An agar diffusion slide assay using a thin layer of calcium caseinate has been developed as a micro-method for the quantitative study of calf rennet and other proteolytic enzymes. The number and width of the precipitation zones formed as a result of proteolytic activity depended upon the enzyme used, the concentration of calcium ion, pH and temperature. The assay was highly reproducible and particularly suitable for the measurement of large numbers of samples. It was considerably more sensitive than the milk clotting technique and, in general, more sensitive than other standard methods for measuring proteolytic activity.

Methods for measuring the reflectance of milk are described. The effect of heating is to reduce the reflectance of whole and separated milk progressively towards the blue end of the visible spectrum. The reflectance of cream is almost unaffected. The measurement of reflectance at the blue end of the visible spectrum is shown to be a useful method for the determination of the brownness of milk. The variation of rate of browning with temperature and with pH and the effect of storage on brownness are given.

1. A diffusion cup technique using Micrococcus lysodeikticus has been found suitable for lysozyme estimation. By this method raw milk gave only small zones of inhibition. The inhibitory substance of milk was shown not to be lysozyme.

2. Dose-response curves (dilution v. pH) were obtained with Streptococcus pyogenes as the test organism. They indicated that the effect of milk on this organism was the result of the interaction of one stimulatory and at least two inhibitory substances.

3. The existence of two inhibitory substances was confirmed by dilution assays, one, L1, occurring mainly in colostrum, the other, L2, occurring mainly in milk. The inhibition is greatest when L1 and L2 act simultaneously.

4. The pH and heat stabilities of the two substances are slightly different. L1 is most stable at pH 6−6·5, 90% being destroyed at 68° C. L2 is most stable at pH 7 and 95% is destroyed at 74°C.

As moderate dilution causes little change in the surface tension of milk, it is shown to be advantageous to use σ-dilution curves in place of the σ-values of the undiluted fluid as a characteristic of the surface properties of milk. The complications arising from the presence of the milk fat are described, and it is suggested that the influence of the fat is of a physical rather than of a chemical nature. A study of the role of the various milk proteins shows the casein to be of great importance, whilst the heat-coagulable proteins have little influence. The serum obtained after removal of the casein and heat-coagulable proteins contains a residual fraction of protein-like material which is markedly surface active though constituting only about 3% of the total milk proteins. The surface-active material (σ-proteose) has been concentrated and isolated, and its properties are described in some detail.

Macrophages isolated from the involuted bovine mammary gland were cultured in vitro. Phagocytosis of opsonized Staphylococcus aureus occurred rapidly, but intracellular killing of bacteria was slow. Many intracellular staphylococci survived for up to 4 d exposure to extracellular cloxacillin and emerged from within the macrophages to multiply extracellularly when the antibiotic was inactivated. Rifampicin was significantly more efficient than cloxacillin in killing intracellular S. aureus after 18 h incubation, but it too failed to sterilize the cultures within 3 d. Staphylococci, which had remained viable within macrophages during 20 h incubation with extracellular cloxacillin, showed an increased sensitivity to dilute lysostaphin on subsequent exposure. A 3 d course of intramammary therapy with cloxacillin, commencing simultaneously with an infecting inoculum of ∼108 colony forming units (c.f.u.) S. aureus, apparently eliminated the infection from one quarter of the udders of each of three lactating cows, but bacteria were re-isolated from two cows after a delay of several days. However, when other quarters of the same cows were infected with ∼108 c.f.u. S. aureus which had been phagocytosed by autologous mammary macrophages, similar simultaneous antibiotic therapy failed to affect these infections. The in vitro and in vivo findings indicate the significance of intracellular survival of S. aureus as a factor contributing to failure of antibiotic therapy.

Similar, marked variations in the freezing-point depressions of jugularvenous blood and of milk throughout a day were observed in cows when drinkingwater was offered for a single, short period each day. Values for milk were found to agree more closely, however, with those for mammary-venous blood than with those for jugular-venous blood. It appears that milk is in osmotic equilibrium with the blood flowing through the udder continuously throughout the period the milk remains within the udder and not only during its formation, and that milk secretion causes a slight alteration in the osmotic pressure of fluids within the immediate locality of the mammary gland. Changes in the milk composition that occurred in association with the observed changes in freezing-point depression were consistent with a movement of water into or out of the udder in response to any change in the osmotic pressure of blood.

