A homo-tetrameric ∼160-kDa cystathionine γ-lyase was purified to homogeneity from Lactobacillus reuteri DSM 20016 by four chromatographic steps. The activity was pyridoxal-5′-phosphate dependent and the enzyme catalyzed the α,γ-elimination reaction of L-cystathionine, producing L-cysteine, ammonia and α-ketobutyrate. The enzyme was active towards a range of amino acids and amino acid derivatives, including methionine. The pH and temperature optima were found to be 8.0 and 35 °C, respectively. Isoelectric pH (pI) was ∼5.0 as determined by two-dimensional electrophoresis. Sensitivity to chemical inhibitors was typical of lactococcal cystathionine γ- and β-lyases, except it was inhibited by sulphydryl reagents. The N-terminal sequence was MKFNTQLIHGGNSED, which had 100% homology with cystathionine β-lyase of Lb. reuteri 104R (Accession Number CAC05298). Lb. reuteri DSM 20016, together with 10 other strains of non-starter lactic acid bacteria, was used as adjunct starter in the production of miniature Canestrato Pugliese-like cheeses. After 40 d ripening, the water-soluble extract of the cheeses with added Lactobacillus fermentum DT41 and Lb. reuteri DSM 20016 contained the highest enzyme activities on cystathionine and methionine substrates. Determinations of methanethiol, dimethyl sulphide, dimethyl disulphide and dimethyl trisulphide in the miniature cheeses confirmed the findings of enzyme activities.

Galacto-oligosaccharides (GOS) are prebiotics that have a beneficial effect on human health by promoting the growth of probiotic bacteria in the gut. GOS are commonly produced from lactose in an enzymatic reaction catalysed by β-galactosidase, named transglycosylation. Lactose is the main constituent of whey permeate (WP), normally wasted output from the cheese industry. Therefore, the main goal of this work was to optimise the synthesis of GOS in WP using β-galatosidase from Aspergillus oryzaea. WP and whey permeate enzymatically treated (WP-GOS) were used as culture media of Lactobacillus plantarum 299v. Lb. plantarum 299v attained the stationary phase in approximately 16 h, reaching 3·6 and 4·1×108 CFU/ml in WP and WP-GOS, respectively. The in situ synthesised GOS were not consumed during growth. No significant differences were observed in the growth kinetics of microorganisms in both media. After fermentation, microorganisms were dehydrated by freeze-drying and spray-drying and stored. The recovery of microorganisms after fermentation, dehydration and storage at 4 °C for at least 120 d was above 108 CFU/g. These studies demonstrated that WP is an appropriate substrate for the synthesis of GOS and the obtained product is also adequate as culture medium of Lb. plantarum 299v. The coexistence of GOS and dehydrated viable probiotic microorganisms, prepared using an effluent as raw material, represents the main achievement of this work, with potential impact in the development of functional foods.

A routine method giving accurate results was required for the determination of the glyceride content of eluate fractions obtained in the silicic acid chromatography of lipid mixtures. The technique of determining glyceride glycerol by saponification, oxidation of the liberated glycerol to formaldehyde, reaction of the formaldehyde with chromotropic acid and measurement of the resulting chromogen at 570 mμ has been investigated. Procedures based on this principle have appeared in the literature but none of these has been found to be entirely satisfactory. Accordingly, a modification of the method has been developed that is capable of giving results of a high degree of accuracy.

1. The acid phosphatase content of milk is reasonably constant from quarter to quarter in normal uninfected cows.

2. The acid phosphatase content of milk from infected quarters is enhanced in comparison with the uninfected quarters. The differences found were from 4·1 to over 50 enzyme units per 100 ml. milk.

3. It has been shown that a Streptococcus agalactiae suspension is capable of hydrolysing phenylphosphate at pH 4–1, and it is suggested that this organism is largely responsible for the enhanced enzyme content of the milk from the infected udder.

Studies on camels' milk, whether with respect to concentration or bioavailability of trace elements from this milk, are limited and warrant further investigation. The object of this study was to analyse the concentration and distribution of zinc, copper, selenium, manganese and iron in camel milk compared to those in human milk, cows' milk and infant formula under similar experimental conditions. Camels' milk and cows' milk were collected from local farms, human milk samples were obtained from healthy donors in Kuwait and infant formula was purchased locally. Milk fractionation was performed by ultra-centrifugation and gel-column chromatography. The concentration of trace elements was analysed by atomic absorption spectrometry and that of protein was determined spectrophotometrically. The concentration of manganese and iron in camels' milk was remarkably higher (7–20-fold and 4–10-fold, respectively) than in human milk, cows' milk and infant formula. The zinc content of camels' milk was higher than that of human milk but slightly lower than in cows' milk and infant formula. The concentration of copper in camels' milk was similar to that of cows' milk but lower than in human milk and infant formula. The selenium content of camels' milk was comparable to those of other types of milk. Approximately 50–80% of zinc, copper and manganese in camels' milk were associated with the casein fraction, similar to that of cows' milk. The majority of selenium and iron in camels' milk was in association with the low molecular weight fraction. It is recommended that camels' milk be considered as a potential source of manganese, selenium and iron, perhaps not only for infants, but also for other groups suspected of mild deficiency of these elements. Further investigations are required to confirm this proposal.

