Proteolysis in aseptic, chemically acidified (GDL) cheese and in starter cheese made under controlled bacteriological conditions (i.e. free of non-starter micro-organisms) was measured by gel electrophoresis, the formation of pH 4·6- and 12% TCA-soluble N, gel filtration and the liberation of free amino acids. The results show that rennet was mainly responsible for the level of proteolysis detected by gel electrophoresis, pH 4·6-soluble N and gel filtration i.e. large, medium and small peptides. However, rennet alone was capable of producing only a limited range of free amino acids; only methionine, histidine, glycine, serine and glutamic acid were produced at quantifiable levels (> 0·2 μmoles/g) in GDL cheese; it is suggested that free amino acids in Cheddar cheese are mainly the result of microbial peptidase activity. The levels of free amino acids in the starter cheese were considerably lower than values reported for commercial Cheddar.

The effects of heat treatment of milk, and a range of rennet and glucono-δ-lactone (GDL) concentrations on the rheological properties, at small and large deformation, of milk gels were investigated. Gels were made from reconstituted skim milk at 30 °C, with two levels each of rennet and GDL. Together with controls this gave a total of sixteen gelation conditions, eight for unheated and eight for heated milk. Acid gels made from unheated milks had low storage moduli (G′) of < 20 Pa. Heating milks at 80 °C for 30 min resulted in a large increase in the G′ value of acid gels. Rennet-induced gels made from unheated milk had G′ values in the range ∼ 80–190 Pa. However, heat treatment severely impaired rennet coagulation: no gel was formed at low rennet levels and only a very weak gel was formed at high levels. In gels made with a combination of rennet and GDL unusual rheological behaviour was observed. After gelation, G′ initially increased rapidly but then remained steady or even decreased, and at long ageing times G′ values increased moderately or remained low. The loss tangent (tan δ) of acid gels made from heated milk increased after gelation to attain a maximum at pH ∼ 5·1 but no maximum was observed in gels made from unheated milk. Gels made by a combination of rennet and GDL also exhibited a maximum in tan δ, indicating increased relaxation behaviour of the protein–protein bonds. We suggest that this maximum in tan δ was caused by a loosening of the intermolecular forces in casein particles caused by solubilization of colloidal calcium phosphate. We also suggest that in combination gels made from unheated milk a low value for the fracture stress and a high tan δ during gelation indicated an increased susceptibility of the network to excessive large scale rearrangements. In contrast, combination gels made from heated milk formed firmer gels crosslinked by denatured whey proteins and underwent fewer large scale rearrangements.

The consumption of food and beverages containing high amounts of biogenic amines (BA) can have toxicological effects. BA found in foods and beverages are synthesized by the microbial decarboxylation of certain amino acids. This paper reports the concentrations of BAs in a number of commercial cheeses, as determined by HPLC. The cheeses studied were made from raw and pasteurized milk of different origin, and were subjected to different ripening periods. BA concentrations were lower in short ripening period than in long ripening period cheeses, and higher in cheeses made from raw milk than in those made from pasteurized milk. The highest BA concentrations were recorded in blue cheeses made from raw milk. Tyramine was the most commonly recorded and abundant BA. The presence of tyramine-producing bacteria was determined by PCR, and a good correlation obtained between the results of this method and tyramine detection by HPLC. These methods could be used to complement one another in the detection and quantification of tyramine in cheese prevention of tyramine accumulation in cheese.

Milks were prepared at 1·7- to 4-fold the initial concentration by combining skim-milk concentrated by ultrafiltration with cream, and used for Cheddar cheese-making. Starter growth was unaffected, but the increased buffering capacity in the more concentrated milks resulted in a slower decline in pH and a higher pH value in the cheese. Curd formation was faster despite the use of reduced amounts of rennet. With milk concentrated more than 2-fold, large amounts of fat were lost in the whey, so that the cheeses had less fat than normally. Fat losses may be partly related to the lower degree of aggregation of the casein micelles when the curd was cut. As the concentration factor of the milk increased, the rate of casein breakdown, the intensity of Cheddar flavour, and the levels of H2S and methanethiol in the cheese decreased. These factors may relate to the reduced concentration of active rennet retained in the curd at pressing.

