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"'1': 11; 1' 11111; 111.11; '11: 1.111.131 '"1'1' "’3' '1 3‘1 1L 1:"1 111,111,341.» 11;“.1'J1 311%; '1 21111. .1111 111111 1" 1‘11"? '111 11"11‘111 171111 “"1","1' '211111 1111111111111'11111' ""1"'11'1" '2111' “'1111'11'1'111‘1‘11111L1 111111111" ' " 11111" 11111' ‘- c: ‘— "‘ — 6"3‘0'! I’ll’h‘i"“‘§' 1 E x -. or; z.- ’ 3' 1 Xvi; '.v'..a.0‘ u- . a it dloloaniap -’;.-‘ 6' 1 d .u.. .. pa. ‘I D ‘4 ‘rfl'o‘.la,. ! R O 04 '-' . -.-_n~ n w: $3, L’lh- d ‘..-an" ‘Wd CT This is to certify that the thesis entitled An Assessment of the Effects of Aging of Milk on Cheese Yield presented by Elizabeth Cara La Duke has been accepted towards fulfillment of the requirements for Masters degree inFood Science _ fiezfimm Major professor Date May 14, 1986 0-7639 MS U is an Affirmative Action/Equal Opportunity Institution MSU LIBRARIES m » RETURNING MATERIALS: Place in book drop to remove this checkout from your record. FINES will be charged if book is returned after the date stamped below. AN ASSESSMENT OF THE EFFECTS OF AGING OF MILK ON CHEESE YIELD BY Elizabeth Cara La Duke A THESIS Submitted to Michigan State University in partial fulfillment of the requirements for the degree of MASTER OF SCIENCE Department of Food Science and Human Nutrition 1986 ABSTRACT AN ASSESSMENT OF THE EFFECTS OF AGING OF MILK ON CHEESE YIELD BY Elizabeth Cara La Duke The effects of aging milk at 4 c on cheese yield and hydrolysis of p-casein were examined. With the partial hydrolysis of p-casein, protecse-peptone components and Y-caseins are expected to increase after 48 h of milk storage. No significant differences in the nitrogen distribution of casein and protecse-peptone fractions were observed at 0, 24, 48 and 72 h (P<0.05). Curd tension decreased from 0 h to 48 h then increased slightly, indicating the possibility of partial hydrolysis of p-casein. Casein yield decreased slightly over 72 h. Electrophoretic assays showed an increase of components 3 and 5 in protecse-peptone. Components 8-fast and 8-slow, and proteins of the casein fraction did not change significantly. The percentage of protein in experimental cheeses decreased at 48 h and increased slightly at 72 h of milk storage. ACKNOWLEDGMENTS The author wishes to express her sincere gratitude to her major professor, Dr. J.R. Brunner for his guidance and support throughout the course of this study and for his aid in the preparation of the thesis manuscript. Appreciation is also extended to Dr. J.A. Partridge, Dr. ILA. Uebersax and Dr. R.W. Luecke for reviewing the thesis manuscript and for serving on the examination committee. The author would also like to thank Me. U. Koch for all her valuable assistance. The author also acknowledges the fine interaction and aid of the many fellow graduate students during the course of this study. Finally, special thanks are extended to the author's parents for years of love and support. ii TABLE OF CONTENTS Page LIST OF TABLES.................................... v LIST OF FIGURES................................... Vi INTRODUCTION...................................... 1 LITERATURE REVIEW................................. 3 MATERIALS AND METHODS............................. 27 Preparation of Milk Fractions for Analysis.. 27 Nitrogen Analysis by Kjeldahl................ 28 Comparison of Rennet Curd Tension............ 28 Comparison of Casein Yield................... 29 Sample Preparation for Polyacrylamide Gel Electrophoresis............................ 29 Polyacrylamide Gel Electrophoresis in a Discontinuous Buffer System................ 31 Staining for Glycoproteins in the Proteose- Peptone Samples on Acrylamide Gels......... 32 Densitometric Scanning of Stained Polyacryl- amide Gels................................. 32 Microbiological Analysis of Milk for Meso- philic and Psychrotrophic Bacteria......... 34 Cheesemaking Process......................... 34 Experimental Design.......................... 36 RESULTS.O0.00.00.00.00...OOOOOOOOOOOOOOOOOOOOOO0.0 37 Kjeldahl Analysis of Protein Fractions of MilkOOOOOOOO0.000000000000000000.0.0.0....O 37 comparison of Rennet Curd Tension............ 37 Comarison of Casein Yield.................... 37 Discontinuous Polyacrylamide Gel Electro- phoresis of Proteose-Peptone Samples....... 38 Staining of Glycoproteins of Proteose- Peptone Following Electrophoresis.......... 38 Discontinuous Polyacrylamide Gel Electro- phoresis of Casein Samples................. 39 Microbiological Analysis for Mesophilic and Psychrotrophic Bacteria.................... 39 Laboratory Scale Cheesemaking Process........ 39 iii Page DISCUSSION 0 O O O O O O O O O O O O O O O O I O 0 O O O 0 O O O O 0 O O 0 0 O O O O O O O 7 0 CONCLUSION O 0 O O O O O O O O 0 O O O O O O O O O O O O O O 0 O O O O O O O O O O O O O O 8 8 BIBLIOGMPHY.0......OOOOOOOOOOOOOOOOOOOOOOO0...... 89 iv Table 1. 10. LIST OF TABLES Procedure for staining of glycoproteins in acrylamide gels............................. Nitrogen distribution of ten individual milksamp1380000oeoooeooeoooeoeooooooeooeeoo Comparison of total nitrogen distribution in mg/100 ml of ten milk samples stored at Comparison of casein nitrogen distribution in mg/100 ml of ten milk samples stored at 4 COOOOOOOOOOOOOOOOOOOOCOOOOOOOOOOOOOOOOOOOO Comparison of proteose-peptone nitrogen distribution in mg/100 ml of ten milk samples stored at 4 C....................... Mesophilic and psychrotrophic Standard Plate Counts of three individual milk samples StorEd at‘COOOOOOOOOOOOOOOOOOOOO000......O Changes over time in PAGE densitometric trace area percentages of the protein- stained zones of proteose-peptone isolated from Cheese milk....IOOOOOOOOOOOOOOOOOOOOOIO Changes over time in PAGE densitometric ’trace area percentages of the protein zones otwney Bamp1330000000000.0.000...0.0.0.0... Average values of casein nitrogen and non- protein nitrogen reported as a percentage of total nitrogen by several authors........... Casein nitrogen and Ca" concentrations in the soluble phase of bulk milks stored at 4 C for up to 7 d (Ali et al., l980a)....... Page 33 41 42 43 44 45 46 47 71 74 LIST OF FIGURES Figure 1. Dilution diagrams for mesotrophic and psychrotrophic bacterial analysis of milk samples stored at 4 C for up to 72 h........ Comparison of rennet curd tension of indi- vidual milk samples stored at 4 C........... Comparison of casein yield from rennet curd made with individual milk samples stored at 4 c.OO...0....0.000.0000000000000IOOOOOOOOOO Electropherograms and corresponding densi- tometric traces of proteose-peptone samples made from milk stored at 4 C for A) O h, B) 24 h, C) 48 h and D) 72 h on acrylamide gels of T:8%, C:2.5% concentration.......... Change in percent area over time of peaks representing protein concentration of proteose-peptone component 3 from densito- metric traces of three samples of proteose- peptone made from individual milks stored up to 72hat4c.0000...OOOOOOOOOOOOO._OOOOOIOO Change in percent area over time of peaks representing protein concentration of an unknown intermediate component between com- ponents 3 and 5 from densitometric traces of three samples of proteose-peptone made from individual milks stored up to 72 h at 4 C... Change in percent area over time of peaks representing protein concentration of com- ponent 5 from densitometric traces of three samples of proteose-peptone made from indi- vidual milks stored up to 72 h at 4 C....... vi Page 35 48 49 51 52 53 54 Figure 10. 11. 12. 13. 14. 15. Change in percent area over time of peaks representing concentration of the inter- mediate zone between components 5 and 8-fast & 8-slow from densitometric traces of 3 sam- ples of proteose-peptone made from individ- ual milks stored up to 72 h at 4 C.......... Change in percent area over time of peaks representing protein concentration of com- ponents 8-fast & 8-slow from densitometric traces of three samples of proteose-peptone made from individual milks stored up to 72 h at‘CI.0.00.000.00.00...OOOOOOOOOOOCOOOO... Electropherograms and corresponding densi- tometric traces of proteose-peptone samples made from milk stored at 4 C for A) 0 h, B) 24 h, C) 48 h and D) 72 h on acrylamide gels of T:12%, C:2.5% concentration......... Proteose-peptone samples on polyacrylamide gels stained with A) a fuchsin-sulfite sol- ution to yield fuchsia zones containing glycoprotein and B) Coomassie Brilliant Blue 6-250 to yield blue zones containing pro- tein...0.0.0.000...'