Six Friesian cows were milked daily, and arterial and mammary venous blood samples were taken between day 266 of gestation and day 6 after parturition. Samples of blood plasma were analysed for their content of acetate and progesterone, and mammary secretion for potassium, isocitrate, 2-oxoglutarate and triglyceride fatty acids.

The onset of secretion of medium-chain triglyceride fatty acids was preceded by, or coincided with, increases of potassium and isocitrate concentration in the secretion. The onset of fatty acid secretion was not accompanied by any change in the gland's extraction of acetate from the circulation, and did not occur at a consistent time relative to parturition or changes in plasma progesterone concentration.

Cells were obtained by centrifuging the mammary secretion of healthy udders of 19 cows during the dry-period and during mid-lactation. The suspended cells were incubated in plastic wells. Those adhered cells classified as mammary macrophages were incubated with pokeweed mitogen (PWM). Autologous peripheral blood lymphocytes were added to wells containing untreated macrophage cultures or cultures pretreated with PWM. In seven cows autologous dry-period mammary lymphocytes were added instead of blood lymphocytes. The macrophages + lymphocyte cultures were subjected to the lymphocyte stimulation test (LST). For comparison, peripheral blood lymphocytes and dry-period secretion lymphocytes were also subjected to the LST in the presence of PWM. In all cases, mitogenic responses were higher in pretreated macrophage cultures than in background control cultures.

The stimulation indices (SI) showed that PWM-pretreated dry-period mammary macrophages enhanced the proliferation of autologous peripheral blood lymphocytes to a greater extent than did blood lymphoeytes plus PWM (49±10 v. 30 ± 6; P ≤ 0·05). Mammary macrophages taken from the same cows but during midlactation also clearly induced proliferation of autologous peripheral blood lymphocytes but to a lesser extent than dry-period macrophages (16 ± 2 v. 49±10; 16±2 v. 30±6; P ≤ 0·01 and P ≤ 0·05).

The PWM pretreatment of mammary macrophages increascd the proliferation of autologous dry-period mammary lymphocytes by at least a factor of three (28±8 v. 8±2 P ≤ O·05).

The present results indicate that bovine mammary macrophages pretreated with PWM enhance proliferation as well as modulation of mammary and peripheral blood lymphocytes. The modulation of lymphocyte stimulation as demonstrated here in vitro, has great significance regarding aspects of local immunostimulation related to modern treatment of mastitis.

Daily quarter-milk yields of eight high-yielding cows (24–28 kg/d) and eight heifers (14–24 kg/d) were measured to examine compensatory changes in milk production between quarters within an udder. Either one, two or three quarters per cow were left unmilked for 12 d, in early or late lactation, and then all quarters of all cows were milked normally for a further period of 12 d. Concentrate feeding levels were constant throughout the experiment.

The mean daily yield per cow fell to 26, 59 and 75% during the period of milking either one, two or three quarters respectively. Twelve days after resumption of normal milking the total daily yield per cow was the same for cows continuously milked in two, three or all four quarters. Daily yield in cows with only one quarter milked continuously recovered to only 78% of the level of the pretreatment period.

During the treatment period, the mean daily yield of the continuously milked quarters increased by almost 14% for cows milked in one quarter only, by more than 10% for two quarters milked, and by 4% per quarter if three quarters were milked. Milk yields of these quarters remained above their pretreatment levels when milking was resumed in the adjacent quarters.

There was no difference in the compensatory effect between cows and heifers in early lactation, but the compensatory effects were lower in late lactation heifers.

