VARIATIONS IN THE COMPOSITION OF COWS' MILK AND THE EFFECT OF ACIDITY AND HEAT TREATMENT ON THE SALTS, HEAT STABILITY AND CURD TENSION OF MIXED MILK By JOHN THOMAS osaomm A THESIS Submitted to the School of Graduate Studies of Michigan State College of Agriculture and Applied Science in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Dairy 1953 4.. n- a-) ‘DJ' 1 31 ESE: ACKNOWLEDGMENTS The writer desires to express his sincerest appreciation to Dr. J. R. Brunner, Assistant Pro- fessor of Dairying, for his encouragement and con- structive criticism in the preparation of this thesis and for his guidance in the direction and scope of the material covered in this study. The author is also greatly appreciative to Dr; Earl Weaver, Professor of Dairying and Head of the Dairy Department, and to Michigan State College for providing the facilities and equipment used in this study. Grateful appreciation is also due to the College Creamery for furnishing the raw milk from different herds which was used in this study. 317521 TABLE OF CONTENTS II‘ITRODTJCTIOII o e o O o o o o e o o o e e e e IIEVIEUV OF LIEMTYJRE o o o o o e o o e 0 Natural Variations in the Composition and Droperties of Cowsrlu'ilk . . . . . . . . Influence of Breed . . . . . . . . . . Fat and total solids . . . . . . . Protein . . . . . . . . . . . . . . Minerals . . . . . . . . . . . . . Curd tension . . . . . . . . . . . Heat stability . . . . . . . . . . Influence of Seasons . . . . . . . Pat and total solids . . . . . . . Protein . . . . . . . . . . . . . . Minerals . . . . . . . . . . . . . Curd tension . . . . . . . . . . . Heat stability . . . . . . . . . . Natural Variations in the Composition and Properties of CowsTCMilk—Eesulting from Induced FadtOrs . . . . . . . . . . . . . Influence of Heat Treatment . . . . . Mineral distribution . . . . . . . Nitronen distribution . . . . . . . Heat stability . . . . . . . . . . Page ’73 10 IO A 329.334 . " iv Page C‘klrd tens ion 0 e o o e o e o o e e o O 1";- Influence of Developed Acidity . . . . . . 1k Mineral distribution . . . . . . . . . 1h Nitrogen distribution . . . . . . . . . 16 Heat stability . . . . . . . . . . . . 16 Curd tension . . . . . . . . . . . . . 17 EXPERIMENTAL PROCEDURE . . . . . . . . . . . . . 18 Preparation and Selection of Whole Milk Samples to Show Effect of SeasonaI Variations Due to Acidity and Heat Upon the Properties and Constituents of‘Nilk . . . . . . . . . . . I8 Preparation of the Milk Serum-- the Dialyzable PoFEIBn of_the Milk . . . . . . . . . . . . . 21 Chemical and Physical Methods Used to Estimate Changes in the Properties and Constituents of the Selected‘Milk Samples and Their ReapeEtIVe DEaIygabIe Portions . . . . . . . . . . . . . 22 Fat and total solids . . . . . . . . . . . 28 Alcohol number . . . . . . . . . . . . . . 22 Acidity (Titratable) . . . . . . . . . . . 22 Curd tension . . . . . . . . . . . . . . . 22 I):I . O O O O O O O O O O O O O O O O O O O 23 {{eat stability 0 O O O O O O O O O O O O O Lt..3 Citric 801:1 O 0 O C O O O O I O O O O O O O L‘.3 Total nitrogen . . . . . . . . . . . . . . an N'inerals o o o e o o o o o o e o o o o e e 32,. RESULTS 0 e o o o o o e e o e e e o e e e o o e e (j? '1, H5515 Variations in Some of the Constituents and Physical Properties of Cows' Milk in Relation to Seasons of the Year . . . . Titratable acidity and pH . . . . . . . Curd tension . . . . . . . . . . . . . . Heat stability . . . . . . . . . . . . . Citric acid . . . . . . . . . . . . . . Nitrogen . . . . . . . . . . . . . . . . iinerals . . . . . . . . . . . . . . . . Variations in Constituents and Some of the Properties of Whole Milk When Subjected to Various Periods of Heating . . . . . Fat and total solids . . . . . . . . . . Alcohol number . . . . . . . . . . . . . Titratable acidity and pH . . . . . . . Heat stability . . . . . . . . . . . . . Citric acid . . . . . . . . . . . . . . Total nitroren . . . . . . . . . . . . . Minerals . . . . . . . . . . . . . . . . The Effect of Heat Upon Some of the Con- stituents and Properties of Milk with Abnormal Acidity . . . . . . . . . . . . Fat and total solids . . . . . . . . . . Alcohol number . . . . . . . . . . . . . Titratable acidity . . . .'. . . . . . . pH . . . . . . . . . . . . . . . . . . . Curd tension . . . . . . . . . . . . . . Heat stability . . . . . . . . . . . . . vi Page Citric acid . . . . . . . . . . . . . . . 3P Nitrogen . . . . . . . . . . . . . . . . . 38 Minerals . . . . . . . . . . . . . . . . . 39 The Effect of Induced Acidity Upon Some of the Constituents and Properties of heated Milk . . . . . . . . . . . . . . . M1 Eat and total solids . . . . . . . . . . . hl Alcohol number . . . . . . . . . . . . . . Ml Titratable acidity . . . . . . . . . . . . Ml pfil . . . . . . . . . . . . . . . . . . . . ill Curd tension . . . . . . . . . . . . . . . k1 Heat stability . . . . . . . . . . . . . . he Citric acid . . . . . . . . . . . . . . . h? Nitrogen . . . . . . . . . . . . . . . . . N2 Minerals . . . . . . . . . . . . . . . . . H3 Effect of Changes in Titratable Acidity Upon Some of the Constituents and Properties of Raw Whole Nilk . . . . . . . NS Eat and total solids . . . . . . . . . . . NS Titratable acidity and pH , . . . . . . . LS Alcohol number and heat stability . . . . AG Curd tension . . . . . . . . . . . . . . . N5 Jitrogen . . . . . . . . . . . . . . . . . MS Citric acid . . . . . . . . . . . . . . . he Minerals . . . .'. . . . . . . . . . . . . N6 DISCUSSION . . . . . . . . . . . . . . . . . . . hfl was» a, vii Page Influence of Breed and the Seasons of the Year Upon Some Properties and Distribu- tion of Various Normal Constituents of P" i l 1": e e o o e o s e e e e e e o o o o e 2+ 9 Effect of Heat Upon Some of the Constituents and Properties of Cows' Milk . . . . . . SM Effect of Various Degrees of Acidity Upon Some of the Properties and Constituents of Cows' Nilk . . . . . . . . . . . . . . G9 Effect of Neat-Treatment Upon Some Proper— ties and Constituents of Milk With Various Degrees of Acidity . . . . . . . 62 Effect of Various Degrees of Acidity Upon Some of the Constituents and Proper- ties of Heated Nilk . . . . . . . . . . . 65 SUNJARY AND CONCLUSIONS . . . . . . . . . . . . 68 Influence of Breed and Tie Seasons of the Year Upon Some Properties and Distribu- tion of Various Constituents of Milk . . 69 Effect of Heat Upon Some of the Constitu- ents and Properties of Normal Cows' P'fiilk O o O O 0 O O O O O O O O O O O O 0 70 Effect of Various Degrees of Acidity Upon Some of the Properties and Constituents Of COWS! r'q‘ill‘c e o o o e o e e e e e o e o 71 Effect of Heat-Treatment Upon Some Proper- ties and Constituents of Milk With Various Degrees of Acidity . . . . . . . 72 Effect of Various Degrees of Acidity Upon Some of the Constituents and Properties of Heated Milk . . . . . . . . . . . . . 73 LITERATUIE CITED 0 0 O O O O O O 0 O O O O 0 O O 97 l. "AF—5‘3 was”. TABLE LIST OE TABLES Seasonal analyses of pooled herds milk Seasonal analyses of milk from a Guernsey herd . . . . . . . . . . Seasonal analyses of milk from a IIOlStein herd e o o o o e e o e 0 Effect of heat upon some of the constituents and properties of milk The effect of various heat-treatments upon some of the constituents and properties of milk with various degrees of acidity . . . . . . . Effect of induced acidity upon some of the constituents and properties of heated milk 0 O O 0 O O O O O O 0 Variations in some of the normal con- stituents and properties of whole milk as the acidity is increased as, Page 77 79 81 83 FIGURE 1. LISI‘CNP'EIOURES The fat content of milk as influenced by seasons of the year . . . . . . Total solids of milk as influenced by seasons of the year. . . . . . . . Variations in the total nitrogen content of milk with seasons of the year. 0 O O I O O O O O O O O 0 Comparison of the Ca, P and the P/Ca ratio in whole milk with seasons of the year. . . . . . . . . . . . Variations in colloidal phosphorous with seasons of the year . . . . . Variations in the total and serum nitrogen contents of milk heated at IABOF. for different periods of time. . . . . . . . . . . . . . The influence of heat on the Ca, P and the P/Ca ratio of milk . . . . Variations in the total and serum nitrogen contents in milk of vary- ing acidities. . . . . . . . . . . Comparison of Ca and P and the P/Ca ratio in milk of various acidities 91 92 93 9A \0 U1 INTRODUCTION For many years the instability of cows' milk toward heat has at times been of great concern to the evaporated milk industry. Some constituent or combination of con- I I. "I stituents normally present in cows' milk is believed to be reaponsible for the heat coagulation of milk. This problem was alleviated to some extent by the use of high temperature preheating treatments or by the addition of protein stabi- lizing salts. The first basic research pertainina to the coagu— lation of milk by heat was reported by Sommer and Hart (1926). Since then, various reports have been published on different phases of the heat coagulation problem. Most of the workers have associated'the instability of milk toward heat with the seasonal effect, the effect of acidity, and the effect of an improper cation-anion balance. The purpose of this study was to determine the changes which occur in the composition of milk due to seasonal and breed differences and the effect of these changes on the chemical and physical properties of the milk. The study was subdivided into three parts. The object of the first part of this study was to determine the variations in the composition of milk with respect .4 E915 l, i to the seasonal influence. The purpose of the second part of this study was to determine the effects of various heat treatments upon the constituents of cows' milk. The last part of the investigation was devoted to the changes which occur in compostion and properties of cows' milk due to the combined effects of heat and acidity. PO l. H595 REVIEW OF LITEWATURE Natural Variations in the Composition and Properties of Cows' Milk Influence of Breed Eat and total solids. There are differences in the fat content of milk from different breeds of cows. Ragsdale and Turner (1922) reported that a Cuernsey herd have milk with a consistently higher fat content than did a Holstein herd. This observation was further supported by the work of Jacobsen and Wallis (1939) who found that the milk of the smaller breeds had a higher fat content than milk from the larger breeds. Since the per cent of fat is encompassed in the per cent of total solids, any variation in the fat content of milk will show up as a variation in total solids. Jacobsen and Wallis (1939) showed that the milk of the smaller breeds had a higher percentage of total solids than did the milk of the larger breeds. Protein. Very little information has been reported on the differences in the total protein content of milk of individuals or breeds. A report published by Shahani and Sommer (1951a) showed a slightly higher average for total protein in Guernsey milk than that found in Holstein xi. ‘. kl milk. For mixed herds milk, the average total protein content was lower than that reported for the individual breeds. Rowland (1938), under English conditions, found that the protein content of a high fat, hinh solids milk was consistently higher than the protein content of a low fat, low solids milk. Minerals. The salts which are most nenerally believed to influence the properties of milk are calcium, magnesium, phosphorous and citric acid. Helm, Webb and Deysher (1932), from complete milk analyses, concluded that the smaller breeds of cows showed a consistently higher calcium content, a higher total phosphorous content and very little difference in mannesium content when compared to similar analyses of the larger herds. However. in a comparison of the citric acid content of high fat milk with that of low fat milk, the citric acid content was consistently higher in the low fat milk. Rice and Narkley (192k) reported data which agreed favorably with the observations of Holm et 1 (1932). Curd tension. Hill (1923), during his classical research on the curd tension of milk, observed that if a milk has a curd tension of less than 30 grams then it usually produces a curd of soft character. The average curd tension of Holstein milk was lower than that of Guernsey milk. He also pointed out that the fat content of the milk has no direct relationship with a hich or ’1 Am. 5 \‘l low curd tension, but that the salt content may be re- sponsible for the differences in curd tension. This is in partial agreement with research carried out by fieisbers, Johnson and NeCollum (1933) who found that the softer curd milks contained less salts than did the harder curd milks. Riddell, Caulfield and Whitnah (1936) showed that the curd tension of milk could be correlated with its protein content. Therefore, the different breeds could be ranked in curd tension in the same order as their total protein content. In this manner, the breeds would rank from the lowest to the highest in the following order: Holstein, Ayrshire, Guernsey, Jersey. Heat stability. The heat stability of milk may be defined as that length of time at which milk will resist coagulation at a specified temperature. The influence of the breed on the heat stability of milk is related to the composition