Milk samples from three groups of cows were taken at frequent intervals throughout lactation following autumn-, winter- or spring-calving. The ethanol (EtOH) stability/pH profile was determined for each sample and its characteristic parameters calculated. The lactational trends in these parameters were examined. Asymptotic maximum EtOH stability (Smax) was low in early lactation but rose rapidly to a value which showed no further lactational trends. Asymptotic minimum stability (Smin) for samples from autumn- and winter-calving cows showed a decrease which could be associated with the transition to summer grazing but no obvious lactational effects. The slope parameter increased slowly during lactation. The profile pK value decreased in early lactation, but thereafter increased throughout lactation giving the most obvious effect observed in direct measurement, namely an alkaline shift in the profile as lactation progressed. The EtOH stability calculated at a fixed pH of 6·6 passed through a maximum, characteristic for each cow, in the first weeks of lactation but declined steadily thereafter. This behaviour mirrors the lactational behaviour of the soluble salt balance ratio calculated from the original data of White & Davies (1958).

Seasonal variations were observed in the proportion of long-chain (C18 and over) and medium-chain (C6–14) fatty acids (FA) in milk fat. Long-chain FA were proportionately higher during the period June to October. Changes were also observed in the concentrations of citrate, isocitrate and 2-oxoglutarate in milk. Changes in [isocitrate]: [2-oxoglutarate] correlated positively with changes in the proportion of long-chain FA in milk fat and negatively with the proportion of medium-chain FA.

The strength of binding of the individual caseins and the nature of the bonding within bovine casein micelles were examined through dissociation of the micelles by dialysis of skim milk either against phosphate-free buffers containing 3 or 6 mm-CaCl2, or against buffers that were nearly saturated with respect to micellar calcium phosphate, but which had a free Ca2+ concentration in the range 0·4–5·9 mm. Dissociation was followed by ultracentrifuging the dialysed milks and determining the partition of the total and the individual caseins between the pellet and serum. During dialysis against the phosphate-free buffers both colloidal Ca and Pi in the milks decreased and about 30 % of the Pi could be removed without significant casein dissociation. With further loss of Pi, however, increasing dissociation occurred and the proportions of the individual caseins retained in the casein pellet were in the order αs2- > αs1- > β- ≈ κ-casein. Dialysis against the calcium phosphate buffers resulted in no loss of colloidal Pi but colloidal Ca increased with the free Ca2+ concentration of the buffer. Little change in the casein partition occurred in the presence of more than 1 mm free Ca2+, but serum casein increased markedly at lower levels, and the strength of binding of the individual caseins in the pelleted casein was in the order αs2-> αs1- > β- > κ-casein. In both types of buffer, dissociation is considered to occur through the breaking of linkages between the caseins and inorganic constituents. Analysis of the amino acids in a calcium phosphate-rich material obtained after exhaustive proteolytic digestion of casein micelles suggests that these linkages involve the phosphate centres of the caseins.

Fast protein liquid chromatography was used to study the κ-casein fraction of casein micelles from bulk milk and from milk from individual animals homozygous for κ-caseins A and B. The extent of glycosylation of the κ-casein appeared to have no effect on its distribution in casein micelles of different sizes, nor did it affect the rate at which κ-casein was destroyed during renneting. The rate of breakdown of κ-casein during renneting was also almost independent of micellar size. The results may indicate a difference between methods which analyse for intact κ-casein or for the product macropeptide during renneting.

Purified bovine β-casein was digested in vitro with varying mixtures of purified proteinases and peptidases including trypsin, chymotrypsin, dipeptidyl peptidase IV (DP IV), aminopeptidase M and prolidase. In digestion mixtures without DP IV the yield of free amino acids was considerably lower than in the corresponding assays with this peptidase. Especially, the release of proline increases drastically from almost zero to the theoretical amount in the presence of DP IV. Quantitative results indicated that the specificities of the two microvillar peptidases (aminopeptidase M and DP IV) optimally complemented each other. This effect elucidates the hitherto obscure physiological role of intestinal DP IV. A similar effect may also apply to other caseins and nutritional proteins.

A sensitive, specific radioimmunoassay for bovine α-lactalbumin (α-la) suitable for measurement of serum, tissue culture media and milk was developed. α-La was not detected in serum from prepubertal male or female cattle, but was detected as early as 60 d of gestation in nulliparous Holstein heifers, the level being greatly increased during the last one third of gestation. Serum from cross bred beef heifers contained less α-la and it was not detected until late gestation. Concentrations of α-la in serum samples from pregnant multiparous Holstein cows decreased at drying-off and subsequently increased just before parturition. Secretion of α-la by mammary tissue explants from steroid-primed prepubertal Holstein heifers was induced by the addition of bovine prolactin, ovine prolactin or human growth hormone to tissue culture media.