Milk gels varying widely in firmness were examined by both scanning and transmission electron microscopy, and a close correlation was found between their ultrastructure and firmness. In gels containing 40 and 50% total solids (14 and 17% protein respectively), casein micelles appeared as individual entities linked by some bridging material. At 60% total solids (20% protein), the micelles were fused and offered a considerably higher resistance to a penetrometer probe. Likewise some additives to gels, such as ammonium persulphate and CaCl2, caused the micelles to fuse. These chemicals appreciably increased firmness of such gels. In gels containing hexametaphosphate, a known inhibitor of gelation, most of the casein micelles were disintegrated; these gels were soft.

The susceptibility of the casein in milk to proteolysis was shown to be greatly influenced by its state of aggregation. In normal milk, where the casein is largely in micellar form, the αs1- and β-caseins are almost inaccessible to proteolysis. On removal of the colloidal phosphate, the casein micelles disintegrate, rendering the components, especially the αs1-casein, accessible to proteolysis. The role of colloidal calcium phosphate in the casein micelle is believed to be that of a non-specific aggregating agent which can be effectively replaced by calcium. Dissolved colloidal phosphate can be effectively reformed by elevation of the pH of colloidal phosphate-free (CPF) milk before equilibrium dialysis. Addition of κ-casein to CPF milk also causes aggregation of the component caseins but the micelles formed are smaller than those of normal milk.

The behaviour of micellar β-casein differs considerably from that of micellar αs1-casein. The evidence suggests that part of the β-casein freely dissociates either outside or within the micelle when the temperature is reduced. The temperature dependence of the susceptibility of β-casein to proteolysis was similar in skim-milk and in solutions of sodium caseinate, and increased as the temperature was reduced. αs1-Casein was quite resistant to proteolysis in normal milk but became susceptible when the micelle structure was disrupted on removal of colloidal phosphate.

It is concluded that limited proteolysis may prove a valuable technique in the study of casein micelle structure.

Many psychrotrophic bacteria contaminating raw milk produce phospholipase that withstands pasteurization and UHT treatments. This enzyme acts on the milk fat globule membrane and exposes triacylglycerides to the action of lipase. Phospholipase production by various isolates of Pseudomonas was investigated. The isolates were cultured aerobically at 8 °C in nutrient broth, McKellar's minimal salts medium, Chrisope's medium, and skim milk. Each strain produced phospholipase during the 50 h incubation. Enzyme production varied significantly (P < 0·001) with strain and growth medium. Strains varied significantly (P < 0·001) in their enzyme production in each medium and during the incubation time as well. Strain, incubation time, and the growth medium significantly influenced (P < 0·001) heat stability of the enzyme activity. Pasteurization reduced the activity, but did not eliminate it in skim milk.

Skim milk samples from 126 Friesian and 147 Jersey cows in eight commercial herds were preheated at 85 °C for 30 min and concentrated to 200 g l−1 total solids. A heat coagulation time–pH curve was determined at 120 °C for each treated sample. Heat coagulation times ranged from 1 to 50 min at the non-adjusted pH and 1 to 60 min at the pH of maximum stability. The following statistically significant effects were found. Maximum heat stability was affected by genetic variants of κ-casein (B > AB > A; P < 0·001) and β-lactoglobulin (B, AB>A; P < 0·05) whereas natural heat stability was affected only by κ-casein genetic variants (B > AB > A; P < 0·001). Maximum and natural heat stability were corre-lated positively with β-casein and κ-casein concentrations and were negatively correlated with αs1-casein and β-lactoglobulin concentrations. Milk from Jersey cows had greater maximum and natural heat stability than milk from Friesian cows. Differences were found between herds within breed for natural heat stability, but not for maximum heat stability. Maximum heat stability declined with age of the cow. The heat stability of skim milk samples taken from 40 Jersey cows in one of the herds was determined at 140 °C. A considerable variation was found in the coagulation time–pH curves. There was a difference in natural heat stability between κ-casein variants (B > AB; P < 0°05). Natural and maximum heat stability were correlated positively with urea concentration. No relationship was found between the heat stability of preheated concentrated skim milk and the heat stability of the original skim milk. The pH of skim milk samples was associated with αs1-casein genetic variant, age of cow, stage of lactation and concentration of γ-casein.