of the milk from the indi- viduals. There are few Specific references to the effect of any certain breed upon heat stability. Sommer and Hart (1919) found that the stability of milk toward heat was influenced by the concentration of the salts which were present. Their experiments indicated that in heated-milk, coagulation will not occur when the calcium is removed, but that small amounts of calcium salts, when added to the milk, will cause coagu- .34: 3-1344 o lation. The calcium and magnesium ions were found to be balanced by phoSphates and citrates in almost gram-equiva- lent amounts. Optimum heat stability in the milk is achieved when the proper balance is present between the calcium and magnesium ions and phOSphate and citrate radicals. However, they concluded that coagulation would result if one of the salt combinations was higher than the other. The data presented by Helm gt a1 (1932) showed, generally, that the higher the calcium and phosphorous content the longer was the time required to coagulate milk at 1300 C. These workers found no definite corre- lation between the salt balance and heat stability. Webb and Holm (1932) reported that as the temper- ature of the milk was increased, the time required for coagulation decreased logarithmically. They determined that a temperature of 1200 C. was the most optimum temperature to use in evaluatina the heat stability of milk. Helm, Deysher and Evans (1923) eliminated the presence of a high fat content as being a factor in the coagulation of milk. They reported that the presence of the high fat content raised the coagulation temper- ature only slightly and deduced from this observation that the difference was probably due to heat absorbed by the fat. Influence of seasons Fat and total solids. The season of the year is known to influence the fat content of milk. In research carried out by Overman (19kg) with the Guernsey breed, the fat content decreased durinn the summer season but gradually increased during the fall and remained at a higher level during the winter months. Overman also reported that in the Holstein breed the fat content of the milk was high in late summer but remained at a lower level during the rest of the year. These results were in general agreement with data reported by Jacobsen and Wallis (1939) who demonstrated a downward trend in fat content for all breeds during the summer months and an increase during the fall and winter months. The work by Ragsdale and Turner (1922) further verified these trends in fat content. As stated earlier, since the fat is included in the total solids, any variances in the fat content will result in a corresponding apparent variation in the total solids. Overman (19h5) observed this relation— ship when he found greater differences in the total solids of Guernsey milk than in Holstein milk over a period of a year. He attributed this to the greater variations in the fat content of Guernsey milk as com- pared to the lower range of variations in fat content of Holstein milk. Erotein. Davis, Harland, Caster and Kellner (l9h7a) conducted experiments under Arizona conditions and observed that the protein content of Guernsey milk was consistently higher than the protein content of Holstein milk. Their results showed flucuations in protein content for Guernsey milk during the early summer months while the period of greatest variances for holstein milk occurred in the later summer months. The Holstein milk showed a leveled decrease period. From later experiments on mixed herds, Davis, Harland, Caster and Kellner (19h7b) found that the protein content of mixed herds milk remained at one level over a period of one year. In experiments upon the use of antiscorbutic- free fodder and pasture feeding of Holstein cows, less, Unger and Supplee (1920) found that the protein content of the milk produced on pasture was inherently higher, but that an increase was also noted in the fat and total solids of the milk. Minerals. In analyses of mixed herd milk, Jacobsen and Wallis (1939) reported that the mineral content generally showed slightly lower values during the summer months while winter milk showed a slightly higher mineral content. Sommer and Hart (1926) reported that there was no chance in the calcium or phosphorous content of milk from cows fed on dry winter roughage and oreen pasture grass. Lenstrup's (1926) data showed that the total phosphorous remained at a constant value during the year but that the values were slightly lower durinn the summer months. Investigations by Goldinp, Mackintosh and kattick (1932) showed lower mineral values for the summer months followed by an increase and eventual leveling off durinq the late fall and winter seasons. Supplee and Bellis (1921) reported only a slight increase in the citric acid content of milk when one mixed herd was chanced from winter feed to green pasturage. Holm _t al (1932) found a slight decrease in the citric acid content of milk produced in March over that in milk produced in February. Their data is inconclusive as to the effect of feed on the salt composition of milk since their cows were fed dry hay. hunziker (19H?) stated that, "green feeds increase the citric acid content of milk; dry winter feeds decrease it.‘' This statement was supported by Sommer and Kart (1926) who believed that there was a significant in- crease in the citric acid content of milk when a cow was changed from winter feed to creen pasture. Hess gt 31 (1920) observed that when a holstein cow was changed from vitamin C-free fodder to green pasture the citric acid content of the milk showed a very significant increase. \ H E5] 10 Curd tension. There are very few research reports related to the influence of seasonal variations on the curd tension of milk. Hill (1923) observed no significant differences in curd tension over a six months period. He believed that the more important salts were responsible for variations in curd tension I i and that a decrease in the insoluble calcium might tend to cause a softer curd or reduced curd tension in a milk. This statement is supported by Weisberg, Johnson and MCCollum (1933) who observed that a soft curd milk contained less calcium and phOSphorous than a hard curd milk. Heat stability. The investigations of Holm gt a; (1932) showed that the heat stability of milk in- creased and reached a maximum during March and April. According to their data, there is a decrease in citric acid content of milk and a slight decrease in calcium and phosphorous. Cole and Tarassuk (19h6) observed that when a high stability of milk to heat occurred that the best stability was encountered during March and April. In general, the same assuptions as stated earlier, under the heat stability as affected by breed, would also be true when applied to the influence of seasons on the heat stability of milk. ll Variations in the Composition and Properties of COws' Milk Resulting from Induced Factors Influence of Heat Treatment Mineral distribution. Verma and Sommer (1950) observed, from determinations of both total and soluble salts in heated milk, that there was a significant decrease in soluble calcium after pasteurization but that following cool aging for 2h hours the amount of soluble calcium was higher than that determined in the raw milk. In analyses of fresh skim milk heated to 2000, 2500, 3000 and 3250 F., Bernardoni and Tuckey (1950) determined that the contents of calcium and phosphorous in the ultra- filtrates of the heated milk samples decreased as the temperature increased. Sommer and Hart (1926) observed that there were larger amounts of soluble calcium in concentrated skimmilk which had been heated at 1800 F. for 5 minutes. This was accounted for, in part, to the precipitation of albumin. Harman and Slater (1950) observed that in all samples of heated milk, regardless of the temperature used, the rates of diffusion of calcium, phOSphorous and magnesium were significantly reduced and that citric acid appeared to be unaffected. Whittier and Benton (1926) observed from their nae-.515 12 experiments that as milk was heated the titratable acidity decreased momentarily and then rose, while the hydrogen-ion concentration increased consistently. In later experiments, Whittier and Benton (1927) concluded that the acid formed, upon the heating of milk, was in direct correlation with the time and temperature used as well as with the concentration of lactose. Gyorgy (1923) found that as the pH was lowered the amounts of calcium and phosphorous in solution were increased and that as the iso-electric point of casein was reached no " calcium remained in combination with casein. Nitrogen distribution. Few works have been reported on the effect of heat upon the nitrogen distri- bution in milk. Menefee, Overman and Tracy (19k1) showed no significant changes between the total nitrogen content of raw milk and the same milk pasteurized at lkSO P. for 30 minutes. However, when the milk was heated to 2030 F. there was an immediate decrease in total nitrogen which was deduced to be due to a partial hydrolysis of proteins. Shahani and Sommer (1951b) observed that there were no significant changes between the total nitrogen or serum nitrogen of raw milk and of milk pasteurized at 1550 F. for 30 minutes although there were changes in a few of the nitrogen supplying compounds. They also observed a similar pattern in the nitrogen distribution 13 of milk heated at 1’30 F. for 30 minutes. The nitrosen compounds most affected were the albumin and globulin. Heat stability. Most of the research on heat stability has been done on evaporated milk. Webb and Bell (19H3) found that high-temperature short-time heating of a concentrated milk caused an increase in heat stability. Further increases in temperature or time resulted in a decrease in the heat stability of concentrated milk. They also observed that when a raw milk had been forewarmed to 120° c. for o to 10 minutes the milk attained a heat stability conducive to the production of a good evaporated milk. The forewarming treatment increased the stability of the milk toward heat. Webb, Bell, Deysher and Holm (19h3) also found that milk forewarmed from 1100 C. to 1500 C. for one- half to 5 minutes exhibited better heat stabilization than did milk forewarmed to 950 C. for 10 minutes. Further, they observed that a forewarming temperature and holding time of 1200 C. for 3 to h minutes to be optimum. In heat-treated milk, Powell (1935) observed that when.a milk was heated to 850 C. the time required for :rennet coagulation was increased many times over that required to cause coagulation of the raw milk before heating. 1h Curd tension. ienefee et a1 (l9h1) showed that the curd tension of milk was lowered when it was pasteur- ized at 1h5O F. for 30 minutes. Mortenson, Rape and Cannon (1935) observed from curd tension experiments that in all samples of milk tested the curd tension of milk was reduced by all temperatures of heating but to different degrees. At pasteurizing conditions of 1’42O F. for 30 minutes the curd tension was reduced approximately one-third, the curd tension of boiled milk was reduced about three times, while the curd tension of milk heated in an autoclave at 2h2O F. for 15 minutes was so reduced that no curd tension value could be made. The work of Hill (1931) substantiates the work of Nortenson _£ 51 (1935) since he also reported a decrease in the curd tension of milk samples when they were heated to in- creasingly higher temperatures. Miller (1935), in his eXperiments on the curd tension of heated Holstein and Jersey milk, observed that heatinn caused a decrease in the curd tension of both milks and that the decreases were similar for both milks. Influence of DevelOped Acidity Mineral distribution. Sommer and Hart (1926) concluded that when milk coagulates due to a lack of calcium the developement of acid will reduce the excess phosphate by reverting some of the phOSphates to primary 15 phosphates. In turn, they stated that the develocement of acid would increase the amount of soluble calcium due to the acid action upon any di-calcium or tri-calcium phosphate present in the milk. Van Slyke (1928) stated that the formation of lactic acid on the heating of milk converts insoluble di-calcium phOSphate salts into a soluble form such as mono-calcium phosphate. The hinher the acidity is allowed to develope, the lower will be the minerals left in the curd. he also determined that insoluble di-calcium phOSphate was converted completely by lactic acid into soluble mono-calcium phOSphate in approximately 12 hours. However, the calcium in calcium caseinate combined with the lactic acid less repidly than did the calcium of di-calcium phosphate. In earlier work, Van Slyke and Bosworth (1916) determined that insoluble magnesium went into solution completely in 11.5 hours and that the citric acid was completely decomposed into acetic acid and carbon dioxide. MCCammon, Caulfield and Kramer (1933) observed that rennet-type cheddar cheese showed a higher calcium content than did the acid types. This was due to the formation of insoluble calcium paracaseinate which was formed before an increase in acidity took place. Khambatta and Dastur, (1950) working with milk from_Indian cows, found that as sourina progressed there 16 was a consistent decrease in total solids and that this decrease was more pronounced in raw milk than in boiled milk. They also determined that there was little chance in total calcium and phosphorous between the original milk and the soured milk. However, they noted that the calcium and phosphorous content of the dialyzable portion of the soured milk was significantly increased over the dialyzable portion of the original milk. These results are similar to those of Rice and Markley (192h) who found that as acidity decreased there was a general decrease in total solids as well as in phosphorous. Nitrogen distribution. Khambatta and Dastur (1951) reported that as the process of sourind occurred the total nitroqen content of milk did not chance, but that the nitroeen content of the dialyzable portion of the soured milk showed a marked increase during souring. Heat stability. Webb and Bell (19h2) found that excessive acidity was conducive to the coagulation of milk during high temperature and that normal, good quality milk could be treated with temperatures of 1500 C.