Isolation and some properties of extracellular heat-stable lipases from Pseudomonas fluorescens strain AFT 36

Aeration increased the growth and lipase production in milk by Pseudomonas fluorescens strain AFT 36, isolated from refrigerated bulk milk. A heat-stable lipase was isolated from a shaken milk culture of this microorganism by DEAE-chromatography and gel filtration in Sepharose 6B. The lipase-rich fraction from DEAE cellulose contained 3 lipases that were separated by gel filtration; only the principal lipase, which represented approximately 71% of total lipolytic activity, was characterized. The purified enzyme showed maximum activity on tributyrin at pH 8.0 and 35 degrees C; it had a Km on tributyrin of 3.65 mM and was inhibited by concentrations of substrate greater than approximately 17 mM. The enzyme was very stable over the pH range 6-9; it was relatively heat-labile in phosphate buffer in the temperature range 60-80 degrees C, where it was stabilized significantly by Ca2+. It was, however, very stable at 100-150 degrees C: the D values at 150 degrees C were approximately 22 s and 28 s in phosphate buffer and synthetic milk serum respectively; the corresponding Z values in the temperature range 100-150 degrees C were approximately 40 and approximately 42 degrees C and the Ea for inactivation were 7.65 X 10(4) J mol-1 and 6.97 X 10(4) J mol-1 respectively.     

Thermal stability of an extracellular proteinase from Pseudomonas fluorescens AFT 36

A metalloproteinase, isolated from a shaken milk culture of Pseudomonas fluorescens AFT 36 by chromatography in DEAE and CM-cellulose and Sephadex G-150, was very unstable in 0.1 M-phosphate buffer, pH 6.6, being completely denatured above 70 degrees C in 1 min. It was also unstable in a Ca-containing buffer (synthetic milk salts, SMS) between 50 and 60 degrees C (minimum at 55 degrees C), but stability was very high above 80 degrees C in this buffer. D-values were determined at 10 degrees C intervals in the range 70-150 degrees C in SMS from which a Z value of 31.9 degrees C and an Ea of 8.82 X 10(4) J mol-1 were calculated; the half-life at 150 degrees C was 9 s. Instability at 55 degrees C was due to autolysis as evidenced by gel electrophoresis, gel filtration and increase in 2,4,6-trinitrobenzenesulphonic acid-reactive amino groups. The extent of inactivation experienced at 80 degrees C was inversely related to the rate of heating to 80 degrees C, i.e. length of time spent in the neighbourhood of 55 degrees C. Addition of increasing concentrations of caseinate substrate reduced inactivation of the enzyme at 55 degrees C, presumably due to substrate binding. Attempts to stabilize the enzyme at 55 degrees C by addition of EDTA or by adjusting the reaction pH to 4.2, at which the enzyme has little proteolytic activity, were unsuccessful, although autolysis was prevented. Unlike the proteinase from Ps. fluorescens MC 60, AFT 36 proteinase did not inactivate itself on cooling to 55 degrees C from 80, 100 or 150 degrees C, but did regain autolytic activity on cooling to below 50 degrees C to an extent dependent on the duration of holding at the lower temperature. It is suggested that on heating to approximately 55 degrees C, a conformational change occurs which renders the enzyme susceptible to proteolysis by still active enzyme; at higher temperatures the enzyme, although susceptible to autolysis, is inactive; an active conformation is restored on cooling to below 50 degrees C.     

Purification and characterization of three extracellular proteinases produced by Pseudomonas fluorescens INIA 745, an isolate from ewe's milk.

Three proteinases were isolated from culture medium of Pseudomonas fluorescens INIA 745 and purified to homogeneity by a combination of Phenyl-Sepharose, DEAE-Sepharose, and Sephadex G-100 chromatography. Optimal temperature for enzymatic activity was 45 degrees C for all three proteinases. The pH optimum of proteinases I and II was found to be 7.0, while that of proteinase III was 8.0. Divalent metal ions like Cu2+, Co2+, Zn2+, Fe2+, and Hg2+ were inhibitory to proteinase activity while Ca2+, Mg2+, and Mn2+ had little or no inhibitory effect. The three enzymes were strongly inhibited by EDTA and 1,10-phenantroline and partially by cysteine. The three enzymes are metalloproteinases since they were inhibited by chelators and reactivated by Co2+, Mn2+, Cu2+, and Zn2+. The Km values of proteinases I, II, and III for casein were calculated to be 3.2, 2.6, and 5.2 mg/ml, respectively. Proteinases II and III rapidly degraded beta-casein, with preference to alphas1-casein, whereas proteinase I hydrolyzed both casein fractions at a slow rate.     

