Plasminogen activation system in goat milk and its relation with composition and coagulation properties

The activity of plasmin (PL), plasminogen (PG), and plasminogen activator (PA) and their correlation with goat milk components and milk clotting parameters were investigated. Seven late-lactating Saanen goats were used to provide milk samples that were analyzed for PL, PG, and PA activity (colorimetric assay) fat, protein, noncasein nitrogen, nonprotein nitrogen, casein content, and somatic cell count (SCC). Milk clotting parameters (rennet coagulating time = coagulation time; K20 = firming rate of curd; A30 = curd firmness) were measured with a formagraph. Average milk yield and composition were similar to those previously observed in other studies. Plasmin, PG, and PA activity, expressed as units/ml, were, respectively, 20.04 +/- 0.94, 3.21 +/- 0.04, and 1154 +/- 57.61. Plasminogen activity was surprisingly low compared with other species (bovine, ovine), but it was consistent with the high activity of PA. A negative significant correlation was observed between PL and milk casein content. The correlation coefficients between PL and casein/protein ratio and PA and casein/protein ratio were negative and significant. A positive significant correlation was observed between PL and rennet clotting time and PA and rennet clotting time. Also positive was the correlation between PL and K20 and PA and K20. The plasmin activity was negatively correlated with A30. High plasmin and plasminogen activator activity in goat milk appeared to be negatively related with coagulating properties in late lactation, most probably via degradation of casein due to plasmin activity.     

Distribution of plasminogen and plasmin in fractions of bovine milk.

The relative amounts of immunoreactive plasminogen and active plasmin in different fractions of bovine milk were examined. Raw milk was centrifuged to separate skim, cream, and a somatic cell pellet. Skim milk was centrifuged to separate milk serum and casein micelles. Milk fat globule membranes were isolated from the cream fraction of bovine milk. Proteins from somatic cells were isolated following sonication of the cells. Western blot analysis showed the presence of several forms of plasminogen in bovine milk. The predominant forms of plasminogen identified following electrophoresis under nonreducing conditions were proteins with approximate molecular weights of 88,000, 152,000, and 160,000. The predominant forms of plasminogen identified after electrophoresis under reducing conditions were two proteins with approximate molecular weights of 88,000 and 50,000. The highest amount (82% of the total plasminogen), as determined by an ELISA, was associated with the casein fraction. Lower plasminogen concentrations were associated with the serum, cream fractions, and milk fat globule membranes. The SDS-PAGE of the cream and milk fat globule membranes indicated that some casein was present in both fractions. Thus, the low plasminogen concentrations in these fractions may be associated with the caseins there. No immunoreactive plasminogen was present in the somatic cells. Active plasmin was present in the same milk fractions in which plasminogen was detected: casein, serum, and cream.     

Contribution of macrophages to proteolysis and plasmin activity in ewe bulk milk

A total of 225 bulk sheep milk samples were collected from 5 intensively managed flocks during early, mid, and late lactation to assess the contribution of macrophages to the regulation of the plasmin-plasminogen system. Samples were analyzed for composition, somatic cell counts, milk renneting characteristics, and for plasmin (PL), plasminogen (PG), and plasminogen activators (PA) activities. Isolation of macrophages from milk was performed using a magnetic positive separation and mouse antiovine macrophage antibody; separated cells were lysed by several freeze-thaw cycles, and activity of urokinase PA (u-PA) was determined. Plasmin activity decreased during lactation (42.06 ± 0.66, early; 31.29 ± 0.66, mid; 28.19 ± 0.66 U/mL, late). The reduction in PL activity recorded in the mid and late lactation milk matched the increase in PG:PL ratio. The activity of PA increased throughout lactation; the highest value being recorded in the late lactation milk (260.20 ± 8.66 U/mL). Counts of isolated and concentrated macrophages were higher in early and mid lactation milk (3.89 ± 0.08 and 3.98 ± 0.08 log₁₀ cells/mL, respectively) than in late lactation milk (3.42 ± 0.08 log₁₀ cells/mL). Stage of lactation did not influence the activity of u-PA detected in isolated macrophages. The activity of u-PA associated with isolated milk macrophages only minimally contributed to total PA activity detected in milk. Proteolytic enzymes, associated with isolated macrophages, act on α-casein hydrolysis, as shown by urea-PAGE electrophoresis analysis. Somatic cell counts did not exceed 600,000 cells/mL, and this threshold can be considered a good index of health status of the flock and of the ability of milk to being processed. Our results lend support to the hypothesis that macrophages in ewe bulk milk from healthy flocks only slightly contribute to the activation of the PL-PG system.     

