milk containing sulphydryl blocking agents, indicates that the volatile organic sulfides could not have been formed by the aggre­ gation of mercaptans.

The volatile sulfur compounds appeared to arise from a variety of mechanisms of a complex nature. The differences in the concentration of organic sulfides, mercaptans and 2,^-DNPH deriva­ tives isolated from heated milk and milk products suggested that these compounds arise from separate reactions and are not necessarily formed from one another. The specific activity variation in heat released hydrogen sulfide also suggested multi-precursors for the formation of these volatile compounds.

Since it was not possible to determine the concentration of the mercaptans and organic sulfides, it was not possible to determine the specific activity of these compounds. Such information would provide more specific information concerning the mechanism of their

formation.

Some of the results presented in this study are different

from those that had been reported previously, especially the concen­

tration of hydrogen sulfide found in heated rennet whey. The

major difference between this study and others is that the whey was

frozen prior to heat treatment. Freezing may possibly have altered .

the susceptibility of rennet whey to heat, by increasing the quantity

of -SH precursors in the system. SUMMARY

The purpose of this study was to obtain knowledge concerning the chemical nature of radioactive volatile sulfur-35 compounds formed by high temperature heat treatment of milk and milk products and to ascertain the factors affecting their formation. In this investigation, sulfur-35 was utilized to obtain radioactive sulfur-35 labelled milk. The four chemical forms of sulfur-35 radioisotopes employed to obtain radioactive milk were: sodium sulfide, methionine, sodium sulfate and barium sulfide.

Variations in the distribution of sulfur-35 were found in the con­ stituents of the different milks. Specific activity data revealed that more radioactive sulfur per microgram of total sulfur was found in the casein and whey protein fractions of methionine and barium sulfide lab­ elled milk than with sodium sulfate labelled milk, A low level of radio­ active sulfur was distributed in the non-protein fractions of barium sulfide and methionine sulfur-35 labelled milk, whereas, with sodium sul­ fate labelled milk, the radioactive sulfur had higher specific activity in the non-protein sulfur fractions than in the protein components.

Volatile sulfur compounds were separated into hydrogen sulfide, mercaptans, sulfides and/or disulfides and sulfur-containing 2,4-DNPH derivatives by selective trapping with a chemical trapping system consis­ ting of solid lead acetate, aqueous mercuric cyanide, mercuric chloride and 2,4-dinitrophenylhydrazine solutions, respectively.

Volatile mercaptans, organic sulfides, sulfur-35 containing 2,4-

131 132 DNPH derivatives, as well as hydrogen sulfide, were found in the volatiles

of unheated raw milk and milk products by sweeping the milk with nitrogen*

Barium sulfide labelled milk contained more free mercaptans, organic sul­

fides and hydrogen sulfide than the other milks and sodium sulfate milk

contained more free 2,4-DNPH derivatives. More free organic sulfides were

found in skimmilk, cream and rennet whey, whereas, acid whey had more free

mercaptans than any other milk products.

Heat treatment of milk released hydrogen sulfide, mercaptans,

organic sulfides and sulfur-35 containing 2,4-DNPH derivatives. Generally,

hydrogen sulfide was released in the highest concentration when compared

to the other organic sulfur volatiles. The concentrations of sulfur-35 in

the various classes of organic sulfur compounds formed by heating varied

according to the type of sulfur-35 precursor used in securing the radio­

active milk following incorporation of the isotope into the cow, and to the

duration of heating time given to the milk.

Very small amounts of volatile sulfur compounds were released from

heating sulfur-35 methionine labelled milk; traces of hydrogen sulfide,

mercaptans and sulfur-35 containing 2,4-DNPH derivatives but no organic

sulfides were found in this type of milk. When sodium sulfide milk was

heated at 90°C for various lengths of time, only mercaptans and hydrogen

sulfide were detected. Whereas, when sodium sulfate milk was heated, all

classes of volatile sulfur compounds were detected with the concentration

of radioactive mercaptans, organic sulfides and sulfur-35 2,4-DNPH deriva­

tives being in higher concentrations than sulfur-35 hydrogen sulfide;

Heating barium sulfide sulfur-35 labelled milk resulted primarily in the

formation of mercaptans and hydrogen sulfide. Maximum concentrations of 133 these two volatiles occurred after heating at 90°C for 120 and 60 minutes, respectively. For sodium sulfate sulfur-35 labelled milk, maximum concentration of mercaptans were formed after 30 minutes of heating and after 120 minutes for the organic sulfides, 2,4-DNPH deriva­ tives and hydrogen sulfide, respectively.

