Levels of plasmin activity in various commercial cheeses are reported. The effects of some processing conditions (pH, cooking temperature and method of salting) on plasmin activity in cheese were investigated. Neither draining pH nor method of salting affected the activity. However, plasmin activity in cheese, rennet curd and micellar casein dispersions increased with increasing cooking temperature. The results suggest that plasmin activity in cheese depends on cooking temperature used during manufacture.

A commercial neutral proteinase was encapsulated in liposomes (multilamellar) and incorporated into cheese curd via the cheese milk. The milk proteins were protected from proteinase attack during curd manufacture. A significant proportion of the liposome-entrapped enzyme was retained in the curd and its subsequent release into the cheese matrix was inferred from an increased rate of β-casein breakdown.

High pressure homogenisation (HPH) is a novel dairy processing tool, which has many effects on enzymes, microbes, fat globules and proteins in milk. The effects of HPH on milk are due to a combination of shear forces and frictional heating of the milk during processing; the relative importance of these different factors is unclear, and was the focus of this study. The effect of milk inlet temperature (in the range 10–50 °C) on residual plasmin, alkaline phosphatase, lactoperoxidase and lipase activities in raw whole bovine milk homogenised at 200 MPa was investigated. HPH caused significant heating of the milk; outlet temperature increased in a linear fashion (0·5887 °C/°C, R2=0·9994) with increasing inlet temperature. As milk was held for 20 s at the final temperature before cooling, samples of the same milk were heated isothermally in glass capillary tubes for the same time/temperature combinations. Inactivation profiles of alkaline phosphatase in milk were similar for isothermal heating or HPH, indicating that loss of enzyme activity was due to heating alone. Loss of plasmin and lactoperoxidase activity in HPH milk, however, was greater than that in heated milk. Large differences in residual lipase activities in milks subjected to heating or HPH were observed due to the significant increase in lipase activity in homogenised milk. Denaturation of β-lactoglobulin was more extensive following HPH than the equivalent heat treatment. Inactivation of plasmin was correlated with increasing fat/serum interfacial area but was not correlated with denaturation of β-lactoglobulin. Thus, while some effects of HPH on milk are due to thermal effects alone, many are induced by the combination of forces and heating to which the milk is exposed during HPH.

Sixty samples of raw goat milk intended for Caprino cheese-making were collected from ten farms in the Bergamo area over a 6-month period. Analyses of main microbial groups, somatic cell count (SCC) and pH were performed to determine the effect of origin (farm) and lactation period (April – September) on microbial composition and the incidence of pathogens in milk. Overall mean values were: standard plate count (SPC), 5.0×104 cfu/ml; yeasts, 2.5×102 cfu/ml; coliforms, 9.1×102 cfu/ml; Escherichia coli, 2.9 cells/ml; enterococci, 1.1×102 cfu/ml; lactococci, 3.4×103 cfu/ml; lactobacilli, 3.0×103 cfu/ml; halotolerant bacteria, 8.2×103 cfu/ml; spores of mesophilic aerobic bacteria, 11 cfu/ml; SSC, 9.9×105 cells/ml; pH, 6.63. Moulds and spores of sulphite-reducing clostridia were found intermittently. Neither Salmonella spp. nor Listeria monocytogenes was detected, while Esch. coli O157[ratio ]H7 was isolated from one milk sample (an incidence of 1.7%). Staphylococcus aureus was discovered at a level >102 cfu/ml in 26 samples (43%) with an overall mean of 1.2×103 cfu/ml, whereas coagulase-negative staphylococci were found in 54 samples (90%) with an overall mean of 1.3×103 cfu/ml. Of Staph. aureus strains, 23% proved to be enterotoxinogenic with a prevalence of enterotoxin C producers. Staph. caprae was the coagulase-negative species most frequently isolated; none of the coagulase-negative staphylococci strains synthesized any of the enterotoxins tested for.

Sample source was the major factor affecting the microbial composition of goat milk: significant differences (P<0.01) were observed among samples from different farms for SPC, coliforms, lactococci, lactobacilli and halotolerant bacteria. Period of lactation had a significant effect (P<0.025) on SCC and pH. SPC correlated well with coliforms, lactococci and lactobacilli; SSC did not reveal positive interactions with any microbial groups or pH.