.....OOOOOOOOOOOOOOOOOOO Electropherograms and corresponding densi- tometric traces of casein samples made from milk stored at 4 C for A) O h, B) 24 h, C) 48 h and D) 72 h......................... Change in percent area over time of peaks representing protein concentration of 7-CN from densitometric traces of three samples of whole casein made from individual milks stored up to 72 h at 4 C.................... Change in percent area over time of peaks representing protein concentration of p-CN from densitometric traces of three samples of whole casein made from individual milks stored up to 72 h at 4 C.................... Change in percent area over time of peaks representing protein concentration of as-CN from densitometric traces of three samples of whole casein made from individual milks stored up to 72 h at 4 C.................... vii Page 55 56 58 59 61 62 63 64 Figure Page 16. Average percent protein in six experimental cheeses made from milk stored at 4 C for 0, 48 and 72 hOOOOOOOOIOOOOOOOOOOOOOOOOOOOOOOOO 65 17. Electropherograms and corresponding densi- tometric traces of proteose-peptone samples made from milk used to make experimental cheese, stored at 4 C for A) O h, B) 48 h and C) 72 ho.0..O0000'...OOOOOOOOOOOOOOOOOOI. 67 18. Electropherograms and corresponding densi- tometric traces of whey samples from exper- imental cheesemaking process using milk stored at 4 C for A) 0 h, B) 48 h and C) 72 no.0...OOOOOOOOOOOOOOOOOOOOO0.000.000. 69 19. Schematic representations of casein samples on polyacrylamide gels in a discontinuous system obtained A) by Eigel (1977b), B) in this study and C) by Melachouris (1969)..... 81 20. Schematic representations of whey samples on polyacrylamide gels in a discontinuous sys- tem obtained A) in this study and B) by Melachouris (1969).......................... 87 viii INTRODUCTION Recent work done by Ali et al.(l980a) indicated that milk held at refrigerated temperatures for 48 h yields . less cheese than the milk supply held for shorter or longer periods of time. The low yield was coincident with the highest apparent levels of soluble casein. After 48 h, there was a period of time in which the level of soluble casein, especially p-casein, decreased. This indicated a possibility for increasing cheese yields if high quality milk were kept under proper storage conditions for a period of time greater than 48 h. Because the dissociation of p-casein from the micellar state during milk storage appears to be a key factor in the differences of this phenomenon, the following hypothesis is suggested. ‘When p-casein dissociates from the casein micelle during cold storage, it is partially proteolyzed by plasmin, an endogenous casein-associated proteinase. Possibly, during the initial 48-hour storage period, the plasmin does not have enough time or the inactive zymogen, plasminogen, is not sufficiently activated to proteolyze the casein significantly. Then, as the cheese milk is warmed.to the setting temperature, the caseins migrate back into the micellar state. Possibly, 2 p-casein moves back onto the surface of the micelle in a different arrangement from that of the fresh milk. This configuration could cause previously available cleavage sites on K-casein to be obscured from the action of chymosin. .A reduction in cheese yield could result. How- ever, after 48 h of storage, plasmin might have enough time to proteolyze a portion of the dissociated p-casein. An increase in proteose-peptones and Y-caseins would result. When the milk is heated for cheesemaking after 48 h of cold storage, there are fewer p-casein molecules to migrate back onto the micelle, leaving more cleavage sites available to the action of chymosin. This rationale might explain the subsequent increase in cheese yield that results when milk has been stored for longer than 48 h. To evaluate the aforementioned hypothesis, a study was initiated to elucidate the components and properties of milk stored at 4 C for up to 72 h as compared to fresh milk; The action of plasmin on p-casein yields Y-caseins and proteose-peptones which are the C-terminal and N-terminal fragments of p-casein, respectively; ‘This study followed.the levels of proteose-peptone.over time with the idea that after approximately 48 h of milk storage, dis- sociated p-casein would be partially proteolyzed by plasmin, resulting in an increase in proteose-peptone levels. Other observations which reflect the partial - hydrolysis of B-casein hypothesis, ine., curd tension and casein yields, were also pursued. LITERATURE REVIEW Raw milk is a heterogeneous system which.contains emulsified fat globules, colloidally dispersed casein micelles and dissolved proteins, lactose, and salts. There are several principal casein components which are phospho- glycoproteins that comprise about 80% of the total protein content of milk (Brunneryl977). The American Dairy Science Association and the Committee on the Nomenclature and Methodology of Milk Proteins grouped the caseins into the following families: 05,-, (15,-, B- and x-caseins, and suggested the respective abbreviations: «1,, -CN, al.,-CN, p-CN, and K-CN (Eigel et al., 1984) There are also some minor caseins present in milk frequently referred to as 7- and A-caseins. Thecnrcaseins consist of one major and one minor component both with the same amino acid sequence (Eigel et al., 1984). The minor component, previously classified as aw-casein (Whitney et al., 1976), has the same amino acid sequence ascu,-CN but contains one additional phosphoryl- ated serine residue. Thecn.-caseins are insoluble in the presence of calciumlII) at concentration levels occurring in milk (Swaisgood, 1973). Milk protein components previously classified as a”-, 4 03,-, and as, -casein by the Committee on the Nomenclature and Methodology of Milk Proteins (Rose et al., 1970; Whitney et al., 1976), and au-casein by Annan and Manson (1969), appear to be components of the 052‘“ family. Evidence strongly suggests that all have the same amino acid sequence but differ in phosphate content (Brignon et al., 1976; Brignon et al., 1977). ogz-caseins are also sensitive to the calcium ion (Brunner, 1981). p-CN has two characteristics which clearly distinguish it from the other caseins. First, it exhibits a strong temperature dependent association, and second, its solubil- ity in the presence of calcium(II) is also temperature dependent. The temperature at which p-casein precipitates is decreased as the concentration of calcium(II) increases (Swaisgood,1973). The K-CN molecule has several unique features. It remains soluble in calcium(II) solutions under conditions that precipitate all other casein components. It has the capacity to stabilize other caseins in the presence of calcium(II) through formation of colloidal micelles. In addition, it is specifically hydrolyzed by several enzymes (particularly chymosin) causing destabilization of the micelle and subsequent curd formation. Finally, it is the only casein in which some species are variably glycosylated (Swaisgood, 1973). _ A critical concentration of calcium(II) is required to form micelles in model mixtures of x- and arm-caseins, 5 hence, these three components represent a minimum require- , ment for micelle formation. The extent to which properties of these model systems represent those of native micelles is somewhat uncertain. Removal of calcium(II) from the micelle does not change the hydrodynamic radius until a critical level is reached at which complete dissociation occurs. Abovethe critical level, removal of calcium(II) results in the; appearance of soluble x- and p-caseins (Swaisgood, 1973). Addition of calcium(II) to native micelle suspensions causes an incorporation of the soluble caseins into the micelle without increasing the hydro- dynamic radius (Lin et al., 1972). The 7- and A-caseins arise as a result of proteoly- sis of the B- and a,,-caseins, respectively. The liquid remaining after casein has been removed from milk is designated as whey or milk serum. Whey proteins represent about 20% of the total milk proteins. The two principal components are B-lactoglobulin (p-LG) and a-lactalbumin (oz-LA). The other principal proteins of whey include blood serum albumin, immunoglobulins, and the proteo’se-peptones. Numerous enzyme proteins and proteins with specific metabolic functions have also been identified in whey (Brunner,1977). The proteose-peptone fraction of bovine milk has been characterized as a mixture of heat-stable, acid soluble (pH 4.6) phosphoglycoproteins which are insoluble in 12% trichloroacetic acid (Whitney et al., 1976). The major 6 fractions of proteose-peptone were first designated as components 3, 5, and 8 based upon their ascending mobili- ties in free-boundary electrophoresis (Larson and Rolleri, 1955). The latter fraction was subsequently separated into components 8-fast and 8-slow (Kolar and Brunner, 1970). Components 5 and 8 were reported to exist in equilibrium. between micellar casein and milk serum. Component 3 could only be found in milk serum and was thought to be of blood origin (Kolar and Brunner, 1969). More recent data suggest that component 3 is of milk fat globule membrane origin (Kester and Brunner, 1982). Along with the Y-caseins, the proteose-peptones have also been found to be the products of proteolysis of the p-CN molecule (Andrews 1978a,b; Eigel, 1981). All the principal milk proteins exhibit genetic poly- morphism, usually due to substitution of one or two amino acids, and less frequently to a deletion of up to eight residues. The frequency of occurrence of genetic poly- morphs is genus and breed dependent (Aschaffenburg, 1968: Bell et al., 1981: Swaisgood, 1973). In normal milk, approximately 95% of the casein exists as coarse colloidal particles or micellesm‘While the detailed structure of the casein micelle is not known with certainty, it is generally accepted that it has a porous structure being composed of spherical submicelles (Fox and Mulvihill, 1982). Numerous models of the casein micelle structure have been proposed. Schmidt (1982) suggested a 7 subunit structure linked by colloidal calcium phosphate (CCP) in which the subunits contain variable levels of surface x-CN. x-CN deficient submicelles are located within the micelle while x-CN rich submicelles are concen- trated at the surface. This gives the intact casein micelle a surface rich in x-CN, thus, stabilizing the other caseins against precipitation in the presence of calcium. There are, however, other caseins on the surface of the micelle as well. Caseins, along with the entire milk system, can be easily destabilized by improper handling. Cold storage of bulk milk is one of the most suitable procedures used to avoid undesireable quality changes by inhibiting the growth ' of contaminating microorganisms. In modern practice of milk processing, raw bulk milk is stored at refrigerated temperatures before and during its transport to the dairy and also at the dairy before processing. The introduction of cold storage of bulk milk was necessary for economic and practical reasons related to centralization in the dairy industry. However, it has resulted in several unexpected problems. iSome.physica1 and chemical changes occur, including dissociation of casein from the micelle, proteol- ysis of the caseins, changes in the rennet coagulation time (RCT) and curd firmness, and reduced cheese yields (Reimerdes, 1982). The effects of cold storage on the processing paramee ters of raw milk.are related to the micellar character of 8 the casein fraction of milk proteins. Many authors have described the dissociation of micellar components, espe- cially of the highly hydrophobic B-CN, during cooling of raw milk (Reimerdes, 1982). In 1955, Sullivan et a1. suggested that p-CN is removed from casein micelles when skim milk is chilled. Rose (1968) later demonstrated that as-, 0-, and x-caseins dissolved in the serum when milk was cooled to 4 C, with p-CN accounting for 55% of the total increase in serum casein. The temperature dependent dissociation of p-CN from the casein micelle was investigated by Downey and Murphy (1970) using high speed centrifugation and gel filtration. The percentage of the total casein in supernatants prepared ° by centrifugation of mid-lactation milks was found to in- crease from approximately 6 to 15% on cooling the milks from 30 to 5 C. p-CN accounted for about 46% of this increase, while 01,- and K-caseins constituted 30 and 23% respecitvely. 0n gel filtration, maximum amounts of free p-CN (60% of the total) were obtained at 5 C. The remain- der of the p-CN appeared to be more strongly bound to the 0:,— and x-caseins, and they thought that it might be involved in the internal cohesion of casein micelles. The free B-CN appeared to be in equilibrium with the bound p-CN. Creamer et a1. (1977) later undertook to determine the effect of temperature on the extent and rate of this disso- ciation of p-CN from the micelle into the serum, and to 9 determine whether some of the micellar p-CN is fixed and cannot exchange with serum p-CN: 'Ultracentrifugation and gel filtration chromatography were used to separate micel- lar and serum caseins from milk which had been subjected to various temperature treatments. When milk was.he1d at lOw temperatures, the concentration of p-CN in the serum increased with time. The amount of p-CN that dissociated from the micelle on standing for 18 hours increased with a decrease in holding temperature. To follow the movement of B-CN in the milk system, Creamer and his co-workers utilized the properties of I‘C-labelled p-CN and the A and B genetic variants of p-CN; These experiments showed that when milk.was cooled to 0-4 C, the p-CN in the serum was able to interchange with.the p-CN in the micelle. However, complete equili- bration.between the serum and micellar components was not attained. The interpretation of these data resulted in the following model of casein behavior: p—CN in the interior of the micelle can exchange with that on the surface, which in turn can exchange with p-CN in the serumn— Thus, when milk.is cooled, p-CN dissociates from the surface sites on the micelle and there follows a redistribution of p-CN within the micelle. The investi- gators felt this indicated a continual flow of p-CN into and out of the micelle. Warming the casein micelles in the presence of serum that contained soluble p-CN results in a transfer of the p-CN from the serum to the outside of the 10 micelle. The p-CN can then redistribute itself within the micelle at any temperature in the range 0-37 C. ‘ Creamer and.his co-workers proposed this model of casein behavior would predict that, in cold milk, micellar and serum B-CN would slowly interchange as the p-CN on the micelle surface associates and dissociates, and as the p-CN monomers move within the micelle from its sur- face. The model would also predict that whenever milk is cooled, the p-CN initially released into the serum comes, from the micelle surface. The subsequent change in the protein composition of milk serum reflects the distribution of p-CN between the micelle surface and the micelle interior. Ali et al. (1980a) also studied the overall pattern changes followed by soluble casein concentration in cold- stored milk. Bulk milks and samples from individual cows, with Na benzylpenicillin added to prevent bacterial growth, were stored for up to 72 h at 4, 7, 10, 13 and 15 C. Samples were taken every 24 h and centrifuged at 38,000 g for 2 h at the temperature of storage. Storage temperature was shown.to play an important role in the distribution of casein between the micellar and soluble phases in milk. Dissociation into the soluble phase resulted from storage at 4 and 7 C to give soluble casein percentages as high as 42% of the total casein. p-CN was responsible for much of this increase: 30-60% could be found in the soluble phase. Up to 30 and 40% of an- and x-casein, respectively, could 11 also occur in the soluble phase. Association of the ca- seins predominated at higher temperatures (13 to 15 C). The initial dissociation of micellar casein into the soluble phase at low temperatures (4 and 7 C) reached a maximum after 48 h, and was followed by a reversal of this change so that a minimum was seen after storage for 72 h. These results were seen in all individual animal milk samples, regardless of the stage of lactation, and for all bulk milk samples examined. After storage at 4 C for 3 d, losses in all casein components due to proteinase activity were very small (LO-2.1%). The whey proteins were not affected under these storage conditions. In addition to cold storage of milk, several other factors have been observed to affect micelle stability and the protein equilibrium in milk. Rose (1968) found that at a fixed level of calcium caseinate, the calcium phosphate content of the micelles and the degree of polymerization of the temperature-sensitive casein components (especially p-CN) are the major factors controlling the proportion of casein present in micellar form. Ali et al. (1980b) assessed the influence of a number of factors, in conjun- ction with cold storage time and temperature of milk, on the equilibria between soluble and micellar phases. .After storage at 4 C both decreasing and increasing pH caused increases in soluble casein. Both soluble calcium and phosphate showed a steady pH dependence, being lowest at the highest pH used. Increasing calcium levels in milk 12 reduced soluble casein concentrations while treatment with EDTA caused increases in soluble casein. In most cases the pattern of casein dissociation from the micellar phase was followed by a partial reassociation at 4 C over a 3d period (Ali et al., 1980a). Addition of low levels of urea led to some dissociation of all casein components, calcium and phosphate into the soluble phase while treatment of milk with urease resulted in small decreases. During storage milk proteins may be subject to proteo- lysis, either by endogenous enzymes (Andrews and Alichanidis, 1983: Reimerdes, 1982) or enzymes produced by microorganisms present in the milk system (Visser, 1981: Adams et al., 1976: Skean and Overcast, 1960: Cousin and Marth, 1977). The presence of a natural protease