Effect of psychrotrophic bacteria and of an isolated protease from Pseudomonas fluorescens M3/6 on the plasmin system of fresh milk

In the present study, we investigated the effect of Pseudomonas spp. growth on the plasmin enzymatic system in casein and whey fractions of fresh milk. Two bacterial strains, Pseudomonas spp. SRM28A and Pseudomonas fluorescens M3/6, were inoculated at a level of approximately 10(3) cfu/ml into fresh milk and incubated at 7 degrees C for 3 d. Bacterial counts were approximately 10(8) cfu/ml by d 3. Samples collected every 24 h were treated to separate the casein from the whey fraction. Casein and whey fractions were subjected to electrophoresis to visualize protein breakdown and plasmin activity and to colorimetric assays to quantify plasmin-related activities. With psychrotrophic bacterial growth, plasmin levels in casein fractions decreased significantly and in whey fractions increased then decreased significantly. Fresh milk results were similar for the two strains and were similar to earlier results with reconstituted nonfat dry milk. A transmission electron microscopy study by immunocytochemistry showed the presence of plasmin in casein micelles and its disappearance upon microbial growth in the milk. We hypothesized that extracellular microbial proteases produced by psychrotrophic microorganisms are responsible for this effect. To confirm this, an extracellular bacterial protease was isolated from Pseudomonas fluorescens M3/6 by ammonium sulfate fractionation and ion-exchange chromatography and incubated with fresh milk. Milk samples analyzed during incubation with the protease had significantly increased plasmin and plasminogen activities in the whey fraction within 5 h of incubation, while differences in activities in the casein fraction occurred at time 7.5 h for plasmin activity and 10 h of incubation for plasminogen activity. These quantitative data were supported by plasmin activity as visualized by casein-SDS-PAGE. These results suggest that growth of the Pseudomonas strains in fresh milk, and particularly their production of extracellular proteases, may be a causative factor in the release of plasmin from the casein micelle. Such plasmin release could affect the quality of cheeses and other food products that utilize dairy ingredients.     

Contribution of somatic cell-associated activation of plasminogen to caseinolysis within the goat mammary gland

Functional regression of the mammary gland is partly reflected by proteolysis of milk protein and tissue protein. The involvement of the plasminogen activation system in degradation of milk protein and mammary tissue damage has been demonstrated under inflammatory conditions. In this study, mammary secretion from 23 dairy goats primarily grouped as lactation (milking twice daily) or involution (milking once daily or less) was used to determine the ratio of gravity-precipitated casein to total milk protein (casein ratio) as an index of caseinolysis, and activities of components of plasminogen activation system as well as their expressions on somatic cells. Based on the casein ratio, lactation goats were subcategorized as very active (71.8 ± 1.0%) or less active (29.9 ± 1.0%) in mammary function; involution goats were subcategorized as gradual (21.7 ± 1.0%) or acute (5.9 ± 0.2%) involution. This result suggests that caseinolysis occurred during regular lactation as well as during involution. On the other hand, activities of components of the plasminogen activation system in mammary secretion were increased along with the decreasing casein ratio, in contrast to the similar activities of their counterparts in circulation throughout various mammary statuses. Correlation analysis between casein ratio and activities of plasminogen activation system of goat milk indicated a significant negative relationship for plasmin (r = −0.64), plasminogen (r = −0.69), and urokinase-type plasminogen activator (uPA; r = −0.78) during involution but not during lactation. As for the cellular components of plasminogen activation system, there was an increase in immunoreactivity on somatic cells toward both monoclonal antibodies of human uPA and human uPA receptor under involution conditions suggesting their upregulation relative to lactation condition. Collectively, these results suggest that plasminogen activation system within the mammary gland differentially contribute to milk caseinolysis along the various stages of goat lactation. Meanwhile, a somatic cell-mediated local elevation of plasmin activity may be committed to extensive caseinolysis during involution.     