Proteolysis in milk: the significance of proteinases originating from milk leucocytes and a comparison of the action of leucocyte, bacterial and natural milk proteinases on casein

The caseinolytic activities at pH 6.8 of polymorphonuclear (PMN) and mononuclear leucocyte homogenates (equivalent to a level of 10(6) cells/ml milk) were less than the levels of natural milk proteinase activity found in milk from healthy cows. Bulk milks contained approximately 4 times more milk proteinase activity than the composite milks from individual healthy cows. Isolated blood leucocytes, when added to raw milk of good bacteriological quality and stored at 5 degrees C, did not readily degenerate and had no detectable effect on the milk proteins even when these cells were completely disrupted by homogenization of the milk. Pasteurization of milk which contained leucocytes caused loss of cell vitality. Extracellular proteinases of psychrotrophic bacteria growing in milk were not detected until the early stationary phase of growth. The total viable count at which this occurred varied greatly. Proteinase production by a pure culture of Pseudomonas fluorescens was not detected in milk stored at 5 degrees C until a viable count of approximately 10(9) colony forming units (c.f.u.)/ml was obtained, whilst normal bulk milks stored at 5 degrees C produced detectable levels of extracellular proteinase(s) when the psychrotrophic flora reached 10(7)-10(8) c.f.u./ml. Casein proteolysis by PMN and mononuclear leucocyte homogenates resulted in similar polypeptide maps, but plasmin and bacterial proteinase isolated from a strain of Serratia marcescens resulted in polypeptide maps different from each other and from that produced by the leucocyte proteinase(s). The rate of proteolysis of caseins by the different proteinase sources appeared to be in the order alpha s1- greater than beta- greater than greater than kappa-casein for the leucocyte extracts, beta- greater than alpha s1- greater than greater than greater than kappa-casein for bovine plasmin and beta- approximately kappa- greater than alpha s1-casein for for S. marcescens proteinase.     

Purification and partial characterization of psychrotrophic Serratia marcescens lipase

Serratia marcescens isolated from raw milk was found to produce extracellular lipase. The growth of this organism could contribute to flavor defects in milk and dairy products. Serratia marcescens was streaked onto spirit blue agar medium, and lipolytic activity was detected after 6 h at 30 degrees C and after 12 h at 6 degrees C. The extracellular crude lipase was collected after inoculation of the organism into nutrient broth and then into skim milk. The crude lipase was purified to homogeneity by ion-exchange chromatography and gel filtration. The purified lipase had a final recovered activity of 45.42%. Its molecular mass was estimated by SDS-PAGE assay to be 52 kDa. The purified lipase was characterized; the optimum pH was likely between 8 and 9 and showed about 70% of its activity at pH 6.6. The enzyme was very stable at pH 8 and lost about 30% of its activity after holding for 24 h at 4 degrees C in buffer of pH 6.6. The optimum temperature was observed at 37 degrees C and exhibited high activity at 5 degrees C. The thermal inactivation of S. marcescens lipase was more obvious at 80 degrees C; it retained about 15% of its original activity at 80 degrees C and was completely inactivated after heating at 90 degrees C for 5 min. Under optimum conditions, activity of the enzyme was maximum after 6 min. The Michaelis-Menten constant was 1.35 mM on tributyrin. The enzyme was inhibited by a concentration more than 6.25mM. Purified lipase was not as heat-stable as other lipases from psychrotrophs, but it retained high activity at 5 degrees C. At pH 6.6, the pH of milk, purified lipase showed some activity and stability. Also, the organism demonstrated lipolytic activity at 6 degrees C after 12 h. Therefore, S. marcescens and its lipase were considered to cause flavor impairment during cold storage of milk and dairy products.     

Kinetics and mechanism of bacterial inactivation by ultrasound waves and sonoprotective effect of milk components

Inactivation of Escherichia coli and Listeria monocytogenes were investigated in buffer and milk upon treatment with ultrasound waves (USW). In addition, sonoprotective effect of milk components and ultrasound-induced changes in bacterial cells were investigated using scanning electron microscopy (SEM). Bacterial cells were added to phosphate buffer, whole milk, skim milk, or simulated milk ultrafiltrate (SMUF). To determine the sonoprotective effect of milk components, lactose (5%), casein (3%), or β lactoglobulin (0.3%) was added to SMUF. Samples were sonicated with 24 kHz pulse USW while maintaining the system temperature between 30 to 35 °C. Aliquots were drawn at set times during sonication and bacteria were enumerated by surface plating appropriate dilutions on selective and nonselective media plates. Escherichia coli exhibited significantly higher D values in whole (2.43 min) and skim milk (2.41 min) than phosphate buffer (2.19 min). Listeria monocytogenes also showed higher D values in whole (9.31 min) and skim milk (8.61 min) compared to phosphate buffer (7.63 min). Data suggest that milk exerts a sonoprotective effect on these bacteria. Escherichia coli exhibited a log-linear inactivation kinetics followed by tailing whereas L. monocytogenes showed 1st-order kinetics throughout. Among the milk components tested, presence of lactose in SMUF resulted in significantly higher D values than SMUF for both organisms suggesting that lactose was exerting a protective effect on bacteria. SEM images showed that USW caused mechanical damage to the cell wall and cell membrane of bacteria leading to their inactivation.     