in bovine milk was first documented by Babcock and Russell in 1897. It is now generally accepted that the milk protease, pre- viously identified as alkaline milk proteinase (Kaminogawa et al., 1972), and known to be transferred from blood into milk (Eigel et al., 1979), is actually blood plasmin or its zymogen plasminogen (Kaminogawa et al., 1972: Eigel et al., 1979: do Rham and Andrews, 1982: Andrews, 1983). This protease is a trypsin-like enzyme which belongs to the group of serine proteinases (Reimerdes, 1982). Transformation of plasminogen (the inactive form of the enzyme) to active plasmin requires a specific peptide - bond cleavage by plasminogen activators, serine protein- ases, that are present in the mammary gland and in milk 13 (Christman et.al., 1977: Okamoto et al., 1981: 0ssowski et al., 1979). Results of a study by Korycka-Dahl et a1. (1983) pointed out that most of the proteolytic activity in freshly drawn milk was in the form of inactive plasminogen. Rollema et a1. (1983) drew similar conclusions from their own work. They also suggested that large amounts of plas- minogen could influence the storage life of milk products. The activation of a fraction of this zymogen could lead to a significant increase in proteolytic activity. The final plasmin activity of milk, and subsequent casein hydrolysis would depend not only on the amount of plasminogen and plasminogen activators but also on the quantity of inhibitors. The occurrence of blood serum trypsin inhibitors in milk.has been documented.(Horkanen- Buzalski and Sandholm, 1981: Lindberg, 1979: Reimerdes et al., 1976). Presumably these inhibitors would interfere with the function of serine proteinases, and therefore, with plasmin and plasminogen activator activity’(Korycka- Dahl et alq,l983). Plasmin activity is variable: the more significant variations are found between individual animals, accounting for up to 82% of the total variance. Stage of lactation has also been observed to have a significant effect on plasmin activity. Enzymic activity decreases as stage of lactation increases (Humbert et al., 1983). Rollema et a1. (1983) observed a strong variation in the levels of plasmin and plasminogen in samples of bulk milk and particularly in 14 samples of milk from individual cows. Eigel (1977a) studied the proteolytic effect of bovine plasmin on al.,-Ct! B and x-CN. The extent of proteolysis was monitored by disc gel electrophoresis. No significant changes could be detected in the electrophoretic pattern of x-CN. The electrophoretic band corresponding tolau-CN B practically disappeared from the gel after 30 min incuba- tion with plasmin at 37 C. Eigel (1977b) also reported that the Y,-, 72-, and'n -caseins, which are identical to the C-terminal amino acid residues 29-209, 106-209, and 108-209, respectively, of the p-CN molecule (Gordon et a1" 1972: Ribadeau Dumas et al., 1972), could be produced in vitro by degradation of bovine B-CN with plasmin. ‘Whether actual proteolysis of B-CN took place in the mammary gland or after milking remained to be determined. Reimerdes (1982) surmised that the transfer of milk proteinases and B-CN into milk serum during cooling provides a mechanism for V-casein formation. 0n the basis of advances in knowledge of the origin of the Y-caseins the Committee on the Nomenclature and Methodblogy of Milk Proteins (Eigel et al., 1984) recom- mended that fragments resulting from proteolytic cleavage be named as derivatives of the parent polypeptide from which they were derived. Thus, 7, -, Y2 -, and 7, -caseins would be called p-CN-lP (f29-209), p-CN (f106-209), and B-CN (f108-209) respectively (for convenience the trivial names will be used throughout this paper). For % -casein, 15 1P determines the number of phosphates in the molecule, and (f29-209) refers to the amino acid residues of the p-CN molecule of which the fragment consists. Several investigators (Chen and Ledford, 1971: Kaminogawa et al., 1969) have reported that rates of pro- teolysis of the major caseins by alkaline milk protease, or plasmin, occur in the order: p-»>’as- > x-caseins. These observations,seem to be confirmed by the results obtained by Eigel (1977a), using isolated plasmin. In the course of studies on proteolysis in milk, Andrews (1978a) found the N-terminal segments of the p-CN molecule located in the proteose-peptone fraction. The proteose-peptone fractions denoted as 5, 8-fast and 8-s1ow were subsequently identified as p-CN amino acid residues 1-105/107, 1-28, and 29-105/107 respectively (Andrews, 1978 a,b, 1979: Jenness, 1979: Eigel and Keenan, 1979: Brignon et al., 1977). The demonstration by Andrews (1978a) that components 5 and 8-fast represent the N-terminal portions of the p-CN molecule while lG-, Y,— and 7,-caseins repre- sent the corresponding C-terminal portions provides very strong evidence that both groups of minor proteins are formed by’a proteolytic breakdown mechanism from B-CN. The Committee on the Nomenclature and Methodology of Milk Proteins recommended.proteose-peptone fractions 5, 8-fast and 8-slow be renamed B-CN-SP (f1-105/107), p-CN-4P (fl- 28), and p-CN-lP (f29-105/107), respectively, corresponding to the fragments of p-CN from which they were derived (for 16 convenience the trivial names will be used throughout this paper). ‘Whether proteolysis of B-CN by plasmin occurs primarily in the mammary epithelial cells, during temporary storage in the alveolar and glandular lumina, during refri- gerated storage, or during protein isolation and purifica- tion remains yet unknown (Eigel et al., 1984). Schaar (1985) analyzed the variation in plasmin activity and proteose-peptone content. Proteose-peptone samples were prepared from fresh milk and from the same ‘milk samples after 72 h cold storage (5 C). IPlasmin activity increased with parity and stage of lactation and differed between breeds. iBreed differences were found to be mainly due to differences in milk casein content with plasmin activity decreasing with increasing casein content. Stage of lactation was found to be the most important factor influencing plasmin activity. Proteose-peptone from both fresh and stored milks were significantly higher in milks containing the BB genotype of p-LG (which contain a higher proportion of casein) than in milks with the AA genotype. The reason for the effect of p-LG genotype on proteose-peptone samples is not clear. It is possible that the p-LG genotype, through its effect on the relative amounts of the casein and whey protein frac— tions, influences the distribution of p1asmin.between ca- sein and whey and thus the access of plasmin to its casein substrate (Schaar, 1985). In the study by Schaar (1985) it was concluded that 17 only a part of the casein-derived proteose-peptone can be accounted for by post-secretory plasmin activity. Donnelly and Barry (1983) made a similar observation when they concluded that the proteinase activity of freshly drawn ‘milk was insufficient to account for the Y-casein content at milking. The amount of proteose-peptone formed will increase with time but in direct proportion to the amount of plasmin present. Therefore, Schaar felt that some of the casein breakdown products do not arise from post secretory proteolysis but are actually secreted into the alveolar lumen. Andrews and Alichanidis (1983) presented evidence that the fragment comprising'p-CN amino acid residues 29-105 had been incorrectly identified (Eigel and Keenan, 1979) as proteose-peptone component 8-slow. They felt it was im- probable that a small molecule such as component 5 could lose the small, fast-moving acidic N-terminal portion, identified as proteose-peptone fragment 8-fast, and still leave another small fast-moving acidic fragment as the residue. Treatment of B-CN with plasmin, using single dimension 12.5% polyacrylamide gel electrophoresis separa- tion, led to formation of the Y-caseins, component 5, com- ponent 8-fast, and a number of other bands. ‘When purified component 5 was treated with p1asmin.component 8-fast was formed along with a few other'bands, all having mobilities less than component 5. In either case, there was no compo- nent having a mobility appropriate for component 8-slow. 18 Since component 5 represents residues 1-105 and 1-107 of p-CN, Andrews and Alichanidis felt this was strong evidence that component 8-slow is derived neither from p-CN nor component 5 and it cannot be equated with residues 29-105 or 29-107 of p-CN. Andrews and Alichanidis (1983) also. showed that many of the peptides which arise from caseins by the action of plasmin are subsequently recovered in the proteose-peptone fraction, and since many of these products are transient in nature the composition of the fraction as a whole is both time and temperature dependent. Available immunological evidence indicates that proteose-peptone component 3 is identical with a soluble glycoprotein.prepared