SOME FACTORS AFFECTING THE SOLAR-ACTIVATED FLAVOR OF HOMOGENIZED MILK AND THE ISOLATION AND CHARACTERIZATION OF A MINOR WHEY PROTEIN FRACTION WHICH IS CAPABLE OF BEING SOLAR-ACTIVATED By BERNARD WEINSTEIN wBwwaitswiBBi A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfilment of the requirements for the degree of DOCTOR OF PHILOSOPHY Department of Dairy 1951 ProQuest Number: 10008697 All rights reserved IN FO R M ATIO N TO ALL USERS The quality o f this reproduction is dependent upon the quality of the copy subm itted. In the unlikely event that the author did not send a com plete m anuscript and there are m issing pages, these w ill be noted. Also, if m aterial had to be rem oved, a note will indicate the deletion. uest. ProQ uest 10008697 Published by ProQ uest LLC (2016). C opyright o f the Dissertation is held by the Author. All rights reserved. This w ork is protected against unauthorized copying under Title 17, United States Code M icroform Edition © ProQ uest LLC. ProQ uest LLC. 789 East Eisenhow er Parkw ay P.O. Box 1346 Ann Arbor, Ml 4 8 1 0 6 - 1346 ACKNOWLEDGMENTS Sincere appreciation is hereby extended to Dr* G. M. Trout, Re­ search Professor of Dairying, for his intense interest and encourage­ ment throughout this study, and for his invaluable aid in the prepa­ ration of this manuscript; to Dr. Earl Weaver, Professor of Dairying, for the Graduate Assistantship and for the availability of the facilities required in this study* The writer is indebted to Mr. C. W. Duncan, Research Associate in Agricultural Chemistry, and his associates for the microbiologi­ cal analyses; to Dr. Carl Redemnn, Research Instructor in Agricul­ tural Chemistry, for the elementary analyses, and to Dr. H. A* Lillivik, Assistant Professor of Chemistry, for the electrophoretic analysis presented herein. iii TABLE OF CONTENTS page INTRODUCTION................................................ 1 REVIEW OF LITERATURE ........................................ 3 PROCEDURE................................................... 9 Source and Treatment of M i l k .... • ....... Isolation of MLnor-protein Fraction ••••............ *..... 9 9 Amino Acid Determinations .................................. 10 RESULTS..................................................... 12 I* Susceptibility of Individual Cows* Milk to the Develop­ ment of the Solar-activated Flavor ......... .............. 12 Susceptibility of individual cow and breed milk to solar activation......... ........... 12 Influence of time of exposure and period of storage on solar activation ••••»*».••••............. 14 Relation of percentage fat •••••....................... 14 II* Role of Oxidation and the Effectiveness of Certain Treat­ ments on the Solar-activated Flavor of Homogenized Milk • Ascorbic acid »............................. ..... 24 Nordihydroquaiaretic acid *•••••.............. 25 Nordihydroquaiaretic acid in combination with ascorbic acid ......... 25 Alpha tocopherol and hydroquinone................. 26 Hydrogen peroxide........ ....................... 26 High-temperature heat treatment 27 ................ Cysteine hydrochloride....... III* 24 Effect of Deaeration, Surface Area of Fat Globules and Relations of the Kreis Test ..... Deaeration ........... ........................ 2S 41 41 iv page IV# V# Increase in fat-globule surface a r e a .............. 1*2 Relation of the Kreis test 43 ................. Isolation and Characterization of a Whey Constituent Ca­ pable of Producing the Solar-activatedFlavor ......... 43 Characterization of the confound............ 43 Amino acid composition of the minor-protein fraction 50 Minimum molecular weight of the minor-protein frac­ tion ............... 51 Electrophoretic Analysis of a Contributing Minor-protein Fraction ....................... 57 Electrophoretic analysis of the minor-protein frac­ tion ............................................ 57 Electrophoretic analysis of whey proteins and heatcoagulated-whey serum proteins .......... 53 DISCUSSION.................... SUMMARY AND CONCLUSIONS .............. LITERATURE CITED ............................................ 63 67 70 V LIST OF TABLES page Susceptibility of milk from cows on pasture to solar activation when homogenized, by breeds........ .*............. 15 Cows not on pasture to solar activation when homogenized (Hol­ stein breed) •*•#.#.................................. •#*,..**♦ 21 Influence of ascorbic acid on the development of the solar—acti­ vated flavor in homogenized milk •««.................. #....... 30 Influence of nordihydroguaiaretic acid in the absence of added ascorbic acid on the development of the solar-activated flavor in homogenized milk #.«•#••• *#.... #.. #........ 32 Influence of nordihydroguaiaretic acid in the presence of 35 mg#/ liter of added ascorbic acid on the development of the so­ lar-activated flavor in homogenized m i l k ..................... 34 The influence of alpha tocopherol and hydroquinone on the devel­ opment of the solar-activated flavor in homogenized milk *.#»•••« 36 The effect of treating homogenized milk with hydrogen peroxide and the subsequent addition of ascorbic acid and riboflavin on the development of the solar-activated flavor ••.•••••••••••••#«• 33 The effect of high temperature treatment (176° F.-5 min#) and the subsequent addition of ascorbic acid and riboflavin on the development of the solar-activated flavor in homogenized milk 39 The effect of solar radiation on the sulfhydryl group of cysteine hydrochloride as measured by the nitroprusside test and organo­ leptic flavor determinations........... ...................... 40 The effect of deaeration on the development of the solar-acti­ vated flavor in homogenized m i l k ............ 44 The effect of surface area of the fat globule as influenced by homogenization, on the development of the solar-activated flavor 45 The relation of the Kreis test to the development of the solaractivated flavor in homogenized m i l k ..... .................... 46 The effect of solar radiation on the development of the acti­ vated flavor in aquasols prepared from the minor-protein fraction In whey (after 1—hr# exposure and 24—hr# storage at 40^ F*) »•••• 53 Comparison of analytical data of the minor-protein fraction with that of sigma—proteose, casein, lactalbumin, beta—lactoglobulin, pseudoglobulin and euglobulin............ *............ 54 vi page The essential amino acids, cystine and glutamic acid content of the minor-protein fraction, casein* lactalbumin, lactoglobulin, pseudoglobulin and euglobulin (all amino acid values expressed as grams per 100 g# of anhydrous, ash-free protein) .......... 55 The number of amino acid residues in the minor-protein fraction .......... as compared to casein and beta-lactoglobulin 56 vii LIST OF FIGURES page Flow sheet diagram for fractionation of milk proteins ••«••••*•• 60 Electrophoretic patterns from one percent solutions of minorprotein fraction at various pH and buffer media ..... 6l Electrophoretic patterns of -whey and heat—coagulated-whey serum proteins ............ .......................... ..... . INTRODUCTION The solar-activated flavor is one of the major problems in the dis­ tribution of homogenized milk* The flavor defect occurs frequently, and gives rise to consumer complaints* At the present time no easy or prac­ tical means for preventing its development in commercially produced homo­ genized milk is available* The term solar-activated flavor as used throughout this study embraces a number of flavors such as 11sunshine,” "burnt,” "burnt—feather,” ”burnt—protein,” "cabbage," "mushroom,” and/or even "medicinal*" Many investigators have shown that homogenized milk was far more susceptible to off—flavors as a result of exposure to daylight than the same milk not homogenized* The occurrence of the solar-activated flavor in homogenized milk exposed to daylight was rather disheartening in view of the gratifying discovery that homogenization retarded or inhibited the development of the copper-oxidized flavor* Thus, while homogenization re­ tarded susceptibility of milk to one undesirable flavor, it enhanced the development of another off-flavor, if and when certain conditions prevail* Although the solar-activated flavor has caused much concern among the com­ mercial dairy plant operators, the available data regarding the various factors involved in the development of the solar-activated flavor and an easy and practical method to prevent its inception are limited* The purpose of the work reported herein werer (a) to study the var­ ious factors related to the off-flavor development* (b) to attempt to find a practical method to prevent the activated flavor development and -2further; (c) to attempt to establish the identity of the constituent affected when a solar—activated flavor develops* -3- KEVIEW OF LITERATURE The deleterious effect of sunlight upon the flavor of milk has been recognized for many years, Browne (1899) early observed that oxidative rancidity was catalyzed by the exposure of the butt erfat to the light# Since this early observation, various investigators, Hammer and Cordes (1920), Frazier (1928) and Henderson and Roadhouse (1934) have studied the effect of light upon the flavor of milk as being associated with a lipid oxidation. Hammer and Cordes (1920) reported that ,,off,, flavors developed in certain milk samples after 10-minute exposure to light and further, that a definite tallowy flavor appeared after 45 minutes expo­ sure to light# They noted also that an abnormal flavor developed rapidly in skimmilk upon exposure to sunlight* The flavor that developed in the exposed skimmilk was not the typical tallowy flavor. These workers dis­ covered that the “off" flavor development could be retarded by the use of brown glass bottles# However, their use resulted in increased milk tem­ peratures, thus favoring bacterial growth. In the light of our present knowledge it appears that the noff" flavor that Hammer and Cordes (1920) observed in exposed skimmilk was in reality the activated flavor so com­ mon today in commercially produced homogenized milk# Frazier (1928) found that diffused daylight acted as a catalyst in the oxidation of the butt erfat# He postulated that although the heavy glass of the milk bottle screened out the ultra-violet rays, it did, never­ theless, allow to pass through the longer rays which exerted the catalytic effect# —4Tracy and Ruehe (1931) found from their studies of flavors in mar­ ket milk that milk esqposed to sunlight for short exposure periods in uncolored glass bottles developed the typical tallowy flavor. They ob­ served that as the period of exposure of the milk to sunlight was in­ creased, a point was eventually reached where the tallowy flavor was masked by the burnt flavor. They believed that the presence of metal­ lic salts was not a factor in the development of the burnt flavor, and that skimmilk and low*-fat milks were more susceptible to the burnt fla­ vor than were whole milk and cream. The burnt flavor described tey Tracy and Ruehe (1931) was attributed to the action of sunlight upon the milk proteins. Henderson and Roadhouse (1934) stated that cream exhibited a great­ er susceptibility to oxidation when exposed to diffuse or direct sun­ light, Doan and Myers (1936) observed that skimmilk, whole milk and but­ termilk could be protected from the catalytic action of sunlight by the use of paper milk containers. However, they found that paper milk bot­ tles offered no protection against the tallowy flavors caused by sun­ light. The authors postulated from the above findings that the photo­ chemical reactions producing the tallowy flavor and the burnt flavor in milk were separate and distinct. The study of the solar-activated flavor prior to the acceptance of homogenized milk as a marketable product received little attention. Since the solar-activated flavor was not a serious flavor defect in nonhomogenized milk, but rather more noticeable in skimmilk, the urgen­ cy for a complete study of the activated flavor was not deemed necessary. The phenomenal acceptance of homogenized milk in America during the past decade (to the extent that in some plants 100 percent is homogenized) has extended the possibilities of the occurence of the defect# Hood and White (1934) early pointed out that homogenized milk was far more susceptible to off-flavors as a result of exposure to daylight than the same milk not homogenized# This observation has since been substantiated and/or reported by many workers# (Doan 1937a, 1937b, 1938, 1943; Tracy 1936, 1938, 1948$ Corbett and Tracy 1937, 19395 Flake, Weckel and Jackson 19395 Dahle 1941; Babcock 1942$ Henderson 1944; and Burke 1948#) Tracy (1936) in a discussion of homogenized milk stated that homoge­ nization was a factor in the case of milk exposed to sunlight since homo­ genized milk would acquire an off-flavor sooner than nonhomogenized milk# He found that when a bottle of each of the two milks was exposed to either direct or indirect sunlight for 10-15 minutes, the nonhomogenized milk developed a slight burnt flavor whereas the homogenized milk had developed a much more pronounced burnt flavor* Tracy (1936) theorized that while light rays would oxidized the butt erfat, the effect of the sunlight on the milk proteins was responsible for the activated or burnt flavor# Although the development of the solar-activated flavor gives rise to consumer complaints in the homogenized milk product, much of the work has been carried out using skimmilk or whey# Weckel, Jackson, Haman and Steenbock (1936), in an effort to deter­ mine the effect of irradiation on the flavors of milk, exposed milk to irradiated energy for various periods# The flavor that developed follow­ ing irradiation in the incipient stage was described as 11flat#11 Upon con­ tinued exposure, this flavor gradually changed into what the authors -6described as ”burnt,” ‘‘burnt feather,” “burnt protein” or "mushroom fla­ vor#” They concluded, that the activated flavor must be distinguished from the papery, cardboardy, tallowy flavors which they believed resulted from the action of radiant energy or metals on the lipids in milk# Weckel, Jackson, Haman and Steenbock (193&) in reference to the division of the spectrum responsible for the off-flavor stated: The effect is due to radiation from that part of the spec­ trum which is known to have an antirachitic effect as well as to parts of the spectrum devoid of such properties# An analysis of the emission of the various arcs permits the conclusion that energy ranging in wave length from 2,600-3,100 Angstrom units is less active in flavor production than energy of wave length less than 2,600 Angstrom units# Josephson (1946) is in disagreement as to the wave length responsible for the off-flavor development# He stated that the sunlight flavor would develop on very cloudy days when little, if any, ultra-violet light penetrated the outer atmosphere# Josephson (1946) employed specially pre­ pared exposure cells and light filters of known transmission and concluded that no protection against the off-flavor development was afforded even when all light below 5,900 Angstrom units was excluded# He reported that for complete protection against the off-flavor development all light be­ low 6,200 Angstrom units must be excluded. He believed the active wave lengths of light were within 5,900 and 7,400 Angstrom units# Since the heavy glass of the milk bottle would tend to filter out the ultra-violet light rays, it seemed logical to assume that the range proposed by Josephson (1946) was the effective range# Weckel and Jackson (1936); Flake, Weckel and Jackson (1939); and Flake, Jackson and Weckel (1940) are chiefly responsible for the few facts we know concerning the chemistry of the activated flavor compound# -7The authors concluded that the constituent affected when a solar-activa­ ted flavor develops is