The objective of the studies reported in this research communication was to investigate differences in composition and enzymatic activities in bulk milk samples provided from Swedish dairy farms with different management systems, i.e. automated (AMS) and conventional milking systems (CMS). A bulk milk sample was collected from each of 104 dairy farms, 51 using AMS and 53 using CMS, located in the same geographical region. Sampling took place within two consecutive days during the indoor period (October). Milk samples were analysed for contents of total fat and protein, free fatty acids (FFA), caseins and whey proteins, somatic cell count (SCC), pH, plasmin and plasminogen derived activities, and total proteolysis. Our results showed a lower protein content and higher SCC in bulk milk from AMS herds compared with milk from CMS herds. Plasmin, plasminogen and total plasmin/ plasminogen derived activities were lower in milk from AMS herds but despite this, total casein and the ß-casein fraction as % of total protein were lower in milk from AMS herds than in milk from herds using CMS. Total proteolysis was higher in milk from AMS herds, suggesting that other proteases than plasmin, e.g. cellular and bacterial proteases, contributed to the degradation of casein. This was supported by a positive correlation between SCC and total proteolysis (P < 0·01), as well as a negative correlation between total proteolysis and ß-casein fraction (P < 0·05). In conclusion, comparing the quality of bulk milk from commercial dairy herds using AMS and CMS, respectively, several differences were observed, suggesting a significant effect from management system.

This study investigated the antibacterial properties of plasmin, the plasmin hydrolysis of bovine κ-casein and the fractions (named κC1, κC2, κC3, κC4, and κC5) liberated from it using RP-HPLC. The target bacteria were Escherichia coli, Staphylococcus aureus (pathogenic), Lactobacillus casei and Lactobacillus acidophilus (probiotic). Three peptides (kC1, kC3, and kC4) were found to have antibacterial activity, with κC3 peptide being the most active. The plasmin digest of bovine κ-casein proved to be stronger than any of its fractions in terms of antibacterial potential. Measurement of the minimum inhibitory concentration (MIC) showed that Gram-positive bacteria are generally more sensitive to antibacterial activity than Gram-negative bacteria. The MIC of nisin, as a bacteriocin peptide, was also measured. The three antibacterial peptides were identified using LC-Mass. The molecular mass of kC1, kC3, and kC4 corresponded to the f(17–21), f(22–24), and f(1–3) of bovine κ-casein, respectively. It was also found that the positive charge and hydrophobicity of a peptide are not key factors in antibacterial activity. On the whole, the present study demonstrated that the plasmin digest of κ-casein has a high antibacterial potential and can be considered as a natural antibacterial agent in the food chain.

The aims of this study were to test the assumption that tissue-type plasminogen activator (t-PA) and plasminogen (PG) are closely associated with the casein micelle and form a functional complex that rules casein degradation. This assumption was essentially verified for bovine milk under conditions wherein the plasmin system was activated by treatment with casein hydrolysate. It was also shown that urokinase-type PA (u-PA), the second type of plasminogen activator present in milk, was not involved in casein degradation. In agreement with previous studies, we show that treatment with casein hydrolysate precipitously reduced mammary secretion, disrupted the tight junction integrity (increase in Na+ and decrease in K+ concentrations), induced hydrolysis of casein, and activated various elements of the innate and acquired immune system. In the present study, we have identified t-PA as the principal PA, which is responsible for the conversion of PG to plasmin. It was found that t-PA and plasminogen are present in freshly secreted milk (less than 10 min from its secretion), suggesting that they are secreted as a complex by the mammary gland epithelial cells. Further research is needed to provide the direct evidence to verify this concept.

Sensitive methods that are currently used to monitor proteolysis by plasmin in milk are limited due to their high cost and lack of standardisation for quality assurance in the various dairy laboratories. In this study, four methods, trinitrobenzene sulphonic acid (TNBS), reverse phase high pressure liquid chromatography (RP-HPLC), gel electrophoresis and fluorescamine, were selected to assess their suitability for the detection of proteolysis in milk by plasmin. Commercial UHT milk was incubated with plasmin at 37°C for one week. Clarification was achieved by isoelectric precipitation (pH 4·6 soluble extracts) or 6% (final concentration) trichloroacetic acid (TCA). The pH 4·6 and 6% TCA soluble extracts of milk showed high correlations (R2 > 0·93) by the TNBS, fluorescamine and RP-HPLC methods, confirming increased proteolysis during storage. For gel electrophoresis, extensive proteolysis was confirmed by the disappearance of α- and β-casein bands on the seventh day, which was more evident in the highest plasmin concentration. This was accompanied by the appearance of α- and β-casein proteolysis products with higher intensities than on previous days, implying that more products had been formed as a result of casein breakdown. The fluorescamine method had a lower detection limit compared with the other methods, whereas gel electrophoresis was the best qualitative method for monitoring β-casein proteolysis products. Although HPLC was the most sensitive, the TNBS method is recommended for use in routine laboratory analysis on the basis of its accuracy, reliability and simplicity.