The aim of the present study was to examine how different types of early interaction between dairy cows and their calves influence milking/suckling-related hormone release in the cows and sucking/bucket-drinking related hormone release in calves. Eighteen cows of the Swedish Red and White breed were studied during the first week after parturition. The cows were machine milked twice daily, and allotted to one of three treatments: [1] cow and calf were kept together and the cow was allowed to suckle the calf for 30 min about 1 h before each milking; [2] cow and calf were kept together and the calf was bucket fed twice daily; and [3] cow and calf were separated immediately after parturition, and the calf was kept in a single box and was bucket fed twice daily. Blood samples were collected around day 7 from both cows and calves. The plasma levels of oxytocin, prolactin, cortisol, gastrin, cholecystokinin (CCK), somatostatin and insulin were analysed. In the cows the levels of oxytocin, prolactin and cortisol were influenced by all three treatments, except for the level of cortisol which did not respond to suckling. The main finding was that the release of oxytocin was significantly greater during suckling compared with machine milking. In the calves, the hormone levels were also influenced by the different milk feeding routines. The plasma concentrations of oxytocin, gastrin, CCK and insulin increased in response to milk ingestion in all treatments. However, during sucking, the increase of oxytocin was significantly greater than during bucket drinking. In addition, a strong correlation between oxytocin and insulin was found in response to sucking. Further, significant increases in prolactin and somatostatin, and a decrease in cortisol were found during sucking. The level of somatostatin also increased in response to bucket feeding when calves were kept separately. During bucket feeding, no significant correlation was found with oxytocin, but strong correlations between the gastrointestinal hormones gastrin, CCK, somatostatin and insulin were seen. Together these data suggest that different hormonal patterns were triggered in the cows by suckling and milking, and in the calves by sucking and bucket drinking. This is further supported by different correlation patterns observed in the calves in response to sucking and bucket feeding. The present findings imply that management routines for cows and calves during the first week after parturition have consequences for the physiology of the animals.

Twenty-nine multiparous cows of each of the Jersey and Friesian breeds, all κ-casein AB phenotype, were grazed together and managed identically. On three occasions during 10 d in spring (early lactation), milk was collected from all cows at four consecutive milkings and bulked according to breed. On a separate occasion, milk samples were also collected from each cow at consecutive a.m. and p.m. milkings to form one daily sample per cow. The bulked milks (800–1000 l per breed on each occasion) were standardized to a protein[ratio ]fat (P[ratio ]F) ratio of 0·80, and 350 l from each breed was made into Cheddar cheese. The solids content of the remaining Friesian milk was then increased by ultrafiltration to a solids concentration equal to that of the Jersey milk. This solids-standardized Friesian milk and a replicate batch of P[ratio ]F standardized Jersey milk were made into two further batches of Cheddar cheese in 350-l vats. Compared with Friesian milk, Jersey milk had higher concentrations of most milk components measured, including protein, casein and fat. There were few difference in milk protein composition between breeds, but there were differences in fat composition. Friesian milk fat had more conjugated linoleic acid (CLA) than Jersey milk fat. Jersey milk coagulated faster and formed firmer curd than Friesian milk. Concentrations of some milk components were correlated with coagulation parameters, but relationships did not allow prediction of cheesemaking potential. Jersey milk yielded 10% more cheese per kg than Friesian milk using P[ratio ]F standardized milk, but for milks with the same solids concentration there were no differences in cheese yield. No differences in cheese composition between breeds were detected. Differences in cheesemaking properties of milk from Jerseys and Friesians were entirely related to the concentrations of solids in the original milk.