protein in nature* Flake, Jackson and Weckel (1940), in an attempt to determine specifically the constituent affected, subjected casein, lactalbumin and various amino acids to ultra-violet radiation* They concluded that albumin, and to a lesser degree casein, acquired a flavor and odor typical of that which develops in milk similar­ ly exposed to radiation* Of the amino acids studied, cystine, methionine, tryptophane and histidine were found to develop flavors suggestive of irradiated milk* The authors1 attempts to measure the effect of ultra­ violet light upon the protein structure by means of the Van Slyke amino nitrogen and nitrogen distribution determinations were not successful since the analytical procedures used were not sensitive enough to show a measurable structure change* Flake, Jackson and Weckel (1940) obtained concentrated solutions of dialyzable substances from milk and exposed them to irradiation* These dialyzable solutions upon irradiation gave rise to a disagreeable flavor and odor which were definitely not typical of the activated flavor of excessively irradiated milk* The authors also carried out dialytic experiments and concluded that the removal by dialy­ sis of a large portion of the dialyzable substances of milk did not alter its susceptibility to the development of the activated flavor and odor* Ansbacker, Flanigan and Supplee (1934) suggested the possibility that sulfur compounds might be involved in the activated flavor formation* Upon subjecting a foaming agent of milk to ultra violet light they noted an odor which was similar to that of over-irradiated milk* These workers theorized that the basic mechanism involved the mobilization of sulfhydryl groups* Flake, Jackson and Weckel (1940) further indicated the possible -8role of sulfur in the development of the activated flavor when they ob­ served that the intensity of the activated flavor was accentuated by heat­ ing milk to approximately 180°F* which is in the temperature range at which the cooked flavor becomes evident* Doan and Myers (1936) believed that the burnt flavor due to solar ra­ diation originated in the casein-and albumin-free serum of milk* Flake, Jackson and Weckel (1940) were able to concentrate a 11fraction11 that they believed responsible for the activated flavor development* analyse the concentrated fraction. They did not However, they did secure a positive nitroprusside test after reduction with potassium cyanide, which indicated the presence of disulfides* The authors reported that when a very small amount of the concentrated fraction was added to milk it imparted to the milk a flavor and odor very similar to that of milk exposed to radiation* Keeney (1947) was able to induce the activated flavor in a casein—, albumin—, and globulin-free milk serum* He succeeded also in concentrating a heat— flocculable, dialyzable substance which he believed to be one of the com­ ponents responsible for the sunshine flavor common to homogenized milk* PROCEDURE The milk used in these studies was obtained from the Michigan State College dairy and experimental b ams and the college creamery* All milk was holder pasteurized at 143°F* for 30 minutes, after which the milk was homogenized at 2,500 pounds pressure in a laboratory model, 25 gallon-perhour homogenizer* The homogenizer was flush-washed with one-half gallon of 150° to l60°F* water after processing sible intermixing of samples* each sample to prevent any pos­ Following homogenization,the samples were ice colled at once to 50°F* In an attempt to keep the source of radiant energy uniform all exposed samples were exposed to clear solar radiation between the hours of 10 a*m* and 2 p.m. Isolation of Minor-Protein Fraction The isolation procedure of Aschaffenburg (1946) was modified to meet the experimental problems encountered in this study* One gallon of fresh­ ly separated skimmilk was rennet-coagulated at 26*7° C. (80° F*)* The coagulum was cut into 0*5-inch cubes and heated to 50° C* (122° F*) to expel the whey* The whey was separated by vacuum filtration, transferred to a 4-liter beaker and heated to 95° C* (203° F*) for one hour to remove the heat-coagulable proteins* A mechanical stirrer was employed during the heating process to insure uniform heat distribution* Following the heat treatment, the mixture was filtered through a milk filter cloth and the resulting serum was clarified by centrifugation at 50,000 r*p.m. for 15 minutes in a Sharpless steam-turbine supercentrifuge* The centrifuged -10serum was crystal-clear and was assumed to be free of the heat-coagulable proteins* The clarified serum was treated with 34*5 g* of C* P. ammonium sulfate per 100 ml* of serum. After standing for three hours, the precip­ itate was separated by centrifugation at 2,000 r.p.m. for 30 minutes* The resulting precipitate was washed in about one-fourth of its volume of dis­ tilled water and again re-separated by centrifugation. The washed precip­ itate was dispersed again in a small volume of distilled water, transferred to a cellophane dialyzing bag and dialyzed for 24 hours against running tap water and then for an additional 2h hours against slow-dripping dis­ tilled water* The non-dialyzable fraction was removed and dried from the frozen state under a high vacuum. was 3*768 g. The weight of the freeze-dried sample The yield was 0.10 percent. Amino Acid Determinations Amino acid determinations were carried out microbiologically by using Lactobacillus arabinosis, Streptococcus faecalis and Leuconostoc me sentero ide s P-60* The media used in the various determinations were essentially the same as those described by Sauberlich and Baumann (1946) with the excep­ tion of those used for isoleucine and methionine, which were prepared ac­ cording to the method of Kuiken et al. (1943) and Lyman et al. (1946). The hydrolyzates for the determination ofarginine, cystine, glutamic acid, histidine, isoleucine, leucine, lysine, methionine, phenylalanine, threon­ ine and valine were prepared according One gram of the material to the method of Stokes et al* (1945)* was dispersed in 25 ml. of 6n HCl and autoclaved for eight hours at 15 pounds pressure* The hydrolyzates were cooled, neu­ tralized to pH 6 .6-6.8 with 18N NaOH, made up to 100 ml., filtered, covered with a few drops of toluene and stored in the refrigerator until analyzed. -11The enzymatic digestion procedure of Wooley and Sebrell (19U5) was used for the tryptophan assay. One gram of the material was weighed into a 100 ml. volumetric flask, 20 mg. of pepsin and 1*0 ml. of 0.1N HgSO^ were added and the flasks incubated at 37° 0 . for 21* hours with constant shak­ ing. The contents of the flasks were then transferred to 100 ml. beakers and 3*0 g. of I^HPO^ were added to each beaker and the pH adjusted to 8.1* with 3N HaOH. The solutions were transferred to 100 ml. volumetric flasks and 20 mg. of trypsin were added and allowed to incubate at 1*0° C. for 21* hours with constant shaking. The contents of the flasks were cooled, ad­ justed to pH 7«0, diluted to 100 ml., centrifuged, filtered, preserved with toluene and stored in the refrigerator* DL-Configurations of isoleucine, leucine, methionine, phenylalanine, threonine.and valine were used in the preparation of standards for these amino acids, whereas, the L-configurations were used for the preparation of the standards for arginine, cystine, glutamic acid, histidine, lysine and tryptophan. In all cases the assays were run after the proper dilu­ tions had been made. -12- RESULTS I. The Susceptibility of Individual Cows1 Milk to the Development of the Solar-activated Flavor Quart samples of morning*s milk collected from individual cows of the milking herds at the Michigan State College Dairy and Experimental Barns were pasteurized, homogenized and cooled as outlined under proce­ dure. Each sample was then divided into three lots: Lot 1 served as a controli Lot 2 was exposed to clear solar radiation for 30 minutes