The formation of heat-released volatiles from heating skim­ milk, cream, buttermilk, acid and rennet whey showed that in general, hydrogen sulfide was formed in the highest levels for all fractions except in cream, whereas, organic sulfides were produced in approxima­ tely the same concentration. Buttermilk was the only product which released all four types of volatiles, upon heating. For these products, the time of maximum formation of mercaptans occurred after 60 minutes of heating for skimmilk and buttermilk, and after 120 minutes for cream, acid and rennet whey. Maximum formation of organic sulfides, for buttermilk, cream and skimmilk occurred after 30, 60 and 120 minutes of heating, respectively. No organic sulfides were formed from heating either acid or rennet whey. Maximum concentration of 2,4-DNPH deriva­ tives were formed for buttermilk after 6o minutes of heating. No appreciable amounts of these volatiles were detected in any of the other fluid milk products, at all heating times. The greatest concentration of hydrogen sulfide was formed from heating skimmilk and cream after

60 minutes and after 120 minutes, for buttermilk, acid whey and rennet whey, respectively.

The formation of heat-released volatile sulfur compounds were affected by the pH of the milk. When the pH of the milk was raised from

6.7 to 7.7 and 9*7 respectively, the formation of hydrogen sulfide 13^ increased by 1.6 and 6-fold. The specific activity of hydrogen sulfide formed from pH 7*7 and 9.7 milk indicated that some of the hydrogen sulfide was formed from non-protein sources. More mercaptans and organic sulfides were released from heated pH 7.7 than 9.7 milk.

The addition of non-rtdioactive cystine to milk retarded sulfur-35 hydrogen sulfide formation by heat treatment, but promoted the formation of organic sulfide by four fold. Similarly, when non­ radioactive cysteine was added to milk, the levels of sulfur-35 hydro­ gen sulfide increased by four fold, sulfur-35 mercaptans and organic sulfides by 3 fold each, respectively. The quantity of sulfur-35 hydrogen sulfide increased by 55 fold in raw milk containing added non­ radioactive cysteine. The results suggested that non-radioactive cysteine acted as a catalyst in promoting the formation of radioactive hydrogen sulfide.

The addition of 5 micromoles of p-chloromercuribenzoate per liter of milk and the milk heated to 90°C for 60 minutes resulted in the retardation of mercaptans and hydrogen sulfide. However, the formation of organic sulfides was unaffected, in fact, there was a three­ fold increase in concentration of this type of volatile.

Aspiration of sulfur-35 labelled milk with air prior to heat­

ing affected the formation of hydrogen sulfide and organic sulfides only by retarding their formation; whereas, there was an increase of

2 fold in the levels of mercaptans. Milk aspirated with nitrogen gas prior to heating, resulted in the formation of all four classes of volatile sulfur compound, demonstrating that these volatiles were heat

released. 135

Specific activities of hydrogen sulfide data revealed that the radioactive hydrogen sulfide probably came from the same source for

skimmilk, cream, buttermilk and rennet whey which were heated at 90°C for various lengths of time. However, formation of radioactive hydrogen

sulfide for milk and acid whey appear to come from more than one source.

The determination of heat formed thiamine__disulfide reducing materials in milk and milk products which had been stored at -1^°C up

to 21 days, showed fluctuations in TDS values were obtained.

Thin layer chromatography separation of 2,k-DNPH derivatives,

showed more than twenty different compounds, however, because of the

small amount of material used and the low concentration of radio­

activity in these derivatives, no radioactivity was detected in any

of the separated compounds, whether by direct count or by radio­

autographic techniques. APPENDIX

136 137

Table 33. Information relating to the type of isotope used, the size of dosage, method of administration and the number of cows invol­ ved in obtaining radioactive sulfur-35 milk

Cow Milk Size of Date of Method of No. labelled dose incorporation incorporation by Sulfur-35 MC of isotope of isotope