Aseptically drawn milk was made into small cheeses (100 g) under aseptic conditions. The influence of the cow's feeding régime on the composition of the cheese fat, and the hydrolysis of the cheese fat and protein by bacterial and native milk enzymes, was studied.

In the absence of a strongly lipolytic bacterial cheese flora, the free fatty acid (FFA) content of cheese appeared to depend on several variables:

1. The initial FFA content of the milk varied according to the mode of milking (the lowest was obtained by cannulation of the udder) and the feeding régime of the cow. When one of a pair of monozygotic twin cows was maintained on pasture and the other on winter feed (hay and concentrates), the cheese made from the milk of the latter contained less FFA than did that from the former.

2. The major changes in the composition of the milk fat of the cows on winter regime were an increase of myristic and palmitic acids from 8 to 11% and 21 to 28%, respectively, and a decrease in stearic and oleic acid from 11 to 7·5% and 38 to 28%; minor changes in the other fatty acids were also observed.

3. The milk lipase in raw milk cheese liberated substantially more FFA than did the weakly lipolytic lactic acid streptococci. Milk lipase was active at the low pH value of cheese, but was inactivated when the milk was pasteurized at 63°C for 30 min.

4. Although rennet did not produce any amino acids in the mature cheeses it hydrolysed some of the cheese protein to nitrogen soluble in water (WS), trichloroacetic acid (TCA-S) or phosphotungstic acid (PTA-S). Some of the WS-N appeared to be utilized by the lactic acid streptococci. The native milk protease resisted the heat treatment used and liberated amino acids at the low pH value of cheese, but its contribution to the proteolysis was relatively less important than the contribution of the milk lipase to the fat breakdown in cheese.

5. The free amino acids of the milk, which appeared in the whey, were not recovered in the cheese. The cheese made from unheated milk with δ-gluconic acid lactone contained low amounts of free amino acids apparently due to the action of milk protease. The proteolytic activity of the lactic acid bacteria increased the amino acid content appreciably.

A holistic approach of a mould cheese ripening is presented. The objective was to establish relationships between the different microbiological and biochemical changes during cheese ripening. Model cheeses were prepared from pasteurized milk inoculated with Kluyveromyces lactis, Geotrichum candidum, Penicillium camemberti and Brevibacterium linens under aseptic conditions. Two cheese-making trials with efficient control of environmental parameters were carried out and showed similar ripening characteristics. K. lactis grew rapidly between days 1 and 6 (generation time around 48 h). G. candidum grew exponentially between days 4 and 10 (generation time around 4·6 d). Brevi. linens also grew exponentially but after day 6 when Pen. camemberti mycelium began developing and the pH of the rind was close to 7. Its exponential growth presented 3 phases in relation to carbon and nitrogen substrate availability. Concentrations of Pen. camemberti mycelium were not followed by viable cell count but they were evaluated visually. The viable microorganism concentrations were well correlated with the carbon substrate concentrations in the core and in the rind. The lactose concentrations were negligible after 10 d ripening, and changes in lactate quantities were correlated with fungi flora. The pH of the inner part depended on NH3. Surface pH was significantly related to NH3 concentration and to fungi growth. The acid-soluble nitrogen (ASN) and non-protein nitrogen (NPN) indexes and NH3 concentrations of the rind were low until day 6, and then increased rapidly to follow the fungi concentrations until day 45. The ASN and NPN indexes and NH3 concentrations in the core were lower than in the rind and they showed the same evolution. G. candidum and Pen. camemberti populations have a major effect on proteolysis; nevertheless, K. lactis and Brevi. linens cell lysis also had an impact on proteolysis. Viable cell counts of K. lactis, G. candidum, Pen. camemberti and Brevi. linens were correlated with the environmental conditions, with proteolytic products and with carbon substrate assimilation. NH3 diffusion from surface to the cheese core during ripening was highly suspected. Interaction phenomena between microorganisms are discussed.