-160O C. without inducing coaqulation. Mclnerney (1920) reported that as the acidity of milk increases the temper ture required for coagulation of the milk sample will decrease, and that this characteristic was due to a transformation of the calcium salts in milk. 17 Rogers, Deysher and Evans (1921) supported these reports when they found that a low pH in milk decreased the time required to coagulate a sample of milk at a given temperature. Qteuart (1920) has shown that a milk of three per cent acidity will curdle when boiled and if the same milk is allowed to attain 0.6 to 0.8 per cent acidity it will curdle spontaneously. He also demon— strated that milk, pasteurized at 1650 F. for 5 minutes and held 3 weeks, would curdle at 0.18 per cent acidity. Sommer and Hart (1926) concluded that the developement of acidity, in milk which coagulated in the heat test due to a low calcium content or citrate and phosphate excess, prevented the coagulation of the milk because of the liberation of soluble calcium and an accompanying reduction in secondary phosphate in the milk. Curd tension. Very little has been reported upon the curd tension of soured milk. However, a common observation in cheesemaking has been that as more acid is produced the curd will become harder. Weisbera _t _l (1933) supported this observation with their eXperiments on soft curd milk. They concluded that in addition to the possible effect of salts on the curd tension, the amount of fat present in the milk may also affect the curd tension by interrupting the growth of micellar threads in the protein. EXPERIMENTAL PROCEDURE The milk samples used in this study were obtained from two individual herds and the pooled herds milk as received by the collefie creamery. One of the individual milk sources was from a Holstein herd and the other source was from a Guernsey herd. The herd samples were obtained from the weiah tank as they were received at the college creamery. -The pooled milk samples were obtained from the raw-milk bulk-storaee tank. All samples were immediately cooled to h50 F. after collection. Preparation and Selection of Whole Milk Samples to Show Effect of_§easonal Variations and Variations Due to Acidity and Heat Upon the Properties and Constituents of Milk The samples of milk used for seasonal variations were obtained frequently throughout the various seasons (5 ol the year. They were collected as stated above. Analyses of the samples were started on the same day that the samples were taken. To study the effect of heat upon milk, raw pooled herds milk was divided into five one-quart portions. One portion of raw milk was kept as a control. The other four portions of milk were put into covered containers g H as 19 which were placed in a hot water bath and were heated to 191,3O F. and succeeding portions were removed after intervals of five minutes, ten minutes, thirty minutes and sixty minutes of holding. Each portion of milk was immediately cooled to at least h50 F. after its 1—-4..- \ u removal from the water bath. The analyses of the control and the heated portions of milk were started at once. To study the effect of heat upon milk of differ- ent acidities six one-quart portions of raw pooled herds whole milk were obtained by the means stated herein. One portion of the raw milk of normal acidity was analyzed as a control. Two other portions of the normal acidity milk were subjected to various decrees of heat treatment. The heating was accomplished as before, with a circulating hot water bath. The second portion was heated to lit5O F. and held at this temperature for thirty minutes and the third portion was heated to 1900 F. and held at this temperature for ten minutes. Analyses of these three portions were started immediately after cooling to k50 F. The remaininr three portions of milk were put into one container with three m1. of fresh buttermilk starter and the acidity was allowed to develops, at room temperature to an arbitrarily chosen acidity of three-tenths per cent acidity after reaching the desired acidity the milk was immediately cooled to 1v,v——- {has 1. 3’1" 20 0 k0 F. or below. The aciditY induced milk was aggin divided into three portions; the first portion analyzed as a control and the remaininm portions subjected to different decrees of heat treatment. The second portion was heated in the hot water bath to a temperature of lkso r. and held for thirty minutes and the third portion of acid-milk was heated to 1900 r. and held for ten minutes at this temperature. Analyses were started immediately after coolina. 1 I 1‘ ,‘.... Pooled herds milk was also used to study the effect of acidity upon milk which had been subjected to heat treatment. A six-quart portion of the normal milk was divided into three two-quart portions. The first of these portions was further divided into two one-quart portions and an analysis made upon the normal milk which served as a control. The second portion was allowed to develope acid, in the manner described earlier, to a titratable acidity of three-tenths per cent prior to being analyzed. The second two-quart portion was subjected to a temperature of 1&5O F. and held for thirty minutes. This portion was then divided into two one-quart portions and an analysis made of one of the normal heated portions and the other portion was analyzed after it had acquired an induced acidity of three-tenths per cent. The third two-quart portion was subjected to a temperature of 1900 F. for ten minutes and then carried through in the same 21 manner as before. A one-quart portion of the normal milk was analyzed as a control. The remaining milk was inoculated with three ml. of fresh buttermilk starter and the acidity was allowed to develope at room temper- ature. One-quart portions were taken from the acid— induced milk at arbitrary acidities of 0.22 per cent, 1 0.30 per cent and O.hO per cent. Analyses of the individual portions were started immediately after they had been cooled. Preparation of the Milk Serum-the Dialyzable Portion of the Nilk From the portions of milk used in this study, 35 ml. aliquots were pipetted into prepared 28 mm. cellophane dialyzing sacs. The dialyzins bags containing the samples of milk were suspended in erlenmeyer flasks which were evacuated by a water vacuum pump. The flasks were partially immersed in a water bath at 500 F. or below to retard the developement of acidity during the experimental period. The ultrafiltration of the milk samples was allowed to continue for not longer than twelve hours. Immediately after removing the dialyzing bass from the erlenmeyer flasks, the ultrafiltration- serum-portions were put into test tubes which were then stoppered and placed in the refrigerator. Analyses of the serum portions were carried out as soon as possible after their removal from the ultrafiltration setup. In 22 the dialyzinq of milk samples with abnormal induced acidity present, several drops of chloroform were added to prevent further bacterial action and acidity increases. Chemical and Physical Methods Used to Estimate Changes in the Properties and Constituents g£_the Selected Milk Samples and Their Respective Dialyzable Portions All samples of milk and their corresponding ultrafiltrates were examined in duplicate by each of the following tests: Fat and total solids. All fat and total solids determinations of the whole milk samples were made by the Mojonnier method (1925). Alcohol number. This test was run on all whole milk samples using five ml. whole milk and an equal amount of 70—75 per cent alcohol as sugnested by Hunziker (19h9). Acidity (titratable). The titratable acidities of all whole milk samples were determined by the acidity method adopted by Hunziker (19kg). Curd tension. Curd tensions of the whole milk samples were determined according to the recommendations of the American Dairy Science Association committee on methods of determininn curd tension (lghl). The apparatus used was the Submarine Signal Company's curd tensiometer calibrated to read curd tension directly in grams. 23 pg. The pH of the whole milk samples were determined by use of a model C, Beckman glass electrode pH meter. heat stability. Heat stability determinations were run on all whole milk samples. Glass tubes of 6 mm. inside diameter and approximately 100 mm. in length were made with one end sealed. A two ml. aliquot of whole milk was pipetted into the tube and the open end was sealed. The tubes were then inserted in specially designed holders mounted on a rotary shaft which was submerged in a constant temperature oil bath at 1200 C. The shaft was allowed to rotate at a slow but constant rate of speed until the first sinn of coaqulation was noticed. Observations were made at regular five minute intervals. Citric acid. A new method devised by Babad and Shtrikman (1951) for determining citric acid in milk was used in determining the citric acid contents of the whole milk samples and the correspondinr dialyzable portions of the milk. A calibration curve was prepared for the procedure as it was used in this study. This was necessary because a Cenco-Sheard Photelometer and a hSO mu filter was used whereas Babad and dhtrikman (1951) used a Photoelectric Lumitron Colorimeter with a uZO mu filter. Otherwise, the procedure was followed exactly as outlined. 2h Citric acid contents of the serum portions were determined by the same procedure except that one ml. aliquots of the serum samples were used directly in the determinations. Total nitronen. Total nitronen contents of the whole milk samples were determined by a semi-micro Kjeldahl method similar to one reported by henefee and ' Overman (late). A five ml. aliquot of the whole milk a sample was pipetted into a 100 ml. flask, diluted to volume and thorourhly mixed. A ten ml. aliquot, - representing 0.5 ml. of milk, was diaested in a 300 m1. Kjeldahl flask. Serum nitrowen was determined by the same procedure except that a two ml. aliquot of the serum was used directly in the nitroren determination. Minerals. Samples of whole milk were prepared for total calcium, total phosphorous and total mannesium assay by usino the ash from 25 crams of milk. The ash was dissolved in hydrochloric acid and evaporated to dryness, then dissolved in dilute hydrochloric acid and hot water, made up to a volume of 50 ml. and filtered. Ten ml. of the filtrate was pipetted into a 100 m1. flask and made to volume. This dilution represents 0.05 grams of milk. The total calcium content of whole milk was determined, using a ten ml. aliquot of the final ashinn- dilution, by a modified method of Kramer-Tisdall as .. on ‘II\ o I 25 suarested by ehohl (1922). Soluble calcium was determined in a two m1. aliquot of the serum accordinn to a modification of the Collip and Clark method as advanced by hawk, ser and Summerson (lQMQ). Total phosphorous of whole milk was determined by a modification of a method of Tiske and Suhbarow (1925) using a ten ml. aliquot of the ash filtrate made to a volume of 250 ml. and representinn 0.05 am. of milk. Soluble inorpanic phosphorous found in the serum portion was determined usinc one ml. serum diluted to 50 ml. and five m1. of this dilution in 25 ml. distilled water, giving a representation of 0.1 ml. of serum. The same procedure was used as for total phOSphorous. Colloidal phosphorous was determined as the difference between total and soluble inorganic phosphorous according to the definition given by Pyne and Ryan (1950). To determine the total inorganic phosphorous in milk, a ten m1. aliquot of milk was added to forty m1. of a ten per cent solution of trichloracetic acid and allowed to stand at room temperature for two hours in order to allow for complete precipitation of the protein. The solution was filtered and a five m1. aliquot of the filtrate made to 50 ml. and 5 m1. of this dilution was pipetted into a 25 m1. flask for the final dilution which represented 0.1 gram of milk. The modified procedure of Fiske and Subbarow (1925) for phosphorous estimation was then followed. “+53 26 The mavnesium content of whole milk was determined accordinc to a method advanced by Briggs (192M) usinn fifteen m1. of calcium-free solution from the calcium determination. The final aliquot represented 0.2 gram 1‘. Oi milk. Soluble magnesium in the serum was determined by the same method