1482 Sodium 1.0 1-27-63 Intraruminally Sulfate

1482 Sodium 1.0 4-5-63 Intraruminally Sulfate

1507 Methionine 1.0 10-27-63 Intravenous drip

1502 Sodium 2.0 2-27-64 Orally 1406 Sulfide

1555 Barium 9.8 9-17-64 Orally Sulfide

1507 Barium 10.2 11-20-64 Orally Sulfide

1577 Barium 10.4 4-17-65 Orally Sulfide 138

Table 34. Summary of radioactivities of different lots of barium sulfide-sulfur-35 labelled milk

Wet counts per minute per ml of Lots of Milking barium sulfide-sulfur-35 milk milk intervals Lot (hours) 1 2 3

1 12 8515 3676 8978

£ 24 16,922 10,470 8714

3 36 13,886 12,016 5892

4 48 12,222 9944 4034

5 60 9,227 8626 3078 139

Table 35- Radioactivities of consecutive lots of milk obtained at six hour intervals following oral administration of sodium sulfide sulfur-35 labelled milk

Milking NCPM Milking NCPM Milking NCPM No. per ml No. per ml No. per ml

1 — 13 620 25 752

2 1716 14 990 26 3266

3 1613 15 IV78 27 4145

4 1399 16 2178 28 4005

5 1457 17 1954 29 5190

6 1484 18 1703 30 44-32

7 1383 19 1518 31 3300

8 1119 20 1307 32 3727

9 805 21 1108 33 1307

10 726 22 1016 34 964

ll 686 23 990 35 832

12 752 24 858

Pooled milk (lots 26 to 32 inclusive) 3783 cpm per ml

Pooled skimmilk 6008 cpm per ml 140

Table 3 6 . Radioactive counts of milk obtained following intravenous infusion of sulfur-35 labelled methionine

Lots Hours after incorporation Net count per minute of isotope per milliliter of milk

1 3 5132

2 6 7475

3 9 9344

4 12 11531

5 15 9167

6 18 5054

Pooled milk (lots 3,4 and 5) 9230 1*1

Table 37- Summary of radioactivities of different batches of sodium sulfate-sulfur-35 labelled milk

Net counts per minute per ml of Milking Milking sodium sulfate'-sulfur-35 milk No. intervals (hours) Trial 1 2

1 6 k36 ----

2 12 1176 ----

3 18 1160 106k

k 2k 1380 2262

5 30 1355 207k

6 — ---- 2110

Pooled milk 113^ --- 142

Table 38* Total sulfur and sulfur-35 content of various milks and milk fractious

Type of sulfur-35 Total Net count per labelled milk Sulfur minute per gram ppm. dry weight

Sodium sulfate

milk 4010 9380 3870 8978

casein 7180 17060 7540 19760

whey protein 10500 16208 11100 18710

Methionine

milk 4240 32302 4500 36198

casein 8630 240760 7410 198740

whey protein 10710 321284 11210 341830

Barium sulfide

milk 3270 62922 3440 78102

casein 6830 362466 7290 369980

whey protein 10930 550253 10120 536191 1^3

Table 3 9 . The effect of heat treatment at 80 and 90°C on the formation of volatile sulfur compounds from sodium sulfate sulfur-35 milk

Trial Treatment Net count per minute per liter of milk No. Temperature Time organic 2,4-DNPH °C minute mercaptans sulfides derivatives

1 80 10 47 0 42

2 80 10 65 0 15

3 80 10 47 0 24

Average 53 0 27

1 90 10 615 785 1502

2 90 10 704 758 1340

3 90 10 520 815 1376

Average 613 786 1406 Table 40. The effect of high heat treatment at 90°C on the forma­ tion of volatile sulfur-35 compounds of sulfur-35 sodium sulfide labelled milk

Net count per minute per liter of milk Treatment Temperature Time Organic 2,4-DNPH Hydrogen °C minutes Mercaptans sulfides derivatives sulfide

Unheated — 18 195 444 0 CO 90 30 - 252 - i46 0 1

90 6o - 106 - 259 - 120 - 285

90 120 0 0 - 61 - 145 145

Table 4l. The effects of heat treatment at 90°C on sodium sulfate sulfur-35 labelled milk on the release of volatile sulfur compounds

Net counts per minute per Treatment liter of milk Trial Temperature Time 2,4-DNPH No. °C Minutes Mer- Organic deriva­ Hydrogen captans sulfides tives sulfide