between the hours of 10 a.m. and 2 p.m. during the first weeks of September5 and Lot 3 was exposed under the same conditions, but for 60 minutes. Imme­ diately after exposure the samples were dark stored at 1*0° F. for 1*8 hours. Flavor examinations were made at 21* and 1*8 hours by experienced judges who did not know the identity of the samples. Susceptibility of individual cow and breed milk to salar activation. The susceptibilities of milk from cows on pasture to the solar activated flavor are shown in table 1. The results comprise three trials on each cow at three-day intervals and include milk from nine Ayrshires, 12 Brown Swiss, 10 Guernseys, 11 Holsteins and four Jerseys. The data indicate that of the 1*6 cows whose milk was studied, 11* of them, or 30 percent, were non-susceptible to the solar-activated flavor in all trials after homogenization and exposure to solar radiationj while the milk from two Guernsey cows, Nos. 71 and 73* exhibited a non-susceptible tendency in two of the three trials. The correlation between breed and susceptibility of milk from individual cows in regard to the solar-activated flavor seems slight. Of the milk studied, 36* 333 23 and 20 percent of that from the Holstein, Aryshire, Brown Swiss and Guernsey breeds respectively proved non-susceptible to solar activation. Studies on Jersey milk were limited since only four Jerseys were in lactation. However, of these four, two of them, or 30 percent, yielded milk susceptible to solar activation. From the data obtained there appears to be no correlation between so­ lar activation and stage of lactation. The results outlined in table 1 show that the cows producing non-susceptible milk represented, in general, all stages of lactation. Fortunately, during the course of this study milk from the experimen­ tal herd of 20 Holsteins that were on dry feed for a period of time was available for investigation. Since approximately 30 percent of the cows on pasture yielded milk which upon pasteurization, homogenization, and sun exposure was stable to solar activation, this herd afforded the opportun­ ity to study the relation of non-pasture or dry feed on the solar-activated flavor of the milk produced. given in table 2. The results ofthis phase of the study are The data indicate that milk from cows on dry feed is more susceptible to the activated flavor than from cows on pasture. Milk from the Holstein breed on pasture, reported above, exhibited a non-sus­ ceptible tendency to solar activation in 36 percent of the milks studied. However, all the milk studied from cows not on pasture feeding showed a susceptibility to solar activation* During the course of this study an off-flavor developed in a few so­ lar-exposed samples which was not typical of the true solar-activated fla­ vor. It is noted by symbol Z in table 1. This atypical flavor is best described as "unclean," "nauseating," and as of a decomposed-protein nature suggesting that of milk sometimes noted from cows having a serious -ll*physio logical disturbance* This off-flavor has been noted previously, in samples brought to the laboratory for flavor examination, but heretofore was not associated with solar activation* The off-flavor is so distinct and different from the easily recognized true sunshine flavor that no sug­ gestion is forthcoming that it is light induced* It is not improbable that some of these off-flavors in milk such as encountered in this study and which have heretofore given rise to consumer complaints are due to ex­ posure to daylight* Influence of time of exposure and period of storage on solar activa­ tion* The time of exposure and length of storage seem to have some effect on the intensity of the activated flavor. A 30-minute exposure to solar radiation in most cases resulted in a more intensified flavor development than did a 60-minute exposure* of the flavor component. This may indicate a partial decomposition From the data obtained and presented in tables 1 and 2 the Intensity of flavor development after 21* hours appears to be very slight. However, some inconsistencies were noted. This would seem to indicate a maximum reaction rate is reached within the first 21* hours after exposure* Relation of percentage fat. There seemed to be no correlation be­ tween the fat percentage and the susceptibility of the milk to the devel­ opment of the activated flavor (tables 1 and 2). noted both in low-and in high-fat milk. Activated flavors were - c •g 15 - + + + + + + + + + 4- 4- + + + + + 4- + + I l l l i I I I I I I I tS3 CO C-3 iS CO u 4* + + + + IS3 £n } Csi C4 tSJ IS3 t i i t i i § X! cd to -41 -P u ca 0 <5 CO ol

+ + + i i i i f t isi iss tsi M + 1 + I ! I i l I CSJ C4 IS ! M £N3CsJ I I i I l 1 I (S3 tsi ♦ COI N £l*H O°lf £ H i I l I t I I I I t •H Xt *H P o\ CD O CO CO S3 o O *H *P ® *8 t>£)43 CM CM vO OA + 4- 4 - III CM CM (V 4- 4- 4- *» N N ,H OA OA W CA - i i i i I I III H I i 1i 1,1 1,1 m + + + t t t t I '+ t ill til + +* + t + 4 III III til It! 111 III + + I I I I I I ++ ++ + tit t t i + + + + + + + + TABLE 1 (continued) - t t t I I I I I I 17 Jl+ 1+ + t 4- t 4- + + ttt + 14 + + + 4- 4* 4* + * t l I I I + 4* + + + I+ + t * § ill til ir \ nO On CO "■s f * CO 02 CO 40 CO co H CM CO f-| til ill lit ill til to O' <50 crs CO OI to CO co 40 CO CO rH 02 CO H -4- 02 CO CO s H 40 40 NO co CO CM c o H O 40 CO 02 CO H 02 i I I 02 02 CO O « CO 40 CM CO HCMcn <50 nO TABLE 1 (continued) -18- J 4 t 4*4 J 4 * 1 1 t 4*4 t J J tit i i i iii j + + + + 4 j + t * 4 t + + *44 i i i iii i i i + CM fr0 -S| H S H C*so iii o'X - 0O" . c ^ CM N ** ** CM rH v • \ - £ 1 1 +11 + tt f I I 4 1 1 + 1 ' | 4 4 111 i i i lit i i i i i i + i i + i i + + $ l i t N N N t 1 1 +** 4*4 *ii i i i i i i i i i i i i i i i vQ tlO *H C— > • , 4 HCM n H "'O -4 * C”— CM O '' 0 s* O '' CM C^\ H vO C*- CM TABLE 1 (continued) - 19- i i i rtf $ + J + t $ t + + * 1 1 iii ++ + i i i i i i H I t+t t+i tit lit + + + i i i i i i tit i i i i i i i i i itt i i i I i t i i i ± + T+ T + + t + "r + + + i i i i i i + + + i+ t+t H- + + + + III III + + H- III III III Itt III 11 i a 1 i t i III 3 cm on NO cr\ rH CM rH CM • -4 - -4 rH CM rH CM OA rH CM « N CM CM ir \ rH ♦ t f\ On ir\ CM - it rH -20- I I I ill III III III + + + + + + i l l H- + + + + + + + + + + + i i t TABLE 1 (continued) + III + H* + III ON NO -4- ON III ttt I I I ' ' ' I I I t + t III 111 + + I I I + + + + t 4- + + III t t t III to I III + + + I III + + + t III III I I I t I I III CM ON NO to •oo ON ON on UN • UN I—I • UN nO CM rH CM ON rH CM ON rH (V C^\ rH CM ON rH CM ON rH CM ON r-H CM ON On C- NO -3" UN sO CNI CM On rH - tf” CO rH CM r rH ON H u \ CM ON rH UN - No activated flavor +++ Pronounced activated flavor + Slight activated flavorZ Peculiar, unclean, nauseating ++ Distinct activated flavor + j + + + + ' ' ' flavor t t t * -21- t i + H- i t t X X H- + X + + XX t XX X + + ++ o O Al + + +++ X X -p cd G| Oi •Hi ■P X5 < U •S 10 cd 0) rH - p O CO rH t£|£ cd t I I I 1 1 1 I I a a XJ +++ i> -P O m cd - I I o o as 1—II—I G *H O O »H •P 0 cd & CM CM CM CM oa oa OA tsO -P cd CM o +a cd CO rH •P cd CM o cd OA CM 'El CM * CM * oa rH CM OA oa rH CM rH CM 8 s CM CM CM OA •H CM CM E-< £ O o UA UA «*! CM xO xO xO «aj CM C- «3J sO C^<*! TABLE 2 (continued) -22- i t i t + i tj t t t t i i i t i t i f + + + t t tt | t | + 11 t i i J 11 ii ii ii it ii ii ii i i I i ii t + + t + i t| ++ tt it t t t + it i ♦ + + t t |t + i $ + i t t i t $ + II I I I I t i l l II II II t i l l CM (* > -s f -4 * U-x >0 vO C ^ - iO O vO >4 IA O C“ * (A W V\ tO r H CM rH CM r H CM rH CM rH CM H r H CM H '•£> CM 0 "\ C^N (A V \ O^ O • • CM rH s£> * » «e*J • o o C ^ - v O CM -3 - “ • CM r H CM CM -T «aj - 4- i * + + + + + + + + II II tt + + x xi p 9 ? 4 4 4 4- 4 4 4 4 4 4 4 4 4 4 4 4 + 4 4 4 4 4 + — 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 — 4 4 4 4 4 4 4 4 4 4 4 4 4 ? ? 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 4 ? 