Search

Plasmin is by far the predominant and most completely studied endogenous protease in bovine milk. Plasmin-induced proteolysis can have either beneficial or detrimental effects on the texture and flavor of dairy products, depending on the extent of hydrolysis and type of dairy product. In cheese, the breakdown of protein can help develop desirable flavors and texture during ripening, whereas in pasteurized milk and ultra-high-temperature milk, proteolysis causes undesirable gelation. Plasmin is part of a complex protease-protease inhibitor system in milk that consists of active and inactive forms of the enzyme, activators, and inhibitors. Considerable research has been done to isolate and characterize components of the plasmin system, determine how they interact, develop and compare quantitation methods, and determine how they are affected by cow characteristics, processing conditions, other milk components, storage conditions, and bacterial proteases. Considerable research has focused on enhancing or minimizing the activity of plasmin system components. The intent has been to control protease activity in casein and whey fractions, depending on the final food or ingredient application. Controlling the activity of plasmin has a great potential to improve dairy product quality and reduce their processing costs.

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.

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.

Assays for bovine milk plasminogen activators were developed. The colorimetric assay for bovine milk plasminogen activators was based on plasmin cleavage of the synthetic substrate Spectrozyme™. This substrate cleavage yielded the yellow chromophore p -nitroanilide, which was quantified spectrophotometrically at 405nm with an ELISA plate reader. Correlation coefficients were >.995 for the linear regions of the standard curves for the assays. Modification of a previously reported casein-plasminogen SDS-PAGE system allowed visualization of plasminogen activators from bovine milk. This system can be used to determine the molecular mass of the milk plasminogen activators and to detect the activity of the plasminogen activators in a sample containing plasminogen activator inhibitors.

Plasminogen activators were partially purified from fresh bovine skim milk by treatments with sulfuric acid, ammonium sulfate, and dimethylformamide, followed by Zn-chelating chromatography, resulting in a purification factor of 2204-fold for skim milk, which contained 340 mU/L of plasminogen activator activity as measured by a colorimetric assay. Further purification with plasminogen activator inhibitor affinity chromatography gave purification factors of about 11,000-fold, but plasminogen activators in this fraction were not stable. The plasminogen activators obtained from Zn-chelating chromatography were characterized for molecular mass, urokinase-type versus tissue-type, and susceptibility to protease inhibitors. Five bands with plasminogen activator activity were detected by casein-plasminogen SDS-PAGE with molecular mass of approximately 93, 57, 42, 35, and 27 kDa. Most or all of the plasminogen activators in bovine milk were urokinase-type; the activity of the bovine milk plasminogen activators was not enhanced by the presence of fibrin. The immunological dissimilarity between bovine milk plasminogen activators and human urokinase-type plasminogen activator and human tissue-type plasminogen activator was shown by antibody quenching tests. Bovine milk plasminogen activators were inhibited by certain serine proteinase inhibitors, endothelial cell-type plasminogen activator inhibitor, plasminogen activator inhibitor from erythrina seed, and α 2 -antiplasmin, but not by α 1 -antitrypsin.

Six milk-derived psychrotrophic microbial cultures were screened for the ability to grow at refrigerated temperatures and produce proteases in reconstituted skim milk. Of these, two cultures, Pseudomonas fluorescens M3/6 and Pseudomonas fragi K122, produced extracellular protease(s) beginning 7 d postinoculation when the cultures had entered late log or early stationary phases of growth. Further work with these two cultures showed that intracellular proteases were present after only 20-h incubation, before detection of the extracellular proteases. Using H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide (S-2251), a sensitive substrate for plasmin activity, P. fluorescens was shown to have greater intracellular proteolytic activity than extracellular activity at 20 h of incubation. The intracellular enzyme activity remained constant while the extracellular and periplasmic activities increased over the remaining 6-d incubation period. The proteases in crude extracellular extracts from both cultures were characterized and were heat stable with broad temperature (7 to 52 degrees C) and pH (pH 5.5 to 8.5) ranges for activity and were inhibited by the metal chelator, EDTA, indicating that they were metalloproteases.

The relationship between proteolysis and gelation was studied in UHT-processed milk following the aseptic addition of the enzyme plasmin at a concentration of .15 mg/L. Individual 250-ml containers of commercially processed (direct steam injection, 134.4°C for 14.2 s) milk were used. The milk was injected with plasmin 1 wk after processing and stored at room temperature (–23°C). Over a 6-mo period, the milk was examined for changes in appearance, pH, apparent viscosity, gel formation, enzymatic activity, and casein breakdown. Control milk samples did not gel during the test period. The milk containers that received the plasmin addition began to form a gel at 90 d of storage, and this gelation was accompanied by an increase in apparent viscosity. In the samples with added plasmin, enzyme activity was detected using the chromogenic substrate, H-D-valyl-L-leucyl-L-lysyl-4-nitroanilide (S-2251), and casein breakdown was apparent as examined by SDS-PAGE. It appeared that the added plasmin preferentially attacked s- and α-caseins over κ-casein. The evidence supports a relationship between a low level of plasmin activity and the gelation of UHT milk.

Proteolysis and gelation were investigated in single strength, 2% fat, UHT-processed milk following aseptic addition of combinations of plasmin, plasminogen, trypsin, trypsin inhibitor (Kunitz), and urokinase (plasminogen activator). Individual 250-ml milk containers processed by direct or indirect methods were examined for the following attributes over 10 mo: growth on slants, appearance, pH, apparent viscosity, gel formation, enzymatic activity, and casein breakdown. Control milk samples in the study did not gel. Addition of trypsin at 1.5 or 7.5mg protein/L of milk or addition of plasmin at .3 or 1.5mg protein/L did not result in gelation. However, containers with plasminogen at .3mg protein/ L began forming a gel at 5.5 mo. Enzyme activity in plasminogen-treated samples was not detected spectrophotometrically using an L-lysine- p -nitroanilide substrate, but extensive casein breakdown was apparent by SDS-PAGE. The evidence suggests plasminogen-derived activity promotes UHT milk gelation.

The efficiency of extracting lactose from NDM with aqueous ethanol was investigated under conditions where four factors were varied. The experiments were conducted in a complete random block design using three values for each of the variables: ratios of solvent to solids 10:1, 15:1, and 20:1 (wt/wt); solvent ethanol content of 62.4, 72.9, and 84.0% (wt/wt); temperature during extraction of 10, 25, and 50°C; and added divalent metal ion chelator (citrate) in the amount of 0, 10, and 20 mmol/100 g NDM. The results were evaluated statistically using response surface methodology with a multivariate linear regression equation. Optimal extraction efficiency was obtained with high ratio of solvent to solid and low ethanol concentration of the solvent. High temperatures and high citrate concentrations tended to solvate proteins and make separation by filtration difficult. Milk protein with residual lactose of 2 to 4% was obtained using this one-step extraction process under optimal conditions.