but using two m1. of calcium-free solution, from the soluble calcium determination, representing an aliquot of 0.5 m1. of serum. RESULTS Variations in home of the Constituents and Physical Properties of Cows' kilk in Relation to Seasons of the Year Fat and total solids. The data nraphically presented (Figure 1) shows a moderately level fat content in pooled herds milk over the entire seasonal ranme. A high in fat per cent occurred in April with a decrease to a lower level in the summer and a slight rise again in the fall. The total solids content of pooled herds milk ( Firure 2) followed approximately the same seasonal trend as the fat but with a smaller range of variations. The data (Tigure 1) also shows the seasonal variations of the fat content observed for milk from a Guernsey herd. For the Guernsey milk, a wider ranne of variations was observed. The high point for the fat per cent was noted to occur in the winter followed by a decrease in the sprine which showed some variations durina the summer but at a lower level than that for the winter. A slimht increase occurred in the fall. It was apparent from the data (Figure 2) that the total solids followed an identical trend of the fat content, the only difference beinn in the ranne of variations. The seasonal variations in the fat content of milk 28 from a Holstein herd are also shown (Figure 1). The fat content showed only slicht variations durinq the seasons until late summer when an increase occurred to show a higher level of fat which was maintained throuch the fall season. The total solids content of the Holstein herd's milk (Figure 2) was found to parallel the trend for the fat over the entire seasonal period. g The fat and total solids contents of the Guernsey herd's milk showed (Figures 1 and 2) consistently higher values and wider variations, than the other milks tested, ; for all the seasons. Titratable acidity and pH. From the data rathered (Tables 1,2 and 3) there appeared to be little evidence of a direct relationship between the titratable acidity and pH of the milks with seasons of the year. However, a marked degree of difference was noted in the titratable acidities of normal milk between breeds. The milk of the Guernsey herd showed consistently higher titratable acidities than either the pooled herds' or Holstein herd's milks. Curd tensiop. The experimental data tabulated in Tables 1,2 and 3 showed no indication that the curd tensions of the cows' milk tested were affected by chances in seasons. It was observed that the curd tension of milk from the Guernsey herd was higher than that of pooled herds or of the holstein herd's milk. 29 Heat stability. The heat stabilities of all three milks showed little relationship to the season of the year (Tables 1,2 and 3). However, all samples of milks were most stable to heat durins the late spring and early summer. Aih_--. - . Citric Acid. The data shows a wide ranee of values for the citric acid content of the milks studied. i The data for the milk from a Guernsey herd (Table 2) _..V_...— -o- shows a slight decrease durinn the late winter and a '__ very material increase in citric acid content in April which was followed by a decrease during the summer and a slight rise in the fall. The citric acid contents of the pooled herds and Holstein herd's milks (Tables 1 and 3) showed a gradual decrease during late winter and early Sprina which was followed by an irregular level until late summer when a slisht upward trend was noted. The citric acid contents of the serum portions of the three milks studied (Tables 1,2 and 3) were observed to follow the same general trend with a decrease to a lower irregular level through the spring and a gradual rise occurring in the summer and fall. filtrggen. The seasons of the year were found to exhibit identical general trends of total nitrogen contents in all the milks studied. The data (Figure 3) showed a slight decrease in total nitrogen during the late winter months followed by an irreqular level during O “7... a. (la a» an ...41 the spring and early summer. However, a steady rise in total nitroaen contents of the milks haven in the summer and continued into the fall. A marked difference was noted in the total nitronen contents of milk from the different breeds. The Guernsey herd's milk showed consistently ._ P“ higher total nitroaen values than the other two milks studied. A similarity was noted between the trends 5 observed for the total solids and the total nitrOéen contents of the milks studied for the seasons of the year. The results for the serum nitroden (Tables 1,2 and w 3) for the milks studied show little relationship with any particular season of the year. There were larde increases and decreases in serum nitroeen noted through— out the entire testing period. Minerals. Tigure h shows the effect of seasons upon the calcium and total phosphorous contents of the milk samples studied. The calcium contents for all milks tested were observed to parallel each other over the entire seasonal ranee. A slisht decrease in calcium content was noted in the late winter followed by a sharp rise in the Sprinq which reached a high in April. The calcium contents of the milks then decreased durine the late spring until a point was reached which approximated the results for the winter season. The trend then remained fairly level during the summer and fall seasons. The total phosphorous contents of the three milks in~ D' gun-rm 5‘” ii 31 vestifiated (Fidure H) followed a trend similar to the one observed for the calcium contents except that a more wradual decrease occurred in the summer and fall. The curves for the P/Ca ratios of the three milks (Figure A) show approximately the same trend as for the calcium and phOSphorous contents in the milks but the peaks of the ratios came in mid-summer followed by a eradual decrease in the late summer and fall. The magnesium contents of the milks studied (Tables 1,2 and 3) were found to be within the same ranee of results over the entire seasonal trend. however, a marked decrease was noticed for the mid-summer season and another low value was observed for Farch. There were few variations found in the calcium content of the serum of the milks studied. However, from the results observed for calcium in the serum of the pooled herds and Guernsey herd's milks (Tables 1 and 2), a slight increase was noted in the sprina followed by a uniform level in the summer with a slight decrease in the fall. The serum calcium of the Holstein herd's milk was noticed (Table 3) to remain at a fairly uniform level over the entire seasonal range except for a slinhtly higher value than the normal observed in the fall season. The serum phosphorous contents of the three milks (Table 1,2 and 3) were found to exhibit the same trend.but at differinn concentration levels. A 32 general increase in serum phosphorous was noted in the late winter with a hich peak reached in the summer and followed by a nradual decrease in late summer and fall. The serum maenesium contents from the three milks studied (Tables 1,2 and 3) were observed to be almost the reverse of the trend noted for maznesium in the whole milk. The lowest values of serum mannesium were found in the winter season milks and hihhest values were found in the summer season milks. The total inorcanic phosphorous contents of all three milks were observed to follow an identical trend (Tables 1 2 and 3). A low was observed in the late 3 winter followed by a very marked increase in the sprinO. A sharp decrease was noted in the summer followed by an increase which remained somewhat level through the fall season. The data presented for the calculated values for colloidal phOSphorous (Figure 5) shows a seasonal trend to occur with a high peak observed in the Spring followed by a nradual decrease during the later seasons of the year. 33 Variations in Constituents and Some of the Properties of Whole Milk Nhen Subjected to Various Periods f heating Fat and total solids. Chances in some of the constituents and properties of whole mixed milk as a result of heating the milk to 1:130 P. for varyinq lengths of time are recorded in table h. The percentages of fat and total solids were not changed as a result of heatine the milk. Alcohol number. The alcohol number of the milk was observed (Table h) to become less as the milk was subjected to longer periods of heating. Titratable acidity and pH. The titratable acidities of the heated milk (Table h) were observed to be only slightly hiaher than the oricinal titratable acidity of the unheated milk. In the case of the milk heated at iii}O F. for five minutes, the titratable acidity showed a slightly lower value than the criminal. The pH of the milk was observed to decrease gradually as the milk was heated for succeedingly lonaer periods of time. Heat stability. The heat stability determination (Table h) demonstrated that the times required to cause coagulation of the milk samples were almost directly proportional to the degree of heat treatment. Citric acid. Citric acid determinations on the 3k heated milk (Table h) showed sliehtly lower citric acid values than the oricinal citric acid contents of the unheated milk. The citric acid contents of the heated milk were Generally a little lower. The citric acid determination of the serum portion of the heated milk showed a nenerally lower level of citric acid than was found in the serum of the unheated milk. Total nitrocen. Total nitrocen and serum nitronen g, values were expressed nraphically (tioure 6) and showed that as the milk was heated the total nitronen increased, at first, then decreased as the heating period lengthened. Serum nitroeen values for the heated milk showed slichtly lower values than those observed for the oriainal unheated milk. Minerals. Ficure 7 shows the values for calcium in raw milk and heated milk. No chance in the calcium content occurred between the raw milk and the milk heated for five minutes at 1’43O F. Nilk heated for ten minutes at the same temperature showed a sharp decrease in calcium with little change observed in the calcium content of milk heated lender than ten minutes. Serum calcium results followed the same trend as that observed for the total calcium but with a smaller degree of change. A decrease in serum calcium was shown to occur in the milk which had been heated at lLL3O F. for five minutes, after which very little variation occurred in kg \fl the serum calcium of milk heated for longer times. ons (Figure 7) show w- Total phosphorous oeterminat a slicht decrease in milk heated for five minutes but no further chanee until the milk had been heated for thirty minutes when a sharp decrease was observed. A marked increase was noted in the milk heated for sixtv minutes. Serum phosphorous determinations shown in the same nraph followed almost the same pattern except that there was no apparent chance in serum phOSphorous prior to the decrease in serum phosphorous recorded for milk heated for thirty minutes. From the calculated P/Ca ratio of the milk studied (Figure 7) little change was noted prior to the period of heating for ten minutes when a rise occurred, followed by only slieht chances as the milk was heated for longer periods of time. The calculated P/Ga ratio for the serum increased markedley for the milk heated for five minutes followed by slight variations as the milk was subjected to longer periods of heatine. The data in Table h showed a steady increase in magnesium as the milk was heated for succeedinqlv longer intervals, except for the milk heated for ten minutes when a slight decrease resulted. The maenesium contents of the serums obtained from the raw and heated milks showed but small variations. The total inorganic phOSphorous (Table h) was 36 found to decrease in milk heated for five minutes when compared with the original value for the raw milk. The results for inoreanic phosphorous then increased gradually as the milk was subjected to longer heating periods. An increase in colloidal phosphorous (Table h) was shown in milk heated for five minutes, but the data then showed a gradual decrease as the period of heatine was lengthened. The Effect of heat Upon some of the Constituents and Properties of Milk with Abnormal Acidity Fat and total solids. All values and data for the determinations made in this section of the study are recorded in Table 5. There was no apparent change in the per cent fat and total solids between the normal and the acid-milks. Alcohol number. The alcohol number increased for normal milk which had bee heat-treated (Table 5) while the alcohol number decreased for the heated milk with developed acidity. The alcohol numbers of the acid-milk samples were much lower than those of the non—acid-milk samples. Titratable acidity. The titratable acidity of the normal milk was noted to decrease slightly as the milk was heated, there being no difference between the milk heated at tho F. for thirty minutes and that heated at 37 19C0 F. for ten minutes. However, the titratable acidity of the acid-milk showed an increase when heated with the highest acidity occurrinm in the acid—milk heated at thO '3‘ ‘- . for thirty minutes. pH. The pH of the normal milk decreased when heated, with the hither heat—treated milk showing a T larger p3 decrease. The oh of the acid-milk samples L showed lower values than the normal milk samples. How- 1 ever, tne pH of the heated acid—milk showed a slight increase over that of the control when heated at thO F. for thirty minutes while the acid-milk heated at 1900 r. for ten minutes showed a decrease over the original acid- milk value. Curd tension. The curd tension of normal heated milk showed a decrease and the milk heated at 1900 F. for ten minutes showed the lowest curd tension. The same trend was noted for the heated acid-milk, but the curd of the acid-milk heated at 1900 F. was gassy and flocculent in character. This curd was not cut by the curd knives but it showed no resistance to the pressure applied by the tensiometer. Heat stability. The heat stability of the normal milk showed an increase in the time required-for coagulation when that milk was heated. However, when an acidity was allowed to develope in the milk, that milk showed no stability toward heat. Heating of the acid—milk failed to 38 show any chanre in the heat stahility or that milk. Citric acid. Data in Table 5 shows an increase in citric acid content of normal milk when heated at 1900 F. for ten minutes. The citric acid content of the unheated acid-milk was slightly higher than that of the normal unheated milk. A decrease in citric acid was - -. _..