Milk lipoprotein lipase distribution in the major fractions of bovine milk

Raw, bovine bulk tank milk and milks from selected cows were separated by ultracentrifugation into four major fractions: casein, sloughed membrane material, serum, and milk fat globule membrane. Milk lipoprotein lipase activity was measured by the pH stat method and protein determinations were made by the Lowry procedure for each of the four fractions in order to calculate specific activity (units per milligram of protein). In six farm-cooled bulk milk samples stored less than or equal to 24 h, casein had a significantly higher milk lipoprotein lipase total activity, 35.66 units/ml of milk, than all of the fractions. Serum had the next highest activity with 11.69 units/ml of milk. Fluff and milk fat globule membrane had activities of .80 and .41 units/ml of milk, respectively. The specific activity of the fluff was 3.3 milk lipoprotein lipase units/mg of protein, which was significantly higher than the casein and serum fractions in pooled milk. Milks from five cows in midlactation were assayed individually for milk lipoprotein lipase activity and protein content immediately after milking and after 12, 24, 48 and 72 h of cold (4 degrees C) storage. Fresh warm milk was characterized by the absence of fluff. Casein had the highest mean activity (29.91 units/ml), followed by serum (10.25 units/ml) and milk fat globule membrane (.26 units/ml) in the warm milk from the individual cows. Upon cooling to 4 degrees C, significant increases in enzyme activity in the fluff and milk fat globule membrane fractions were observed at 12 h.(ABSTRACT TRUNCATED AT 250 WORDS)     

Physicochemical transformation of milk components and release of foot-and-mouth disease virus

Possible mechanisms for protective roles of milk components on foot-and-mouth disease virus present in the milk of infected cows were examined. Light scattering bands collected from Ficoll-sucrose gradient fractions of skim-milk contained membrane-limited structures but these were non-infectious for bovine kidney cells. Infectivity titres in buttermilk higher than those of the original cream or butter suggested association of virus with milk fat globules. Increased infectivity titres in skim-milk after treatment with SDS suggested release of virus particles from dissociated casein micelle subunits. Chelating agents, de-emulsifying agents and trypsin, which alter the structure of the individual milk components casein, lipid and milk fat globule membrane were without effect on infectivity titres.     

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.     

Activity and nature of plasminogen activators associated with the casein micelle

In fresh milk, plasminogen, the zymogen form of plasmin (PL), is the predominant form. Therefore, plasminogen activators (PA) can contribute significantly to PL activity in milk. Both tissue-type PA (tPA) and urokinase-type PA (uPA) exist in milk; however, contradictory findings have been reported for which type of PA is most closely associated with the casein micelles. Little is known about the factors that might lead to variations in the individual activities of the PA. The objective of this work was therefore to investigate possible factors that might affect the association of tPA and uPA with the casein micelle and their activities thereafter. Plasminogen activators were isolated from milk samples with different somatic cell counts following 2 different isolation protocols. Determination of uPA, tPA, and PL activities was carried out quantitatively following chromogenic assays using 2 different substrates, and qualitatively using specialized sodium dodecyl sulfate-PAGE. Different isolation methods and conditions led to differences in uPA, tPA, and PL activities. Urokinase-type PA activity was significantly higher in PA fractions isolated from milk with high somatic cell counts than from milk with low somatic cell counts. Activity results indicated that in pasteurized milk uPA could dissociate from the somatic cells and bind to casein. Moreover, a high level of PL in isolated PA fractions contributed to significantly enhanced PA activities. Overall, results confirmed the association of both uPA and tPA with the casein micelle; however, their amounts, activities, and molecular weights varied based on the nature of the milk and methods of separation, with uPA being the PA with greater potential to affect plasminogen activation in milk.     

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 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.     

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.     

Physical,textural, and rheological properties of whipped cream affected by milk fat globule membrane protein

This work aims at improving the textural and whipping properties of whipped cream by the addition of milk fat globule membrane protein. The determination of particle size distribution and average diameter of whipped cream showed that the small particle size was shifted to a larger range after milk fat globule membrane protein was added. The average particle size (d_3,2) of whipped cream reached a maximum value of 5.05 µm at 1% milk fat globule membrane protein, while slowly decreased with increasing milk fat globule membrane protein levels from 2% to 5%. In addition, the partial coalescence of fat increased with the increase of milk fat globule membrane protein levels, and the correlation between the whipping time and the overrun of whipped cream was positive. The addition of milk fat globule membrane protein also altered the rheological behaviour of whipped cream, resulting in the increase of modulus G′ and the loss modulus G″. The results also indicated that higher milk fat globule membrane protein level decreased the serum loss of whipped cream while improved its stability. While milk fat globule membrane protein levels had no significant effect on viscosity, its increasing levels effectively improved the hardness, consistency, and viscosity of whipped cream.     