Effect of high-pressure treatment at various temperatures on indigenous proteolytic enzymes and whey protein denaturation in bovine milk

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 degrees 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 degrees 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 degrees C and at the pressures 450 and 650 MPa at 55 degrees C. Synergistic negative effects on the amount of native beta-lactoglobulin were observed at 450 and 650 MPa and on the amount of native alpha-lactalbumin at 650 MPa. There were significant correlations between enzymatic activities (PL, PA and cathepsin D) and the residual native beta-lactoglobulin and alpha-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.     

Effects of somatic cell count and stage of lactation on the plasmin activity and cheese-making properties of ewe milk

The experiment was conducted from March to July 2002 using 5 intensively managed flocks of Southern Italy. In each flock, 2 groups of 50 ewes were created. The groups were designated LSCC (low somatic cell count [SCC]) when their milk SCC was lower than 500,000/mL and HSCC (high SCC) when their milk SCC was higher than 1,000,000/mL. Bulk milk and whey samples were analyzed for fat, total protein, lactose, casein, and whey protein contents. Renneting properties of milk were also determined. Moisture, NaCl, and nitrogen fractions were determined in fresh cheese curds. In addition, plasmin (PL) and plasminogen (PG) activities in milk and cheese were monitored. The proteolytic activity of plasmin by urea-polyacrylamide gel electrophoresis and the white blood cell (WBC) differentials were determined. The HSCC resulted in higher pH values in milk and in higher moisture and lower fat contents in fresh cheese curds. Moreover, a lower recovery of fat and whey proteins was obtained from the HSCC than from the LSCC raw milk. The crude protein and casein contents were higher in the HSCC than in the LSCC curds during early and midlactation; an opposite trend was observed in late lactation. Plasmin and PG activities underwent more marked fluctuations in the LSCC than in the HSCC curds through lactation. The results of this experiment demonstrate that the PL activity in ewe milk is markedly influenced by the SCC, although SCC is not the only parameter for predicting PL and PG evolution in ewe milk. The LSCC milk resulted in a higher proteolytic potential of Canestrato pugliese cheese curds.     

Changes in plasmin-plasminogen-plasminogen activator system in milk from Italian Friesian herds

Changes in plasmin, plasminogen and plasminogen activator (PA) throughout the lactation were investigated in individual milk samples obtained from 32 Friesian cows from four commercial herds located in Northern Italy. Herds were chosen to represent four different, yet typical for Italy, diets. Increased levels of plasmin and PA (P < 0.05) were observed with advancing lactation. Plasminogen peaked during the fifth month of lactation. The increased levels of plasmin during the fifth month of lactation are partly due to increased plasminogen, which reflects increased permeability of mammary epithelium. However, the ratio of plasminogen to plasmin decreased with advancing lactation, suggesting accelerated conversion of plasminogen to plasmin. Major differences were observed between herds with respect to plasmin levels. These differences probably reflect differences in diets and management practices. This could be very important for Northern Italy where most of the milk produced is used for cheese manufacture. Plasmin, PA and somatic cell counts (SCC) were negatively correlated with casein/protein with coefficients of −0.38, −0.43 and −0.40, respectively. A significant correlation existed between PA and SCC (r = 0.50). PA was positively correlated with plasmin (r = 0.49).     

Seasonal variations in composition,properties, and heat-induced changes in bovine milk in a seasonal calving system

We determined seasonal variations in the composition and characteristics of bovine milk, as well as heat-induced changes in the physicochemical properties of the milk, in a typical seasonal-calving New Zealand herd over 2 full milking seasons. Fat, protein, and lactose contents varied consistently during the year in patterns similar to those of the lactation cycle. Seasonality also had significant effects on milk calcium, ionic calcium, fat globule size, buffering capacity, and ethanol stability, but not on casein micelle size. The ratio of casein to total protein did not vary significantly over the season, but late-season milk had the highest content of glycosylated κ-casein (G-κ-CN) and the lowest content of α-lactalbumin in both years. We observed significant between-year effects on protein, total calcium, ionic calcium, pH, and casein:total protein ratio, which might have resulted from different somatic cell counts in the 2 years. Compared with heating at 90°C for 6 min, UHT treatment (140°C for 5 s) induced greater dissociation of κ-casein, a similar extent of whey protein denaturation, a lower extent of whey protein–casein micelle association, and a larger increase in casein micelle size. Indeed, UHT treatment might have triggered significant dissociation of G-κ-CN, resulting in aggregation among the casein micelles and increased apparent mean casein micelle diameter. Seasonality had significant effects on the partitioning of G-κ-CN between the micelle and the serum phase, the extent of whey protein–casein micelle association under both heating conditions, and the casein micelle size of the UHT milk.     