Whey protein concentrate (WPC) solutions containing 10, 30, 60 and 120 g dry powder/kg were heated at 75°C and whey protein aggregation was studied by following the changes in the distribution of β-lactoglobulin, α-lactalbumin and bovine serum albumin, using one dimensional and two dimensional PAGE. The one dimensional PAGE results showed that a minimal quantity of large aggregates was formed when 10 g WPC/kg solutions were heated at 75°C for up to 16 min whereas appreciable quantities were formed when 30, 60 and 120 g WPC/kg solutions were similarly treated. The two dimensional PAGE analysis showed that some disulphide-linked β-lactoglobulin dimers were present in heated 10 g WPC/kg solution, but very little was present in heated 120 g WPC/kg solution. By contrast, SDS was able to dissociate monomeric protein from high molecular mass aggregates in heated WPC solution of 120 g/kg but not in 10 g WPC/kg solution heated for 30 min. The rates of loss of native-like and SDS-monomeric β-lactoglobulin, α-lactalbumin and bovine serum albumin during heating increased as the WPC concentration was increased from 10 to 120 g/kg. In 120 g WPC/kg solution heated at 75°C, the amounts of SDS-monomeric β-lactoglobulin in each sample were greater than the quantities of native-like protein. However, in WPC solutions of 10, 30 and 60 g/kg, the differences between the amounts of native-like and SDS-monomeric proteins were slight. The loss of the native-like or SDS-monomeric proteins was consistent with a first or second order reaction. In each case, the apparent reaction rate constant appeared to be concentration-dependent, suggesting a change of aggregation mechanism in the more concentrated solutions. Overall, these results indicate that in addition to disulphide-linked aggregates, hydrophobic aggregates involving β-lactoglobulin, α-lactalbumin and bovine serum albumin were formed in heated WPC solution at high protein concentration, as suggested by model studies using binary mixtures of these proteins.

Rennin acts on a specially sensitive bond in αs1 B-casein to produce a basic peptide containing residues 1–23 of the original protein. At pH 6·4 and 30°C, the action is specific and rapid, the kinetic constants being Km 4·5×10−4M, Kcat 3·8 s−1, and kcat/Km 0·85×104 s−1 M−1. Pepsin, and a protease impurity in the acid phosphatase from wheat germ, have a similar action.

Preheating milk at 140 °C for 1 min at pH 6·6, 6·8, 7·0 or 7·2 shifted the heat coagulation time (HCT)/pH profile to acidic values without significantly affecting the maximum stability. Whey proteins (both β-lactoglobulin and α-lactalbumin) co-sedimented with the casein micelles after heating milk at pH < 6·9 and the whey protein-coated micelles, dispersed in milk ultrafiltrate, showed characteristic maxima–minima in their HCT/pH profile. Heating milk at higher pH values (> 6·9) resulted in the dissociation of whey proteins and κ-casein-rich protein from the micelles and the residual micelles were unstable, without a maximum–minimum in the HCT/pH profile. Preformed whey protein–casein micelle complexes formed by preheating (140 °C for 1 min) milk at pH 6·7 dissociated from the micelles on reheating (140 °C for 1 min) at pH > 6·9. The dissociation of micellar-κ-casein, perhaps complexed with whey proteins, may reduce the micellar zeta potential at pH ≃ 6·9 sufficiently to cause a minimum in the HCT/pH profile of milk.

Probiotic microorganisms, especially lactic acid bacteria, are effective in the treatment of infectious diarrhoeal diseases and experimental colitis. Although the mechanisms by which these organisms exert their anti-inflammatory effects are largely unknown, immunomodulating effects are suggested. The objective of this study was to examine the effect of a 5-week oral administration of Lactobacillus rhamnosus subspecies GG (Lb. GG) on the cellular immune response to intestinal microorganisms in ten healthy volunteers. Peripheral blood cells (PB) were stimulated with either ‘self’ or ‘non-self’ preparations of faecal samples and isolated Bacteroides fragilis group-organisms (Bfg) or Escherichia coli (Esch. coli), and pro- and anti-inflammatory cytokines (IL-10, IL-4, IL-6, IFN-γ, TNF-α) were measured in the culture supernatant. CD4+ T-lymphocyte activation was determined by measurement of intracellular ATP following lysis of the cells. The activational response of CD4+ T-lymphocytes towards isolated and heat-inactivated intestinal organisms was increased after the probiotic treatment. Additionally, TNF-α, IL-6 and in part IFN-γ cytokine secretion by PB cells following stimulation with whole stool preparations and single members of the flora was significantly decreased, whereas the IL-10 and in part IL-4 cytokine secretion was increased at the end of the study. In contrast, the activational response of CD4+ T-lymphocytes following stimulation with whole ‘non-self’ intestinal flora was higher than by ‘self’ intestinal flora, but both responses showed a trend towards a reduction at the end of the study. This study documents a direct effect by Lb. GG on the cellular immune system of healthy volunteers and offers a promising tool to investigate systemic immunomodulation due to oral administration of probiotic microorganisms.