-ea.¢r [Fl noted For the acid-milk heated at thO F. for thirty minutes while citric acid in the acid-milk heated at 1900 F. For ten minutes was the same as for the normal unheated milk. Nitrogen. Hhen normal milk was heated the total nitrogen increased, with the largest increase occurring in the milk heated to 1900 F. for ten minutes. The total nitrosen of the unheated acid-milk was found to be higher than that for the unheated normal milk. A decrease was noted in total nitrogen of the acid-milk heated at thO F. for thirty minutes but a marked increase was noted in the acid-milk heated at 1900 F. for ten minutes. The nitrogen contents of the sera from the normal heated milks showed an increase over that of the original milk. The serum of the normal milk heated at 1900 F. showed the greatest increase in nitrogen. The serum nitrogen of the unheated acid-milk was higher than that of the unheated normal milk. The sera of the heated acid- milks showed an increase over the unheated acid-milk. The increases in serum nitrogen of the heated acid-milk " "U were of less marnitude than those for the heated normal xaill{. Minerals. The calcium content of normal heated milk increased over that of the unheated milk. The calcium showed a higher increase in the milk heated to the higher temperature. The calcium contents of the i heated and unheated acid-milks showed little variation between each other and calcium value reported for the 3 original unheated norma- milk. The phosphorous content f the normal milk increased as the milk was subjected L to a hiaher temperature. The calculated r/Ca ratios for the acid-milk samples were dound to be closer to 1:1 than the ratios for the normal milk samples. The magnesium contents of the normal milk showed an increase when the milk was heated to 1900 F. For ten minutes but ‘ q L H. n marne um was noted in the milk \ ) H0 a slight decrease heatel at thO F. for thirty minutes. flannesium in the acid-milk showed an increase when the acid-milk was heated. The acid-milk heated at tho F. for thirty showed the largest increase. lnoreanic phosphorous of the normal heated milk increased over the value of the original milk irrespective of the degree of heat-treatment. The inorganic phosphorous of the acid-milk showed a succeedinrly higher increase as a higher heat-treatment was used. The inortanic phOSphorous values of the heated and unheated acid-milk were hicher than the values for the normal milk samples. The serum calcium 0? the normal milk showed a succeedingly larger decrease as the milk was heated to a higher temperature. The serum calcium of the acid-milk decreased as the acid-milk was subjected to heat and the greatest decrease occurred when the acid-milk was heated to 1900 F. for ten minutes. It was also noted that the serum calcium values of the heated and unheated acid—milks were higher than those recorded for the sera of the normal milk samples. The phosrhorous contents of the sera of the normal heated and unheated milks showed little channe, but the serum phOSphorous of the acid—milk showed higher values than the normal and these values were noted to decrease as the milk received a higher heat-treatment. The data showed sliqht decreases in serum mamnesium as the normal milk was heated with the largest decrease occurring in the serum of the milk heated to lHSO F. for thirty minutes. The marnesium of the sera from the heated acid-milk showed a decrease over the value for the unheated acid-milk. A greater decrease was noted in the a serum magnesium of the acid milk heated to lHSO r. for thirty minutes. 4 ___.._... H The Effect of Induced Acidity Upon Some of the Constituents and Properties of heated Nilk Fat and total solids. Analyses of the constituents and observations on the properties of the milk samples studied in this part of tne experiment are tabulated in Table 6. The Fat and total solids showed no change between the normal milk and the acid—milk. Alcohol number. The data showed an increase in alcohol number as the normal milk was heated. The acid— m milk showed no stability to the alcohol test but the acid-milk heated to 1900 F. for ten minutes showed a slight increase in alcohol number. Titratable acidity. As the normal milk was heated a decrease in acidity was noted with the larner decrease occurring in the milk heated to 1900 F. for ten minutes. The acidities recorded for the acid-milk were chosen as the end-point for the study of acidity upon heated milk. EE- From the data (Table 6) it was observed that the pH of the normal milk decreased as the milk was subjected to higher temperatures. The pH values of the acid-milks were lower than those of the normal milks, but there was no apparent trend related to the heat— treatment used. Curd tension. The curd tension of the normal milk showed a marked decrease only in the milk heated to 1900 F. for ten minutes. The data showed lower curd tension values For the acid—milk which had been heated than those recorded For the normal milk. However the curd Formed ‘ 9 _‘ in the acid-milk which had been heated to lQCJ b. was nassy and of a flocculent character. The curd was not It" cut by the curd knives and offerred no resistance to u- a m.’ the curd tensiometer. Eeat stability. The heat stability 0? the normal 4A— A; -. .__ Vbn- 4—“ milk showed an increase as the milk was subjected to heat, but the acid—milk showed no stability towards heat. «i Citric acid. The citric acid content of normal milk showed no chance between the unheated milk and the milk heated to thO F. but a lower value was observed for the milk heated to 1900 F. The citric acid values For the acid-milks were found to decrease when the milk had been heated to a hicher temperature. The citric acid values of the acid-milk samples were observed to be generally lower than those for the normal milk samples although the opposite was true For the milks heated at 1900 F. The citric acid contents in the sera of the acid- milk samples were found to be generally lower than in the normal sera. However, the sera From the milk, both normal and acid-milks, showed h aher values when the milk had been heated at 1900 F. Nitrogen. The data shows the total nitrocen of the normal milk to be higher as a higher heat-treatment R.- is used. The total nitrocen values For the acid—milk show a decrease as the milk was heated with a hiqher temperature and also lower values than those reported for the normal milk. The serum nitrocen of the normal milk was observed to decrease as higher heat-treatment was used while the serum nitrocen values 0? the heated acid—milks increased at equal rates. The serum nitrogen values of the acid-milk samples were markedley higher than those of the sera From the normal milk samples. Ninerals. The calcium contents of the normal milks showed no significant change as the milks under- went heat-treatment. The results for calcium in the acid-milk samples showed an increase only For the milk which had been heated at 1900 F. The phOSphorous values of the normal milk showed a slight increase as the milk was subjected to heat. The phOSphorous content of the acid-milk was higher than the original value and as a higher temperature was used on the milk the phosphorous value increased. The calculated P/Ca ratio was noted to be higher in the milks which were heated at tho F. Magnesium values in the normal milk decreased as higher temperatures were used. The macnesium contents of the acid-milk varied with no special trend noted for the heat-treated samples. The inorganic phosphorous results showed a slightly higher value in the normal milk heated at thO F. but no other variation. The inorganic m phosphorous of the acid—milk increased as a higher l . we temperature was used in heat-treatinc the milk. T. calculated colloidal phosphorous values for the normal milk showed slight increases as higher temperatures were used. The calculated colloidal phosphorous values for the acid-milk showed an increase when the milk had 0 . . been heated at 1H5 F. but a decrease when the milk had _ r O n been heated at 120 A. The calcium contents of the sera Trom the normal milks were observed to be lower as a higher heat-treating temperature was used. The data shows that the serum calcium of the acid-milk increased as the milk was heated at a higher temperature. These values for the serum calcium of the acid-milks were much hinher than those recorded for the normal milks. serum phosphorous decreased in the serum of the normal milk as the milk was subjected to higher heat—treatment while the phosphorous content of the serum from the acid—milk increased as the milk was heated at higher temperatures. The serum phosphorous contents of the acid-milks were higher than the reported normal values. The serum magnesium of the normal milk decreased as the milk was heated at higher temperatures but the serum macnesium of the acid-milk increased as higher temperatures were used in heat-treatment of the milk. The serum maenesium values of the heated acid-milk were higher than those a. 4' 30‘. h--¢ .o reported for the normal values. Effect of Changes in Titratable Acidity Upon Some of the Constituents and Properties of Raw whole Milk Fat and total solids. All data For this section of the study is recorded in Table 7. There were no changes in per cent fat or total solids as the milk increased in titratable acidity. Titratable acidity‘and pg. The milk was allowed to develope titratable acidity to the values recorded and samples of milk were analyzed at those acidities. The ph values of the milk decreased as the acidity increased. Alcohol number and heat stability. The values for the alcohol numher and heat stability of the milk were observed to decrease as the milk increased in titratable acidity. Curd tension. The curd tension showed a narrow range of variations until the Final stage of acidity when no curd resistance was encountered by the curd knives. The curd Formed in the milk of O.h2 per cent acidity was noticed to he of a gassy, flocculent character. flitroqen. Viaure 9 shows total nitrogen values for milk with induced acidity. It was apparent that the nitrogen values increased only slightly at the hé first stare of developed acidity, but, as the acidity was allowed to develope further, the nitrOfien values increased at a succeedinfily hichtr rate. The serum nitroeen decreased at a fairly level rate as the acidity of the milk increased. Citric acid. The citric acid content of the 1 milk showed no change until the second staqe of induced 5 acidity when a sharp decrease occurred followed by a e .- —I further decrease as the acidity increased in the milk. ' \r—1n \ fir, However, the citric acid values for the serum showed an aw increase as the milk developed a higher acidity. Kinerals. Figure 10 shows that the calcium of the milk increased almost proportionally with the degree of acidity developed in the milk. The phOSphorous content showed a decrease in milk of the first and second stages of induced acidity followed by an increase to the orieinal value in the final stare of acidity. The P/Ca ratio for whole milk decreased (Figure 10) as the acidity of the milk increased. The magnesium content of the milk (Table 7) showed a wide rance of variations as acidity developed in the milk. A slieht decrease occurred in magnesium present in milk of the first staae of induced acidity, but, an increase was observed in milk of the second induced acidity stage. The macnesium content showed a marked decrease in the final stage of acidity. Values for the inorganic phOSphorous (Table 7) showed no sidnificant chance. The calculated values for the colloidal phOSphorous show a sliaht decrease in the milk of the first and second staces of developed acidity followed by an increase in the final stare to a value slightly higher than the normal The calcium in the serum portion of the milk steadily increased as the milk developed hicher acidity. The phOSphorous content of the serum also increased as the acidity increased but at a areater rate than the calcium. The P/Ca ratio for the serum paralleled the P/Ca ratio of the whole milk but with a smaller decrease as the acidity of the milk developed. The serum mannesium increased as the milk increased in acidity while the hiqhest madnesium value occurred in the final stage of acid developement. . w.