The effect of supplementing a normal dairy feed ration (3–4% fatty acid of DM) with saponified palm acid oil (soap) was investigated in two experiments. The first experiment was carried out in eight herds (four Jersey, four heavy breeds), where the diet was supplemented with 1·0 and 0·5 kg soap for cows in the first and last part of lactation, respectively. Taste and acid degree of milk were not affected, and no breed × soap interaction was found. Fatty acid composition in milk was only slightly but significantly affected. The content of short and medium chain fatty acids was reduced (3·5% unit) and was followed by an increase in the content of 16:0 and 18:1. The increase in 16:0 by the soap supplementation was smaller in Jersey cows than in the other breeds. In the second experiment the effect of supplementing diets either with 0·5 or 1·0 kg Ca-soap to cows in the first part of lactation was investigated in two herds of Danish Black and White cows. Acid degree and time for curd formation on renneting (K20) were significantly lowest at the highest soap inclusion. No general effect on the proportion of short and medium chain fatty acids in the milk fat was observed as a function of level of Ca-soap, but the proportion of 16:0 was increased. It is assumed that the very small effect on milk fatty acid composition obtained when the daily allowance of fatty acids was increased from 600 to ~ 1400 g per cow, is more or less a specific effect of dietary fat rich in palmitic acid and given in excess to the basic diet.

Two cleaning-in-place disinfectant Systems (alkaline–acid, alkaline–CI2) were tested on wild bacteria isolated from a milking line. Most of the isolated strains were Gram-negative and capsule-positive. The effect of milk and cream on the resistance of the wild strains to the cleaning Systems was tested. Suspension tests did not show the resistance of bacteria whereas the surface test showed high resistance towards all cleaning-agents. Milk had a protective effect only on the surfaces whereas cream showed mild protection in suspension and complete protection on the surfaces. It was concluded that the growth pattern of contaminants on surfaces needs further attention and the laboratory suspension tests do not correlate with the practical situation. Disinfectant tests based on bacterial growth on the surface are of importance.

Variations in chemical composition and in some physico-chemical properties of milk from a herd of lactating Alpine goats imported into Greece were determined from week 8 after parturition until the end of lactation (35 weeks, 35 samples). The average composition (g/100 g) of the milk was: fat 3·44, protein 3·35, casein 2·46, lactose 4·30, ash 0·79, total solids 11·76 and solids-not-fat 8·32. The average pH was 6·57 and titratable acidity was 16·76 (°D). The averaged density was 1·030. The mean mineral contents (mg/100 g) of the milk were: Na 53·12, K 157·67, Ca 140·06, P 110·00, Mg 13·59, citrate 106·97. All constituents studied as well as pH and density were significantly (P < 0·05) dependent on the stage of lactation. Titratable acidity was not significantly affected by stages of lactation. There were significant (P < 0·001) differences in Na/K and Ca/P ratios in the milk between lactation stages. Trends with lactation for each of the components and physico-chemical properties determined are reported and correlations between constituents in the milk are given.

In milk samples from 549 cows of the breeds Danish Jersey, Red Danish Dairy Cattle (RDM), and Black and White Danish Dairy Cattle (SDM) the genetic polymorphisms of the αs1, β and K-cascin and β-lactoglobulin (β-Lg) loci were determined by isoelectric focusing in agarose gels. The results of the screening were comparcd with results obtained by Larsen & Thymann (1966). In addition, the genetic linkage of the three casein loci was studied, and the association between milk protein genotypes and yields in first and second lactations of milk, fat and protein were investigated.

The distribution of genotypes of all four milk protein Systems was different from breed to breed.

For Jersey cows, significant differences in the gene frequencies from the results of the 1966 investigation were found for αs1 and K-casein and β-Lg. For SDM cows a change in the K-casein frequency had occurred whereas for RDM cows no changes were found.

Linkage between some of the casein loci was found within ail three breeds. For the RDM breed the possible linkage between αs1-casein and the other caseins could not be tested bccause nearly ail thc cows were homozygous for the αs1-cascin-B genotypes.

β-Casein genotypes were associated with yield parameters in ail breeds. The A2A2 genotype of this protein gave higher yields of milk, fat, and protein in the second lactation than thc A1A1 genotype.

The separation of bovine milk proteins by fast protein liquid chromatography has been studied by ion-exchange chromatography on Mono S and Mono Q columns. The use of a non-ionic detergent, octyl-glucoside, made possible the separation of the four major casein components (αs1, αs2, β and κ) as well as minor caseins on the Mono S column using urea-containing buffers at pH 3·5. There was, however, considerable overlap between the αs1 and αs2-casein peaks. A good resolution was obtained with a low concentration of urea (3·5 M) and detergent (0·1%). These conditions allowed the separation of casein components in their native state in less than 50 min. The purity of isolated κ-casein was further improved by a second separation on a Mono Q column.