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.     

Effects of Some Chemical and Physical Treatments on Proteolysis in Milk

Addition of either a water-soluble or oil-soluble surfactant to milk enhanced proteolysis. This effect was attributed primarily to alteration in the availability of the casein and secondarily to a partial release of proteolytic enzymes from the milk fat globule membrane. Adding milk fat globule membrane to milk also increased proteolysis, presumably by contributing additional proteinase.Proteolysis increased after milk was subjected to Waring blendor treatment, ultrasonic treatment, or temperature fluctuations. These treatments presumably cause dissociation of proteinases from milk fat globule membranes or casein micelles.Addition of carrageenan to milk inhibited proteolysis. Carrageenans may inhibit proteolysis by preventing dissociation of casein micelles or by blocking access to the active sites on casein. Removing either calcium ions or colloidal phosphates from milk by dialysis enhanced proteolysis. This enhancement may be due to dissociation of casein micelles or milk proteinases originally adsorbed on the casein micelles. Removal of calcium ions increased proteolysis more than removal of colloidal phosphates.     

β-Casein-phospholipid monolayers as model systems to understand lipid-protein interactions in the milk fat globule membrane

Phospholipid-protein monolayer films were studied as model systems to mimic the structure of the native bovine milk fat globule membrane (MFGM) and to understand lipid-protein interactions at the surface of the globule. Phospholipids extracted from bovine raw milk, raw cream, processed milk and buttermilk powder were spread onto the air-water interface of a Langmuir trough, β-casein was then added to the sub-phase, and Langmuir-Blodgett films were studied by epifluorescence microscopy and atomic force microscopy. In all films, β-casein was responsible for clustering of the sphingomyelin- and cholesterol-rich microdomains into larger platforms. This suggests that the same phenomenon may happen at the surface of the milk fat globule, where specific MFGM proteins may cause aggregation of microdomains.     

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 the fat globule sizes on the meltdown of ice cream

The meltdown of ice cream is influenced by its composition and additives and by fat globule size. The objective of this study was to examine the effect of fat globule size and fat agglomerate size on the meltdown stability of ice cream. Therefore, an ice cream mix (10% milk fat) was homogenized at pressures ranging from 0 to 30 MPa in single-stage, double-stage, and selective homogenization processes. The ice cream, produced on a continuous ice cream freezer, was characterized by an optimized meltdown test while, in addition, the fat globule sizes and the free fat content were determined in the mix and the molten ice cream. The meltdown was dependent on the fat agglomerate sizes in the unfrozen serum phase. Agglomerates smaller than a critical diameter led to significantly higher meltdown rates. Homogenization pressures of at least 10 MPa were sufficient to produce a stable ice cream. Furthermore, proof was provided that double-stage homogenization is not necessary for fat contents up to 10% and that selective homogenization is possible to produce stable ice creams. Based on these results a model was deduced describing the stabilizing mechanisms during the meltdown process.     

Impact of cream washing on fat globules and milk fat globule membrane proteins

Milk proteins, contained within the aqueous phase surrounding fat globules, should be removed before analysis of the composition of the native milk fat globule membrane (MFGM). The effect of the conditions applied during washing of cream on MFGM integrity has not been fully studied, and factors potentially effecting a modification of MFGM structure have not been systematically assessed so far. In this study, a cream separator was used to investigate the impact of cream washing on milk fat globule stability and the corresponding loss of MFGM proteins. Flow velocity, fat content, and type of washing solution were varied. Particle size measurements and protein analyses were carried out after each washing step to determine fat globule coalescence, removal of skim milk proteins, and efficiency of MFGM isolation. Significant differences in fat globule stability and protein amount in the MFGM isolates were measured using different washing conditions.     

Native milk fat globule size and its influence on whipping properties

The objective of this study was to investigate the influence of native milk fat globule size on the aeration of high fat dairy products with regard to maximum firmness time, gas inclusion and foam stability. The results showed that whipping time to maximum firmness was inversely proportional to mean fat globule size for both unhomogenised and slightly homogenised (2 MPa) creams. Additionally, increasing native mean fat globule size of the creams resulted in increased overrun. No significant differences in serum drainage were found between creams with different native milk fat globule size. Furthermore, when creams with native large mean fat globules were homogenised, the results showed that the maximum firmness time was in accordance with the mean fat globule size of non-aggregated creams. In the present study, cream fractionation was achieved by creaming or in a cost effective and fast manner using a modified centrifugal separator.     