4 4 4 4 4 4 4 4 4 4 4 + 4 4 4 4 4 4 4 4 4 4 4 4 4 + 4 *> 4 4 4 4 4 4 4 4 - ? v 4 4 4 4 4 4 4 4 4 4 4 4 4 -31TABLE 3 (continued) 0 30 60 120 9 10 +KE3T + + + + + + + + + + + + + + + + + + + + + + + + + + + + + ^m 0 30 60 120 ? + + + No activated flavor Questionable activated flavor Activated flavor -32- TABLE 4 Influence of Nordihydroguaiaretic Acid in the Absence Of Added Ascorbic Acid on the Development of the Solar-activated Flavor in Homogenized Milk Activated flavor* in exposed homogenized milk ‘ whan various amounts (mg» / liter) of N»D+G»A» were added: Trial (no *) 1 Expo­ sure (min*) 0 30 60 120 0 30 60 120 0 30 6o 120 Control______ 0_______ 12+5 + + + + + — - Bitter Bitter Bitter + + + + + - Bitter Bitter Bitter + + + + + + — — Bitter Bitter Bitter + + + + — - Bitter Bitter Bitter 0 30 6o 120 + 0 30 6o 120 + + + 0 30 60 120 120 e o 30 60 120 ? + ? Bitter Bitter Bitter + + + + + + - Bitter Bitter Ritter + + + - Bitter Bitter 7 + ? Bitter Bitter Bitter 0 30 60 25 ______ 75 + + + -33TABLE 4 (continued) 9 10 0 30 60 120 + -t* + + + 0 30 + 60 + + 120 + + — No activated flavor ? Questionable activated flavor + Activated flavor - Bitter Bitter Bitter Bitter Bitter -____ Bitter -3 b- TABLE 5 Influence of Nordihydroguaiaretic Acid in the Presence of 21 ss- L Liter of Added Ascorbic Acid on the Development of the Solar-activated flavor in Homogenized Milk Activated flavor* in exposed homogenized milk when various amounts (mg. / liter) of N.D.G.A. were added Trial (no.) 1 2 3 k 5 6 7 8 Expo­ sure (min.) 0 30 60 120 0 30 60 120 0 30 60 120 0 30 60 120 0 30 60 120 0 30 60 120 0 30 60 120 0 30 60 120 Control 0 12.5 25 75 mm + + + + + - + 4- + — + + 4- + + + 7 +■ + 7 + + +• + + 4- - — + + — - — + 7 7 + + + — + 4* 4* — 4- 4* 7 4- 4* + - 4- 4- + — 4* 7 + 4- + + — 4* + + ? + + + + -f + - 4- + + — + + 7 4- + 7 - + + + — TABLE 5 (continued) 9 10 *KET: 0 30 60 + + ? + + ? ? 120 + + + ? 0 30 + + + - 60 + + + — 4- * 120 +__________ + — Mo activated flavor ? Questionable activated flavor + Activated flavor - 36- TABLE 6 The Influence of Alpha Tocopherol and Hydroquinone on the Development of the Solar-activated Flavor in Homogenized Milk Activated flavor* in exposed homogenized milk when alpha tocopherol and hydroquinone were added to homogenized milk Trial (no.) 1 2 Expo­ sure (min. ) 0 30 60 Control (not exposed) Alpha tocopherol Hydroquinone 0 0 50 (mg. / 1-) (i) Alpha tocopherol plus hydroquinone 50 (mg. / 1.) + 0.0005 (mg./l.) w 0.0005 — + + 0 30 60 _ 3 0 30 60 _ k + + + + — — — + ? + — + + + + + + + + + — — — — 0 30 60 + + ? + + + — — — 5 0 30 60 -t- + + + + + + 6 0 30 60 + + o 9• + -f* + *■ 0 30 60 + + ••? + + + — ? - + + + — 7 a 0 30 — — _ ft ? -37- 10 *KEY: 0 30 60 + + 0 30 60 ? + • ? + + + ? + + ? + No activated flavor Questionable activated flavor Activated flavor 4* 9 •■0 TABLE 6 (continued) -38- TABLE 7 The Effect of Treating Homogenized Milk -with Hydrogen Peroxide and the Subsequent Addition of Ascorbic Acid and Riboflavin on the Development of the Solar—activated Flavor Series (no.) I II III Treatment 1 Activated flavor* at 24 hours in trial 5 6 7 2 3 4 — — Exposure (min.) Addition of 0.02& ml. of 30^ H2O2 per liter to milk prior to pas­ teurization and homo­ genization 0 30 60 Addition of 25 mg./ 1* ascorbic acid to perox­ ide-treated milk. 0 30 60 — ■£- — - •* ■it- ■K* •* -K- •> 4* 4- 4- 4- 4- *■ * _ — ■* * -* ■* _ _ 0 Addition of 25 mg*/ 1. 4 44-4- +++ ascorbic acid and 2 30 ++ 4-4*460 4-4- 4mg./l. riboflavin to peroxide-treated milk. - No activated flavor ? Questionable activated flavor * Powder milk-rubber like flavor + Slight activated flavor ++ Distinct activated flavor +++ Pronounced activated flavor + + 4* 4- + 4- + 4-4- ++ 4-4- 4-4- 4-4-4- 4-4- 4-4- 4-4- - 39- TABLE 8 ,221® Effect of High Temperature Treatment (176° F»-j> min») and, the Subsequent Addition of Ascorbic Acid and Riboflavin on the Development of the Solar—activated Flavor in Homogenized Milk Activated flavor* when milk is pasteurized by high temperature treatment on the subsequent addition of ascorbic acid (25 mg./l) and riboflavin (2 mg*/l) Ascorbic Ascorbic acid Control and riboflavin acid 0 added added Trial Expo­ sure ^no*) 1 (min*) 0 60 2 0 60 cooked 3 0 60 cooked h 0 60 cooked 5 0 60 cooked 6 0 60 cooked 7 0 60 cooked 8 0 60 cooked 9 0 60 cooked 10 *KHX: cooked ■f + + + *> + + — + + + + + ? + ? + + cooked 0 — 60 activated flavor - Wo ? Questionable activated flavor -4- Activated flavor -U o - TABLE 9 The Effect of Solar Radiation on the Sulfhydryl Group of Cysteine Hydrochloride as Measured by the Nitroprusside Test and Organoleptic Flavor Determinations Activated* flavor when cysteine HC1 (mg./l#) is added to homogenized milk Trial (no.) 1 2 3 *KET: Expo­ sure (min*) 0 30 60 120 20 40 cooked + + + cooked 0 30 60 120 cooked + + + cooked 0 30 60 120 — ? + + — + + + cooked cooked ? ? + + + + No activated flavor Questionable activated flavor Activated flavor Negative nitroprusside test Positive nitroprusside test Nitroprusside test** before exposure + + + + + + + before exposure — — — — - + + + — — — -la­ in* Effect of Deaeration, Surface Area of Fat Globules and Relation of the Kreis Test The milk used in this study was pasteurised, homogenized and cooled as outlined under procedure* In the deaeration studies, the ice—cooled, £0° F. milk was divided into three lots* Lot I was used as a control; Lot II was not deaerated but was exposed to solar radiation for one hour, then stored at I4O0 F* for I4.8 hours; and Lot III was deaerated in a 500 ml* flask under partial vacuum, then similarly exposed and stored* The milk used in the surface-area studies was mixed, pasteurized non-homogenized milk obtained from the College Creamery. divided into three lots: The milk was Lot I served as a control; Lot II was exposed to the sun for 60 minutes; while Lot III was similarly exposed then homo­ genized after exposure at 2,500 pounds pressure. All samples were stored at I4.O0 F. for 2lt hours and then examined for the activated flavor. The homogenized milk used in the Kreis test was divided into five lots: Lot I served as a control; Lot II was exposed to solar radiation; Lots III and IV were treated with 35 and 50 mg./l of ascorbic acid respec­ tively; while Lots V and VI were treated with 12.5 and 25 mg./l of nordihydroquaiaretic acid (N.D.G.A*) respectively. The milk to be exposed to solar radiation was further divided Into three lots which were exposed for 30, 60 and 120 minutes respectively. After 2k hours storage at J4.O0 F. the samples were examined organoleptically for the off-flavor and chem­ ically, using the Kreis test, to note oxidation. Deaeration* The results, representing five trials, obtained on the effect of deaeration on the solar-activated flavor in homogenized milk are shown in table 10. The data show that the solar-activated flavor did not develop when the milk was deaerated and then exposed to solar radiation. —ij.2—However, when air was incorporated into the deaerated and exposed milk and then reexposed to solar radiation for 60 minutes, the milk developed the typical solar-activated flavor. The exposed milk not deaerated ex­ hibited the typical solar-activated flavor, while that serving as a con­ trol was indistinguishable from that deaerated and exposed. These results would seem to substantiate the observations made under section two to the effect that the development of the solar-activated flavor in homogenized milk results from an oxidative process. Increase in fat globule-surface area, Although many observations have been made showing a greater intensity of activated flavor develop­ ment in homogenized than in nonhomogenized milk similarly exposed to day­ light, no data were noted showing the potential activated flavor develop­ ment of exposed milk when the surface area of the fat globules was increased by homogenization subsequent to exposure. Such a study would seem to show whether surface area of the fat globules was a factor in the develop­ ment of the off-flavor. The results, representing five trials, are