The effect of temperature and time of heating whole milk on the renninclotting time, the primary phase of rennin action and the protein (mainly β-lactoglobulin) soluble in 2% trichloroacetic acid (TCA), have been studied. Considerable changes in these parameters occurred above 60°C. The primary phase was inhibited (the degree of inhibition being both temperature and time-dependent), the clotting time was increased, and the protein soluble in 2% TCA decreased considerably.

It is suggested that the inhibition of the primary phase was due to complex formation between κ-casein and β-lactoglobulin, the increase in clotting time to a combination of complex formation and a change in the distribution of Ca, and the decrease in β-lactoglobulin to both its interaction with κ-casein and its heat denaturation. The relevance of such changes to the heat stability of milk is discussed.

In this research communication I show the effect of various cow–calf contact systems on milk yield and milk composition during the periods when calves where allowed to suckle their dams and after the calves were separated in comparison to cows that were only machine milked throughout their lactation. Analyses were based on four different experiments, but conducted at the same research station and under comparable housing and feeding conditions. Nursing dams had contact to their calves during the whole day, during night-time or only twice per day shortly before milking. A control group of cows that had no contact to their offspring was part of every experiment. Data of the regular monthly milk recordings were analysed with linear mixed models. Results showed a significant effect of the suckling system on the machine milk yield over the whole lactation. While cows with night-time contact reached the milk production level of the control cows, cows with whole day or short-time contact still produced less milk after the calves were separated. Fat content was always lower during the suckling period but not afterwards. The significantly higher milk protein content in dams with calf contact requires further investigation. Somatic cell count in milk of nursing dams was slightly increased, probably due to the exposure of the teats to frequent suckling in addition to machine milkings. In conclusion, cow–calf contact systems influence the performance of cows during and after the suckling period but to varying degrees depending on the system adopted.

Forty-eight Manchega dairy ewes were used during a complete lactation in a 2×2 factorial design to determine the effects of supplementing diets with fat (calcium soaps of palm oil fatty acids, CSFA) and rumen undegradable protein (RUP) on milk production and composition. Factors tested were amounts of CSFA (0 or 200 g/kg) and RUP (300 or 450 g/kg crude protein) in the concentrate. RUP was altered by adding a mixture of maize gluten meal and blood meal. Lactation was divided into one nursing period (period 1, weeks 1–4), and three milking periods (periods 2–4, weeks 5–8, 9–14 and 15–21). Concentrates were given at 0–8 kg/d during periods 1 and 2, and at 0–6 kg/d in periods 3 and 4. Ewes grazed rotationally in an Italian rye-grass pasture and received a daily supplement of 0·8 kg vetch–oat hay during period 1, and 0·3 kg lucerne hay during periods 2–4. For the whole lactation, supplemental fat markedly increased milk fat content (+23%) and yield (+16%), and decreased milk protein content (−9%). The positive effect of feeding CSFA on milk fat content was more evident at the beginning of lactation; however, its negative effect on milk protein was more pronounced in late lactation. Supplementary RUP had little effect, increasing milk protein content only in period 3, when the crude protein content of pasture was lower. Milk yield and lamb growth were not affected by dietary treatments. The results indicated that CSFA can be useful for increasing the milk fat content of dairy ewes at pasture, which may help farmers to produce milk reaching the minimum requirements of fat content for the cheese industry.

It is fairly easy to control the enzymic hydrolysis of proteins in alkaline conditions by measuring the base consumption required to keep the pH constant in the reactor. Unfortunately, however, base consumption is not related in any simple way to the degree of hydrolysis reached at any given moment and to establish this relationship it is essential to find out the mean pK of the α-amino groups released during the hydrolytic process. We have shown here that the correct mean pK value varies according to the pH of the working conditions and that the relationship between these values may depend upon the kind of protein and protease used. We have put forward a method for determining this relationship experimentally by using a given protein–protease system, consisting of an alkaline titration of the raw protein and when partially hydrolysed. We have tested the results predicted by our theoretical model by applying it to the hydrolysis of whey proteins with a bacterial protease from Bacillus licheniformis at 50 °C, pH 8·0. This model can easily be applied to any hydrolytic process involving the appearance of functional groups that are partially protonizable under the working conditions in question in order to follow the kinetics of the reaction via the consumption of the neutralizing agent required to keep pH constant.

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.