Casein phosphopeptide amorphous calcium phosphate nanocomplexes (CPP-ACP) in chewing gum, lozenges and mouthrinses have been shown to remineralize enamel subsurface lesions in human in situ experiments. The aim of this double-blind, randomized clinical study was to investigate the capacity of CPP-ACP added to bovine milk to remineralize enamel subsurface lesions in situ. Ten subjects drank milk containing either 2·0 or 5·0 g CPP-ACP/l or a control milk whilst wearing removable appliances with enamel slabs containing subsurface demineralized lesions. Each 200 ml milk sample was consumed once a day for each weekday over three consecutive weeks. After each treatment and one weeks rest the subjects crossed over to the other treatments. At the completion of the treatments the enamel slabs were removed and remineralization determined using microradiography and microdensitometry. The results demonstrated that all three milk samples remineralized enamel subsurface lesions. However, the milk samples containing CPP-ACP produced significantly greater remineralization than the control milk. The remineralising effect of CPP-ACP in milk was dose-dependent with 2·0 and 5·0 g CPP-ACP/l producing an increase in mineral content of 70 and 148%, respectively, relative to the control milk. The differences in remineralization following exposure to the three milk samples were all statistically significant (P<0·001). In conclusion, this study shows that the addition of 2·0–5·0 g CPP-ACP/l to milk substantially increases its ability to remineralize enamel subsurface lesions.

Changes in chemical composition, proteolysis, lipolysis, texture, melting and sensory properties of low-fat Kashar cheese made with three different fat replacers (Simplesse® D-100, Avicel Plus® CM 2159 or β-glucan) were investigated throughout ripening. The low-fat cheeses made with fat replacers were compared with full- and low-fat counterparts as controls. Reduction of fat caused increases in moisture and protein contents and decreases in moisture-in-non fat substance and yield values in low-fat cheeses. The use of fat replacers in the manufacture of low-fat Kashar cheese increased water binding capacity and improved overall quality of the cheeses. Use of fat replacer in low-fat cheese making has enhanced cheese proteolysis. All samples underwent lipolysis during ripening and low-fat cheeses with fat replacers had higher level of total free fatty acid than full- or low-fat control cheeses. Texture attributes and meltability significantly increased with addition of fat replacers. Sensory scores showed that the full-fat cheese was awarded best in all stages of ripening and low-fat variant of Kashar cheeses have inferior quality. However, fat replacers except β-glucan improved the appearance, texture and flavour attributes of low-fat cheeses. When the fat replacers are compared, the low-fat cheese with Avicel Plus® CM 2159 was highly acceptable and had sensory attributes closest to full-fat Kashar cheese.

The effect of the hygienic quality of milk on changes in microbial counts and biogenic amine content was evaluated during ripening of goat cheeses manufactured from pasteurized and raw milks at 1, 14, 30, 60 and 90 d. The original milk, rennet, curd and whey were also included in the study. The pH, salt content and extent of proteolysis in the cheese were also evaluated. Spermidine and spermine were the main amines in raw milk, while they were minor amines in cheeses. Other amines increased markedly during ripening, tyramine being the main amine in cheese made from raw milk and cadaverine and putrescine in those produced from pasteurized milk. Enterobacteriaceae counts decreased during ripening whereas those of lactic acid bacteria increased, especially lactobacilli and enterococci. Cheese made from raw milk showed higher microbial counts during ripening than those made from pasteurized milk, especially for Enterobacteriaceae and enterococci, counts being 2 or 3 log units higher. Raw milk cheese showed remarkably higher biogenic amines compared with pasteurized milk cheeses. Therefore, pasteurization of milk causes a decrease in final biogenic amine content of cheese as a result of the reduction of its microbial counts.