Partial purification and characterization of native plasminogen activators from bovine milk

At least four native plasminogen activators were detected in bovine milk, and two partially purified plasminogen activators were characterized. The plasminogen activators were dissociated from casein proteins by treatments with sulfuric acid and dimethylformamide. The plasminogen activators in the resulting fractions were partially purified with size exclusion, affinity, or metal chelate chromatographic techniques. Molecular weights of the two partially purified plasminogen activators were 47.2 and 30.5 kDa by gel electrophoresis. Size exclusion chromatography gave a molecular weight of 43.2 kDa for the first plasminogen activator. The isoelectric points of the two plasminogen activators were in the pH range 6.2 to 6.7. Because activity was not enhanced by the presence of fibrinogen fragments in a plasminogen activator assay mixture and decreased when human anti-urokinase Ig were added, at least some bovine milk native plasminogen activators appear to be urokinase-type plasminogen activators.     

Factors affecting the plasmin-plasminogen system in milk obtained from three Greek dairy sheep breeds with major differences in milk production capacity

The purpose of this study was to evaluate the effect of breed, stage of lactation, and health status of the udder on the plasmin-plasminogen system in ovine milk. A total of 38 ewes were used from 3 breeds [Boutsiko (n = 12), Chios (n = 12), and a synthetic breed (50% Boutsiko, 25% Arta, and 25% Chios, n = 14)] with major differences in their genetic potential with respect to milk yield. Milk samples were collected every 2 wk throughout the lactation period and were analyzed for fat, protein, lactose, and somatic cell count (SCC). In addition, milk plasmin (PL), plasminogen (PG), and plasminogen activator (PA) activities were determined. The Chios breed had the greatest average daily milk yield, the synthetic breed had an intermediate milk yield, and ewes of the Boutsiko breed had the lowest milk yield. Milk samples obtained from the Boutsiko breed had similar PL and PA activities, compared with those obtained from the other 2 breeds. The ratio of PG:PL was less in milk samples from the Boutsiko breed compared with the other 2 breeds, indicative of an increased rate of conversion of PG to PL for this breed. There was no correlation between PL activity and daily milk yield in ewes from all 3 breeds. Activities of PL, PG, and PA were greater in ovine milk with elevated SCC (>300,000/mL) compared with activities in milk with low SCC (<300,000/mL). The ratio of PG:PL was less in the high-SCC group compared with the low-SCC group, which indicates an increased rate of conversion of PG to PL for the high-SCC group. There was a decrease in PG and PA activities as well as in the PG:PL ratio in late lactation milk (mo 5 to 6) when compared with early or mid lactation milk (mo 1 to 4). Thus, the PL-PG system is affected by breed, stage of lactation, and the health status of the udder. No relationship was found between PL activity and daily milk yield in the 3 Greek dairy sheep breeds. Plasmin is not a marker for gradual involution in the Greek sheep breeds studied.     

Effect of mastitis on plasminogen activator activity of milk somatic cells.

This study was conducted to examine the effects of mastitis and stage of lactation on plasminogen activator (PA) activity in milk somatic cells. An assay system, which measures the plasmin-mediated hydrolysis of the chromogenic substrate D-valyl-leucyl-lysine p-nitroanilide, was used to assess PA activity present within milk somatic cells. Milk cell associated PA activity was increased (P < 0.05) by 50% in the presence of fibrin fragments. This suggests that milk somatic cells contain tissue PA which, unlike urokinase PA, is preferentially activated in the presence of fibrin fragments. An increase of the milk somatic cell count from < 5 x 10(4) to > 10(6) cells/ml resulted in an 8-fold increase in PA activity per cell. Elevated levels of PA activity were associated with milk somatic cells isolated from mastitic quarters obtained from cows in early (< 4 months in lactation) or late lactation (> 8 months in lactation). We conclude that PA activity is increased during severe mastitic inflammation. Although the physiological function of this enzyme is as yet unclear, we propose that it may be involved in the conversion of plasminogen to plasmin, contributing to the higher levels of plasmin occurring in milk isolated from mastitic quarters.     