In this study, the physicochemical properties of a low-fat dried yogurt paste (kashk) were determined, and the effects of different concentrations (0, 0·1, 0·3 and 0·5% w/w) of gum tragacanth exudates from Astragalus gossypinus on the stability and texture of the samples were investigated by measuring amount of syneresis, turbidity, particle size distribution (PSD), flow behaviour and viscoelastic properties. The flow behaviour index was not very sensitive to the concentration of gum, while a remarkable concentration dependency of the power-law consistency coefficient and Herschel–Bulkley yield stress was observed. The initial increase in the gum concentration at 0·1 and 0·3% levels led to a higher degree of syneresis, which was related to the depletion flocculation mechanism. However, the reduced amount of syneresis in samples containing 0·5% gum tragacanth was attributed to the significant increase in viscosity of the continuous phase, which is also accompanied by trapping of the aggregated casein particles. The presence of 3% salt in the samples may have led to the neutralization of charges on the surface of gum tragacanth; consequently, the non-adsorbing behaviour of high-ionic-strength polysaccharides inhibited the formation of electrostatic protein–polysaccharide complexes. Furthermore, maximum values of polydispersity, syneresis and tan δ at high frequencies were found in samples containing 0·1% gum tragacanth.

Cows managed for extended lactations of 16 months duration were milked on a half-udder basis twice or thrice daily, commencing in lactation week 9. Mammary epithelial integrity (assessed by milk sodium[ratio ]potassium ratio) was greater in the half-udder which was milked thrice daily. This difference was evident throughout the lactation but became greater after week 41. Milk protein composition was assessed during late lactation (52±3 weeks). Casein number (casein as a proportion of total protein) was significantly higher in half-udders milked thrice daily, as were the relative amounts of α- and β-caseins, whilst those of κ- and γ-caseins were reduced. Two days of inverted milking frequency (i.e. thrice-milked udder halves now milked twice, and vice versa) only partly reversed these differences. We concluded that thrice-daily milking will help to prevent or ameliorate the usual decline in milk processing quality associated with late lactation. Part of this effect is due simply to reduced exposure to proteolytic enzymes as a result of decreased storage time in the udder, but part is due to a better maintenance of epithelial tight junction integrity as lactation advances, which restricts leakage of proteolytic enzymes from serum into milk.

At atmospheric pressure, inactivation of lactoperoxidase (LPO) in milk and whey was studied in a temperature range of 69–73 °C and followed first order kinetics. Temperature dependence of the first order inactivation rate constants could be accurately described by the Arrhenius equation, with an activation energy of 635·3±70·7 kJ/mol for raw bovine milk and 736·9±40·9 kJ/mol for diluted whey, indicating a very high temperature sensitivity. On the other hand, LPO is very pressure resistant and not or only slightly affected by treatment at pressure up to 700 MPa combined with temperatures between 20 and 65 °C. Both for thermal and pressure treatment, stability of LPO was higher in milk than in diluted whey. Besides, a very pronounced antagonistic effect between high temperature and pressure was observed, i.e. at 73 °C, a temperature where thermal inactivation at atmospheric pressure occurs rapidly, application of pressure up to 700 MPa exerted a protective effect. At atmospheric pressure, LPO in diluted whey was optimally active at a temperature of about 50 °C. At all temperatures studied (20–60 °C), LPO remained active during pressure treatment up to 300 MPa, although the activity was significantly reduced at pressures higher than 100 MPa. The optimal temperature was found to shift to lower values (30–40 °C) with increasing pressure.

The effects on rumen fermentation and milk-fat secretion of a dietary supplement of protected tallow given to 4 Friesian cows established on a low-roughage ration and with depressed milk fat is reported. The ratios of acetate to propionate in the rumen were unaffected by the supplement and remained typical of those associated with low-roughage diets in that the proportion of propionate was increased. The supplement produced almost complete recoveries in yield and content of milk fat without any increase in intramammary fatty-acid synthesis. The recoveries were due to transfer of about 20% of the total fatty acids of the tallow supplement. These results are discussed in relation to the effects of low-roughage diets on milk-fat secretion and it is concluded that in the ‘low-fat syndrome’ the capacity of the mammary gland to absorb preformed fatty acids is not impaired.