1 24 — 160 213 320 0

1 90 10 133 27 826 227

2 90 10 613 787 4o6 163

Average 373 407 616 195

1 90 30 421 240 150 165

2 90 30 798 46o 533 332

Average 609 350 342 248

1 90 6o 4q o 4oo 420 162

2 90 6o 186 80 506 160

Average 293 240 463 l6l

1 90 120 292 48o 692 110

2 90 120 226 852 752 506

Average 259 666 722 308 Table 42. Effect of heat treatment at 90°C on-barium sulfide-sulfur-35 labelled milk on the release of volatile sulfur compounds

(TDS) Trial Treatment Net countsi per minute per liter of milk m g . of cysteine No ,a Temperature Time organic 2,4-DNPH hydrogen hydrochloride °C minutes mercaptans sulfides derivatives sulfide per liter of milk

1 2b unheated 370 360 153 153 0 2 2b unheated 90 305 62 82 0 3 2b unheated 117 ^33 -- 0 0 b 2b unheated 233 1000 -- 133 0 Average 203 525 100 95 0 1 90 30 1365 142 88 1790 18.6 2 90 30 1215 305 92 4465 18.6 Average 1290 147 90 3107 18.6 1 90 6o 1850 160 20 13420 16.6 2 90 6o 1585 280 26 6915 16.6 Average 1717 220 23 10767 16.6 1 90 120 2215 320 163 6830 20.0

2 90 120 l48o 160 144 9585 20.0 Average 181-7 240 154 8207 20.0

aMilk of trials 1 and 2 was obtained from cow numbered 1555/ trial 3 from cow number 1507/ and trial 4 from cow number 1577*

i— 1 ■p- c s \ Table 43. Effects of heat treatment at 90°C on the types and distribution of volatile sulfur com­ pounds from skimmilk, cream and buttermilk of sulfur-35 sodium sulfide labelled milk

Net counts per minute per liter of product

Organic 2,4-DNPH Hydrogen Treatment______Mercaptans sulfides_____ derivatives______sulfide______Temperature Time Skim- Butter Skim- Butter Skim- Butter Skim- Butter °c minutes milk Cream milk milk Cream milk milk Cream milk milk Cream milk

24 0 12k 0 0 0 0 67 12 0 184 0 0 0

90 30 55^ 0 26 95 0 0 0 0 0 306 0 64

90 6o 64 325 0 669 200 114 0 0 0 367 0 149

90 120 871 252 0 4l8 0 0 0 117 221 237 254 23

H -<5-f=" Table 44. Effect of heat treatment at 90°C on barium sulfide sulfur-35 labelled skimmilk on the formation of volatile sulfur compounds

(t d s ) Net counts per minute of aliquots from traps mg. of cysteine Trial Treatment per liter of skimmilk hydrochloride No. Temperature Time organic 2,4-DNPH hydrogen per liter °C minutes mercaptans sulfides derivatives sulfide of skimmilk

1 24 unheated 630 2155 233 62 0 2 24 unheated 490 2155 164 105 0

Average 560 2155 198 83 0 1 90 30 850 1705 223 6975 10.2 2 90 30 630 1845 325 6830 10.4 Average 740 1770 274 6902 10.3

1 90 6o 960 2155 56 5855 15.4

2 90 60 735 1705 164 8715 12.5 Average 847 1930 110 7285 14.0

1 90 120 735 3550 136 6015 13.9

2 90 120 490 1545 178 7570 13.3

Average 612 2542 157 6792 13.6 Table 4^. Effect of heat treatment at 90°C on barium sulfide labelled cream on the release of volatile sulfur compounds

(TDS) Net counts per minute of aliquots from mg. of cysteine Trial Treatment traps per liter of cream hydrochloride No. Temperature Time organic 2,4-DNPH hydrogen per liter °C minutes mercaptans sulfides derivatives sulfide of cream

1 24 unheated 112 1777 346 186 0

2 2k unheated 446 3869 337 90 0 Average 279 2823 342 138 0

1 90 30 731 5674 331 4451 13.6 2 90 30 589 9000 316 2897 l4.8

Average 660 7337 334 3674 14.3

' 1 90 60 1120 9051 286 7160 15.0 2 90 6o 886 8080 414 6320 13.7 Average 1003 8365 350 6740 14.4

1 90 120 1725 6566 180 2777 13.6

2 90 120 1474 7714 328 7345 13.6

Average 1599 7i4o 254 5065 13.6

VO Table 46. Effect of heat treatment at 90°C on barium sulfide sulfur-35 labelled buttermilk on the release of volatile sulfur compounds