Effects of Pseudomonas fluorescens M3/6 bacterial protease on plasmin system and plasminogen activation

Heat-stable proteases produced by the psychrotroph Pseudomonas fluorescens M3/6 have been shown to affect the plasmin system in milk, which in turn will affect the quality of processed milk. The M3/6 proteases cause dissociation of plasmin from casein in minimally processed milk. The objective of this work was to study the effect of M3/6 protease on the plasmin system, as well as its role in plasminogen activation, under commonly applied cheese-making conditions. Isolated M3/6 protease was added to raw milk, which then was pasteurized, and subjected to pH adjustments and CaCl₂ addition. Casein and whey fractions were separated by chymosin treatment then analyzed for plasmin activity. Individual and interaction effects of M3/6 protease addition, pH treatment, and CaCl₂ addition on plasmin activity were studied. Enzyme activity assays were carried out to study individually the effect of M3/6 protease on plasmin system components. Kinetic parameters were calculated to characterize the effect of M3/6 protease on plasminogen activation. Plasmin activity increased in the curd fractions of the protease-treated milk that was subjected to conditions most resembling cheese-making conditions, indicating that M3/6 protease triggered plasminogen activation rather than dissociation of plasmin from casein micelles. Results from the studies on plasminogen activation confirmed that the observed activation of plasminogen in protease-treated samples subjected to cheese making conditions was attributed to the stimulatory effect M3/6 protease had on plasminogen activators (PA). The M3/6 protease stimulated human and bovine PA by increasing their activity 4.5- and 2.5-fold, respectively. Similarly, the catalytic efficiencies of human urokinase-type PA and bovine PA were increased in the presence of M3/6 protease by 12- and 4-fold, respectively. Our research presented a basic step toward fully understanding the effect of bacterial proteases under different processing conditions, where the gathered information can aid in better control of processing conditions based on the desired outcome.     

Expression of protease nexin-1 and plasminogen activators during follicular growth and the periovulatory period in cattle

Extracellular matrix remodeling occurs during ovarian follicular development, mediated by plasminogen activators (PAs) and PA inhibitors including protease nexin-1 (PN-1). In the present study we measured expression/activity of the PA system in bovine follicles at different stages of development by timed collection of ovaries during the first follicular wave and during the periovulatory period, and in follicles collected from an abattoir. The abundance of mRNA encoding PN-1, tissue-type PA (tPA), urokinase (uPA) and PA inhibitor-1 (PAI-1) were initially upregulated by human chorionic gonadotropin (hCG) in bovine preovulatory follicular wall homogenates. PN-1, PAI-1 and tPA mRNA expression then decreased near the expected time of ovulation, whereas uPA mRNA levels remained high. PN-1 concentration in follicular fluid (FF) decreased and reached the lowest level at the time of ovulation, whereas plasmin activity in FF increased significantly after hCG. Follicles collected from the abattoir were classified as non-atretic, early-atretic or atretic based on FF estradiol and progesterone content: PN-1 protein levels in FF were significantly higher in non-atretic than in atretic follicles, and plasmin activity was correspondingly higher in the atretic follicles. No changes in PN-1 levels in FF were observed during the growth of pre-deviation follicles early in a follicular wave. These results indicate that PN-1 may be involved in the process of atresia in non-ovulatory dominant follicles and the prevention of precocious proteolysis in periovulatory follicles.     

Quality of milk and of Canestrato Pugliese cheese from ewes exposed to different ventilation regimens

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 15d 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 alpha- and beta-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.     

Effect of heat on the distribution of fluorescently labeled plasminogen and plasminogen activators in bovine skim milk

ABSTRACT:  Differentially fluorescently labeled bovine plasminogen (PG-594) and human tissue- and urokinase-type plasminogen activators (tPA-647 and uPA-546) were added to bovine skim milk to track the effect of heat on the location and concentration of these plasmin system components following acid precipitation or ultracentrifugation. In unheated milk, the majority (71.7% to 89.0%) of the added PG and PAs associated with casein micelles or acid curd, and PG-594 in the serum fraction was partially due to associations with nonsedimentable caseins. Heat treatment (85 °C for 16 s) significantly ( P < 0.05) affected distribution of PG-594, tPA-647, and uPA-546, resulting in reduced concentrations of PG and PAs in the serum fractions and reciprocal increases in their levels in the nonsedimentable casein fractions. Overall, almost all of the added PG and PAs (95.9% to 97.5%) became associated with caseins following heat treatment. This is the 1st study to successfully use fluorescent labeling to quantify effects of heat on the location of plasmin components in skim milk.     