Casein micelles in a reconstituted bovine skim milk were dissociated by dialysing them against various concentrations of EDTA. Gel chromatography showed a gradual but limited dissociation when micellar Ca was chelated and two main fractions were isolated. Monolayer technique and gel electrophoresis were used to characterize the interfacial properties and composition of the fractions. The first fraction contained highly cohesive complexes that did not unfold at the air-water interface. From π-A isotherms the close-packing area (A0) was 0·08 m2 mg-1 and was related to an average particle diameter of 9·1 nm. κ-Casein was concentrated in this fraction in an equimolar ratio with β-caseins and a variable ratio with αs-casein (0·55–1·10). These particles are thought to represent submicelles. The complexes of the second fraction were easily disrupted and spread out at the interface like casein monomers, with a close-packing area of 1·05 m2 mg-1. Two different complexes were identified in this last fraction. One of them contained an approximately equimolar ratio of αs- and β-caseins, and the other was composed exclusively of αs-casein. The results are discussed in relation to casein interactions leading to micelle formation.

High performance gel chromatography of the casein micelles disaggregated by 6 m-urea was carried out on a TSK-GEL G4000SW column using 6 M-urea synthetic milk serum as the effluent. The eluate was divided into two fractions. Fast eluted fraction 1 decreased from 67·5 to 57·3% on reduction of casein micelles and was not observed in reduced colloidal phosphate-free casein micelles. Fraction 1 from reduced casein micelles contained 1·7 times as much Ca and Pi bound to casein as did whole casein micelles, while fraction 2 essentially contained only bound Ca. These facts indicated that fraction 1 of reduced casein micelles consisted of the casein aggregates cross-linked by colloidal Ca phosphate (CCP). Polyacrylamide-gel electrophoresis showed that fraction 1 of reduced casein micelles contained more αs1- and αs2-caseins and less β-casein than whole micellar casein. No κ-casein was detected in fraction 1 of reduced casein micelles. It is suggested that the ester phosphate groups of casein are the sites for interaction with CCP.

The effect of heat treatment on the profiles of casein micelles obtained at 340 and 280 nm from controlled pore-glass chromatography of milk was studied in a series of unhomogenized whole milk samples which had been heated in a pilot plant at 75, 90, 105, 120, 135 and 150 °C for holding times of 4, 16, 32, 72, 150 and 300 s. A second experiment was carried out in which whole milk was preheated at temperatures between 70 and 110 °C for 13, 60 and 180 s before being treated at 120 °C for 4 s or 150 °C for 16 s. The average micelle size in some of the samples was determined by photon correlation spectroscopy. Profiles were divided into four fractions in which micelle size decreased from fraction 1 to fraction 4. The dimensions of these fractions were expressed in terms of size relative to that of the total profile. Fraction 4 was not affected by any of the heat treatments. Fractions 1–3 were significantly affected only by temperatures above 120 °C. Between 120 and 150 °C fraction 1 increased significantly while fractions 2 and 3 showed a corresponding decrease. Holding time affected fraction 1 only above 120 °C. For fraction 2 there was no effect in < 32 s at temperatures less than 150 °C. Fraction 3 was the most sensitive to changes in holding time, but no effect was observed below 105 °C. Average micelle size measurements indicated that a substantial size increase only occurred in those milks heated at 135 °C for longer than 16 s and in all the samples at 150 °C. To assess whether this increase in micelle size was reversible, samples were dialysed against a 70 mm-NaCl solution containing 10 mm-EDTA and then separated by column chromatography on Sephacryl S-300. The proportion of materials which was undissociated by dialysis remained unchanged for temperatures below 120 °C, but progressively increased with treatments more severe than 120 °C for 16 s. Column profiles were not influenced by variations in the conditions of preheating

Bovine whole milk was concentrated up to 5-fold by ultrafiltration and separated conventionally to produce protein enriched creams with fat contents ranging from 20 to 50 % (w/w). The measured viscosity of the concentrates increased with the degree of concentration. This led to inefficient centrifugal separation, as measured by the loss of fat into the skimmed milk, which was further worsened when damage to the fat globules occurred during ultrafiltration. The viscosity of the creams, at constant fat percentage, increased as the concentration factor of the milk from which it was separated rose.

AS the break-up of weak curd during processing leads to fines losses, the mechanical strength of acid casein curd is important. When different techniques were investigated to evaluate objectively the strength of acid casein curd a plate extrusion method was found to require the least preparation of the curd and to give rapid and reproducible results. The optimum condition for mechanical testing of samples was after acidulation or after cooling for 2h at 25 °C.