The whole N fraction of six samples of hard and semi-hard pressed cheeses was analysed using PAGE, polyacrylamide gel isoelectric focusing and immuno blotting with polyclonal antibodies against β- and αs1-casein. The origin of some electrophoretic bands corresponding to peptides produced from the enzymic degradation of the casein fractions was established. A number of these peptides were also present in the in vitro hydrolysates of casein with plasmin and chymosin. Thus, it was also possible to determine which casein was the source of each peptide and which enzymes were active in cheese. Compared with the traditional Coomassie staining procedures, immunoblotting is more sensitive and specific, making the interpretation of each electrophoretic profile easy. Thus, it was also possible to obtain a clear picture of the state of each casein fraction in a cheese variety. Two main peptides were isolated from the pH 4·6-insoluble N fraction of Parmigiano-Reggiano using DEAE-cellulose chromatography and identified, from the amino acid sequence of the N- and C-terminal ends, as γ3-casein (β-casein(f108–209)) and αs1-PL1 (αs1-casein(f80–199)). In both cases, a Lys–X bond was hydrolysed, indicating the action of a trypsin-like enzyme in β- and αs1-casein hydrolysis during the ripening of this variety of hard pressed cheese.

The assumption, that metabolites derived from the activity of the mammary gland epithelial cells reflect changes in milk secretion and its coagulation properties, was tested in dairy cows. The experiment included cows with uninfected udders and cows with one of the glands infected by different bacteria specie. Analysis were carried at the cow level (including all four glands), or at the gland level. High and significant correlations among the concentrations of lactose, glucose, glucose-6-posphate, milk related respiratory index (the ratio between the concentrations of citrate/lactate+malate in milk) and milk-derived glycolytic index (the ratio between glucose-6-phosphate and glucose in milk) and milk clotting parameters were found. The physiological basis for these relations and their ability to predict the deterioration in milk quality in subclinically infected glands and in glands previously clinically infected with Escherichia coli are discussed.

The effects on milk composition and processing characteristics of varying grass supply by changing stocking density and of offering a concentrate supplement were investigated. The experiment was conducted over 28 weeks of the lactation (April–October) using 48 spring-calved Friesian–Holstein cows. Three herds each of 16 cows were offered a restricted grass supply, a standard grass supply and a standard grass supply with a supplement of 3 kg concentrate/d. Treatment groups were grazed separately with a residence time of 3 d/paddock. Milk production, composition and processing characteristics such as renneting properties, ethanol stability and plasmin activity were measured weekly. Increasing stocking density above the standard system resulted in significant reductions in milk fat and protein yields, the concentrations of total protein, casein and whey proteins, and a deterioration in most processing characteristics. Imposing concentrate supplementation on the standard system increased total protein, casein and whey protein concentrations but generally did not improve processing characteristics except for ethanol stability. These results suggest that the standard grass supply in a rotational grazing paddock system can support efficient production of quality milk, and concentrate supplementation will not improve processing characteristics when an adequate supply of good quality herbage is available.

Bacteriology, somatic cell counts (SCO) and N-acetyl-β-glucosaminidase (NAGase) activity determinations were conducted on milk samples collected from does in three dairy herds with caprine arthritis–encephalitis virus (CAEV) infection. In two herds, CAEV-infected does were more likely to have a subclinical bacterial infection of the udder than CAEV-free does (P < 0·05). Does with CAEV but no bacterial udder infection had significantly greater mean SCO and NAGase activity than CAEV-free does without udder infection (P < 0·01). In two herds, changes in milk SCC and NAGase associated with CAEV infection were similar to those produced by coagulase-negative staphylococcal infections. The findings confirm that indirect indicators of bacterial mastitis infection may have reduced specificity in dairy goat herds with CAEV.

Microbial diversity of the raw milk for the production of Fior di Latte di Agerola and its changes during cheesemaking were studied. Viable counts showed that at the end of curd ripening, loads of lactic acid bacteria, both mesophilic and thermophilic rods and cocci, higher than those commonly evidenced in similar cheeses produced by using natural or commercial starters, were detected. Identification of 272 isolates, supported by molecular diagnostic aids, evidenced representative cultures of a high number of bacterial taxa of interest as participating in the process, although most of the isolates belonged to Lactococcus lactis and Lactobacillus helveticus species. RAPD-PCR and REA-PFGE biotyping were performed for the isolates of the above species and it was shown that most of the strains isolated from the raw milk occurred during the whole cheesemaking process, and an active role of these strains in the fermentation was supposed. The results offer further proof of the importance of the raw milk as source of technologically interesting strains of lactic acid bacteria capable of driving the fermentation of traditional cheeses.

The rate of acid production by a slow variant of Streptococcus cremoris in milk was raised to that of the fast parent strain when peptone or acid hydrolysed peptone were added. Lactose utilization was similarly affected. However, the stimulation took place without any change in the rate of growth. Other sources of available nitrogen gave similar results.