‘ V .- .... - “.13., ‘~. llloCllhlehI Milk becomes a very unstable product when various external influences are applied or when the normal balance among the constituents of milk is upset. By the same token, milk can he made more stable with the application of external influences and by restoring a more favorable balance amonc the constituents of the milk. These variations and adjustments have been shown to exist by such basic works as those by Sommer and hart (1926). Althourh the minerals of milk are in the lowest quantity, they seem to be the most important Group of constituents to affect the stability of a milk toward the application of external factors. The three most common external influences which seem to affect the constituents of milk are the seasons of the year which cause natural occurrine variations, the formation of acidity due to the presence of normal lactic acid fermentine bacteria and the application of heats used in processing milk for consumption. Other external factors such as health of the cow, the presence of bacteria normally not found in milk and tea presence of chemicals in the milk are not considered as normal external factors. It is penerally spread among dairy research workers ’0 that a certain balance should be present amonc the mineral elements, calcium, magnesium, phosphorous, and a his study was undertaken to acquire information about the distribution of some of the normal constituents ‘l? of milk when the milk was subjected to the three common «1 31—— external factors discussed, alone and in combination with - each other. 2 Influence of Breed and the Seasons of the Year 5. Upon Some Properties and Distribution of a Various Normal Constituents of Milk In this part of the study no attempt was made to control factors of lactation, type of feed other than to know when cows were feeding on pasture or winter rations or individual cows in a herd, but it was found from the producers that the two typed herds were put on pasture within the same week. The pooled herds milk was not checked but it was assumed from the general weather conditions that there would be little difference in the time that herds of the middle section of Michigan were put to pasture. h The trends observed in Pihure l for the fat and total solids of Guernsey milk were found to coincide closely with results reported by Overman (lQhS) and Jacobson and Wallis (1939) that lower values were observed during the summer months with an increase in the fall and hiah values in the winter months. fhe results in Figure 2 for Holstein milk are also similar to those reported by Overman (19H5) in that increases were observed in the late summer while a moderate level was maintained the rest of the year. Guernsey herds' .A-- ‘l milk is decidedly higher in fat and total solids when compared to values for Holstein milk. This was also in Mood agreement with work done by Racsdale and Turner (1732). 1h‘-.. L‘i.-u __~i,, #_._ — guéafi Since the total solids of a milk includes the total nitrogen content it was assumed that it should vary as the total solids varied. This was found to be the case and correlated closely with work reported by Rowland (1938) and Shahani and Sommer (1951) hat a milk with a high fat, high solids content showed consistently hiaher total nitroaen contents. Investi- gations of the total nitroaen content of cows' milk for seasons produced similar results to those found by Davis _t al (l9h7a) over a period of one year. The nitroaen contents generally showed lower values in the winter months with increases noted when the cows were put to pasture, which was also in wood agreement with works reported by Hess at al (1920). Cranfield, Griffiths and Ling (1927) reported that CaO decreases in summer and the P205 increases for the summer period with CaO:P205 ratio reaching a maximum in Aunust. 1is 13 in part contradictory to esults by hess e a. (1920), Ellenberaer, Newlander and Jones (1950), who observed that both the calcium and phOSphorous increased when cows were put on pasturaae. Results obtained in this study indicate an agreement with the work done by Hess and co-workers in that the calcium and phOSphorous increased markedly . in the spring season. These values were shown to be 3 lower durina the late summer which is in agreement with i | results of Golding t al (1932‘. Since the total solids 1 of milk from the smaller breeds are hirner it was expected that the mineral content of high solids milk would be higher. This is in accord with works reported by liolm 31; §_1_ (1932) and Graham and Kay (1933-31,). In the past, investigators have differed as to the effect of seasons upon the citric acid content of milk. One main reason for this has been the lack of a satisfactory method for determinin: citric acid which gives undisputable results. dommer and hart (1925) believed, with Hunziker (l9h9), that the citric acid content increases when a cow noes onto green pasture in the spring. It appears from the data obtained in this study that the citric acid content may increase ofi’ decrease in milk from cows on pasture. An increase occurred in the Guernsey milk sampled in the sprine but a decrease occurred in the Holstein milk. This "\ k 2 U“. fact is in direct disagreement, as concerned with breed differences, with results of Holm et a (1932) who found decreases in high fat milk and i creases in low :5 fat milk. The disarreement in results could possibly be traced to the weather conditions or type of -;—'.-v sur Jun-g. I“ flAa' r 1’. pasturage. The results in this study upon the curd tension with seasons of the year show a relationship between increases in total nitrogen and hicher values in curd f tension. This is supported by investiaations reported by Riddell gt al (1936). No definite relationship was observed between the salt content and the curd tension, but it was observed in some cases that a higher curd tension resulted when a combination of high total nitrogen and high calcium contents were present. The decree of citric acid present in the samples appeared to make no difference upon the results of curd tension. The stability of milk towards heat is an important factor in processinr of the milk. The heat test is one of the best means advanced to give an indication of heat stability. From the results of this study the reasons for a better heat stable milk are not very clear but it seems to require a balance amona the salts and the protein content of the milk. The best stability of the milk toward heat appeared to occur when the differences between the mineral contents and \."l to \ the nitrocen contents of the milk were at their widest limits. This seems to follow the reasoning of Sommer hart (1926) that the coa~ulation of albumin is HJ Hfli nastened when an increasinn amount of electrolyte is added. They have also shown a lower heat stability of milk to occur when the calcium and albumin contents of E milk are high. This may account for the occurrence of a lower trend in heat stability of the spring season E milk when the calcium content was highest alone with a g relatively high protein content. (An assumption must be aw: made that the albumin content of milk increases as the protein content increases as no determination was made in this study of the components of the total protein.) The idea of Sommer and Hart (1926) that an improvement , is inherent in the stability of the milk toward heat when the cows go on pasture in the spring, is in dis- anreement with results in this study as the best stability was noticed to occur in the summer and fall. Colloidal phosphorous appears to be affected by seasonal variations, as shown in results in Figure 5, with a peak noticeable in the spring and agrees favorably with data reported by Davies and Provan (1928) that colloidal phosphorous (total inorcanic) showed an increase as a result of grazing. This is in disagreement with results stated by Ling (1937) that the total inorganic phosphorous did not appear to be affected by cha ges in seasons. As a result of work in this study and the studies of other investieators a closer control of factors which (3 might influence variations 0. milk constituents and standardization of analytical procedures is an end to be ...-—-«. i. desired if results are to be compared between investigations. There seemed to be no relationship of the alcohol 3 number and the season of the year. One interestina obser- vation on all the milk studied was that the alcohol is number improved as the total inornanic phosphorous in- w; creased. This follows the report by Sommer and Binney (1923) that a slight increase in a phosphate salt will increase the alcohol test but an increase in a calcium salt will give a lower alcohol test. Effect of Heat Upon Some of the Constituents and Properties of Cows' Milk rnv w inere has been little work reported on the effect of pasteurizing temperature with various holding times upon changes in properties and constituents of milk. It was realized, after this part of the study was completed, that a forewarming temperature should have been run in conjunction with the pasteurizinn temperature and holding times used in order to estimate the effects of the two more common processing heats. This will be covered to some extent in later investiaations. Nest of the temper- SS atures reported in earlier studies by other investigators were for temperatures near the boiling point of milk and forewarminn temperature of milk in preparation for concentration. Davies (1936) relates that the application of - 0"“ heat to milk acts to cause a slinht decrease in titratable acidity by driving off carbon dioxide and { causing a partial decomposition of bicarbonates present, F but that further heatin: of the milk will cause a slieht f; increase in acidity due to the chaneinc buffer index as resulting from the precipitation of insoluble calcium salts. Results of titratable acidity as obtained in this study upon heating of milk follow the reasoninn of Davies. Investiaations by fihittier and Benton (1927) showed a similar trend in results of milk heated at 95° C. for varying time lengths but with greater increases. In later works Whittier and senton reported the increase in acidity to be directly related to the amount of lactose present in the milk. No corresponding data as to the effect of lactose concentration was determined in this study. Followinn the reasoninn established by fihittier and Benton it appears that the acidity of the milk investipated here should increase with a decrease in pH as milk is heated. Such was the case in this study when the pH was observed to follow a steady decrease as the milk was heated for longer periods of time. The variations in total nitroeen values reported for this study (Pieure 6) on the effect of heat upon the total nitroxen content of milk is in disagreement with other investinators. dhahani and Sommer (1951) and kenefee ,t a (l9h1) have all shown that there were no sisnificant changes in total nitroeen when milk was heated. Jince no attemnt was wade to investinate the S. himh values reported it is assumed that they are due to I r improper samplinn or unsatisfactory analytical technique. However, since it is common knowledce that proteins are denatured by heat, it is possible that the 1k heated for H- low total nitrosen value obtained in the m o o i O . ' Sixty minutes at 113 E. may have been due to the ' denaturation and subsequent coatine ef coaeulated proteins ntified (D on the heat he vessel. Davies (1936) has id the albumin fraction of milk protein as conforming to the characteristics of a water-soluble protein and globulin as a colloidal protein. That albumin and plobulin in milk are coagulated by heat in succeedinely larner amounts as higher temperatures are used was reported by Rowland (1933). This may account for the small decreases in dialyzable nitrogen observed in this study which were possibly due to the coat-3111a tion of water—soluble proteins present in the raw milk. However, this is in contradiction to results reported by Nattick and Kallet (1929) that no chanre occurred in the diffusible portion of milk heated for thirty minutes at a ranre of temperatures from hO-QCO C. The decrease in total calcium iS uneXplainable ercept that in the coanulation of some of the proteins, the calcium was used in the precipitation reaction 5 E caused by the heat and inaccurate sampline resulted. ; I The loss in calcium may be due to the formation of tri- % calcium phosphate snown by Soldner (1989) to occur in i i r '_ the coanulum “hich forms on the surface of the milk when heated. fhe soluble calcium was found to decrease slichtly when the milk was heated at ik3° F. for five minutes and remain level durinc further heating. This is in good agreement with works reported by Verma and Sommer (1950) and Lampitt and Eustill (193%) that soluble salts are precipitated with heat in the presence of water—soluble proteins. Sommer and Mart (1919) have explained the heat stability of milk upon the basis of a balance amonn the major salts found in milk. From the praph in Figure 7 showine the P/Ca ratios for whole milk and the serum and comparing the increase in heat stability results (Table h) and increase in the ratios as the milk was heated it is apparent that the ratio of phOSphorous increased over the calcium at the same point that the heat stability increased. Followina the reasoning of 58 Sommer and Hart it may be suppositioned that the chance in the salt ratio has sore ezfect unon the improvement in the heat stability of the milk. As stated earlier if some of the soluble calcium salts were precinitated to an insoluble form in the surface coagulum formed, the ratio of soluble salts would become higher for the phosphorous over the calcium, as the phosnhorous shows little variation except for increases in inornanic phOSphorous. } t . It has been cenerallv acreed among investigators LWJ that the Firmness of the curd formed between rennin and paracaseinate is subject to the presence of calcium salts in one form or another. The evidence available from this study points to the Fact of a lower curd tension occurring when a lower calcium content was noticed. This follows the reasoninr of Van Slyke and Bosworth (1916) that the firmness of a rennin dormed curd of paracaseinate is due to the amount of calcium salts present, the more calcium present the firmer the curd. Jhen normal milk was heated for longer periods of time the alcohol number was observed to increase, but the heat stability of the heated milk increased along with an increase in acidity and a decrease in soluble calcium. These results are in agreement with reports of Sommer and Binney (1923) that the effect of salts and acidity are additive and that increases in heat will cause a relative increase in stability due to the precipitation of soluble calcium. They did not, however, account for the relative increase and effect of acidity due to the heat. Effect of Various Degrees of Acidity Upon some ot the Properties and Constituents of Cows' Nilk ‘»“\I If ‘7'? _.