(TDS) Net counts per minute of aliquots from mg, of cysteine Trial Treatment traps per liter of buttermilk hydrochloride No. Temperature Time organic 2,4-DNPH hydrogen per liter of °C minutes mercaptans sulfides derivatives sulfide buttermilk

1 24 unheated 0 377 280 312 0 2 24 unheated 0 198 528 124 0 Average 0 287 4o4 218 0

1 90 30 310 1919 510 3172 9.0 2 90 30 465 1342 413 4962 11.2

Average 388 1630 461 4067 10.1 1 90 6o 310 577 825 3937 5-4

2 90 6o 465 765 363 6993 4.8

Average 388 671 594 5465 5*1

1 90 120 135 577 825 5480 4.1

2 9° 120 135 577 355 5738 4.8

Average 135 577 590 5609 4.5 Table Vf, Effect of heat treatment at 90°C on barium sulfide labelled acid whey on the release of volatile sulfur compounds

(t d s ) Net counts per minute of aliquots from rag. of cysteine Trial Treatment traps per liter of acid whey hydrochloride No.a 'Temperature Time organic 2,4-DNPH hydrogen per liter °C minutes mercaptans sulfides derivatives sulfide of acid whey

1 2k unheated 1339 0 3^5 105 0 2 2k unheated 1365 0 318 165 0 3 2k unheated 6kk 1744 ----- 222 0 Average 1116 581 331 l6k 0 1 90 30 1585 0 6k 8 165 O .87 2 90 30 1585 0 180 265 0.87 3 90 30 372 1583 — ^39 5.20 Average 1181 528 klk 289 2.31 1 90 6o 1115 0 193 330 O .87 2 90 6o 1220 0 180 265 0.87 3 90 6o 128 1267 — 517 6.10 Average 1116 581 331 165 2.98 1 90 120 1735 0 166 975 1.7 2 9d 120 1955 0 0 11U0 1.7 3 90 120 516 1583 ----- 733 9.1 Average 1402 581 331 9k9 k.l

aTrials 1 and 2 were conducted from milk supplied by cow number 1555 and trial 3 "was conducted from milk of cow number 1577 Table 48. The effect of heat treatment at 90°C on the type and distribution of volatile sulfur compounds from barium sulfide sulfur-35 labelled rennet whey

(TDS) Net counts per minute per liter of mg. of cysteine Trial Treatment rennet whey hydrochloride No.a Temperature Time organic 2,4-DNPH hydrogen per liter °C minutes mercaptans sulfides derivatives sulfide of milk

1 24 — 467 2,889 — 67 0 2 24 — 351 1,083 — 72 0 Average 409 1,986 — 70 0

1 90 30 120 1,878 — 2,872 12.51

2 90 30 239 1,783 — 3,805 10.47 Average 180 1,830 — 3,338 11.49

1 • 90 6o 117 1,589 — 3,544 13.67 2 90 6o 244 1,366 — 3,167 13.67

Average 180 1,^77 — 3,355 13.67

1 90 120 350 1,878 — 3,272 15.71

2 90 120 494 1,972 — ^,938 15.13

Average 422 1,925 — ^,105 15.42

aTrials 1 and 2 were conducted from milk of cow number 1577 Table 49. The effect of pH on the formation of volatile sulfur compounds from barium sulfide sulfur-35 milk after high heat treatment at 90°C for 60 minutes

(TDS) Net counts per minute per mg. of cysteine Trial liter of milk hydrochloride No. PH organic hydrogen per liter mercaptans sulfides sulfide of milk

1 6.7 139 339 5,977 16.3

2 6.7 302 37^ 10,327 20.7 3 6.7 467 389 8,561 18.9

Average 302 33^ 8,288 18.6

1 7-7 850 1,222 8,027 11.9 2 7-7 1,240 1,383 12,680 8.1

3 7-7 657 1,589 20,080 16.6 Average 916 1,398 13,598 15.5

1 9-7 520 480 51,537 28.5 2 9.7 131 486 56,634 L.A 3 9.7 131 166 39,377 27.0 Average 260 377 49,182 27.8 Table 50. The effect of heat treatment at $0°C for 60 minutes in the presence of cystine and cysteine on the release of volatile sulfur compounds. Milk was labelled with barium sulfide sulfur-35