The objective of the present study was to determine the effect of high pressure (HP) processing (200, 450 and 650 MPa) at various temperatures (20, 40 and 55°C) on the total plasmin plus plasminogen-derived activity (PL), plasminogen activator(s) (PA) and cathepsin D activities and on denaturation of major whey proteins in bovine milk. Data indicated that transfer of both PL and PA from the casein micelles to milk serum occurred at all pressures utilized at room temperature (20°C). In addition to the transfer of PL and PA from micelles, there were reductions in activities of PL (16–18%) and PA (38–62%) for the pressures 450 and 650 MPa, at room temperature. There were synergistic negative effects between pressure and temperature on residual PL activity at 450 and 650 MPa and on residual PA activity only at 450 MPa. Cathepsin D activity in the acid whey from HP-treated milk was in general baroresistant at room temperature. The residual activity of cathepsin D decreased significantly at 650 MPa and 40°C and at the pressures 450 and 650 MPa at 55°C. Synergistic negative effects on the amount of native β-lactoglobulin were observed at 450 and 650 MPa and on the amount of native α-lactalbumin at 650 MPa. There were significant correlations between enzymatic activities (PL, PA and cathepsin D) and the residual native β-lactoglobulin and α-lactalbumin in bovine milk. In conclusion, HP significantly affected the activity of indigenous proteolytic enzymes and whey protein denaturation in bovine milk. Reduction in activity of indigenous enzymes (PL, PA and cathepsin D) and transfer of PL and PA from the casein to milk serum induced by HP is expected to have a profound effect on cheese yield, proteolysis during cheese ripening and quality of UHT milk during storage.

The somatic cell count (SCC) in milk is associated with increasing proteolytic degradation of caseins and it has been suggested that enzymes derived from somatic cells contribute to a lower yield and poorer quality of cheese. It is essential to increase the knowledge on naturally occurring milk proteinase activities to better understand how to improve the technological quality of milk. The aim of this work was to identify peptides actually present in milk as a result of proteolysis at different levels of SCC and to assign these peptides to potential responsible proteases where possible. Peptide fractions were prepared from acid whey by ultrafiltration at a molecular cut-off value of 10 000 Da. The peptides were separated using capillary reversed phase high performance liquid chromatography (RP-HPLC) and identified by matrix-assisted laser desorption/ionization-time of flight tandem mass spectrometry (MALDI-TOF MS/MS). Peptides identified ranged in mass from 1023 to 2000 Da, and originated from αS1-, αS2- or β-casein. Possible responsible proteases that could be suggested when examining the peptide cleavage sites included plasmin, cathepsin B, D and leukocyte elastase. The results indicated that plasmin was primarily responsible for the observed proteolysis in milk at low cell count, whereas the cathepsins and elastase became implicated at elevated cell count. Specificity and activity of cathepsins and elastase has earlier mainly been studied in model systems, whereas less is known about their activities in milk itself. This is also the first indication of involvement of elastase in milk proteolysis through the unequivocal determination of cleavage sites.

Bovine milk contains a number of indigenous proteolytic enzymes, of which plasmin is the most important (Grufferty & Fox, 1988; Bastian & Brown, 1996; Kelly & McSweeney, 2003). Plasmin (EC 3.4.21.7) is a serine proteinase with pH and temperature optima of 7·5 and 37 °C, respectively. In milk, most of the plasmin is present as its inactive precursor, plasminogen, which is converted to active plasmin by plasminogen activators (PA) present in milk, e.g., urokinase-type (u-PA) and tissue-type PA (t-PA) (Bastian & Brown, 1996). Since plasmin, plasminogen and PA are associated with casein micelles, they are incorporated into cheese curd, while plasmin inhibitors and inhibitors of PA are lost with the whey. Plasmin incorporated in cheese curd acts on its substrate, the caseins, contributing significantly to primary proteolysis during ripening (Upadhyay et al. 2004b).