The protective effect of feeding milk fermented with a mixture of Lactobacillus casei sp. and Lb. acidophilus sp. against Salmonella typhimurium infection in mice was compared with that obtained feeding milks fermented with these microorganisms individually. The survival rate obtained after oral infection with Sal. typhimurium was 100% in mice pretreated by feeding during 8 d with the mixture of Lb. casei and Lb. acidophilus fermented milks. Similar treatments with the individual milks were ineffective. Moreover, mice became more susceptible to infection with Sal. typhimurium after such treatment. The colonization of liver and spleen with the pathogen was markedly inhibited by the pretreatment with the mixture of fermented milk, while such inhibition was not observed using the Lb. casei and Lb. acidophilus milks. The highest levels of anti-salmonellae antibodies in serum and in intestinal fluid were found in the group of mice fed with the mixture and with Lb. casei fermented milk respectively. However, this latter milk was not effective in protecting against Sal. typhimurium. When the mice were first infected with Sal. typhimurium and then fed with the mixture of fermented milks, pathogen colonization was not prevented. The results suggest that the augmentation of resistance to salmonellae caused by the treatment with Lb. casei-+Lb. acidophilus-fermented milk was due to the anti-salmonellae protective immunity mainly mediated by the mucosal tissue. Milk fermented with this mixture could be used as an immunobiological method to prevent gastrointestinal infection.

The pressure-induced (100–400 MPa) changes in casein micelles from bovine, caprine and ovine milks were investigated by studying the distribution of minerals and proteins after separation of the micellar and serum phases by ultracentrifugation. The results showed that pressurization markedly increased the levels of non-sedimentable casein in the three species, as well as the levels of Ca, P and Mg in the serum, but led to only small increases in the Ca2+ concentration. The dissociation rates of individual caseins corresponded to the ester phosphate contents, in the order κ-casein, β-casein>αs1-casein >αs2-casein. The concentration of non-sedimentable casein was highest in pressurized ovine milk, which also contained higher levels of pressure-liberated serum minerals than the milks from the other two species. In the case of bovine and caprine milks, maximum dissociation from the micelle was found in milks treated at 300 MPa, while in ewes' milk dissociation increased with pressure up to 400 MPa. Denatured β-lactoglobulin (β-lg) was present in the ultracentrifugation pellets of the pressurized milks of the three species, but pressure-denatured β-lg also remained in the soluble fraction. Despite the increases in the content of soluble proteins, all casein and most β-lg were incorporated in the rennet curds of the pressurized milks from the three species.

Several peptides were isolated from the diafiltration retentate, prepared using 10 kDa membranes, of the water-soluble extract from a commercial mature Cheddar cheese and identified by amino acid sequencing and mass spectrometry. Most of the peptides were from the N-terminal half of β-casein, but peptides from αs1- and αs2-caseins were also identified; the extract also contained α-lactalbumin. Identified peptides showed the important role played by lactococcal cell envelope proteinases in the degradation of primary proteolytic products from αs1- and β- caseins, produced by chymosin and plasmin respectively. Plasmin seemed to be involved in the hydrolysis of αs2-casein. Several phosphopeptides were identified and the action of phosphatase on these peptides was evident.

We have developed a method to quantify the resistance to freezing and frozen storage of lactic acid starters, based on measuring the time necessary to reach the maximum acidification rate in milk (tm) using the Cinac system. Depending on the operating conditions, tm increased during the freezing step and storage. The loss of acidification activity during freezing was quantified by the difference (Δtm) between the tm values of the concentrated cell suspension before and after freezing. During storage at −20 °C, linear relationships between tm and the storage time were established. Their slope, k, allowed the quantitation of the decrease in acidification activity during 9–14 weeks of frozen storage. The method was applied to determine the resistance to freezing and frozen storage of four strains of lactic acid bacteria and to quantify the cryoprotective effect of glycerol.