The effect of arginine on the production of extracellular lipase by Pseudomonas fluorescens 32A was studied. Arginine repressed lipase production when N was supplied partially or totally as arginine. Proteinase production was unaffected under the same conditions. Arginine did not repress lipase secretion when cells were pregrown in an arginine-containing medium; however, repression was found with uninduced cells. Several arginine-analogues were tested for the ability to repress lipase secretion and. of these, L-homoarginine and L-canavanine were the most effective. D-Arginine, L-arginine methyl ester, and L-ornithine did not cause repression. With the exception of glutamic acid and methionine, those amino acids that supplied N but not C for growth were found to repress lipase secretion. It is suggested that the accumulation of metabolic intermediate(s) may cause repression of lipase production.

The present study identifies proline-specific peptidases found in Streptococcus cremoris AM2. These activities were present prcdominantly in the cytoplasmic fraction of the cells with minor amounts in cell membrane fraction. No evidence vvas found for such activities in either the extracellular fluid used to grow the cells, in the wash fraction or in the cell wall fraction. The involvement of these enzymes in release of free proline from proline-containing peptides is considered.

The present study describes the purification of a post-proline dipeptidyl aminopeptidase from the cytoplasm of Streptococcus cremoris AM2. On the basis of its elution from a calibrated Sephadex G200 column, the enzyme had a molecular weight of 117000 and exhibited a broad pH optimum activity between 6·0 and 9·0. The activity was most comprehensively inhibited by phenylmethylsulphonylfluoride and more modestly inhibited by 1,10-phenanthroline and 8-hydroxyquinoline but not by EDTA. A range of peptides containing either proline or alanine as the penultimate amino acid residue could act as substrates. The presence of proline on the carboxy side of the scissile bond prevented hydrolysis. However the enzyme could release Pro-Pro from Pro-Pro-Gly-Phe-Ser-Pro. The significance of this substrate specificity is considered in the context of removal of either single proline residues or prolylproline sequences from oligopeptides during cheese ripening.

The formation of hydrophobic sites on the surface of casein micelles as a consequence of rennet action has been followed through the binding rate of a fluorescent probe and its distribution between a free and an ‘aggregated’ protein fraction. The variation of this parameter has been related to clotting time and curd hardening kinetics.

Results show that a first aggregation of casein through hydrophobic sites interaction began as soon as rennet was added to milk. At the natural pH of milk, the sol-gel transition occurred when all the casein micelles were already involved in large aggregates. This was not the case with slightly acidified milk (pH 6·5 and 6·3) where clotting occurred well before the first aggregation step had been completed. The surface hydrophobicity of casein continued to increase in the curd due to continuing enzymic action and structural rearrangements. When this process has been completed the hardening of curd proceeds at an accelerated pace until it reached its maximum asymptotic value.

The effects of chromatographically pure bovine chymosin and pepsin A on cheese curd firmness have been investigated. The enzymes were studied in parallel experiments, using a Formagraph, under which the following parameters were tested and varied: reconstituted skim milk powder, bulk raw milk, CaCl2 addition, enzyme concentration and temperature. Chymosin had a slightly better rate of curd firming (P ≤ 0·001) and better curd firmness 30 min after gelling point compared to pepsin under all of the conditions investigated. However, as regards curd firmness in cheese manufacture, this difference is only a question of time, since pepsin will give the same degree of curd firmness of the cheesemilk as chymosin after some minutes. The biggest disadvantage with pepsin is its strong pH dependence, which can lead to very long gelling times and weak curd if the pH is too high and the rennet is dominated by pepsin.