Heat-induced inactivation of bovine plasmin, denaturation of β-lactoglobulin (β-lg), the interactions between both species and casein micelles and the subsequent net effect on proteolysis of β-casein was studied in a model system consisting of phosphocasein and β-lg in synthetic milk ultrafiltrate. The inactivation of plasmin and denaturation of β-lg were first order reactions, with the rate of inactivation of plasmin being greater than the rate of denaturation of β-lg. The predominant mechanism involved in the denaturation of plasmin in the temperature range 65–80 °C was its interaction with β-lg (kr at 60 °C, 0·0526; Ea, 176 KJ/mol). At the point of complete inactivation of plasmin ~45% of the β-lg remained undenatured. Thermal inactivation of plasmin through other mechanisms was negligible. The association of β-lg with the casein micelles at 60 °C had a rate constant of 3·71×10−5 min−1 and an Ea of 259 KJ/mol; thermal denaturation of β-lg was of much less importance, with a rate constant at 60 °C of the order of 1×10−10 min−1 and an Ea of 250 KJ/mol. On denaturation of all β-lg in the system, a maximum of ~55% was associated with the casein micelles. The effect of heating on the subsequent hydrolysis of β-casein indicated that the level of plasmin activity was the most important factor affecting proteolysis, while the interaction of β-lg with the casein micelles had limited effect. Overall, thermal stability of plasmin in milk is very much dependent upon its interaction with β-lg.

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.

Effects of ventilation regimen on the quality of ewes' milk and on proteolysis in Canestrato Pugliese cheese during ripening were studied. Cheeses were manufactured from the bulk milk of Comisana ewes subjected to three different ventilation regimens, which were designated low (LOV, 23 m3/h per ewe), moderate (MOV, 47 m3/h per ewe) and programmed ventilation regimen (PROV, 73 m3/h per ewe; fan set to maintain 70% relative humidity). Bulk milk was analysed for chemical and microbial composition, renneting parameters and plasmin-plasminogen activities. At 1, 15, 30 and 45 d of ripening, the cheeses were analysed for gross chemical composition, nitrogen fractions, and plasmin and plasminogen activities. The pH 4·6-insoluble nitrogen fractions were analysed by urea-PAGE. Free amino acid content was determined at the end of ripening. Lower concentrations of bulk milk somatic cell count (BMSCC) and of mesophilic bacteria were found in the MOV group than in the LOV and the PROV groups. A lower plasminogen (PG) to plasmin (PL) ratio (PG/PL) was observed in the MOV and PROV than in the LOV cheeses. Irrespective of treatment, PL activity in cheeses was higher at 15 d of ripening, while a sudden decrease of PL and PG activities was observed at 30 d, which was associated with a marked increase in non-protein nitrogen. The peptide profile characterized in the urea-PAGE showed a greater intensity of α- and β-CN hydrolysis in the MOV than in the PROV and LOV cheeses. The results provide evidence that a proper ventilation regimen is critical for optimizing the hygienic quality of milk and the proteolysis of Canestrato Pugliese cheese during ripening.

High pressure (HP) processing has attracted considerable interest in dairy research in recent years; its effects on cheese have been reviewed by O'Reilly et al. (2001). HP treatment of cheese may influence several of its properties, e.g., for hard and semi-hard cheeses, such as Cheddar and Gouda, the most notable effects are on the texture and rate of ripening. HP treatment increased elasticity and improved organoleptic properties of Gouda cheese (Kolakowski et al. 1998). Messens et al. (2000) reported that, immediately after treatment at 400 MPa, Gouda cheese was less rigid and solid-like and more viscoelastic than untreated cheese. HP treatment also enhances the functional properties of Mozzarella cheese (Johnston & Darcy, 2000; O'Reilly et al. 2002).

We examined the physical and chemical changes in milk during early lactation, and how these changes were affected by leaving one quarter unmilked in either the first or second milking, with the purpose of discriminating between colostrum and normal milk. Milk samples were collected from each quarter of 17 cows during the first 5 d after calving and then after about 7 d and 14 d. Samples were analysed for somatic cell count (SCC), fat, protein, casein, lactose, IgG1, colour, plasmin, pH and coagulation properties. Large variations occurred in both chemical and physical properties throughout the study period. Within six milkings, the concentration of casein decreased by 60%, IgG1 by 94%, and lactose increased by 34%. At milking number 6, rennet coagulation time was lowest and curd firmness was highest. The pH increased from 6·4 to 6·7 over the period of the experiment, and the colour changed from yellow (reddish) to white. Coagulation properties and the pH fell within the range of normal milk after five milkings. Measurement of colour and density appeared to be a potential method for detection of milk unsuitable for the dairy factory. Effects of omitting one quarter in one milking differed between milk components, but seemed to be of little importance to the physical properties.