from the milk.fat globule membrane (Kanno and Yamauchi, 1979). Plasmin has been shown to be associated with the milk fat globule membrane, and.to be capable of degrading associated polypeptides (Hofmann et al., 1979). Eigel (1981) reported the possibility that component 3 is a plasmin derived fragment of a milk fat globule membrane (MFGM) polypeptide. In a study by Hester and Brunner (1982) the proteose-peptone glycoprotein (com- ponent’3) was found to contain a component antigenically similar to one in the saline-soluble‘MFGM glycoprotein fraction 1. Component 3 was established as a principal constituent of the proteose-peptone glycoprotein fraction. It appeared that this glycoprotein was also a component of the MFGM. Thus component 3 was thought to originate in one of three ways: it might be a product of endogenous proteo- 19 lytic degradation of a principal membrane glycoprotein. Or, it could represent a loosely bound membrane component which is desorbed partially into the aqueous phase fol- lowing secretion. iLastly, it could be a serum component that is adsorbed partially to the membrane complex. Easter and Brunner felt the second.possibililty was the most acceptable, however further study was deemed necessary. A-casein appears to consist primarily of components which are actually fragments of cry-casein. It appears that A-casein also occurs as a result of proteolysis of proteins in milk. ‘Whether these peptides are actually , generated intracellularly or result from proteolysis during) storage after milking, or during preparation of various milk fractions is not known (Aimutis and Eigel, 1982). Plasmin does not affect the major whey proteins a-LA (Yamauchi and Kaminogawa, 1972) and p-LG (Chen and Ledford, 1971: Yamauchi and Kaminogawa, 1972). In fact, the latter has been found to act as an inhibitor for the enzyme (Chen and Ledford, 1971: Kaminogawa et al., 1972: Snoeren et al., 1980). The proteins in milk may also be proteolyzed by en- zymes produced from a bacterial source. Psychrotrophic bacteria which are in most raw milk supplies (Foster et al., 1957) can grow readily at refrigeration temperatures. These organisms produce proteolytic enzymes which can.at- tadk milk proteins (Adams et al., 1976). There have been reports suggesting that proteolytic psychrotrophs may de- 20 crease cheese yields by breaking down the milk proteins and causing increased N losses into the whey (Olson, 1977: Yates and Elliot, 1977: Cousin and Marth, 1977: Nelson and Marshall, 1977: Aylward et al., 1980: Hicks et al., 1982). Hicks et al. (1982) found that when pasteurized grade A raw milk was inoculated with psychrotrophic Bacillus and Pseudomonas strains, cheese yield decreased as inoculum increased. All milk samples were stored for at least 6 days at 10 C before processing into cheese. Nelson and Marshall (1977) reported that test psychrotrophic species either had no effect or decreased cheese yield. Yan et a1. (1983) stored two different supplies of grade A raw milk at 4 C and 7 C. After 0, 2, 4, 6 and 8 days of storage, aliquots from each storage temperature were pasteurized and used for the manufacture of direct- acid-set experimental cheese curd. Both aerobic and psy- chrotrophic counts of the raw milk increased from less than 10‘ cfu/ml at day 0 to greater than 10' cfu/ml after 8 days of storage at 4 or 7 C. Proteolysis increased with an increase in storage time. No apparent decreases in cheese yield were observed for milk stored up to 4 days at 7 C and up to 6 days at 4 C. In fact, slight increases in yield were observed for cheese made from these milks. Cousin and Marth (1977) suggested that psychrotrophs can decrease pH of milk.during low temperature storage, thus decreasing the soluble ca- sein. .As soluble casein decreased, micellar casein would 21 increase, which may account for the slight increases ob- served in yield (Yan et al., 1983). {A decrease in yield was shown in cheese made from milk stored 6 days at 7 C and 8 days at 4 C. Rapid decreases in cheese yield occurred only when counts of raw milk were greater than 10' cfu/ml. Partridge (1983), in a study of the effects of lactic culture seeding of raw milk on the yield and quality of Cheddar cheese, found yields of control and seeded cheeses to show a trend toward higher levels on the third and fifth days of manufacture when milk was stored at 3.3 C. Joshi et a1. (1983) investigated the possibility of a correlation between proteose-peptone content of stored pasteurized milks with their bacteriological contents since' this fraction might be released from major milk proteins due to proteolytic action of bacteria. Before storage, milk samples were analyzed for their'proteose-peptone and bacteriological contents. Subsequently, the milk samples were stored at 8-10, 22, and 37 C for 7 days, 42 h, and 18 hrs respectively. .At the end of storage the milk.sam- ples were further analyzed for their'proteose-peptone and bacteriological contents. The proteose-peptone content of the samples stored at 8-10 C for 7 d did not reveal any increase in proteose-peptone level, whereas there was a slight increase in coliform and enterococcal counts. The samples stored at 22 C for 42 h and 37 C for 18 h both exhibited a considerable increase in the level of proteose- peptone with an increase in total bacterial counts. An 22 increase in the level of proteose-peptone was mainly attri- buted to an increase in the coliform counts. Adams et a1. (1976) studied the effects of psychrotro- phic growth in raw milk on proteins of milk. Pseudomonas spp. isolates were inoculated into raw skim milk samples to initial populations of lOl/ml or 103/ml. The standard plate count of the uninoculated milk was always less than 100/m1. The caseins of raw skim milk were attacked readily by the psychrotroph isolates during storage at 5 C. x-casein appeared most susceptible to proteolysis by the psychrotrophic enzymes. The p-caseins were also proteo- lyzed rather rapidly. as-Caseins appeared to under go some' proteolysis, but it was not as significant as the other two ‘ caseins. Adams' results were similar to those reported by Skean and Overcast (1960). However, Adams and his co- workers felt that in both studies the psychrotrophic popu- lations were much.higher than would occur in normal raw milk. Colony counts of uninoculated milk remained below 100/ml, and no proteolysis was detected. Detectable pro- .teolysis of p- and as-caseins required much higher popula- tions,’at least with the isolates used by Adams and and his co-workers. They concluded that the effect of proteolysis on raw milk might depend on the composition of the psychrotrophic flora of the milk since this could influence the extent of proteolysis and the proteins that are . attacked. Law at al. (1979) felt that the reports concerning 23 Cheddar cheese yield loss due to proteolytic bacteria in- volved experiments in which either the numbers of proteo- lytic psychrotrophs in milk had reached 10'/ml before pasteurization, or proteolytic enzymes had been added in amounts which could not be related to an equivalent bacte- rial population. In practice, total psychrotrophic counts do not normally exceed 107 cfu/ml in stored milk and only a proportion of these are caseinolytic (Chapman et al., 1976: Law et al., 1979). Law and his co-workers then experimented with Cheddar cheeses made from milks in which proteolytic, psychrotrophic bacteria had grown to numbers likely to be encountered in practice. Casein breakdown in the milks, stored at 7.5 C for up to 72 h, and the cheeses was mea- sured and yield and qualilty of cheese was compared with results obtained using low-count, stored milk. Despite the growth of the proteolytic psychrotrophs in the cold stored milks to approximately 10° and 107 cfu/ml in 24 and 72 h respectively, very little casein breakdown was evident. Differences between inoculated and control milk samples were either undetectable or only very slight in all cases. Law et’ al. concluded that excessive proteolysis due to psychrotrophs in stored raw milk is unlikely to be a prob- lem in practice. Ali et al. (1980a), in studying the effect of cold storage of milk on cheesemaking parameters, determined that the growth of psychrotrophs to levels of 5 x 105 cfu/ml (milk stored up to 72 h at 4 C) had very little apparent 24 effect on cheese yield or quality. The changes in the physical and chemical properties of milk due to cold storage are known to prolong coagulation times (Fox, 1969) and to lower curd firmness (Klostermeyer and Reimerdes, 1976). significant variations in coagula- tion time and curd firmness have.been observed in relation to individual animals, their stage of lactation, and milk pH (Okigbo et al., 1985). Milk pH was the most significant factor affecting coagulation time and curd firmness. Reducing pH shortened coagulation time and increased the curd firming rate. Variation in curd firmness has also been attributed to mastitis (Ali et al, 1980b), feed.