Based on potential health benefits, there is a need to develop effective strategies for enhancing milk fat concentrations of cis-9 18[ratio ]1, 18[ratio ]3 n-3 and conjugated linoleic (CLA) content in milk without compromising the sensory or storage characteristics of processed milk or dairy products. Sixteen Finnish Ayrshire dairy cows were used in a cyclic change-over experiment with four 21-d experimental periods and a 4×2×2 factorial arrangement of treatments to evaluate the effects of forage conservation method, concentrate level and supplements of propylene glycol (PG), and their interactions on milk fatty acid composition and vitamin content. Experimental treatments consisted of four conserved forages offered ad libitum, supplemented with two levels of a standard concentrate (7 or 10 kg/d) and PG (0 and 210 g/d) fed as three equal meals. Primary growths of timothy and meadow fescue sward were conserved by ensiling with none (NA), an inoculant enzyme preparation (IE) or a formic acid based (FORM) additive or as hay 1 week later. Conservation of grass by drying rather than ensiling resulted in lower forage 18[ratio ]2n-6, 18[ratio ]3n-3, total fatty acid and fat-soluble vitamin concentrations. In spite of lower intakes, milk fat 18[ratio ]2n-6 and 18[ratio ]3n-3 content was higher (P<0·05) for hay than for silage diets (12·1, 9·6, 9·6 and 9·3 and 5·00, 3·51, 4·27 and 2·93 g/kg total fatty acids, for hay, NA, IE and FORM silages, respectively). Forage conservation method had no clear effects on milk trans 18[ratio ]1 or CLA content. Compared with silage, hay diets resulted in milk containing lower (P<0·001) riboflavin, α-tocopherol and β-carotene concentrations, but had no effect on ascorbic acid, thiamine, pyridoxine or retinol content. Feeding more concentrates had no effect on milk fatty acid composition or milk vitamin content, other than lowering (P<0·001) 16[ratio ]0 concentrations from 348 to 338 g/kg fatty acids. Supplements of PG led to small (P<0·05) increases in milk 13[ratio ]0 anteiso and 15[ratio ]0 content from 1·06 and 11·3 to 1·22 and 12·6 g/kg fatty acids and reduced (P<0·05) the concentrations of ascorbic acid (16·1 v. 15·1 g/kg milk).

Samples of curd and cheese, taken throughout cheese-making with milks concentrated to different extents, were examined by light microscopy and by scanning and transmission electron microscopy. The mature cheeses were analysed for textural parameters by instrumental and sensory means. The protein was packed in larger, more compact areas and the fat was more segregated in the curds and cheeses from the more concentrated milks. The protein network increased in coarseness in proportion to the concentration factor of the milk, the relative differences being the same throughout cheese-making into the mature cheese. Thus, the basic structure of the protein network was laid down during the curd firming process and was not fundamentally altered later in cheese-making. The fat/protein interfaeial area in the cheese and the changes in structure during maturation decreased as the concentration factor of the milk increased. The instrumental firmness, cohesiveness and force and compression required to cause fracture, and the sensory firmness, crumbliness, granularity and dryness of the cheese all increased as the concentration factor of the milk increased. The adhesiveness declined and there was little change in elasticity, with increase in milk concentration factor. The textural differences were related to the smaller fat content, lower level of proteolysis, stronger links in the protein matrix and reduced capacity of the fat and protein phases to move relative to each other in the cheeses from the more concentrated milks.

The aim of this study was to evaluate the antioxidant, antihypertensive and immunomodulatory characteristics of skim milk fermented with Lactobacillus delbrueckii ssp. bulgaricus LB340. Supernatants obtained from the ferments after centrifugation were subjected to ultrafiltration and yielded four peptidic fractions of 10–5 kDa, 5–3 kDa, 3–1 kDa, and <1·0 kDa. Peptides in 5–3 kDa range exhibited a good antioxidant activity. The peptides (<1·0 k) was applied to Superdex-30 G column fractionation and produced six fractions (F1–6). Fraction F2 presented the highest angiotensin I-converting enzyme inhibition activity with IC50 of 67·71±7·62 mg/ml. Moreover, fraction F6, which displayed a good immunomodulatory activity, had a positive effect on murine spleen lymphocyte proliferation with Stimulation Index of 0·729±0·123. The present data showed the potential of the milk fermented with Lactobacillus delbrueckii ssp. bulgaricus LB340 as a functional food, however, further research is needed to evaluate the biofunctional activity of this fermentation product in vivo using model animal.

The microflora of ten batches of La Serena cheese, made from raw milk of Merino ewes and using vegetable rennet as coagulant, was monitored throughout a 60 d ripening period. After 15 d, lactic acid bacteria predominated in the interior of the cheese, whilst lactic acid bacteria and lactic acid-utilizing species of yeasts and moulds predominated on the cheese surface. Coagulase-positive staphylococci were not detected in the interior or on the surface of any of the cheeses after 45 d and faecal coliforms were not found after 60 d. Higher death rates of staphylococci and coliforms were recorded for cheese made in spring than for the cheese made in winter.