The link between oxidation and increased proteolysis in raw milk was studied. To accelerate oxidation, H2O2 (1 mM) was added to raw milk, resulting in enhanced proteolysis by up to 11·2% after 24 h incubation at 5 °C. Addition of Cu2+ (10 μM) to milk or exposure of milk to light (60 min) likewise increased proteolysis. To explain the mechanism responsible for increased proteolysis as a result of oxidation, the effect of lipid oxidation products on plasmin-induced proteolysis was tested. Addition of malondialdehyde to skim milk increased the formation of γ-caseins, a proteolysis product from plasmin hydrolysis of β-casein. The same observation was made in a model system containing 4·5 g β-casein/l sodium tetraborate buffer at pH 8 and plasmin. Addition of a plasmin inhibitor blocked the formation of γ-casein. The results indicate that aldehydes accumulated from lipid oxidation can modify β-casein and thereby increase susceptibility of the proteins to proteolysis. Furthermore, the data suggest that proteolysis in raw milk may be connected to oxidative processes.

Lyme borreliosis is a systemic infection caused by the spirochaete Borrelia burgdorferi, which is transmitted by tick bites and maintained in a delicately balanced ecological cycle. Recent increases in the population densities of tick hosts, the abundance of ticks and the proximity of man to natural tick habitats have led to an escalating worldwide incidence of Lyme borreliosis, and nonspecific clinical manifestations have yielded significant misunderstanding of the disease. After entry, B. burgdorferi activates local inflammation, yet evades host defences and facilitates dissemination by potentially masquerading with host components such as plasmin and complement. The extent of tissue injury is determined by the aggressiveness of host inflammation and immunological reactions, as well as by genetic attributes of the spirochaete. The clinical presentation can be highly varied, including early manifestations that are limited to erythema migrans and ranging to disseminated infection with arthritis, carditis, cranial nerve palsy, peripheral neuropathy, meningitis, or other manifestations. Diagnostic tests have improved, but are unhelpful during certain stages of infection. Therapy varies depending on the degree of involvement, and recovery is usually rapid and complete. Post-treatment clinical manifestations in the absence of evidence for active infection are still poorly understood. The understanding of how B. burgdorferi survives in the environment and interacts with human and mammalian hosts has improved. However, further advances in prevention and therapy depend on continued investigation of the ecological risks and improved understanding of the pathobiology of this obligate bacterial parasite.

The fate of a forming thrombus is determined through the delicate balance between the coagulation cascade, favouring clot formation, and the fibrinolytic system, favouring clot lysis. These processes occur simultaneously, and enhancement of fibrinolysis has been shown to reduce occlusive thrombus formation in animal models. This review examines the roles of the major fibrinolytic factors involved in clot lysis. The regulation of plasmin activity by plasminogen activators, α-2-antiplasmin, plasminogen activator inhibitor 1, and thrombin-activatable fibrinolysis inhibitor, and their effects on thrombus formation in vivo are discussed. Since alterations in fibrinolytic capacity appear to affect thrombus formation in animal models, there is considerable interest in the pharmacological manipulation of fibrinolysis.

Human plasminogen undergoes a large tertiary structural change in the presence of lysine derivatives (e.g. ε-amino caproic acid, EACA). This change facilitates human plasminogen activation by human plasminogen activators, resulting in elevated blood plasmin levels. It is hypothesized that this structure-function relationship is similar for bovine plasminogen. The objectives of this study were to investigate the effect of the ligand EACA on the secondary structure of plasminogen (bovine, human, and rabbit) and the tertiary structure of bovine plasminogen using Fourier-transform infrared spectroscopy (FTIR). Spectra of plasminogen, EACA, and a mixture of plasminogen and EACA in water and deuterium were collected using FTIR. Fourier-self deconvoluted spectra in the amide I region (1700–1600 cm−1) were used to detect changes in secondary structure of plasminogen after EACA addition. Change in bovine plasminogen tertiary structure was determined by comparing ratios of amide II (1600–1500 cm−1) to amide I bond intensities over time for samples in deuterium. No differences in secondary structure were observed for any plasminogen in the presence of EACA; however, addition of EACA significantly changed tertiary structure of bovine plasminogen. This tertiary structural change indicates a transition from a folded to an unfolded state, which could be more easily converted to plasmin. These results are consistent with reported human plasminogen studies using neutron scattering (tertiary structure) and circular dichroism (secondary structure) methods.