(Hi11,v 1931), milk composition (Garnot et al., 1982), temperature history of the milk, and season (McDowell et al., 1969). Curd firmness at cutting influences the texture (Lyal, 1969) and yield.(Bynum and Olson, 1982) of cheese. The process of milk clotting involves a primary and a secondary phase. The secondary phase of the overall coagu- lation process is the phase in which the coagulum actually forms. This phase is known to involve interactions between calcium ions and.ay-q B-, and para-x-casein to form an insoluble coagulum (Mehaia and Cheryan, 1983). Milks stored at 4 C were found by Ali et al (1980a) to prolong rennet coagulation time (RCT) and processing times. The extent of this increase reached a maximum after 48 h of storage. After 72 h the increase in RCT was slightly less than after 48 h. Ali and his co-workers felt that the 25 increase in RCT could be explained either by the changes occurring in the calcium phosphate equilibrium (Fox, 1969), or by the changes occurring in the casein micelle, or by both. Yan et al. (1983) also found the time required for rennet to develop a firm coagulum was increased as storage time of milk at 4 and 7 C was increased. It was concluded that extensive proteolysis during extended storage appar- ently caused damage to the casein micelles, causing the delayed coagulation time and.preventing the coagulum from reaching the desired firmness. The many factors discussed above must all be consid- ered when studying the effect of certain parameters on cheese yield and quality. .Ali et al. (1980a) found changes: in cheese quality and yield correlated well with the changes in casein distribution on cold storage. In labo- ratory as well as larger-scale experiments, milk stored at 4 C for 24, 48, and 72 h was used to make cheese curds with the standard procedure for Cheddar cheesemaking (Chapman and Burnett, 1972). Over a storage period of up to 48 h the percentage of moisture in the curd increased while cheese—yields decreased. On further storage there was a reversal of these trends and all these changes reflected the pattern of changes occurring in serum casein concentra- tions of stored milks. Dzurec and Zall (1985) found that cottage cheese yields increased as a result of heating (74 C, 10 s), cooling (3 C), and storing (7 d) milk prior to cheese- 26 making. Heating prior to storage apparently caused part of the p-CN to be trapped physically in the micelle, leading to increased yields since less casein would be lost in the whey. There is evidence that cheesemaking properties of milk are also affected by genetic variation of milk proteins (Mariani, 1983). The linkage between 05,-, p-, and x-casein loci, and variations in gene frequencies between cattle breeds must be considered. Cow's milk containing x-CN BB has better coagulation properties than milk con- taining x-CN AA. Variation at the B-CN locus also af- fects cheesemaking properties: the p-CN B variant is supe- rior to the p-CN A variant. Polymorphism at the 6-1.6 locus has an indirect effect on cheesemaking properties of milk: cows with the BB variant yield more casein than cows with the AA variant (Mariani, 1983). However, evidence indicates that while B-LG genotype AA is associated with higher casein, and perhaps milk 'yield, milk from the BB genotype is better for cheesemaking (Sartore and Stasio, 1984). MATERIALS AND METHODS The experiments conducted in this study involved the analysis of raw whole milk subjected to four different treatments. Fresh raw milk was taken immediately after milking, cooled to 20 C and divided equally into four containers. The first portion, representing the control, was analyzed immediately. The remaining portions were stored at 4 C for 24, 48, and 72 hours and subjected to the. same analyses as the control. All milk was obtained from Holstein cows of the Michigan State University dairy herd. The bulk of the fat in the milk used for protein fractionations and.polyacrylamide gel electrophoresis was removed by centrifugation. Preparation o_f Milk Fractions for Analysis Protein and non-protein fractions of raw whole milk were prepared according to the procedure used by Shahani and Sommer (1951a). The fractions prepared included total nitrogen (TN), non-casein nitrogen (NCN), proteose-peptone plus non-protein nitrogen(PP+NPN) , and non-protein - nitrogen (NPN). The nitrogen content of these fractions 27 28 was determined by a semi-micro Kjeldahl method. Nitrogen Analysis by Kjeldahl Nitrogen analyses were performed using a micro- Kj eldahl apparatus. The digestion mixture consisted of concentrated H280, and employed CuSO, and Sec, as cata- lysts. Samples were digested for one hour in the digestion mixture, H202 was added and the samples digested for a second hour. The digestion mixture was neutralized with 40% NaOH and the ammonia released was steam distilled into 4% boric acid. The ammonium borate complex was titrated with .02N HCl using an indicator consisting of 0.1% methyl red and 0.03% methylene blue in 60% ethanol. Tryptophan was used as a standard with an average recovery of 102.51%. Comparison o_f Rennet Q13 Tension Rennet curd was prepared using the method of Ashworth and Nebe (1970). Four replicate 225 ml samples of raw whole milk were placed into 250 ml beakers, and warmed in a water bath to 35 C. Each sample was mixed with 4.5 m1 of a 1/50 dilution of commercial rennet extract (Dairyland Food Laboratories Inc.) in water, and returned to the water bath at 35 C for 30 min. Curd tension was measured on a Raytheon curd tens iometer. 29 Comparison 25 Casein Yield Rennet curd was again prepared using the method of Ashworth and Nebe (1970). Four replicate 80 ml samples of raw whole milk were placed in centrifuge tubes and warmed to 35 C. Diluted commercial rennet extract (1.6 ml of 1/50 dilution)‘was mixed with each sample which was held in a water bath at 35 C for one-half hour. The curd was cut and the samples centrifuged at 1000 g for 15 min to ‘ pelletize the casein matrix. The final weight of the entire pellet was determined using the procedure for total solids on the Mojonnier Milk.Tester. Dried samples were placed under vacuum and reweighed until the weight of the dish was stabilized. Kjeldahl nitrogen determination of protein fractions, polyacrylamide gel electrophoresis of the proteose- peptone fraction, and microbiological analysis for meso- philic and psychrotrophic bacteria were performed on several milk samples from individual cows. Sample Preparation £53.: Polyacrylamide G_el E1ectrbphoresis Proteose-peptone was prepared from 100 ml of raw milk by heating in a 95 c water bath for 40 min, cooling promptly to 20 C, and adjusting to pH 4.6 with 1N HCl. The serum was filtered through Whatman No. 1 filter paper. Thirty milliliters of the proteose-peptone solution desig- nated for electrophoresis was dialyzed overnight in a solu- 30 tion of polyvinylpyrrolidone to remove most of the water. The contents of the dialysis tubing was rinsed with a small amount of distilled water into a small vial. This sample was then evaporated under nitrogen and the open vial placed in a dessicator containing anhydrous CaSO, to remove resid- ual moisture. Prior to electrophoresis the sample was rehydrated with stacking gel buffer. Casein samples were prepared by adjusting a 100 ml sample of milk at 20 C to pH 4.6 with 1N HCl and filtering with Whatman No. 1 filter paper. The precipitated casein was washed three times with acetate buffer (pH 4.6) and blotted dry. Samples used for electrophoresis were solubi- A lized with stacking gel buffer. The protein content of the proteose-peptone and casein samples was determined using the colorimetric Pierce BCA protein assay. In this assay proteins react with alkaline copper II to produce copper I. BCA protein assay reagent reacts to form an intense purple color at 562 nm with copper I. The BCA protein assay reagent consists of: ’ Reagent A - sodium carbonate, sodium bicar- bonate, BCA detection reagent and sodium tartrate in 0.1N NaOH Reagent a - 4% ouso.