On heating casein micelle systems containing β-lactoglobulin (β-lg) at 90°C for 10 min, β-lg complexed with casein micelles at pH < 6·9, probably as a result of interaction with κ-casein via sulphydryl-disulphide interchange, and co-sedimented with the micelles on ultracentrifugation. Complex formation with β-lg appeared to prevent the dissociation of micellar κ-casein on heating. However, at pH ≥ 6·9, κ-casein/β-lg complexes dissociated from the micelles on heating, thus enhancing the release of micellar κ-casein. High concentrations of β-lg (≥0·8%) induced coagulation at pH 7·3, essentially by promoting the dissociation of micellar κ-casein. It appeared that αs1-, αs2-, β- and κ-caseins dissociated from serum protein-free casein micelles to equal extents, but the presence of β-lg specifically enhanced the dissociation of κ-casein at pH values ≥ 6·9. Micelle hydration increased slightly when casein micelles were heated in the presence of β-lg at pH 6·7, while at pH 7·3 β-lg decreased the degree of hydration of casein micelles. Formation of a complex between β-lg and κ-casein appeared to stabilize the micelles in the pH range 6·5–6·7, possibly via increased micellar charge or degree of hydration or by preventing the dissociation of κ-casein.

The combined effect of ultrasonic waves (20 kHz, 150 W) and heat treatment applied simultaneously (thermoultrasonication) on the survival of a strain of Staphylococcus aureus was studied in 0·05 m-phosphate buffer pH 6·8 and ultra-heat-treated whole milk. This combined process decreased by 63% the decimal reduction times for the heat treatment when the organism was suspended in buffer and by 43% when suspended in milk. These effects were much greater than the additive effect of the two agents considered independently.

Isolates of various species of coagulase-negative staphylococci (CNS) from extramammary swab samples were compared with isolates of bovine mastitis CNS species. Swab samples were taken from perineum skin and udder skin, teat apices and teat canals of lactating dairy cows of the research dairy herd of the University of Helsinki in 1999 and 2002. In addition, hands of herd staff and liners of teat cups were sampled for CNS. CNS isolates from milk samples of subclinical or clinical mastitis in the same herd were collected during 1998–2002. Species identification was performed using phenotyping (API Staph ID 32 test) and by constructing a 16 and 23S rRNA RFLP library (ribotyping). Based on phenotype, 84% of mastitis isolates and 57% of extramammary isolates were identified at species level with >90% probability. Ribotype patterns formed 24 clusters, and 15 of them included a CNS type strain. If the ribotype clusters contained isolates of both extramammary and mastitis origin, they were further typed using pulsed-field gel electrophoresis (PFGE). The predominant CNS species in mastitis, based both on phenotyping and genotyping, were Staph. chromogenes and Staph. simulans. Phenotyping failed to identify half of the extramammary isolates. Based on phenotyping, Staph. equorum and Staph. sciuri, and based on ribotyping, Staph. succinus and Staph. xylosus, were the predominant CNS species in extramammary samples. The most common species in milk samples, Staph. chromogenes, was also isolated from several extramammary samples, and five out of ten pulsotypes were shared between mastitis and extramammary isolates, indicating that strains from udder skin are highly similar. The second commonest mastitis species, Staph. simulans, was isolated only from three extramammary samples, indicating that Staph. simulans may be more specifically associated with mastitis. Consequently, the origin of CNS mastitis may vary depending on the causing CNS species.

The effects of κ-casein and β-lactoglobulin (β-lg) genetic variants on cheesemaking were examined in two laboratory scale experiments. Variants of κ-casein had highly significant (P < 0·001) effects on milk citrate content (A > B) and rennet clotting time (A > B). When curds were cut at constant firmness there were only minor effects of κ-casein variants on milk component recoveries and cheese yield and composition. A tendency to higher fat recoveries from milks containing κ-casein B was reflected in significantly (P < 0·05) higher fat contents in cheese after 3·5 months of ripening. Genetic variants of βlg had highly significant (P < 0·001) effects on casein number (B > AB > A) and protein recoveries (B > AB > A) and significant (P < 0·05) effects on yield of cheese dry matter (B > AB > A). Green (unripened) cheeses made from the different milks also differed in fat, protein and moisture contents, probably in part due to differences in fat/casein ratios of the cheesemilks. The compositional differences were reflected in differences in the extent of proteolysis during ripening (A > AB > B). In the experiment with β-lg genetic variants the effect of time of cold storage of the cheesemilk was also investigated. No differences were found in milk composition and component recoveries or cheese yield, composition and quality when milks were cold-stored for 24, 48 or 120 h before cheesemaking.