Effects of heat and β-lactoglobulin on distribution of fluorescently labeled tissue- and urokinase-type plasminogen activators in a model milk system

A fluorescent labeling method was developed to determine the effects of heat and β-lactoglobulin (BLG) on the concentration distribution of added fluorescently labeled tissue-type (tPA-647) and urokinase-type (uPA-546) plasminogen activators (PAs) in a model milk system. Prior to heating, the majority of the added PAs (87% of tPA-647, 72% of uPA-546) became associated with casein micelles and could be dissociated by acidification or NaCl. Addition of 0.5% BLG to the unheated system had no significant effect on the distribution of PAs. Heat-induced binding of uPA-546 to micelles was shown by an increase of uPA-546 from 72% to 88% or 95% in the micelle fraction of systems without or with BLG, respectively. Although heat alone had little effect on the total amount of tPA-647 associated with caseins (88%), addition of BLG to a heated system shifted 32% of the tPA-647 to a non-sedimentable casein fraction. After heating, casein–PA interactions became more complex.     

Transmission Electron Microscopy Study of Casein Micelle in Raw Milk with Different Somatic Cell Count Levels

The relationship between elevated raw milk somatic cell count (SCC) and casein micelle dimension was investigated by transmission electron microscopy (TEM). Milk samples collected from the dairy cattle with three different levels of SCC (<200,000, 200,000 to 800,000, and >800,000 cells/ml) were studied by TEM. The results indicated that an increase in SCC resulted in a decrease in the casein micelle size with an increase in their aggregation. The present research supported the hypothesis that elevated proteolytic activity, reduced secretary ability of the mammary glands, lower electrostatic and steric repulsion as well as different mineral contents of mastitic milk could affect casein micelle properties.     

The liquid-state 31P-nuclear magnetic resonance study on microfiltrated milk

To gain further insight into diversiform phosphorus in bovine milk, we separated skim milk into casein micelle and serum fractions by microfiltration and subjected them to liquid-state 31P-nuclear magnetic resonance (NMR) spectroscopy. As previously reported, the skim milk spectrum showed a broad and indistinct peak from phosphoserine residue (SerP) of casein. In the casein micelle spectrum, however, the SerP peak was more clearly observed with a phosphate peak that may be from micellar calcium phosphate (MCP). The serum spectrum was the same as skim milk spectrum, except for SerP peak. Furthermore, two types of casein micelle fractions, with 0.90 and 1.04 of [beta-casein + kappa-casein]/[alpha(s1)-casein + alpha(s2)-casein] ratios were generated by different temperature microfiltrations, occurring because beta-casein is released from the micelle at a low temperature. The shape of SerP peaks changed dramatically in both the casein micelle spectra, when the temperature dropped from 35 to 5 degrees C. Deconvolution analysis indicated that each SerP peak comprised the same set of four peaks. Half-width and composition discriminated between the two types of casein micelle fractions. As a consequence, there was significant interaction between casein micelle and milk serum, causing cloudiness of SerP in the liquid-state 31P-NMR spectrum of milk. Casein composition influenced the SerP-MCP interaction in micellar structure. Shape changing of the SerP peak was discussed in connection with beta-casein-release phenomenon.     

Effects of incubation temperature and salt concentration on plasminogen activators in cheese curd

Plasmin (PL) can play a major role in the proteolysis of dairy products, especially during cheese ripening. The effects of cook temperature and salt concentration on PL in cheese systems have been studied; however, the effects of cheese making conditions on plasminogen activators (PAs) have not. Therefore, the objective of this study was to determine the effect of curd cook temperature and salt concentration on PL, plasminogen (PG), and PAs. PL, PG, and urokinase-type PA activities extracted from cheese curds cooked at 21 °C were significantly ($P⩽0.05$) lower than those extracted from cheese curds cooked at 37 or 55 °C. Activity assays showed that PG was the only component in the PL enzyme system that was significantly affected by salt concentration, with higher PG-derived activity at 0% NaCl than 5% NaCl. Specialized electrophoresis confirmed that PL and PA activities increased with increasing cook temperature, and, unlike the activity assays, indicated that increase in salt concentration (to a certain extent) could increase PL and tissue-type PA activities.