- 5:120 The BCA working reagent is a 50:1 mix of BCA reagents A and B. 2 ml of the BCA working reagent was added to a 100 sample containing 1-120 #9 protein. The sample and reagent mixture was incubated at 37 C for 30 min. The absorbance 31 of the samples was then read against a reagent blank at 562 nm. Standard curves were prepared using lyophilized samples of proteose-peptone and Na caseinate (obtained from J.R. Brunner). Kjeldahl nitrogen determinations were per- formed on these standards to determine protein content. From the results of the BCA.protein determinations of the proteose-peptone and casein samples, the size of each sample to be used for electrophoresis was calculated to provide the same amount of protein in each assay. Thus apparent differences in the electropherograms would be reflected in the protein distribution. Polyacrylamide Gel Electrophoresis in a Discontinuous Buffer System Polyacrylamide gel electrophoresis was performed according to the method of Ornstein (1964) and Davis (1964). All electrophoretic studies were performed in glass gel tubes measuring 0.5 x 7.5 cm. All gels were allowed to age at least 18 h before use to assure complete and consistent polymerization. Two different gel concen- trations (T-8.0%, c-2.5% and T-12%, c-2.5%) were employed for proteose-peptone samples and T-10%, c-2.5%, 7M urea gels were used for casein samples. Protein staining was by Coomassie Brilliant Blue G-250, according to Reisner et a1. (1975). {All gels for each milk sample, consisting of the control and milk stored for 24, 48, and 72 h at 4 C, were stained and destained for the same amount of time, such that the staining/destaining procedure would be uni- 32 form and not contribute to any differences in dye reten- tion. Staining for Glycoproteins in the Proteose-Peptone Samples in Acrylamide leg Glycoproteins were visualized by the technique described by Zacharius et a1. (1969). Following electro- phoresis, acrylamide gels were washed thoroughly with distilled water to remove the buffer ions before beginning the staining procedure. Table 1 shows the steps in the staining procedure. The trichloroacetic acid fixes the protein, and complete removal of the periodic acid is imperative to obtain a stained gel with a clear background. The fuchsin-sulfite (Schiff's reagent) was prepared as follows: 2 g basic fuchsin were dissolved in 400 ml water with warming and then cooled and filtered. 10 ml 2N HCl and 4 g R S O were added, and the solution was kept cool and dark overnight in a stoppered bottle. 1 g activated charcoal was stirred in and the solution was filtered, and sufficient 2N HCl (10 ml or more) was. added until a drop dried on a glass slide did not turn red. Densitometric Scanning g; Stained Polyacrylamide Gale A Shimadzu dual-wavelength, thin-layer chromato scan- ner, model CS-930, was used to assess the polyacrylamide gels stained with Coomassie Brilliant Blue G-250 at 580 nm. 33 Table 1. Procedure for staining of glycoproteins in acrylamide gels Staining Procedure Time (min) 1) Immerse gels in 12.5% trichloroacetic acid 30 2) Rinse with distilled water 0.25 3) Immerse in 1% periodic acid in 3% acetic 50 acid 4) Wash 6X for 10 min in distilled water or 60 or wash overnight with a few changes of overnight distilled water 5) Immerse in fuchsin-sulfite stain in dark 50 6) Wash 3x for 10 min with freshly prepared 30 0.5% meta-bisulfite solution 7) Wash with frequent changes of distilled overnight water until excess stain is removed 8) Store in 5% acetic acid 34 Microbiological Anal sis of Milk for Mesophilic and Psychrotroph c Bacteria The Standard Plate Count method as described by Di Liello (1982) was used to enumerate mesophiles and psy- chrotrophs in the milk samples. Plate count agar was the :medium.used. Sample dilutions for mesophilic and psychrotrophic bacteria are shown in Figure l. Cheesemaking Process The cheesemaking process was performed according to the method used by Chapman and Burnett.(l972). However, it was modified to an experimental, bench-scale procedure in which six replicate 1000 ml beakers containing 900 g of raw whole milk were used. The beakers of unpasteurized milk were heated in a warm water bath. Pro-cheese treatments included milk that had been stored at 4 C for 0, 48, and 72 h. A direct vat-set starter culture, obtained from Hansen's Lab., Inc. via the Michigan State University Dairy Plant, was used. Starter (1.4 ml) was diluted in 8.6 ml milk for a total volume of 10 ml. One milliliter of this mixture was added to each of the six beakers to give .0164% starter in each sample. One milliliter of a 1/50 dilution of commercial rennet extract in water was added when the samples had reached 30 C and pH 6.58. Temperature and pH were followed over time rather than the development of titratable acidity. In this way curd "fines” present in the whey, which would contribute to the final weight of the cheese, were not lost. ‘When the cooking stage was 35 Mesophilic Bacteria raw whole milk - 1 ml 1 ml—l 1 m1-1 .1 9 m1 l 9 ml 9 ml blank blank blank ' 1' 1 1 m1 1 ml m plate dilutions: O O 0 1:10 1:100 1:1000 Plates were incubated at 32 C for 48 h. Psychrotrophic Bacteria raw whole milk ——> 1 ml 1i ’ 7U... blank blank \\\\. 1 m1 l‘hl plate dilutions: O O 0 1:1 1:10 1:100 Plates were incubated at 7 C for ten days. Figure l. Dilution diagrams for mesophilic and psychro- trophic bacterial analysis of milk samples stored at 4 C for up to 72 h. 36 finished, at 39 C and pH 6.15, whey was drained and filter- ed to catch the fines. The curds and fines from each batch of cheese were combined and wrapped in cheese cloth and held under pressure for 15 h. The final cheeses were weighed and a sample was taken from each for a Kjeldahl‘ nitrogen determination from which the percentage of protein of each cheese sample was calculated. A sample of the cheese milk from each treatment was retained for proteose- peptone analysis by gel electrophoresis. A similar assay was performed on the whey produced in the cheesemaking process. The nitrogen content was determined by Kjeldahl. The milk used for cheesemaking was also analyzed microbio- logically for the presence of psychrotrophic bacteria. Eyperimental Design Statistical analysis of the data accumulated in this study was performed using a randomized complete block design (Steel and Torrie, 1980). IMilk samples from individual cows were identified as blocks, and the treatments involved storage of milk at 4 C for 0, 24, 48 and 72 h. The data were analyzed by the following statistical model: Yii 11+ 3: + Ti + Si where: u - overall mean, 1} - fixed effects of the jth treatment (t-4), 3, - Random effects of the ith block trial, 6” - residual error. 'Data were analyzed using the factor'procedure of the MSTAT program from Michigan State University. RESULTS Kjeldahl Analysis 95 Protein Fractions pg‘Milk TN, NCN, PP+NPN, and NPN were the fractions analyzed. Casein nitrogen was calculated by subtracting NCN from TN. Proteose-peptone nitrogen was determined by subtracting NPN from PP+NPN. Table 2 shows the nitrogen distribution of~ 10 individual milk samples subjected to the four treatments used in this study. Tables 3, 4,and 5 demonstrate the trends followed over time by the TN, casein nitrogen, and proteose-peptone nitrogen fractions,respectively, of each individual milk sample. Comparison pg Rennet Curd Tension Rennet curd tension values for individual milk samples stored at 4 C are represented in Figure 2. Comparison 9; Casein 21219 The final weights of casein curd obtained from rennet- treated individual milk samples stored.at 4 C are reported in Figure 3. 37 38 Discontinuous Polyacrylamide Gel Electrophoresis pg Proteose-Peptone Samples Polyacrylamide gel electrophoresis of a preparation of proteose-peptone, utilizing T:8%, C:2.5% and T:12%, C:2.5% gels, showed a number of protein bands. Figure 4 depicts electropherograms and corresponding densitometric traces of a proteose-peptone sample on gels of T:8%, C:2.5% acryla- mide concentration. Figure 4 A, B, C, and D represent protease-peptone samples isolated from raw milk stored at 4 C for 0, 24, 48 and 72 h, respectively. Figures 5, 6, 7, 8 and 9 show graphic representations of changes in the area percentages over time on the densitometric traces for each major peak. Figure 10 represents the electropherograms and corre- sponding densitometric traces of the same proteose-peptone sample on T:12%, C:2.5% acrylamide gels. The densitometric traces in Figure 10 A, B, C, and D show changes in the dye retention of each protein band, which is proportional to changes in concentration of the proteins over time. Staining pg Glycoproteins pg Proteose-Peptone Following Electrophoresis Carbohydrate-containing proteins were detected in acrylamide gels by the technique described by Zacharius et a1. (1969). In this method, protein bands which contain glycoproteins are identified by a distinctive fuchsia colored zone. IFigure 11 A shows one principal band as well as several minor bands on the gel. 39 Discontinuous Polyacrylamide Gel Electrophoresis pg Casein Samples "——‘ PAGE of the casein preparation on T:10%, C:2.5% acryl- amide , 7M urea gels produced electropherograms and densi- tometric traces such as those shown in Figure 12. Changes over time in protein concentration, determined by changes in the area percentage of the peaks on the densitometric traces of three major bands are depicted graphically in Figures 13, 14 and 15. Microbiolggical Analysis for Mesophilic and Psychrotrophic Bacteria Individual Standard Plate Counts are reported in Table Laboratory Sgglg Cheesemaking Process The final protein content, expressed as a percentage of the total cheese weight, of the cheeses made with milk stored at 4 C for 0, 48, and 72 h is represented in Figure 16. Microbial analysis of the cheese milk samples for psy- chrotrophic bacteria yielded no more than 10 cfu/ml at up to 72 h of storage at 4 C. PAGE of proteose-peptone samples made from the milk used for cheese produced the electropherograms and corre- sponding densitometric traces depicted in Figure 17. Changes in the area percentages of the peaks are reported in Table 7. The electrophoretic assay performed on cheese whey 4O resulted in the electropherograms and corresponding densi- tometric traces shown.in Figure 18. Corresponding changes in peak areas are reported in Table 8. 