presented in table 11. The data show that the nonhomogenized milk exposed to the sun (Lot two) did not develop an activated flavor in two of the five trials and only a slight off-flavor In the remaining three trials. On the other hand, portions of the same milk homogenized immediately following exposure (Lot three) and then stored developed a very strong activated flavor in all five trials. These results would seem to indicate that total surface area of the fat globules may be an important factor in the development of the solar-activated flavor. -U3~ Relation of the Kreis test. Since the solar-activated flavor has been shown to be oxidative in nature, a clue in regard to the constituent oxidized was sought by the use of the Kreis test. The chief phospholipid of milk, lecithin, is postulated to be the constituent oxidized when the copper—induced oxidized flavor develops, and further that the milk fat itself becomes oxidized during this process, following its induction period# The Kreis test denotes oxidative rancidity with the formation of color due to the presence of epihydrin aldehyde# Although the absence of color formation in itself is not definite proof of the absence of oxidative ran­ cidity, the Kreis test remains a very good presumptive test. To this end studies were made# The results on the relation of the Kreis test to the development of the solar-activated flavor in homogenized milk, representing 10 trials, are given in table 12# The data indicate that a positive Kreis test did not develop in any milk not exposed to solar radiation# A comparison of the flavor and the Kreis test examinations seems to indicate a trend be­ tween a positive organoleptic taste sensation and a positive Kreis test# The failure of a positive Kreis test to correspond with a positive solar- ^ activated flavor in all cases does not, of course, preclude the possibil­ ity of a "lipid-fraction'* oxidation, but it does, however, leave the way clear for further studies on the isolation and characterization of the constituent affected when a solar-activated flavor develops# -SUI­ TABLE 10 The Effect of Deaeration on the Development of the Solar-activated Flavor in Homogenized Milk Lot Treatment of milk (no*) 1 2 3 4 ■#KEIt Control - not deaerated or exposed Trial (no*) 1 2 3 4 5 Not deaerated — exposed to solar radiation for 60 min* Deaerated and exposed to solar radiation for 60 min* Deaerated and exposed to solar radiation for 60 mint, then treated so as to incor­ porate air and reexpose for 60 min* - No activated flavor ++ Distinct activated flavor +++ Pronounced activated flavor Activated flavor# at 46 hours 24 hours .. — — — - — — — - +++ +++ ++ -W-+ +++ +++ +++ +++ -)—I—h +++ 1 2 3 4 5 — — — — — “* 1 2 3 4 Z +++ -+•++ +++ +++ +++ +++ +*+*+ +++ +4-+ +++ 1 2 3 4 5 — -USTABLE 11 *^ie Effect of Surface Area of the Fat Globule as Influenced by Homogenization. on the Development of the Solar-activated Flavor Lot Treatment of milk (no*) 1 Control - not exposed 2 3 ■*KET: Trial (■noTT 1 2 3 4 5 Exposed to solar radiation for 60 min*- not homogenized (Normal surface area) 1 2 3 4 5 Exposed to solar radiation for 60 min* and then homogen­ ized (Increased surface area after esq)osure) 1 2 3 4 .JL- + -M+++ Wo activated flavor Slight activated flavor Distinct activated flavor Pronounced activated flavor Activated flavor* i 24 hours — — — + + + +++ +++ ++ +++ +++ -U6- co *H -P CD CO JU CD « -P tr\ CM I d •rl P a> P t> Vi ► CO *H *P CD CO P CD » lr\f2 -p «*! • * O CM l d * rH H P I cd • PO -P aJ H «aj P* Vi o rH a) o Eh > cd CO rH •rH CD U d « a> ,p a> cd X p ,P Vi O cd G O *H P cd rH CD P5 II I O* I I o- + I I I I I I 1 I I I I I I I+ I 1 t + I + 4-4-4- I H~ + 4-4-4- 4-4-4- + + + 4-4-4- + + + + + + + + + + + 4-4-4- + + H- + c-* 4- 4-4-4- + H- + + + + + 4-4-4- + + + CO •H +3 CD GO -P © *1 s G o d 1—1 CD CD N > ♦H CD C Q CD fe£ CD Q X e P !o ,ae O P c P i to u JH 0) d C\i I I c~ | cd f-t O (SH O A cd rH O CO 0-i j> H- to P CO CD P O -d l tn © p P 0 CQ *H -P CD CO U a> cd ITSW -p O d c^i 1 d *H CD I •H ♦H X> o -P P Vi O O o CO CO cd •9 •H CD t CD o o o « -P CO O •eaX 1 •ri -P O «3j d •w o I + ( c-« I I I+ I C-* + 4* + 4* I+ + H* 4-4- + + + + C-* + 4-4-4* 4-4-4- + + + + H- + 4- 4- + + + 4* + 4- 4- 0) •H P rH CD CO O G a> MW p I d c o *H CD CId o PO PCd rH P f-4 O .«; > Vl > o C^NSO o o cm o a) & o* ^G a« w H O O O O CM O rH rH cd ti Eh O rH G O Q O o r\v£> cm CM < r\ -1*7- I l i e- t I I I + I I e-* + I +1+ +11 I I I I I I § •H -P TABLE 12 (continued) g o O cd £ o •H 35 .H + + ++ I+ + I+ + + o~ + l^"+ ++ I++ i o» + I+ + + + + + + + + + + + C-» + + + + + + t k- * + + + + + + + + + + •8 ^ -8 05 .H © Sh © U W ^U CO I + + O 0>-8 6 cd Odd) + + + *> © > •H *H *rl +»+»+> 05 03 «H tkO p o + + + 53 & (U +1 + I O- + + + + ++ + + + + + + + + + I + + C-* + + ++ + + + I+ + P* O'* + + + + + + + ++ p* + + p> + + + p* + + + + It C-++ + + + + ++ + + + + + + + + + + + + c-» + + + + €^*++ +^-+ + + + + + + + + + + + + + + + + + + I HH C xf -H cd -P rH O O cd > cd t ( a) h ffl H •P rQ g g -g 3 -3 8 043 > cd 05 -H n CM rH « on UN -jf On xj|« » «H •PI'H UN UN O NO NO On CJN • CM • nO UN rH O • rH UN O • O CM to • o o UN NO ON rH F} C? rH «• CM rH •• CM P-. On 5 ctf -p ,a -POO on • fe d CM CM * on u \ 9 9 UN i—I ON rH CM CM CM O • ON CM CM a UN • CM O rH • UN O on e C- un iD to • • UN rH u\ 'mo|IP©I Q| ON -S t UN vO• UN 1— 1 o* UN On • ON 3 rH © O <13 -P CM CM ON * rH CM ■oo to o o to CM NO o 1 UN ♦ * o rH ** UN rH •• CM CM CM CM S ON —-t” CM i—I *• CM «ho||5© t o aJ o Vi CM UN ON • NO O o o I —I l> 7 CM O ON O rH • * On NO NO -4 " On to On * o ON ON tf O rH t*"\ O 'P o n o w o i—I r-i g rH • rH >8 fed i s p .Q H r \ o Oi rH on S O « 0 Q n o W O O 4 Q o to ^ CM un un Z4h5 -4"CM Un cx5 o^toso NCMOrHtOifNHCnc^^HUMA rH rH rH rH O O W>rH • •I on •H -4 O O O O p Q O O fraction ■P O . *f, O •O* t»• nD• ON « ON « *O ( . I CM u\ O n CM u n u n cm -4 CO O C in anhydrous minor-protein O|0h vO O -4* Q O O O O Q O Q O O ON H^HHHWC\?«OCKWCVxO ~4tv 4 b^CO O to IN t - N ry 4 CM NO H O r \0 r iV M A r l ^ c n 4 Q nH feO « S Oh -56- TABLE 16 The Number of Amino Acid Residues in the Minor-protein Fraction as Compared to Casein and Beta—lactoglobulin Constituent Yield % 2 1 Minor-protein fraction Moles per 10^ g. Molar ratio when protein Tryptophan = 5 4 3 BetaLacto— Casein-*globulin^ 6 7 Arginine 1*70 9.80 2.30 7 24 7 Cystine (l/2) 0.86 7.20 1.69 5 3 8 Glutamic acid 13.30 90.40 21.20 64 38 24 Histidine 0.89 5.70 1.34 4 20 4 Isoleucine 3.66 27.90 6.55 20 47 27 Leucine 5.71 43.60 10.20 31 70 50 Lysine 5.74 39.30 9.20 28 56 33 Methionine 1.34 9.00 2.10 6 19 9 Phenylalanine 2.35 14.20 3.30 10 30 9 Threonine 6.56 55.10 12.90 39 41 21 Tryptophan 0.87 4.26 1.00 3 6 4 Valine 9.74 83.30 19.60 59 61 21 Sulfur 1.40 43.60 10.30 31 - 21 70.300 12.800 42.020 (M min.) ^k^ordon (1949) Brand (1945) -57V. The Electrophoretic Analysis of a Contributing Minor-protein Fraction It has been reported previously (section IV) that a minor-protein fraction had been isolated from skimmilk* after the major proteins had been removed* which is capable of being photosensitized to produce the typical solar-activated flavor of homogenized milk* Furthermore* the elementary analysis and the percentage composition of the amino acids in the minor-protein fraction differed markedly from the other characterized whey proteins* Since the characterization and properties of this com­ pound indicated that it was not a previously recognized whey protein* it was thought desirable to make an electrophoretic examination of the mi­ nor-protein fraction* Various other investigators* Deutsch (1947)* Smith (1946* 1948) and Stanley et al. (1950)* have studied whey proteins electrophoretically* but their methods of preparing the whey proteins varied considerably and none was comparable to the method of preparation employed in this study* Freshly separated skimmilk obtained from the Michigan State College creamery was rennet-coagulated according to a