(TDS) Compound Net counts per minute per mg. of cysteine Trial 0.5 gm/liter liter of milk hydrochloride Wo. of milk organic hydrogen per liter mercaptans sulfides sulfide of milk

1 None 139 339 9,977 16.3 2 None 302 374 10,327 20.7 3 None 467 389 8,561 18.9 Average 302 334 8,288 18.6 1 Cystine (unheated) 139 0 166 0

1 Cystine 497 920 223 14.5 2 Cystine hsn 1,697 297 10.4 Average 497 1,308 260 12.5 1 Cysteine (unheated) 68 165 4,286 267.7 2 Cysteine (unheated) 68 85 6,714 250.2 Average 68 125 5,500 258.0

1 Cysteine 948 1,337 34,245 192.0 2 Cysteine 817 674 33,051 186.2

Average 883 1,005 33,648 I8 9 .O Table 51* The effect of sulphydryl blocking agent, p-ehloromercuribenzoate on the release of volatile sulfur compounds after high heat treatment at 90°C for 60 minutes

(TDS) p-chloromercuri- Net counts per minute of 0.5 ml mg. of cysteine Trial benzoate of solution per liter of milk hydrochloride No.a 5 micromoles organic hydrogen per liter per liter mercaptans sulfides sulfide of milk

1 none-unheated 150 156 75 0 2 none-unheated 129 186 85 0 Average 137 171 80 0

1 present-unheated 95 333 0 0 2 present-unheated 158 585 0 0 Average present-unheated 127 459 0 0

1 none-heated 139 339 5,977 16.3 2 none-heated 302 37^ 10,327 20.7 3 none-heated 1*67 38 9 8,561 18.9 Average 302 33^ 8,288 18.6 1 present-heated 0 672 322 5.24 2 present-heated 0 781 126 5.24

3 present-heated 126 1250 lb 4.24 Average k2 901 xjb 4.91

aTrials 1 and 2 were conducted from milk of cow number 150? and trial 3 vas con­ ducted from milk of cow number 1577• Table 52. Effect of air on the formation of volatile sulfur compounds after high heat treatment at 90°C for 60 minutes of barium sulfide sulfur-35 milk

(TDS) Net counts per minute of aliquot mg. of cysteine Trial from traps per liter of milk hydrochloride No.a Airb organic hydrogen per liter mercaptans sulfides sulfide of milk

1 Control 1^67 389 8,561 18.9 2 Control 302 37^ 10,327 20.7 3 Control 139 339 5,977 16.3 Average 302 33^ 8,288 18.6 1 Air 1,200 183 2,039 9.3 2 Air 750 120 1,833 8.1 3 Air 250 ^72 3, Ml 15.7 Average 735 258 2,if28 11.0

aTrials 1 and 2 were conducted from milk obtained from cow number I507 and trial 3 was conducted from milk by cow number 1577 Milk samples were aspirated with air for 2 hours prior to heat treatment 157

Table 53. The formation of volatile sulfur-35 compounds from sodium sulfate-35 labelled milk aspirated with -Ng for two hours prior to heat treatment

Trial Treatment Net counts per minute per liter of milk Wo. Temperature Time organic 2,4-DNPH hydrogen °C minute mercaptans sulfides derivatives sulfide

1 unheated — 0 0 0 0

2 unheated — 0 0 0 0

Average 0 0 0 0

90 10 1370 0 557 95

90 10 1560 0 295 163

Average 1^65 0 426 129

90 30 434 685 515 214

90 30 392 381 285 402

Average 413 533 4oo 308

60 60 550 200 428 162

90 60 542 120 532 104

Average 546 160 480 133 Table 54. The relationship between nonradioactive and radioactive hydrogen sulfide of barium sulfide sulfur-35 milk and milk products

B^S produced after heat treatment at 90°C for different time intervals (minutes)______Fraction Trial Raw 30 120 No. specific specific specific specific ug/l activity ug/l activity ug/l activity ug/l activity

Milk 1 0 0 354 380 530 2 0 0 327 15.47 426 31.50 478 14.28 3 0 0 327 13.65 380 18.19 530 18.08 Average 0 0 336 9.56 395 24.84 512 16.18 Skimmilk 1 0 0 300 283 560

2 0 0 425 16.41 425 13.77 530 11.34

3 0 0 380 17.97 425 20.50 530 14.23 Average 0 0 368 17.19 377 17.13 540 12.78

Cream 1 0 0 283 426 635 642 9.84 4.00 2 0 0 530 8.39 1 694

3 0 0 478 6.06 692 10.31 694 10.56 Average 0 0 430 7.22 587 10.07 674 7.28 Table 54 (contd.)