The influence of chromatographically pure bovine chymosin and pepsin A on cheese curd syneresis has been studied. The enzymes were compared in experiments using a microscale syneresis assay, where the light absorbance of the released whey was used as a measurement of the syneresis. The following parameters were investigated: reconstituted skim milk powder (RSM), bulk raw milk, CaCl2 addition and cutting time of the curd. The gelling (clotting) time was held constant under all experimental conditions to eliminate its influence on syneresis. The differences in influence on syneresis of the two enzymes were neither obvious nor uniform. As regards the syneresis of RSM, the influence of chymosin and pepsin seemed to be very similar, while the syneresis of bulk raw milk seemed to be more enhanced by chymosin. However, results were not sufficiently clear to prove any distinct differences between the enzymes. As long as the cheese curd is cut at a proper firmness, there are probably no practical problems in using bovine rennets with varying chymosin: pepsin ratio.

Twenty Lactococcus lactis subsp. lactis strains isolated from cheese made from raw milk have been, studied and compared with well known starter strains. Many of them produced acid bacteriocins resembling those of other lactic streptococci and their patterns of antimicrobial agent susceptibility were also similar to previously studied dairy strains. On the basis of their ability to clot milk they could be divided into two groups. Some strains were rapid fermenters, with high β-phosphogalactosidase and proteolytic activities. The other group had low β-phosphogalactosidase activity and coagulated milk slowly, but seemed to have proteolytic activity. All the strains had plasmid DNA in amounts and sizes similar to starter strains. A β-phosphogalactosidase probe was constructed with plasmid DNA from a Lc. lactis subsp. lactis NCDO 712 derivative. Southern hybridization located homologous sequences in chromosomal and plasmid DNA from the wild isolates that fermented lactose efficiently, but the degree of homology was lower than that observed between starter strains.

Although proteolysis is regarded as the most important biochemical event in the ripening of most cheese varieties (Rank et al. 1985; Grappin et al. 1985; Law, 1987), current methods for the fractionation of cheese N, yield very heterogeneous fractions (Rank el al. 1985; Grappin et al. 1985; Fox, 1989). This communication describes the results to date of a study undertaken to develop a comprehensive scheme for the fractionation of cheese N.

Lactodynamographic analysis is commonly employed to evaluate the clotting aptitude of milk (Frentz, 1965; Tarodo de la Fuente et al. 1969). The individual lactodynamographic response can therefore be used in selection as a quantitative trait. The aim of this work was to estimate either the biological repeatability of the lactodynamographic response or the repeatability of the analytical method itself.

Milk contains indigenous plasmin-like and thrombin-like proteinases and also exogenous proteinases, mainly extracellular enzymes of psychrotrophic bacteria. Their activities lead to protein hydrolysis which may have important effects on milk quality and cheese yield. It is also useful to estimate proteolysis and its development during cheese ripening.

Milk opacity or turbidity due to calcium phosphocaseinate micelles and fat globules necessitates preliminary sample treatment, i.e. precipitation and filtration before spectrophotometric measurement. It is possible to eliminate these firs steps of sample treatment by dispersing the colloidal constituents. Some authors have already used different dissolving mixtures for sample analysis. Nakai & Anh Chi Le (1970) were the first to determine protein contents of milk and dairy products by absorbance measurement at 280 nm after clarification. Bosset & Blanc (1977) used the Biuret method. Owen & Andrews (1984) measured free amino groups with 2,4,6-trinitrobenzene sulphonic acid (TNBS). Humbert et al. (1982) and Stead (1987) used synthetic or specific substrates for proteinase determination.

In this work, we report the application of a new dissolving reagent to measure free amino groups in milk and cheese.

Titration is an old method used for the determination of acidic and basic groups of a molecule. Several metabolic end-products in cheese are compounds with acidic or basic groups, e.g. propionic, acetic and lactic acids, amino acids, amines and ammonia. A number of colorimetric and fluorometric methods have been used to study proteolysis in cheese; some are time-consuming with several steps, and certain of the reagents are hazardous.

We have used acid-base titration for analysis of the products of proteolysis in Swiss-type cheese. The titration system was that originally developed for use in silage analysis (Moisio & Heikonen, 1989) and applied to cheese it has proved to be useful, easy and rapid with an accuracy similar to that achieved by colorimetry.