Cheddar cheese was produced free of non-starter bacteria, acidified with starter or glucono-δ-lactone and containing active coagulant (chymosin or pepsin) or inactivated coagulant (pepsin). The level and type of proteolysis in the experimental cheeses was monitored by protein solubility at pH 4·6 and in 12 % TCA, polyacrylamide gel and high voltage paper electrophoresis, gel filtration and paper chromatography. The results show that the coagulant was primarily responsible for the formation of large peptides while small peptides and free amino acids were produced principally by the starter, possibly from coagulant-produced peptides.

It has been shown by Hipp, Groves, Custer & McMeekin (1952) that the differential solubility in urea of the casein components of cow's milk can be utilized for preparative purposes. Fractionation is achieved by step-wise dilution of a solution of all the casein components in strong (6·6 molar) urea. In connexion with the work on the three genetically distinct β-caseins discussed in the preceding paper, the need arose to prepare the variants A, B and C from the milk of appropriate homozygotes. This led to the development of the modified procedure described in this note, a procedure more sparing in urea and more effective in the initial isolation of the β-casein fraction than the original method of Hipp et al.

The proteins of the milk-fat globule membrane in homogenized cream have been studied using polyacrylamide-gel electrophoresis and electron microscopy. The effects of pasteurization and subsequent storage of the homogenized cream on the strength and composition of the membrane have also been investigated. Caseins and undenatured whey proteins are adsorbed to the fat–serum interface during homogenization; the caseins are the more dominant group, but with no apparent preference for any individual protein. After homogenization, but before pasteurization, whey proteins are more easily removed by washing than are the casein components. After subsequent pasteurization, and on storage, whey proteins become more tightly bound and are no longer readily removed by washing. Electron micrographs showed that the interfacial membrane between fat droplets and the serum phase consists of a protein composite material containing casein micelles, casein micellar sub-units and molecular, or non-micellar protein.

Unidentified constituents in fresh pasture increase milk fat cis-9, trans-11 conjugated linoleic acid (CLA) concentration, and prevent milk fat depression, even though ruminal conditions conducive to reducing milk fat synthesis exist. One possible explanation is vitamin E (α-tocopherol), a constituent high in fresh pasture, but naturally low in conserved/dried forages and cereal grains. Twenty late-lactating dairy cows previously consuming a total mixed ration (TMR) were randomly allocated to one of two dietary treatments for 21 d: TMR (control; n=10); and TMR plus an additional 10000 i.u. α-tocopherol/d (VIT E; n=10). These cows were simultaneously compared with 13 late-lactation dairy cows previously grazing fresh pasture (PAS) balanced for age, parity and genetic merit. Average daily α-tocopherol intakes were approximately 468, 10520 and 1590 i.u./cow for the control, VIT E and PAS treatments, respectively. Dietary α-tocopherol supplementation (VIT E v. control) slightly increased milk fat content by 0·23 percentage units, but did not significantly alter milk fatty acid composition. Plasma trans-11 18[ratio ]1 (VA) content tended to increase and trans-10 18[ratio ]1 levels numerically declined following α-tocopherol supplementation suggesting possible changes in rumen biohydrogenation products. In addition, increased α-tocopherol intake in TMR-fed cows decreased serum urea levels and tended to alter milk fat 15[ratio ]0 suggesting changes in rumen microbial populations. However, when compared with cows grazing pasture, TMR-fed cows supplemented with α-tocopherol, still produced milk with lower cis-9, trans-11 CLA and VA, and higher trans-10 18[ratio ]1 concentrations suggesting α-tocopherol is not a primary reason for milk fatty acid profile differences between pasture and TMR-fed cows. Therefore, additional unknown pasture constituents favour production of fatty acids originating from the cis-9, trans-11 instead of the trans-10, cis-12 CLA biohydrogenation pathways.

TWO models of lactating dairy cows were constructed by combining mechanistic models of digestion and metabolism reported previously and by adding elements to allow simulation of changes in tissue metabolic capacities over time. One model (day version) had an integration interval of 0·005 d and was suitable for simulation of within-day dynamics of nutrient supply and partition. The other (lactation version) had an integration interval of 1 d and was suitable for simulation of full lactation cycles. A number of simulation analyses were conducted to characterize and evaluate the models, to examine quantitative and dynamic properties of mechanisms which influence partition of nutrients, to identify aspects requiring further study, and to illustrate the potential usefulness of mechanistic, as compared to empirical, models in analyses of energy balance in lactating dairy cows.