I^S produced after heat treatment at 90°C for different time intervals (minutes) Fraction Trial Raw 30 60 120 No* specific specific specific specific ug/l activity ug/l activity ug/l activity ug/l activity

Buttermilk 1 0 0 543 ---- 1062 --- 1114 ---- 2 0 0 530 5.98 955 4.12 1087 5.04

3 0 0 589 8.42 1034 6.76 1034 5.o 4

Average 0 0 554 7.20 1017 5.44 1078 5.29

Acid whey 1 0 0 216 1.22 268 1.23 ill 8.78

2 0 0 177 0.93 216 1.22 ill IO.27 Average 0 0 131 1.07 242 1.22 ill 9.57

1 0 483 5.94 567 6.25 650 5.03

2 0 510 7.46 567 5.58 650 7.59

Average 0 490 6 .81 567 5.91 650 6.31 i6o

Table 55* Effect of pH on the formation of hydrogen sulfide of milk heated to 90°C for 60 minutes

pH Trial H2S Microgram per Specific activity No. liter of milk cpm/ug.

6.7 1 380 15.72

6.7 2 426 * 24.24

6.7 3 380 22.52

Average 395 20.98

7.7 1 1440 5.57

7.7 2 1387 9.14

7.7 3 1499 12.55

Average 1475 9.21

9.7 1 9557 5.39

9.7 2 9034 6.26

9.7 3 8510 4.62

Average 9033 5.44 l6l

Table 56. The effect of added cystine and cysteine to milk on the formation of hydrogen sulfide after heat treatment at 90°C for 60 minutes

Trial No. Added compound KbjS Microgram per Specific activity 0.5 gra/liter liter of milk cpm/ug. of milk

1 None (heated) 380 15.72

2 None (heated) 426 24.24

3 None (heated) 380 22.52

Average 395 29.98

1 Cystine (unheated) 0 0

2 Cystine (unheated) 0 0

Average 0 0

1 Cystine (heated) 111 2.00

2 Cystine (heated) 111 2.67

Average 111 2.34

1 Cysteine (unheated) 589 7.27

2 Cysteine (unheated) 642 10.45

Average 615 8.94

1 Cysteine (heated) 8173 4.19

2 Cysteine (heated) 8173 4.04

Average 8173 4.11 Table 57. The formation of volatile sulfur-35 compounds from barium sulfide sulfur-35 labelled milk aspirated with air for two hours prior to heat treatment

Hydrogen sulfide Specific Trial Treatment microgram per activity Ho. with air liter of milk cpm/mg.

1 none 380 15.72

2 none 11-26 2k.2k

3 none 380 22.52

Average 395 20.98

1 air 325 6.3

2 air 310 5.9

3 air 371 9.1

Average 335 7.1 163

Pig. 5* Chromatogram of 2,^-DKPH derivatives from sodium sulfate sulfur-35 labelled milk heated to 90 C for 30 minutes. 1 6 4

2,4-DNPH Derivatives From Milk Heated 90°C For 30 Minutes 165

Fig. 6 . Chromatogram of 2,4-DNPH derivatives from sodium sulfate sulfur-35 labelled milk heated to 90°C for 1 hour. 1 66

Q . & o

2,4-DNPH D«rivatives From Milk Heated 90°C For I Hour 1

167

Fig. 7 . Chromatogram of 2, U-DMPH derivatives from sodium sulfate sulfur-35 labelled milk heated to 90 C for 2 hours. 168

o o

0

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i I, Ronald Roy Pereira, was b o m in Hong Kong, December 12, 1931-

I received my secondary school education at St. Joseph*s College,

Hong Kong. I pursued my undergraduate training at The University

of Manitoba, Winnipeg, Manitoba, which, in 1959# granted me the

Bachelor of Science degree in Agriculture. I received the Master of

Science degree from the same institution in 1961. While completing

the requirements for the Doctor of Philosophy degree here in the

Department of Dairy Technology, The Ohio State University, I was

appointed to the position of Research Assistant and held this

post until the completion of my graduate program.

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