Whey protein complexed and became co-sedimentable with casein micelles after heating milk at ≥ 90°C for 10 min at pH ≤ 6·9 while at higher pH values (7·3) whey proteins and κ-casein-rich protein dissociated from the micelles on heating. κ-Casein-deficient micelles were more sensitive to heat, Ca2+ or ethanol than whey protein-coated or native micelles and were readily coagulable by rennet. Isolated κ-casein added to skim milk before preheating (90°C for 10 min) did not associate with the micelles at pH ≥ 6·9. Sodium dodecyl sulphate increased the level of both non-sedimentable N (NSN) and N-acetylneuraminic acid (NANA) and shifted the NSN-pH and NANA-pH curves to more acidic values while cetyltrimethylammonium bromide had the opposite effect. It is suggested that the pH-dependent dissociation in micellar κ-casein, which appears to be reversible, depends on the surface charge on the micelles; at a certain negative charge, disruption of hydrophobic and electrostatic forces could result in the dissociation of κ-casein from the casein micelles.

Three groups of coryneform bacteria isolated from the rind of nine soft and semi-hard cheese varieties, i.e. the orange, white and yellow strains, were studied with regard to morphological and physiological features. The 115 orange isolates of the Brevibacterium linens type formed only one homogeneous phenon. The 96 white and 161 yellow pigmented strains were grouped into 15 phena. Five phena (58% of the strains) belong to the species ‘B. ammoniagenes’, Arthrobacter variabilis and A. nicotianae. The other strains were arranged into five Arthrobacter spp. and four Corynebacterium spp. groups as well as a Rhodococcus sp. group. Usually specific forms for the individual cheese varieties were involved. In cheeses of poor surface-smear quality B. linens was found in reduced numbers.

The precipitation stage in acid casein manufacture controls the initial strength of the casein curd, which is an important factor in determining fines losses during processing. A plate extrusion method was used to determine the mechanical strength of casein curd, precipitated batchwise at temperatures from 25 to 53 °C over a pH range of 3·9 to 5·1. Curd strength was determined either immediately after acidulation or after cooling for 2 h at 25 °C. Curd was stronger immediately after acidulation than after cooling and increased in strength as precipitation temperature and pH were increased. Curd precipitated at high temperature and high pH was mechanically strong, had a large particle size, and had high total solids and Ca contents. The relationship between these curd properties is discussed, and it is concluded that Ca retention at high precipitation temperature and pH is largely responsible for the characteristic curd properties under these precipitation conditions.

The irreversible heat denaturation of α-lactalbumin (α-la) in 0·1 M-phosphate, pH 7·0, at 100 °C was studied using polyacrylamide-gel electrophoresis (PAGE). PAGE revealed two groups of bands, one moving faster than native α-la and one slower, in addition to some denatured protein which remained at the origin and some residual native α-la. The faster group had unchanged molecular weight, but an increase in charge, partly due to hydrolysis of glutamine and asparagine residues. The slower group was shown by two-dimensional sodium dodecyl sulphate-PAGE to be oligomers of denatured α-la; formation of the smaller oligomers preceded the larger ones. The oligomers reverted to monomers in the presence of dithiothreitol, showing that they were disulphide-linked aggregates of denatured α-la. Immuno-blots of the gels showed that both fast and slow groups of bands had irreversibly lost most of the antigenicity of the native protein.

Taking as an example a tryptic hydrolysate of bovine β-casein, it was shown that the method used for the determination of optimal elution conditions for isocratic systems also applied to the separation of complex peptide mixtures by reversed-phase high-performance liquid chromatography with linear gradients. Using a C18 column and the same solvents, successive studies of the influence of flow rate, pH and temperature allowed a satisfactory separation of the sample in less than 30 min. Valuable information on the specificity of the action of trypsin on β-casein was deduced from the yield of the eluted peptides.

A typing scheme was used to test 15 strains of Streptococcus uberis according to their production of (P-type) and sensitivity to (S-type) bacteriocin-like inhibitory substances. Twelve of the strains were inhibitor producers and nine different P-types were detected. All of the strains were typable according to inhibitor sensitivity, ten different S-types being distinguished. Both the P-type and S-type designations of the strains were reproducible on repeated testing. By combination of P-typing and S-typing, highly discriminatory inhibitor ‘fingerprints’ of the strains could be obtained. This scheme would appear to have considerable potential for typing isolates of Str. uberis as an aid to investigations into the epidemiology of Str. uberis mastitis in dairy cattle.