-‘ . An unusual increase in total nitrocen, when 1'- acidity is allowed to develope in milk, was noted which -w was unac,ountable. Other investicators have found no chance in the total nitrocen of fresh and acid milks. This was shown in data reported by Khambatta and Dastur (1951). however, they reported an increase in the ammonia nitroaen content of soured milk. Roy and Bhatnasar (19h8) believed that increases in the total nitrocen were due to microbial actisn. These reports may aid in accounting for the unusual increase in total nitronen found in the acidified milk in this study. The decreases in the dialyzable nitroaen contents as reported in these experiments disaoree with the reports of Khambatta and Dastur (1961) that dialyzable nitrogen increases in sourinc milk. 3 The increases in calcium with increasing acidity are not in agreement with results reported by Khambatta and Dastur (1950) althounh results on the total phOSphorous 60 were found to be similar in that no larne variations occurred with channes in acidity. The results for the serum calcium and phosphorous agree with works of Van Slyke (1928) in that these dialyzable minerals increase as the acidity is allowed to increase. Van slyke and Bosworth (1916) also report results similar to those i found in this study. Dialyzable maenesium was observed to increase with increases in acidity. The decrease in the citric acid content of sourinq milk occurred due to the fermentation of citric acid by various organisms. _“4 The increase in dialyzable citric acid is unaccountable and in disagreement with a general opinion of other workers that a decrease should occur due to fermentation. More data is needed on this constituent with emphasis upon the formation of a suitable analytical determination procedure. The results of this experiment appear to lend some support to the ”salt balance" theory of Sommer and Hart (1926) that a change in the balance will cause a decrease in the stability of milk. When the heat stability results are compared with changes in the P/Ca ratio shown in Figure 9, the heat stability decreased very markedly with a decrease in the ratio of total phOSphorous to the total calcium. Of course, it must also be realized that the presence of the casein and minor proteins should also be included in 61 explaining the heat stability of an acid—milk. Such a relationship has been demonstrated by Van Slyke and Bosworth (1916) when they found the maximum solubility of calcium to occur when casein was at its isoelectric point. dommer and Hart (1926) expressed the same reasoning when they observed that an optimum calcium content will furnish optimum stability to the casein. -.__...-._. -<-s_-..a.e:n ' Hammarsten (1896) and Palmer and Richardson (1925) observed from experiments on rennin coaqulation n.‘ k that a flaky precipitate, such as was observed in the a rennet coagulated curd of milk at O.h2 per cent acidity in this study, is found when only calcium paracaseinate is present and no calcium phosphate; but a clot will form when calcium phosphate is present. It is possible to assume that the flocculent curd observed in this study was due largely to the absence of calcium phOSphate which is thought to be soluble in the presence of in- creased acidity. The factors responsible for varying curds were too varied and incomplete to express any definite conclusions at this point. i In this portion of the study the alcohol number of the normal milk was found to decrease as the acidity was allowed to develope. Along with the decrease in alcohol number with decreasing acidity an increase in soluble salts was noted. The lowered alcohol numbers may then be explained by reports of Sommer and Binney ()2 (1923) that addition of soluble salts will decrease the alcohol test. Effect of Heat-Treatment Upon Some Properties and Constituents of Milk flith Various Degrees of Acidity The effect of heat and acidity upon milk was i studied and discussed earlier. In this part of the study g the effect of heat upon milk of hich acidity was investi- 5 gated to determine changes which mipht occur under processing conditions. The increases obtained for the nitrosen values of the heated milk are reparded with suspicion. According to other investigators, Verma and Sommer (1950) and Nattick and Hallet (1929), no increases should occur when normal milk is heated. The increases in nitrogen values for acid-milk may be considered valid following the reasoning of Roy and Bhatnaqar (19MB) that nitrogen increases may be expected as a result of increased bacterial action. The heat-treatments serve to increase certain bacterial action. This probably takes place as the temper- ature of the milk is beinq increased to the desired processing temperature and during the period of acid developement in the milk preliminary to the heat-treatments. Another reason for the increases may have been due to poor sampling technique of the flocculent proteins which formed due to the acid-heat denaturation. Nelson (1953) 63 has observed that the serum nitroden found in whey will decrease as heat is applied. he also stated that the denaturation may play an important part in the heat stability because of a serious loss of charae resulting in a larger particle size and aidinr in causinc an in- stability between thc calcium and phosphorous salts. In this study the dialyzable nitrogen was found to increase in the milk as hirher heat-treatments were used. Then acid developement was allowed to take place the dialyzable ’ ._.W ”a. lum‘“, nitrogen increased but when the milk was subjected to heat the dialyzable nitrogen was lower than those recorded for the normal heated milks. These results are in fair agreement with results reported by Khambatta and Dastur (1951). From results discussed e rlier upon the effect of heat on milk, a decrease in soluble calcium was expected with no change in the other soluble salts. Also, from an earlier discussion of effects of pH (acidity) upon milk, a marked increase in soluble salts could be expected to be found in a milk with a lowered pH. These trends occurred in the heat—treated acid-milk. However, the increases of soluble salts were found to be checked by the application Of heat to the acid-milk. Sommer and Hart (1926) reasoned that acid developement aided the heat stability by the fact that the soluble calcium increased from the insoluble di- and tri—calcium phosphate of milk. However, it appears that since the increase in soluble (prinary; pacephates was of such a marked rise compared to the increase of soluble calcium, the P/Ca ratio increased at .4 CD ’3 ate as to cause a lowered heat stability test 0 due to a lack of secondary phOSphates. This also adheres r: y / to the reasoninfi of oommer and hart (1913) that acid . _—T_“-. I s developement in milk will cause a decrease in secondary phosphates and increase in primary phosphates which have no effect on the salt balance. But in this study, since ! the calcium and phOSphorous did not increase at the same 4- rate the difference between the two salts was too great to be in good balance. Since few chanaes in citric acid contents were observed, it would appear that there were few citric acid fermenting bacteria present or active at the time of samplinn. Probably, the apolication of heat stopped the action of these bacteria. The curd tensions of the normal and acid-milk appear to show the same effect whether high acidity was present or not. However, the lower curd tension values observed for the heated acid—milk may be attributed to a lower insoluble salts content in combination with the coagulum formed by the rennet used in the determination. The alcohol number decreased in milk with developed acidity and also with an increase in soluble salts. An increase in alcohol number was also noted as the heat 6g stability increased in the heated milk. These results follow the reasoninr of dommer and dinney (1923) that acidity increases will brine about increased amounts of soluble salts and it is the relation of soluble salts which are responsible for the reaction in an alcohol test. Effect of Various Degrees of Acidity Upon dome of the Constituents and Proper ies of Heated Milk This section of the study was concerned with the effect of heat and of chanses which occur when acidity is allowed to develope in the heated milk. The increase in total nitroaen of the heated milk is unaccountable but the decrease in serum or dialyzable nitrogen miaht be attributed to the denaturation and coagulation of soluble proteins in the serum. This is in accord with reports by Rowland (1933-3h) that water soluble protein of milk are coagulated by heat. he decrease in total nitroaen found to occur in the acid—milk as temperatures of processing were increased are in agreement with observations of Roy and Bhatnagar (lth) who stated that the decrease could be due to the breakdown of the colloidal protein by proteolytic bacteria. This may also aid in explaining the marked decrease in total nitroaen along with the fact that inaccurate sampling would result due to coagulated proteins. The results for total amounts of the salts show 66 sliaht increases which mifht be attributed to a concentration of the milk durine heatinc although the volumes of the heated milk and the unheated milk were not compared to determine the amount of evaporation which had taken place. The state'ent above would also hold for the UV; A\4 increases noted for the results observed. The dialyzable ‘2‘-" :Kmusx i. " alcium and phosphorous both showed marked increases in (I) V heated milk where a lowerinn of pH occurred. This was 'a—nfimn :~ I > in good agreement with works reported by Lampitt and Bushill (193k). The citric acid contents show little variation with heat-treatment of the milk samples, which follows an observation reported by Arup (1939) who stated that there were negligible chanres in citrates when milk was heated ”1‘ .L in an autoclave at succeedinaly higher temperatures. he decreases in citric acid values in the acid-milk are similar in trend to decreases shown to occur in experiments 13y Van Slyke and Bosworth (1910). The decreases are possibly due to the utilization o? the citrates by the bacterial flora. The presence of a lower pH in the acid-milk appears to Ex: the deciding factor in the lower curd tension of the rmilk because an increase in acidity lowered the curd t0n8i1n1 to a value less than that observed in normal heatxxilnilk. This is probably due to the changing of 67 insoluble salts to a soluble form from the denatured proteins. That the soluble salts are precipitated by heat has been shown in reports of Sommer and Hart (1926) while insoluble calcium and phosphorous salts are shown to be solubilized by the work reported by Lampitt and Bushill. In ceneral, the alcohol number of the milk samples in this portion of the study follow the same trends discussed in earlier parts 0P this investigation. The alcohol test may be as effective tool in forecasting the heat stability of a milk as it appears that the test shows whether an abnormal reaction has occurred in he salt and acidity present in the milk. I n.