procedure outlined pre­ viously under procedure. Figure 1* however* shows graphically the frac­ tionation procedure employed in the isolation of the various whey protein fractions which were examined electrophoretically. Electrophoretic analysis of the minor-protein fraction* One percent solutions of the minor-protein fraction (fig. 1, fraction III) were pre­ pared by dissolving a definite amount in four different buffer solutions and then dialyzing them to osmotic equilibrium at 5° C. for 24 hours* or until constant conductivity was reached on both sides of the membrane. -58The electrophoretic resolution of the minor-protein fraction in the var­ ious buffers was then studied at pH ranges from 3*3 to 9*3 with a PerkinsElmer Tiselius Electrophoresis apparatus at 1.2° C. The electrophoretic patterns of unit magnification of both the ascending and descending boun­ daries obtained from the electrophoretic examination of the minor-protein fraction are illustrated in figure 2 9 The horizontal arrows in figures 2 and 3 indicate the direction of migration* The diagrams on the left side of figures 2 and 3 represent ascending boundaries while those on the right represent descending boundaries. The tail of the arrow in both cases signifies the position of the starting boundary* Electrophoretic analysis of whey proteins and heat-coagulated-whey serum proteins. In an effort to follow the various steps in the isolation of the minor-protein fraction electrophoreticallyj a series of patterns were obtained from the whey proteins and heat-coagulated-whey serum pro­ teins* These two protein fractions were obtained in the course of the isolation of the minor-protein fraction and were removed and dried from the frozen state under high vacuum (fig. 1* fractions I and II). It should be mentioned that prior to lyophilization a precipitate was formed during the dialysis of the whey against water which was discarded. Samples of the whey proteins (fig. 1, fraction I) were made up in concentrations of 0*22, 0.2i* and 1.0 percent in three different buffer solutions and dialyzed to osmotic equilibrium at 5° C. for at least 2k hours• The serum protein fraction (fig. 1* fraction II) was made up in one percent concentration in veronal-citrate buffer and dialyzed as indicated above. The veronal-citrate buffer suggested by the work of Stanley et al. - 59- (1950) was used because poor electrophoretic resolution was evident from the use of acetate and phosphate buffers (fig. 3 ). The resulting patterns at unit magnification of both the ascending and descending boundaries obtained from the electrophoretic analysis of the whey and heat-coagulated-whey serum proteins are shown in figure 3« Any screening effect due to opalescence has been eliminated in the photographic reproductions of both figures. - 60- Fig.l FLOW SHEET DIAGRAM FOR FR AC TIO N A TIO N OF M ILK PROTEINS Skim M ilk 1 (Rennet Coagulated) f .... Whey ! Divided into 2 lots , A and B ) ■ Casein (Discarded) i Lot A li (Dialyzed 2 4 hrs.; and lyophylized) 1 F r a c tio n ! (Whey Proteins) Lot B 1 1 (Heated a t 9 5 ° C . 1 hr.; centrifuged c le a r ) * Supernatant 1 (Divided into 2 lots, C and D ) Lot C i (Dialyzed 2 4 hrs.; and lyophylized) 1 T F ractio nIL (Serum Proteins) Precipitate (Discarded) Lot D ( 3 4 . 5 g. ammonium sulfate added to 100 ml serum; centrifuged clear) \ Supernatant (Discarded) P recipitate I (Dialyzed ion F re e ; and lyophylized) Fraction m " Minor -P r o t e in - Fraction" -61 Fig.2 - ELECTROPHORETIC PATTERNS FROM |% SOLUTIONS OF MINOR -PROTEIN-FRACTION AT VARIOUS pH AND BUFFER MEDIA ASCENDING ,. _ pH 7 . 6 ; Veronal 1 VBuffer-, u = 0 .0 9 : Citrate J DESCENDING 4 5 0 0 sec.: 8.7 volts cm. pH 6 . 8 ; 0 . 0 5 M Phosphate Buffer + 0 . 0 5 M sec.; 5 . 6 N a C I; u = O .I; 5400 volts cm."1 pH 3 . 3 ; 0 . 0 5 M Acetate Buffer + 0 . 0 5 M N a C I; u = 0.l; 7 2 0 0 sec. 18.2 volts cm. '* pH 9 . 3 ; 0.1 M Ammonia B u ffer; u = O .I; 3 6 0 0 sec.; 7.8 volts c m . ' 1 - Fig.3 62- ELECTROPHORETIC PATTERNS OF WHEY AND HEAT COAGULATED WHEY SERUM PROTEINS. ASCENDING DESCENDING pH 7 6 ; Veronal - Citrate B u ffer; u = 0 . 0 9 ; 5 5 0 0 sec.; 9.1 volts c m . 1; Whey Protein Concentration 1 .0 % . ^ ---------------1 I ------------------ h pH 6 . 8 ; 0 . 0 5 M Phosphate Buffer + 0 .0 5 M N aC I; u = 0 .l; 1800 sec. 8.1 volts cm."1; Whey Protein Concentration 0 . 2 4 % . pH 3 .3 ; 0 . 0 5 M Acetate Buffer -I- 0 . 0 5 M Na C l; u = 0.1; 5 0 0 0 sec.; 5 . 8 volts c m .'1; Whey Protein Concentration 0 . 2 2 % . pH 7 .6 V e ro n a l-C itra te B u ffe r; u = 0 . 0 9 ; 4 5 0 0 sec.; 9 . 1 volts cm."1; Serum Protein Concentration I % . -63- DISCUSSION Many data are found in the literature relative to the factors re­ sponsible for and associated with the copper-induced oxidized flavor* Recorded data, however, relative to those factors associated with the so­ lar-activated flavor such as susceptibility of individual cow* s milk, antioxidant treatments, deaeration and surface area of the fat globules were not noted. The data presented in section I showed that of the ij.6 cows on pas­ ture feeding whose milk was studied 30 percent, were non-susceptible to the production of the solar-activated flavor after homogenization and ex­ posure to solar radiation* However, all the milk from cows not on pasture feeding showed a susceptibility to solar activation. This observation is similar to that reported by many workers on the copper-induced oxidized flavor. The variations as regards the solar-activated flavor development within individual cows both of the same breed and of different breeds cannot be fully explained. However, it may indicate that under certain conditions some of the mammary secretory areas may not function properly. The higher incidence of off-flavor development found when cows are on dry feed can be attributed to the lack of green feed in the diet. The use of antioxidants and other treatments (discussed in section jjJ seemed to indicate that the development of the solar-activated flavor was oxidative in nature. studies. This fact was substantiated by the deaeration The results presented in section XXX showed that the total sur­ face area of the fat globules appears to be an important factor in -6Uaccentuating the intensity of the activated flavor development. The Kreis test for oxidative rancidity did not confirm a lipid-fraction as the con­ stituent oxidized by solar radiation, giving rise to the activated flavor development. This reaction, however, did not definitely preclude the pos­ sibility that a lipid-fraction was involved in the oxidation. Since non­ homogenized milk does not develop the solar—activated flavor to the same intensity in a given time as does homogenized milk, and since the lipidfraction has not been shown definitely to be the constituent affected, the key to the development of the activated flavor may be found in the selec­ tive rearrangement of the fat globule membrane following homogenization. The constituent affected may be a protein constituent adsorbed on the fat globule. Since homogenization gives rise to increased protein adsorption on the fat globule, this possible selective protein adsorption on the in­ creased fat surface may explain the marked increase in the intensity of the solar-activated flavor in the milk that was homogenized following ex­ po sure • Isolation and Characterization of a Minor-protein Fraction A whey protein fraction has been isolated and characterized (section IV) which possesses the ability, after being photosensitized, of produc­ ing the characteristic solar-activated flavor commonly found in homogen­ ized milk. These findings are in accord with the observations of Doan and Myers (1936) and Keeney (19U7) that the solar-activated flavor origi­ nates in a serum component that remains after the removal of all of the major milk proteins. Keeney (19U7) was able to produce the solar—acti­ vated flavor in a casein—, lactalbumin— and globulin—free serum and