Plasmin activity in pressurized milk

The effects of pressure (up to 400 MPa), applied at room temperature, on native proteinase activity of milk were investigated by means of plasmin activity, plasmin-derived activity after plasminogen activation and their distribution in different milk fractions, micelle microstructure, beta-LG denaturation, and casein susceptibility to proteolytic attack. The pressure conditions assayed did not lead to plasmin inactivation and only decreased around 20 to 30% total plasmin activity after plasminogen activation. However, pressure caused severe disruption of the micellar structure, releasing high levels of caseins, plasmin, and plasminogen to the soluble fraction of milk. High levels of soluble denatured beta-LG were also found in the ultracentrifugation supernatants of pressurized milks, particularly in those treated at 400 MPa. Probably as a result of micellar disintegration, caseins became more susceptible to proteolysis by exogenous plasmin. However, no enhanced proteolytic degradation was observed when we compared the evolution of pressurized and unpressurized milks during refrigerated storage. Serum-liberated plasmin may become more vulnerable to the action of proteinase inhibitors leading to a reduced proteolysis on refrigerated storage, despite the increased susceptibility of caseins to proteinase action.     

Microfiltration of buttermilk and washed cream buttermilk for concentration of milk fat globule membrane components

Buttermilk, the by-product from butter manufacture, has gained much attention lately because of the application potential of its milk fat globule membrane (MFGM) components as health ingredients. Microfiltration (MF) has been studied for buttermilk fractionation because of its ability to separate particles from dissolved solutes. However, the presence in this by-product of skim milk solids, especially casein micelles, restricts concentration of MFGM. The use of cream washed with skim milk ultrafiltrate to produce buttermilk with lower casein content was studied as well as fractionation of this buttermilk by MF. Results have shown that washing the cream prior to churning yields buttermilk with 74% less protein than normal cream buttermilk. Analysis of the protein profile of washed cream buttermilk revealed that caseins and whey proteins were the main classes of proteins removed. The MF of washed cream buttermilk resulted in permeation fluxes 2-fold higher than with normal cream buttermilk. The second separation of the cream induced high losses of phospholipids in the skim phase. However, retention of remaining phospholipids in washed cream buttermilk by the MF membrane was higher resulting in a phospholipids concentration factor 66% higher than that of normal cream buttermilk. The results presented in this study highlight the impact of casein micelles on the separation of MFGM components as well as their effect on permeation flux during MF.     

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).     

Preliminary investigation of the properties of somatic cell proteases

Effect of the plasmin inhibitor 6-amino-n-hexanoic acid on somatic cell proteases (equivalent to 2.3 X 10(6) cells/ml) was determined using a model system of casein micelles dispersed in Jenness/Koops buffer (1.5% wt/vol). Sodium dodecyl sulfate-polyacrylamide gel electrophoresis was used to quantitate casein. There was no effect of 120 mM 6-amino-n-hexanoic acid on casein proteolysis by somatic cell proteases. Molecular weights of casein proteolysis products produced by somatic cell proteases were different from those produced by plasmin. Quantitative and qualitative analyses of pasteurized mastitic milk by SDS-PAGE indicated that a portion of the somatic cell proteolytic activity survived pasteurization. Because 6-amino-n-hexanoic acid does not inhibit somatic cell proteases, it can be used to establish the relative contribution of somatic cell proteases and plasmin to total proteolytic activity in mastitic milk.     

Gravity separation of raw bovine milk: fat globule size distribution and fat content of milk fractions

This project determined effects of time and temperature on changes of fat globule size distribution and fat content in milk fractions during gravity separation. Fresh raw bovine milk was gravity separated at 4 or 15 degrees C. After 2, 6, 12, and 48 h, seven fractions, from bottom fraction (F1) to top fraction (F7), were successively drained from a separation column. Higher temperature resulted in a faster rate of fat separation. Within 2 h, large fat globules had already moved to the top, and the volume mean diameter of F7 increased from 3.13 microm (without separation) to 3.48 and 3.64 microm, respectively, at 4 and 15 degrees C. In F7, there was little change in globule size distribution after 2 h, but fat content continued to increase with separation time. The fat content of F7 reached 26.6% after 48 h at 4 degrees C, achieving a 58.8% creaming capacity. For F1 to F6, longer separation time resulted in smaller fat globule sizes and lower fat contents, especially for F1. After 48 h at 4 degrees C, the volume mean diameter of F1 decreased from 3.23 microm (without separation) to 1.16 microm, and fat content decreased from 3.75% (without separation) to 0.20%. Gravity separation may have unique applications in the dairy industry today. Its simplicity makes it an effective procedure for small-scale dairy product manufacturers to produce milks with a range of fat contents without using a centrifugal cream separator.