41 Table 2. Nitrogen distribution of ten individual milk samples Storage 0 (mg/100ml milk) Time (h) Component Low High Average 0 Total N 494.0 635.3 555.9t 46.1 Casein N 364.3 489.1 429.0i 35.1 P-P N 13.5 29.5 20.5* 4.7 24 Total N 489.5 638.7 560.1* 47.6 Casein N 375.9 497.8 433.7i:34.9 48 Total N 499.6 625.9 557.3 #44.5 Casein N 371.2 486.1 432.2 1: 32.2 P-P N 15.0 35.2 22.2 t 5.4 72 Total N 497.0 645.4 570.3 148.4 Casein N 358.7 502.4 440.0 i=38.6 P-P N 13.5 39.0 23.4 it 6.4 °Mean of two determinations. 42 Table 3. Comparison of total nitrogen distribution in mg/100ml of ten milk samples stored at 4 C Hours of Storage Sample 0 24 48 72 1 546.52100f’ 527.5(96.5) 512.0(93.7) 549.0(1oo.5) 2 529.5(100) 544.5(102.7) 529.5(100) 551.0(104.1) 3 540.0(100) 545.0(100.9) 536.5(99.4) 584.5(108.2) 4 494.0(100) 489.5(99.l) 515.0(104.3) 497.0(100.6) 5 567.5(100) 593.0(104.5) 579.5(102.l) 580.0(102.2) 6 578.9(100) 569.6(98.4) 580.1(100.2) 579.5(100.l) 7 615.7(100) 615.1(99.9) 613.1(99.6) 625.2(101.5) a 494.9(100) 503.0(101.6) 499.6(100.9) 497.6(100.5) 9 635.3(100) 638.7(100.5) 625.9(98.5) 645.4(101.6) 10 -556.3(100) 575.4(103.4) 582.0(104.6) 593.8(106.7) “Mean of two determinations. bValues in parentheses reported as percent of 0 h N. 43 Table 4. Comparison of casein nitrogen distribution in mg/100 ml of ten milk samples stored at 4 C Hours of Storage Sample 0 24 48 72 1 421.62100? 411.5(97.7) 404.0(96.0) 424.0(100.7) 2 416.5(100) 424.5(101.9) 419.0(100.6) 426.0(102.3) 3 422.5(100) 424.0(100.4) 417.0(98.7) 455.0(107.7) 4 402.5(100) 402.0(99.9) 425.5(105.7) 411.0(102.1) 5 434.0(100) 459.5(105.9) 445.3(102.5) 447.6(103.l) 6 445.6(100) 437.1(9a.1) 448.4(100.6) 447.6(100.4) 7 473.2(100) 467.9(98.9) 463.0(97.8) 473.4(100.l) a 364.3(100) 375.9(103.2) 371.2(101.9) 358.9(98.5) 9 489.1(100) 497.8(101.8) 486.1(99.4) 502.4(102.7) 10 421.0(100) 436.3(103.6) 442.3(105.1) 452.9(107.6) °Mean of two determinations. Values in parentheses reported as percent of 0 h N. 44 Table 5. Comparison of proteose-peptone nitrogen distribution in mg/100 ml of ten milk samples stored at 4 C Hours of Storage Sample 0 24 48 72 1 17.62100? 23.0(135.3) 18.0(105.9) 23.0(135.5) 2 22.5(100) 27.0(120.0) 22.0(97.8) 25.0(111.l) 3 24.0(100) 20.2(84.2) 25.5(106.3) 20.5(85.4) 4 13.5(100) 16.0(118.5) 15.0(111.1) 13.5(110) 5 19.1(100) 21.0(109.9) 22.9(119.9) 21.0(109.9) 6 16.7(100) 16.8(100.6) 21.0(125.7) 22.4(134.1) 7 29.5(100) 35.8(12l.4) 35.2(119.3) 39.0(132.2) 8 16.8(100) 18.3(108.9) 18.8(1ll.9) 21.5(128.0) 9 22.3(100) 25.4(113.9) 21.9(98.2) 24.9(lll.7) 10 23.7(100) 21.6(91.1) 21.8(92.0) 22.8(96.2) °Mean of two determinations. bValues in parentheses reported as percent of 0 h N. 45 Table 6. Mesophilic and psychrotrophic Standard Plate Counts of three individual milk samples stored at 4 C Mesophilic Count Psychrotrophic Count (cfu/ml) (cfu/ml) Hours of Storage Hours of Storage Sample 0 24 48 72 0 24 48 72 1 1000 1200 1300 1700 20 15 26 50 2 1400 1600 1700 2900 110 320 150 450 3 18000 20000 19000 18000 730 680 970 1000 46 Table 7. Changes over time in PAGE densitometric trace area percentages° of the protein-stained zones of proteose-peptone isolated from cheese milk Protein Zone Storage Time (hours) 0 48 72 Area under peak (%) Component 3 20.3 15.8 19.7 Intermediate between ”3" and 13.7 25.3 18.4 ”5!! Component 5 45.0 47.9 49.3 Intermediate between ”5" and 3.9 4.4 4.1 "8-fast & 8-slow" Components "8- fast and 8- 6.1 6.6 6.3 slow" °Area reported is average of three determinations. 47 Table 8. Changes over time in PAGE densitometric trace area percentages° of the protein zones of whey samples Protein Zone Storage Time (hours) 0 48 72 Area Under Peak (%) BSA 11.2 11.2 12.1 Unknown proteinb zone between BSA 4.9 4.2 4.2 and a-Lactalbumin a-Lactalbumin 43.7 43.8 44.3 p-Lactoglobulin 40.2 40.8 . 39.4 °Area reported is average of three determinations. bUnknown protein zone did not compare to proteose- . peptone zones. 48 szuw>wocw mo cowmcmu vino became mo :om_smasou masses. cam» somaowm . r .uoe um esteem mwpasmm x__s .N mt=m_m a. an 8 ‘Ili ._ ._‘ / , mm /_ _ 1 ov . , ~_m. 18 F3 . «:23 a Nona.” .‘ «onion 4. 8 (sueJO) uotsual pang 49 .8.4 a. were». m._asmm x_.s F.=u_>_u=1 33.; mums utzu became sage ups?» cwmmmo we comwsmqsou an on .m shamed mass as sumac .W 5 3 Wm v [ $3.... a. nuns-am n «33$. 1 (seeJO) ugasea )0 infigan 50 Figure 4. Electropherograms and corresponding densitometric traces of proteose-peptone samples made from milk stored at 4 C for A) 0 h, B) 24 h, C) 48 h and D) 72 h on acrylamide gels of T:8%, C:2.5% concentration. _————- l0 START 51 52 .o e on a N5 cm a: bemoan oxafie Hmsoa>fioca Scum moms ocouooolomoououo mo moaosmm oounu mo moosuu oauuosouamsoo Eouw m ucosooaoo osouooolomoououo wo cowumuu Icoosoo Camacho wcwusomouoou mxmoo mo mafia Ho>o some accused cw omsmnu .m shaman Amazes. sump oaoaoum x 9 cm a _ . a U. i... a 12 l, L-i r: 33 6 ~33 a. 388 4 r8 53 .o e on c mm on on monoum oxaws Hmovfi>fiocfi Scum owns ocouooolomoououo mo moaosmm ooucu mo moomuu owuuoEOuwmcov scum m can m mucocooEoo coo3uob moocoosoo sunsposhoqu ssosxss no mo soaumuu Isoocoo caououo meaucomosoou oxmoo mo osfiu uo>o mono bemused CH owcmno .o osswfim .maaoz. sump moaaosm me a“ em a a 0. Nu 1m M" 2 m— is~ rm“ 18 «:38 a «:88 a. 338 4 8 54 cwmuoca m:_u:ommcams mxmwa eo ms_u cm>o mote accuses cw manage .5 s .oos s. 5 Ne as a: cocoon mx—_s Fmauw>wucw soc» mums mcouama-mmoouoca eo mmpasmm mots» Co mount» uwcamsobwmcmu sot» m neocoasoo mo cowuogucwucoo v mason. meme comes“ .5 misuse m cm was» 4 none-am n «ca-om 4. im~ ram rs eaav x 55 .u e on 5 mm cm on oououm mxafie Hosow>aosfi scum coma ocouooolomoououo mo moaoaom mounu mo moomuu oHouoEouHmcoo scum BonIw w uwmmlw use m monocoQEoo coosuon econ ouowcoSMouca one no soauouu Icoocoo cwououo wcfiuoomouoou oxooo mo mafia uo>o sous unmouoo cw owsmno Amazon. meme oaoaoum . .1 a .m muswwm ~33 4. «Sign 6 ~33 4 eaav z 56 .0 e an 5 Nu cu o: pououm mxHHE Hospw>wch Eoum moms mcouoooiomoououo mo mmHoEmm mouse mo moomuu ofiuuoaouamcoo Scum soawiw can ummwiw mucosooaoo mo coaumuu Icoocoo summons mcfiusomouoou mxmoo «0 mafia Ho>o some ucoouoo ca owcmno .maaog. meme mossesm W .1 .1. a. .m osswwm mung o 33 4. Gui-m 4 1m in rs In 3 88JV I 57 Figure 10. Electropherograms and corresponding densitometric traces of proteose-peptone samples made from milk stored at 4 C for A) 0 h, B) 24 h, C) 48 h and D) 72 h on acrylamide gels of T:12%, C:2.5% concentration. 58 10 L -- \lllv 3 E. :. Figure 11. U1 ‘3 Proteose-peptone samples on polyacrylamide gels stained with A)'a fuchsin-sulfite solution to yield fuchsia zones containing glycoprotein and B) Coomassie Brilliant Blue G-ZEO to yield blue zones containing protein. 60 Figure 12. Electropherograms and corresponding densitometric traces of casein samples made from milk stored at 4 C for A) 0 h, B) 24 h, C) 48 h and D) 72 h. 62 .u e an n as ou o: vououm oxawe Hosofi>fiocw Scum some cfiommo oHog3 mo woaosmm moans mo mousse ofiuuosouamcoo scum 20:» mo cofiumuu Isoocoo cfiououo wsausomonoou mxooo mo mafia uo>o some accused CH owomco .mH ouswwm .maaog. acme comacbm .x a“ 4m 9 1. 93W I «283 n in ~33 4. «238 4 63 .u e as g - cm a: cocoon mx—_s Fmsuw>wu=w scam moms :mwmmu upon: eo mmpqsmm on» Co mounts owguosouwmcmu sate zunm we cowamgucmocoo :wmuota mcwucmmmgamc mxmma mo mews em>o mote accuses cw omcmzu .ea oc:m_m mil 3 .Amazoc. use» mamacwm o~ . «02.8 6 ~33.» 4 «uni-m 4 new 1 64 .er as s - so a: notopm mLFWE pszuw>wucw sage owes :_mmmu o_osz co mmpgsmm mecca Co mounts uwcuosouwmcmu sage zu- a $6 cowumcucwocou :_muoca mcwucmmmcamc mxmoa we we?» co>o mote accuses as manage assoc. camp comaowm 45 a“ .7 .ms massed cm 23.8 6 ~38...“ 4 388 4 93k“!!! 65 um .g N. as. me .o toe 0.4 5. eat... apes 50.4 meme mommmzo paucmswgmaxm xwm cw :wmuoga accuses mmmcm>< 3 Amazon. mama mamacwm .mH alas.u m~ Ulalon Z 66 Figure 17. Electropherograms and corresponding densitometric traces of proteose-peptone samples made from milk used to make experimental cheese, stored at 4 C for A) O h, B) 48 h and C) 72 h. 67 : 68 Figure 18. Electropherograms and corresponding densitometric traces of whey samples from experimental cheesemaking process using milk stored at 4 C for A) 0 h, B) 48 h and C) 72 h. 69 <4