it may have been construed by some to mean that the flavor originated from -65a protein-free serum. The isolation of a minor-protein fraction from the "protein-free11 serum stresses the possible importance of milk proteins other than the major protein fractions. Since biological systems are dy­ namic rather than static, the possibilities and relationships of other minor proteins and/or protein transformation products in milk and whey are of fundamental interest* The minor-protein fraction obtained from the "protein-free11 serum may be a normal constituent of milk, or it may have been due to an abnormal functioning of the mammary glands, or it may conceivably be accounted for by some degradation product of milk pro­ tein due to heat. Electrophoretic Analysis of the Minor-protein Fraction An inspection of the four patterns obtained for the minor-protein fraction shows that this fraction does not necessarily represent a homo­ geneous compound (fig. 2). The curves seem to indicate that at least two components or complexes are present in the so-called minor-protein frac­ tion. There was no pH range or buffer system used in this investigation that appeared to suggest otherwise. The best resolution and enantiography for this fraction occurred when the veronal-citrate buffer system was used at pH 7.6 rather than at any other pH or buffer system. This conclu­ sion is also evident from studies reported by Stanley et al. (1950) and Heutsch (19U7)• Preliminary calculations showed that when mobility values of the ma­ jor components were plotted as ordinates against the pH values of the buf­ fer system as abscissas an isoelectric zone of the minor-protein fraction was evident at pH 3«7 to U.lu This range is in good agreement with the -66isoelectric range of I4.I to I4.*3 previously obtained by minimum solubility-pH relationships (section IV). Inasmuch as this study was intended only to establish preliminary data on the electrophoretic character of the minor-protein fraction, it was not considered desirable to measure the individual mobilities and per­ centage composition for each component until further controlled work could be completed. The patterns obtained on both the whey and heat-coagulated-whey ser­ um proteins (fig. 3 ) in veronal-citrate buffer show conclusively that some of the components are lost after heat treatment of the whey. This would indicate that the heat labile components of whey are relatively well separated by this fractionation procedure* As was mentioned previously (section V), some precipitation occurred when the whey (fig. 1, fraction I) was dialyzed against water. The discarding of this precipitate may account for the small number of components in this whey fraction than would otherwise be anticipated. The degree of resolution and enantiography was not affected notice­ ably by the concentration of the whey proteins when the veronal-citrate buffer was used* A preliminary pattern (not shown) of this fraction at a concentration of 0 .2Lj. percent showed essentially the same resolution as that obtained when the concentration was one percent. A comparison of the electrophoretic pattern obtained at pH 7*6 in the veronal-citrate buffer of the serum protein (fraction II, fig. 3) with the pattern of the minor—protein fraction in the same buffer and at the same pH (fig. 2) shows that the two complexes are dissimilar. -67- SUMMARY AND CONCLUSIONS Approximately 30 percent of the milk from individual cows on pasture failed to develop a solar-activated flavor following pasteurization, homo­ genization and 30— and 60-minutes sun exposures. Homogenized milk from all of the cows on dry feed was susceptible to solar activation. There appears to be no correlation between breed, stage of lactation, and fat percentage and the susceptibility of milk to develop this off-flavor. Milk from some cows on summer pasture, which upon pasteurization, homoge­ nization and exposure to sun, yielded a nauseating flavor distinctly un­ like the true activated flavor. The addition of ascorbic acid had no preventive effect on the devel­ opment of the solar-activated flavor of homogenized milk. The addition of 25 mg./l of nordihydroguaiaretic acid alone, or 75 mg./l in combination with ascorbic acid, prevented the activated-flavor development in homogenized milk after 60-minutes exposure to solar radia­ tion* Alpha tocopherol and hydroquinone added separately or in combination did not offer complete protection to homogenized milk against the cffflavor development. Homogenized milk treated with hydrogen peroxide to destroy the nat­ urally occurring ascorbic acid rapidly, prior to pasteurization and homo­ genization, did not develop a typical solar-activated flavor when exposed to solar radiation. - 68- The development of the solar—activated flavor was not retarded or prevented by high-temperature (176° F. for.five minutes) heat treatment* The typical cooked odor noted in adequately heat treated homogenized milk was dissipated after a 60-minutes exposure to solar radiation* "When the cooked flavor was produced by the addition of cysteine hydrochloride and exposed to solar radiation for various periods of time, oxidation of the sulfhydryl group of the cysteine hydrochloride was indicated by a negative nitroprusside test. Deaeration studies on homogenized milk indicate that the development of the solar-activated flavor results from an oxidative process. Increasing the surface area of the fat globules of milk by homogeni­ zation subsequent to exposure accentuates the development of the solaractivated flavor and indicates that the surface area of the fat globules is a factor in the development of the off-flavor. A definite correlation between a positive organoleptic taste sensa­ tion of the solar-activated flavor in homogenized milk and a positive Kreis reaction was not obtained in all cases. The identity of the con­ stituent affected when the solar-activated flavor develops in homogenized milk was not established by the Kreis test. A minor-protein fraction has been isolated from skimmilk after the major proteins had been removed, which is capable of being photosensi­ tized to produce the typical solar-activated flavor of homogenized milk. The elementary analysis, amino acid composition, isoelectric zone and the average minimum molecular weight (M min.) have been determined. From the data, a (M min.) of 70,300 was calculated along lines of ortho­ dox organic chemistry* The determination of 12 amino acids shows that -69- "the minor—protein fraction contains the following amino acid residues: Argy, Cy S ^ Glu^, His^, Ileu20, L e u ^ I^s2g, Met^, Phe10, Thr^, Try^ and V a l ^ (the first three letters of each amino acid is used as the sym­ bol) . Electrophoretic analyses of the minor-protein fraction in various buffers and at various hydrogen-ion concentrations indicate that this frac­ tion is composed of at least two components or complexes. Best electrophoretic resolution and enantiographic patterns of all of the whey proteins were effected with veronal-citrate buffer at a pH of 7.6, Electrophoretic patterns obtained with whey and heat-coagulatedwhey serum proteins indicate that the heat labile whey components were effectively removed by the fractionation procedure employed in this work. -70- LITERATURE CITED (1) (2) Ansbacher, S., Flanigan, G.E., and Supplee, G.C. 193U. Certain foam producing substances of milk. Sci., 17:723-731. Aschaffenburg, R. 19H6. Surface activity and proteins of milk. 1^:316-329. Jour. Dairy Jour* Dairy Res., (3) Babcock, C*J. 19U2* Effect of homogenization on the curd tension, digestibil­ ity and keeping quality of milk. U.S. Dept. Agr., Tech. Bui. 832, 2i| pp. (!}.) Block, R.G., and Bolling, D. 19l|$. The amino acid composition of proteins and foods. 398 pp. plus xiv. Charles C* Thomas:Springfield, 111. (5) Brand, E., Saidel, L.J., Goldwater, V7.H«, Kassell, B., and Ryan, F.J. 19U5. 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