The objective of this study was to determine the effect of high pressure homogenisation (HPH) on alkaline phosphatase activity and plasmin and plasminogen-derived activities in raw whole bovine milk. Milk (approximately 4% fat) was treated by two-stage conventional homogenisation (18 MPa) or single or two-stage HPH at 50, 100, 150 or 200 MPa. Inactivation of plasmin and plasminogen-derived activities was evident in conventionally homogenised samples, and increased as HPH pressure increased. Two-stage HPH reduced both activities to a greater extent than single-stage HPH. Milk inlet temperature had a significant effect on residual plasmin and plasminogen activities of HPH-treated milk samples, especially those treated at 50 MPa. Inactivation of plasmin and plasminogen on HPH-treatment (150 MPa) of milk samples of varying fat contents (0–10%) was also investigated; there was a curvilinear relationship between residual plasmin and plasminogen-derived activities and fat content in the ranges 0–2% and 0–4%, respectively, with little additional inactivation at higher fat contents. Thus, indigenous proteolytic activity of milk is clearly affected by HPH. However, all homogenised milk samples retained active alkaline phosphatase, indicating that thermal conditions during HPH did not equate to that of conventional high temperature short time pasteurisation, and that the wide range of forces experienced by milk during HPH treatment does not inactivate the latter enzyme.

Milk contains the alkaline proteinase plasmin and lysosomal proteinases; the significance of the latter is ill-defined. The objective of this study was to investigate composition and activities of several different proteolytic enzymes in milk samples of varying somatic cell count (SCC). Increasing milk SCC was correlated with increased plasmin, cathepsin D and cysteine protease activities, with concomitant increases in proteolysis in milk. Addition of plasmin inhibitors confirmed the heterogeneity of proteinase activities in milk, as urea-PAGE analysis of milk samples showed casein hydrolysis in milk after 7 d storage even in samples with inhibitors added; extent and heterogeneity of proteolysis was correlated with milk SCC. Rennet coagulation properties were not significantly correlated with SCC, or activities of measured enzymes. Milk of increasing SCC also exhibited decreased physical stability during incubation of milk at 37 °C. Pasteurized milk was more stable than raw milk, suggesting that the enzyme(s) or mechanisms leading to such instability are impaired by pasteurization. Overall, milk has a very heterogeneous proteolytic enzyme population, with a higher significance of non-plasmin enzymes, such as cathepsin D and cysteine proteinases, than perhaps previously recognised.

The objective of this study was to determine the effects of stage of lactation (SOL) and time of year on plasmin-derived proteolytic activity in the milk of pasture-fed dairy cows in New Zealand. Four herds of 20 Friesian cows were used, one herd calving in each of January, April, July and October. Cows grazed ryegrass/white clover pasture only, except during June (winter) when all cows received supplementary pasture silage. Milk samples were collected on four occasions during the year (spring, summer, autumn and winter) from each cow in milk, to give a total of three samples per cow (early, mid and late lactation; c. 30, 120 and 220 days after calving, respectively). Milk samples were analysed for plasmin-derived proteolytic activity. There was no effect of either SOL or time of year on plasmin activity and therefore yields of plasmin followed patterns in milk yield (highest in early lactation and in summer). There were effects of both SOL and time of year on plasminogen-derived and total plasmin plus plasminogen-derived activity, both of which were highest in late lactation and in spring. Changes in plasminogen-derived activity and total plasmin plus plasminogen-derived activity due to SOL were not only due to the decrease in milk yield associated with advancing lactation, because enzyme yields were also increased with advancing lactation. Similarly, effects of time of year on plasminogen-derived activity and total plasmin plus plasminogen-derived activity could not be attributed solely to concomitant changes in milk yield, and may be influenced by the variation in the quality and quantity of feed during the year inherent in a pasture-based dairy system. Effects of SOL on proteolytic activity were greater than, and independent of, effects of time of year.