Multiple regression analysis has been used to relate contributions of individual flavour components to the flavour of Cheddar cheese. Equations have been derived that relate Cheddar flavour to the headspace concentrations of 2-pentanone, methanethiol and methanol. The concentrations of ethanol and H2S found in cheese headspace have been related to the flavour defects ‘fruity’ and ‘sulphide’ respectively

We describe a case of listerial mastitis in a flock of 130 sheep. The animals were housed at a farm where the bulk raw ewe milk was processed to produce raw milk soft cheese. List. monocytogenes was shed from the right mammary complex. Shedding was observed over a period of 99 d. A mean level of 4·56×104 cfu (colony forming units) Listeria monocytogenes/ml was recovered from the raw milk originating from the infected udder. The numbers ranged from 9×101 to 2·95×105. The bulk milk was contaminated by approx. 5·7×103 cfu/ml. In the cheese product, 2·0×102 cfu List. monocytogenes/g were constantly detectable for a period of 7 d post manufacture. The starter culture used for coagulation had a pivotal influence on the behaviour of List. monocytogenes during cheesemaking. Using the same mesophilic buttermilk culture as used by the farmer allowed numbers of Listeria to increase 60-fold within 12 h owing to a delayed acidification of the bulk milk. Addition of a thermophilic yogurt culture reduced the numbers of Listeria within 8 h of incubation.

Evaluation of eleven candidate probiotic Lactobacillus strains isolated from human milk showed that some of the strains were well endowed with desirable cell surface and attachment attributes. The cell surface properties (hydrophobicity, auto-aggregation, attachment to collagen and HT-29 monolayer) of probiotic Lactobacillus species of human milk origin were compared with reference probiotic/ non-probiotic species and pathogenic strains. The bacterial adhesion to hydrocarbons (BATH) was determined using three aliphatic (Chloroform, n-Hexane and n-Octane) and two aromatic (Toluene and Xylene) solvents. Maximum affinity of Lactobacillus strains towards chloroform and toluene indicated the presence of low electron acceptor/ acidic surface components on cell surface of most of the strains. The highest value of per cent hydrophobicity was recorded with chloroform in HM1 (L. casei) (97·10 ± 3·35%) and LGG (98·92 ± 1·24%). A moderate auto-aggregation attribute was observed in all of our Lactobacillus isolates. Only HM10, HM12 and HM13 exhibited comparatively enhanced precipitation rate after 7 h of incubation period. The adhesion potential to collagen matrix was highest in LGG (26·94 ± 5·83%), followed by HM1 (11·07 ± 3·54%) and HM9 (10·85 ± 1·74%) whereas, on HT-29 cells, HM8 (14·99 ± 3·61%), HM3 (13·73 ± 1·14%) and HM1 (11·21 ± 3·18%) could adhere effectively. In this manner, we noticed that although the cell surface properties and adhesion prospective of probiotic bacteria were strain dependent, five of our isolates viz. HM1, HM3, HM8, HM9 and HM10 exhibited promising cell surface properties, which could be further targeted as indigenous probiotic.

The relationship between proteolysis and texture in Feta-type cheese made from ultrafiltered cows' milk was studied using capillary electrophoresis, chemical analysis and uniaxial compression. For this purpose cheeses were made with almost identical gross chemical compositions, but wide variations in texture, obtained by varying the amount of rennet and the coagulation conditions. αs1-Casein (CN) 8P, αs1-CN 9P and αs1-CN 8P-I were degraded during ripening, while β-CN A1, β-CN A2 and para-κ-CN were slightly degraded at the highest rennet additions and longest storage time tested (39 weeks). αs1-CN 8P, αs1-CN 9P and αs1-CN 8P-I were degraded even in cheeses made without rennet, and this was ascribed to cathepsin D activity. αs1-CN 9P disappeared faster than αs1-CN 8P, which suggests that milk acid phosphatase was active during storage. During ripening, stress at fracture (σf), strain at fracture (εf), the deformability modulus (E) and work to fracture (Wf) all decreased. Since the gross chemical composition was essentially constant during storage, these changes could be ascribed purely to proteolysis. Although εf could be predicted fairly well from capillary electrophoresis results (correlation coefficient 0·85), there were no unique relationships between degradation of casein components and σf when both storage time and the amount of rennet used were varied. In the young cheese a high level of proteolysis could coexist with high values of σf. This suggests that high levels of rennet caused σf to increase, via an effect unrelated to general proteolysis, whereas general proteolysis caused the reverse effect.