“ mau‘w “‘mt . I 1 I SUMMARY AND COWCLUSIONS Studies were made upon the chanses which occur in some of the normal constituents and properties of Guernsey holstein and mixed herds milk durina seasons of the year. Experiments were also conducted upon normal mixed herds milk to determine the effects of heat-treatment and develOped acidity upon some of the cons ituents and properties of cows' milk. Influence of Wrsed and the Seasons of the Year Upon Some Properties and Distribution of Variou. Constituents of Milk J) The seasons of the year were found to show definite trends for some of the milk constituents. The trends were similar in almost all cases for the milks investi- gated. It was found that the fat and total solids were nenerally lower in the summer season and hicher durinc the winter months. There was a tendency for the total and dialyzable nitrocen values to increase steadily throuah the sprins and summer seasons after tine cows were put on pasture. The nitrogen values also varied as the total solids contents varied. The citric acixi showed no steady trends but the values were generally hiqher for milk produced in the summer months. jMinerals of the milks were found to increase markedly in .M“:“‘ 'A “-3’3} .v.“ i f)? 0 J— l tne sprine with lower values “ound in tne winter months. The heat stahility and curd tension were pound to he indirectly related to seasons of the year by an associ- ation with the seasonal chanses in the calcium and total nitrceen present in the milk. iioh curd tension values were Found in the milk when hirh calcium and total ; nitroeen contents were present in the milk due to in- 1 at stahilitv of (D creases in the snrina season. The h the milks showed an improvement in the summer when the . -Cp ‘2“. it. calcium and the total nitroeen contents were Found to uww be at the'r widest difference. The alcohol number was found to have no definite relationship to seasonal change but when the total inorcanic phosphorous in— creases were noted, there was a drcided improvement in the alcohol test. In comparinr analyses of the Guernsey and holstein milks, the Guernsey milk showed consistently hicher averane concentrations of fat, total solids, total nitrogen, citric acid, calcium, but lower average concentrations of phosphorous and macnesium salts. The curd tensions of the Guernsey milk were decidedly ’hiqher than those of the Holstein milk. ) \J 1" \v of‘ fleet Upon. some of the tuents and Properties 0? Kormal Cows' Milk Heatins of milk causes an increase in acidity and a decrease in pH. The variations in total nitromen values were discussed. The total nitroren content of heated milk showed an increase during the initial staee of heating with cecreases as the milk was heated for lonoer periods of time at 1&30 F. to marked variations -mu-mm- -u.gvfl.£-‘- f. ”J were observed or the dialyzable nitrogen. Total , . calcium and phosphorous decreased slinhtly as heat was applied, but the masnesium content increased. The dialyzable salts in heated milk showed little variation over the normal values, except for the dialyzable calcium which decreased sliehtly durine the initial stare of heating. As the milk was subjected to loncer ods of heatine the heat stability of the milk was 5—1. '31" r(‘ roved. The curd tensions of the heated milk were H- I. Al: "(3 found to he lower when lower levels of dialyzable calcium.were found. The stability of the milk toward the alcohol test increased as the milk was suhLected to loneer periods of heatinn. 71 Effect of Various Denrees of Acidity Upon dome of the Properties and Constituents of Cows' Milk ne titrat- H. A decreasing pH accompanied an increas able acidity. The total nitroren content "ncreased at a steady rate as the acidity increased while the dialyzable . Th, total calcium increased slightly nitrogen decrea ‘BC L I with increases in acidity but the other salts showed little variation from the oricinal values. The dialyzable salts increased with increasing acidity with soluble phosphorous showing the areatest increases. Citrates are known to be utilized by certain bacteria, therefore the resulting decreases in citric acid of whole milk were eXpected. But the increases in dialyzable citric acid cannot be accounted for. The stability of the milk to- ward heat may be indirectly related to the acidity present in the milk but directly related to the dis- turbing of the equilibrium among the salts and proteins of milk. The curd tension varied little in the early stares of developed acidity but at the highest acidity the character of the curd appeared to be completely changed thus showing to some extent the range of equi- librium which will tolerate the destabilizing effect of acidity. .._.__.____ .. W_._.__._. Effcct of Heat—Treatment Upon Home Properties and Constituents of Uilk Uith Various Degrees of acidity In studying the effect of processing heat- treatments upon acid-milk it was found that the total nitroren values increased as the normal and acid-milks i 'fl-‘ ' were heated. The total nitrojen values of acid-milk showed a smaller increase with heat-treatmeit. ihe f . J dialyzable nitronen also increased as hinher procesSinc i t , l temperatures were used. The dialyzable nitroeen of ? 5 “a ..--..1 the acid-milk showed only slivhtly higher values after heat—treatment. Sliaht increases in calcium and phosphorous were hotel in the heated normal milk but little chanmc occurred in these salts when the acid—milk was he'ted althoueh the level of salt contents were ( hirher than for the normal heated milk. The concentration of dialyzable salts increased markedly with acidity hut decreased sliéltly when the milk was heat-treated. Evidently the acidity had not increased to a point where the citrates were affected or there was a destruction of citric acid fermenting bacteria present in the acid- milk at the processine temperatures. Slirhtly lower etuwi tensions were observed in the acid-milk than in the 'normal milk, but both milks showed decreasing curd tunnsions with higher processinr temperatures. The lower *malues mish be attributed to the decrease in the in- «J \A.) solutle salts in the acid-milk. OF Acidltv Upon Effect of Various ees latituents and Lea Some of the Properties oi It was found that when heated milk is allowed to develope acidity the total nitrrrsn content decreased as a hirher orocessin: temperature 'a) used out the ,‘j 3 '~ . ‘— rfi . -‘) t y ‘ ‘ ' 2 ‘ ' " dialyzable nitro-en increased in tflQ acid-milk when ' rowan was nicher in heated DJ CD 1‘.) CT O K‘. )‘J O *3 D—vJ r (3 :1; H0 C) H ‘1 N (‘3 (I. ',_.I «D :3 '_. K .- acid-milk than in normal tented milk. The calcium, phosphorous and mannesium varied little upon heat- trcatment of the milk but the total salts of the acid— milk were hisher than those of the normal milk. The were hidher U) dialyzable calcium and phosphorors value in the acid-milk than in the normal milks; increasina proportionallv with the heat treatment. However, the dialvzahle calcium and phosphorous of the normal milk decreased with hiaher temperatures of heat-treatments. The citric acid contents were found to vary little with reference to the processing temperatures but a marked decrease was found to occur in the milk when the acidity was allowed to develope. The develOpement of acidity in milk appears to lower the curd tension of milk. From all indications, the most important factors found to cause detrimental effects in milk in relation to heat stability are the salts and proteins present in 7br milk and the developement of an abnormal acidity. The salt and protein contents are definitely related to season: of the year and mav be altered to form an un- balanced combination of constituents in the milk. 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MAY JUNE JULY AUG. SEPT. Figure l. Tne fat content of milk as influenced by seasons of the year. A- Milk from a Guernsey herd. .1- ? Pooled herds milk. C- milk from a Holstein herd. l J I l l l l J l JAN. FEB. MAR. APR. MAY. JUNE JULY AUG. SEPT. Figure 2. Total solids of milk as influenced by seasons of the year. A- Milk from a Guernsey herd. B- Pooled herds milk. C- Milk from a Holstein herd. DOKAL AUNROGEW'fimmIV IOOO 800 600 200 90 J J l l l l l l l JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. Figqre 3. Variations in the total nitrogen content of milk with seasons of the year. A- Nilk from n @uernsey herd. B- Nilk from a Holstein herd. C- Pooled herds milk. I80 ISO GaIWnLfiU 5 ‘0 I30 109 uflflZLE'AflMJV P lmg.%/ 91 JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. Figure A. Comparison of the Ca, F and the P/Ca ratio in whole milk with seasons of the year. A- Milk from a Guernsey herd. 8- Milk from a Holstein herd. C- Pooled herds milk. 601L0fl74RH0$RH0flwMFflnmflv 80 60 4O 20 92 JAN. FEB. MAR. APR. MAY JUNE JULY AUG. SEPT. —- \X “LO—O... \ X \ __ “~§M~ -.-:::":Ei \\ I l l 3 l l l l I 1 Figure 5. Variations in colloidal phosphorous with seasons of the year. A- Milk from a Guernsey herd. B— iilk from a Holstein herd. C- Pooled herds milk. TUTZL AUKROGEWV {nuz96) 700 650 600 550 500 -— -45 -— -{ 4o _' 4-d35 X#———— \ ‘/ X‘T" 1 1 l L i l 1 25 0 IO 20 30 4O 50 60 TIME OF HEATING (minutes) Figure 6. Variations in the total and serum nitrogen contents of milk heated at lh3” F. for different periods of time. A- Whole milk. 8— Serum portion of the milk. SERUM ”/7306! IV (my. 96/ I" “OO.-u- ~I‘ ' WHOL E MIL K 00 (my. x) P finaiil P/Ga 9h- IO 20 3O 4O 5O 60 'TIME OF HEATING (minutes) 90 P— 7 80 ._ _. 6 [A 70 —- - 5 60 I— x B —_ 4 \‘~‘_-_x’-’X 96 —- -‘44 94 — d 42 92 hr -*40 90 _' "n38 SBt- -‘36 86*— -134 84r— -432 X L4 -— x’ \~ _ __...—x B A 9.8 I.2 - *‘A - 9.6 L0 _- l -SL4 .98 — I — 9.2 X 0 Figure 7. The influence of heat on the Ca, P and the P/Ca ratio of milk. A- Thole milk. h w- Serum. 60 (mg. 96) P (mg. 96) SERUM P/Ca r0 TA]. NITROGEN I my. 7.) 600 550 500 450 400 — —J A _' -T — ‘1 r—— x — ‘\~~ I i 1 \MB 1 0 .IO 20 .30 .40 .50 TI TR ATA BLE ACIDITY We) Fiqure 8. Variations in the total and serum nitrogen contents in milk of varying acidities. A- Whole milk. 8- Serum portion of the milk. 55 50 45 4O 35 5mm! NITROGEN (mg. %) ‘1'. .-.-.-.-.1 WHOLE MIL K Pliny. g) 00 (my. 9‘) P/Co 344770 (my. %) H0 I00 90 80 70 94 92 90 as as 84 82 so .96 .94 .92 .90 .88 _. A _ /'/ — x — x/’/ A —1 ._ x B L. ¢/ —+ — "/ — /’ +— X -— _ .J F— J _ 0 J0 20 .30 .40 TITRATABLE ACIDITY (We) Figure 9. Comparison of Ca and P and the P/Ca ratio in milk of various acidities. A- Whole milk. 8- Serum. ()6) 30 5 '9me 6'0 (my. VJ P/Oa RAT/0 (mg. ‘36) (6) I v e o ‘-)o J 935. Citric acid leT““”"”” CII I.)II.I\ L'.1I_.4 Lahad, J., and Shtrikmnn N. thl The estimation 0 Cl /) o nrwkhicts. .I 7 I I1 P1 l _ ,. (T1 ‘ O. n.’ 4nd .HCkcy, chan~es produced calcium, phasphorous o? ;kim milk Jurinn heat cxchnnner. (Ab 3109-311C. Brinns, A. P. lQQH. Some apnlications of {fliosgfliaixe n.8m1ncni. .T. Commit tee on lth. Final report of ‘—.~» 1. J5 determinations in milk and milk produces. Analyst, 63: S. L. in the ultrafiltcrzkle , and nitrouen comnonents processing in a Fallory st.) J. De ry Sci., 2;; the colorimetric Y / niol. Chem., SQ: 225—20M. Methods of Determininn the Curd Tension of Lilk: American Dairy Science Association. determininn the curd Dairy Sci. a1: Cranfield, h. T., Griffiths 1927. J. 5*r. 601., 17: 72 Cited by Pevies, 3} Mi 1k. J01 10, 522 he trand Co., Inc. Davies, W. L. 1936: The 1 :‘.1 Davies, 5. L 1928, N631 Cit 171- committee on methods of tension of milk. J. 2 3-8:? and Linn, E ’ . (Original not 3 L. The Chemistry __ pp. New York: D. 9n R. een. 0? Chemistry of Iilk. Vol. 10, 522 pp. .: D. W n Nostrend C0,, Inc. ° 9 -1WL. 1937.) and Provan, ___J sh J. Aer E: 11k. (Griminal not seen ed by Linn, E. R. J. Dairy Rese Harcl c: (1c) (11) (15) Dayle R. K., Harland, s. :. Caster (3 Iavis 1. fi., Harland, P. 7., Caster, A. 3., and Icllnor, Q. N. IQMTH. \hariations iri the otnnstitnents (mirnilk under firivona conditions. 1. Variations o” individual cows within breeds hy calendar month . J. Dairy sci., 39: 115-L2H. ‘ “ A. 3., and , :{.11. 10M7b. Variations in the constituents of wilk under Arizona conditions. 111. Variations in milk From Jersey, Guernsey, Holstei ani Fixed herds. J. Dairy dci., 39: I . o ll_3rJ-"+}+L. . Ellenhercer, H. 8., Newlander, J. A., and Jones, a U ‘ 1950. Variations in calcium and phosphorous contents of cow's milk. Univ. of Vermont and State Aer. Collere Aar. bxpt. eta. Bull. 550. 26 pp. Piste, C. H., and Subbarow, Y. 1925. The colorimetric determination of phospho— rous. J. Biol. Chem., éé: 375-hOO. Cold'ro J., Hackintosh, J., and Mattick, E. C. V. Graham, J. R Gyorsy, 1032. Investiaations on the milk of a typical herd of shorthorn cows. I. J. Dairy Research, h: h8-73. ., Jr., and Kay, H. D. 1933. Phosphorous compounds in milk. I. The phosphorous partition in milk, with preliminary observations on-milk phoSphatase. J. Dairy Research, g: Sh—hZ. 1923. Biochem. Zeitsch. 1H2: 1. (Original not seen. Cited by Allen, L. A., J. Dairy Research, 3: 1-51. 1931.) 99 (lo) Lam11r'. “g" . '. . . ,h -. K‘ ) ‘ j I -.' a ,I I . W - . . . "i . , _ -- t'. ' .v ’. ’1 " | - r "_‘... 1' '~ I. ‘I K . ‘l I . :17 ' ‘ p\ J r .‘J‘, f‘LV ‘ v f. " 7 ‘Ffi ' 9-,, '- ‘ f‘ ‘IF' . .‘ x ; ..._ O - ‘ . 'L .. ~. ’ ' \.'< I. - ‘y'- -- - b"~a ' ‘ ‘- ;3' v — - _ . . '—-. _r_ “ :. I3 ,9 ‘. _ ,o -. I . . __, ‘. .J _ . "7.1- I "m . .‘ l ‘ . .._. ‘3 , 4 ‘It' . _ bl“ I A . 4. C x ¢c ' "d‘. ' ' ' _‘ an; . , , . ?._‘l ‘3," . . . "1:". ' ‘ ' .r,‘ .'o -. - . ‘ o - '1 ‘ l- l . , ; . x "- '- . . N); " I- R" - ' ' r - " ‘ '- _.'<. ,— . ‘_ L‘ [- .'. ‘ . 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