It is concluded that the change from a fast to a slow culture which occurs on continued transfer is due to the loss of ability to utilize the nitrogen compounds of milk.

Several peptidase activities in the 3 species of Group N streptococci were partly separated by gel filtration on Sephadex G-200. The peptidases identified were a general aminopeptidase of wide specificity, a tripeptidase, a proline iminopeptidase (prolyl-β-naphthylamidase), a proline iminodipeptidase and an aminopeptidase-P. The effects of temperature and pH on the stability of the enzyme activities, and the influence of the type of N source used in the growth medium on the elution pattern of the enzymes were examined.

A randomized, controlled field trial was performed in The Netherlands to determine the therapeutic efficacy of parenteral penethamate hydriodide (Leocillin®) against naturally occurring, chronic, streptococcal mastitis during lactation. Quarter milk samples were collected from subclinical cases of Streptococcus uberis or Streptococcus dysgalactiae mastitis to determine the effect of treatment on bacteriological cure and somatic cell count (SCC) at quarter level. A quarter was considered to be cured when the bacterial species, isolated prior to treatment, was not isolated from the quarter milk samples taken on days 10 and 20 post-treatment (bacteriological cure), or when a quarter milk SCC (QMSCC) was <250000 cells/ml on days 10 and 20 post-treatment (SCC cure). Longitudinal data analysis was performed to determine the effect of antibiotic therapy on SCC and milk yield at cow level. Bacteriological cure occurred in 59% of 29 treated quarters, while no cure was observed in any of the 21 untreated control quarters. Treatment resulted in a significant decrease in SCC at cow and quarter level in comparison with untreated controls. There was no significant effect of treatment on milk production. Antibacterial treatment of subclinical streptococcal infections during lactation also prevented clinical mastitis. Furthermore, the treatment may contribute to reduction of bulk milk SCC and to prevention of pathogen spread in dairy herds.

The double use of cheese whey (culture medium and thermoprotectant for spray drying of lactobacilli) was explored in this study for adding value to this wastewater. In-house formulated broth (similar to MRS) and dairy media (cheese and ricotta whey and whey permeate) were assessed for their capacity to produce biomass of Lactobacillus paracasei JP1, Lb. rhamnosus 64 and Lb. gasseri 37. Simultaneously, spray drying of cheese whey-starch solution (without lactobacilli cells) was optimised using surface response methodology. Cell suspensions of the lactobacilli, produced in in house-formulated broth, were spray-dried in cheese whey-starch solution and viability monitored throughout the storage of powders for 2 months. Lb. rhamnosus 64 was able to grow satisfactorily in at least two of the in-house formulated culture media and in the dairy media assessed. It also performed well in spray drying. The performance of the other strains was less satisfactory. The growth capacity, the resistance to spray drying in cheese whey-starch solution and the negligible lost in viability during the storage (2 months), makes Lb. rhamnosus 64 a promising candidate for further technological studies for developing a probiotic dehydrated culture for foods, utilising wastewaters of the dairy industry (as growth substrate and protectant) and spray drying (a low-cost widely-available technology).

In this study, the chemical composition, proteolysis and in vitro angiotensin-converting enzyme-(ACE)-inhibitory and antioxidant activities of white cheeses made using probiotic adjunct cultures (Bifidobacterium bifidum DSMZ 20456 and Lactobacillus acidophilus DSMZ 20079) were investigated. The cheeses were ripened in a vacuum package or brine for 120 d at 4 °C. The cheese samples maintained the probiotic characteristics of the viable cells as >106 cfu/g even after ripening for 120 d. The proteolysis degrees in terms of water-soluble nitrogen/total nitrogen (WSN/TN), trichloroacetic acid-soluble nitrogen/total nitrogen (TCA-SN/TN) and phosphotungstic acid-soluble nitrogen/total nitrogen (PTA-SN/TN) values in the cheeses increased throughout the ripening. The highest levels of proteolysis were found in cheese made using Lb. acidophilus DSMZ 20079 and ripened in a vacuum package. ACE-inhibitory activity of the water soluble extracts (WSEs) of the cheeses increased significantly (P < 0·05) throughout the ripening (IC50 values 82·78–140·99 μg/ml). Use of Lb. acidophilus DSMZ 20079 and packaging under vacuum significantly increased the percentage of ACE inhibiting activity. WSEs had DPPH scavenging activity (the IC50 values were 2·41–5·39 mg/ml and the inhibition values were 5·10–10·38%), increasing up to 60 d ripening. In the present study, it was observed that Lb. acidophilus DSMZ 20079 was more effective than Bifido. bifidum DSMZ 20456 in terms of the cheese characteristics investigated.