The effect of heating on plasmin activity in various media, including phosphate buffer pH 7·0, skim milk, blood plasma, solutions of casein and solutions of whey proteins were investigated. Plots of log residual activity υ. heating time were linear at all temperatures from 63 to 143 °C. In buffer solutions the presence of casein led to substantial substrate protection, the Arrhenius plots being linear both in the presence and absence of casein. The activation energy, Ea, for the inactivation reaction, was 62·4 kJ/mol in buffer alone and 58·4 kJ/mol with casein present at 25 mg/ml. In skim milk, despite the presence of casein at a similar concentration, plasmin was no more stable to heat than in buffer alone, and a curved Arrhenius plot was obtained indicating a more complex inactivation mechanism. Heating in the presence of proteins having free -SH groups accelerated the inactivation of plasmin. The role of -SH groups was confirmed by experiments with added α-lactalbumin, in which no free -SH groups occur, and reduced carboxymethylated β-lactoglobulin, both of which were without effect. In blood plasma, plasmin was less stable to heat than in buffer (pH 7·0) or in skim milk. Plasminogen behaved very similarly to plasmin either when activated to plasmin with urokinase before heating or when activated afterwards. A hypothesis is presented to describe the heat inactivation and denaturation of plasmin. Technologically important findings are that in skim milk plasmin was largely unaffected by pasteurization conditions and 30–40% of its activity remained even after ultra high temperature processing conditions.

Investigation of the effect of pH on the formation of deposit from milk during ultra high temperature treatment using a plate-type plant showed that deposit formation was greatly increased when the pH of whole milk was reduced to 6·54, irrespective of whether the adjustment was made through the addition of HCl or lactic acid. Most of the increase in deposition took place in the higher temperature sections of the plant. Conversely, an increase in milk pH to 6·8 using NaOH resulted in considerably less deposit being formed during heat treatment. Reducing the pH of whole milk increased the deposition of both protein and fat, but reduced the deposition of minerals. Despite very high concentration of fat in the deposits, it is unlikely that fat per se was responsible for increased deposit formation. Deposition also increased when the pH of skim milk was reduced to 6·51 before processing. Electron micrographs of the milks after heat treatment indicated that pH reductions caused the formation of large aggregates containing casein micelles during heating. Fat globules were also present in aggregates formed in whole milk with reduced pH. Slight reductions in the pH of milk before processing appear to enable the pH during heat treatment to fall below a critical value at which coagulation of milk takes place at the heated surfaces.

The report that addition of KI03 (0·1 mm) to milk before ultra high temperature (UHT) treatment induces extensive proteolysis during subsequent storage at 37 °C was confirmed. None was produced by addition of H202 KMn04 or K2Cr207. The pH optimum for KI03-induced proteolysis was between 7·0 and 8·0 and the temperature optimum 37—45 °C. β-Casein was particularly susceptible and the proteolysis pattern was similar to that caused by indigenous alkaline milk proteinase (MPA, plasmin). Addition of plasmin to milk before UHT treatment (140 °C/10 s) caused slight proteolysis during subsequent storage but addition of 0·1 mm-KI03 and plasmin caused extensive proteolysis which was prevented by addition of soyabean trypsin inhibitor, indicating the probable involvement of plasmin in KI03-induced proteolysis in UHT-treated milk. Equally extensive proteolysis occurred in serum protein-free casein micelle systems (SPFCM), with or without KI03, during storage at 37 °C following UHT treatment, indicating a role for whey proteins in KI03-induced proteolysis. Addition of β-lactoglobulin (β-lg) to a SPFCM system inhibited proteolysis, but extensive proteolysis occurred in a SPFCM system containing both β-lg and KI03. MPA-free Na caseinate (prepared by heating at 140 °C for 7 min) underwent extensive proteolysis when treated with plasmin before UHT treatment; proteolysis was inhibited by addition of °-lg to this system and KI03 reversed the inhibitory effect of β-lg. Plasmin proteolysis of isolated αs1-casein was inhibited by denatured β-lg (90 °C/30 min) at a level of 4 mg/ml but not by native β-lg. When denatured in the presence of KI03, β-lg had a lower free SH content than the control and was less inhibitory for plasmin in proteolysis of isolated αsl-casein. The results show that denatured β-lg inhibits plasmin proteolysis of caseins in UHT milk and that inhibition is prevented by KI03. This inhibition may occur via thiol–disulphide interchange, which is prevented if the SH group of ²-lg is oxidized by KI03, thus permitting the stimulatory effect of KI03 on proteolysis in UHT-treated milk.