Heat inactivation of exogenous proteinases from Pseudomonas fluorescens. I. Possibility of inactivation in milk

The inactivation reaction of the proteinase of a P. fluorescens strain of biotype I in milk was investigated at 130-150 degrees C, also in milk and in buffer with and without added CaCl2 at temperatures below 100 degrees C. The decline in activity corresponded to first order kinetics in the UHT region; Ea = 115 kJ/mol. D values were 290 (130 degrees C), 124 (140 degrees C) and 54 s (150 degrees C); therefore, the usual temperature time combinations of UHT treatment are not sufficient to achieve the required rates of inactivation. At temperatures below 80 degrees C, inactivation corresponded increasingly to second order kinetics with considerably higher reaction rates; at 55 degrees C, an inactivation reaction corresponding to that induced by UHT treatment could be achieved at a thermal stress lower by a factor of 500. This "low temperature inactivation" was observed in a further 20 strains representing the spectrum of P. fluorescens. The average rates of inactivation following heat treatment in milk for 20 min are 47% at 55 degrees C and 44% at 60 degrees C. This can be regarded as the most effective temperature range for the inactivation of the proteinases in milk. Clear connections can be seen between the biotype groups and the optimum temperature for inactivation: biotype group I ca. 55 degrees C, group II (with a few exceptions) less than or equal to 50 degrees C and group III greater than or equal to 60 degrees C. The inactivation reaction is systematically influenced by the proteins and Ca++ ions present in milk.     

Heat resistance of Yersinia enterocolitica grown at different temperatures and heated in different media.

In the range of 4-20 degrees C, growth temperature did not influence the heat resistance at 54-66 degrees C for Yersinia enterocolitica at pH 7 in citrate phosphate buffer. However, when cells were grown at 37 degrees C. the D62 increased from 0.044 to 0.17 min. This increase was constant at all heating temperatures tested (z = 5.7-5.8). Growth temperature did not influence the proportion of heat-damaged cells after a heat treatment, as measured by their response to a 2% of sodium chloride added to the recovery medium. The sensitivity of heat treated cells to nisin or lysozyme depended on growth temperature: Whereas the number of cells grown at 4 degrees C surviving heat treatment was the same regardless of the presence of 100 IU/ml of nisin or 100 microg/ml of lysozyme in the recovery medium, that of cells grown at 37 degrees C was, in these media, lower. The pH of maximum heat resistance in citrate phosphate buffer was pH 7 for cells grown at 37 degrees C, but pH 5 for those grown at 4 degrees C. In both suspensions the magnitude of the effect of pH on heat resistance was constant at all heating temperatures. For cells grown at 4 degrees C the heat resistance at 54-66 degrees C, in skimmed milk or pH 7 buffer, was the same. For cells grown at 37 degrees C this also applied for heat treatment at 66 degrees C but at 56 degrees C the heat resistance in skimmed milk was higher.     

Milk ultrafiltrate analysis by ion chromatography and calcium activity for SMUF preparation for different scientific purposes and prediction of its supersaturation

In milk, the serum phase is in a dynamic equilibrium with insoluble salts within the casein micelle and therefore saturated with respect to calcium phosphate depending on pH, temperature and other compositional factors. In this study, simulated milk ultrafiltrate (SMUF) was therefore considered as a dynamic medium that approximates ultrafiltration (UF) permeates of a certain milieu condition resulting in changes in calcium and phosphate content as detected by ion chromatography. Due to reported spontaneous precipitation of existing SMUF solutions and interference with measurements and experiments, SMUF solutions were developed according to the chemical composition and physical parameters of UF permeate at different temperatures. SMUF solutions were proven to be as stable as their corresponding ultrafiltration permeates as they were similar in composition and calcium activity. Calcium activity was considered as a useful parameter to estimate the onset of supersaturation and spontaneous precipitation depending on calcium concentration and pH.     

Thermal inactivation kinetics of bovine cathepsin D

Cathepsin D, the principal indigenous acid proteinase in bovine milk, is a lysosomal proteinase, which exists in milk in four forms, including the inactive zymogen procathepsin D. The thermal inactivation kinetics of bovine cathepsin D, isolated from spleen and milk, were studied under isothermal conditions, using a specific HPLC assay to determine residual activity. Inactivation of the blood enzyme preparation followed first order kinetics, with z-values in phosphate buffer (pH 6.7) and skimmed milk of 6.5 and 7.6 degrees C, respectively, the enzyme being far more stable in the latter environment. Inactivation kinetics of the enzyme purified from milk were more complex, and could be best approximated by a double exponential model. Again, stability was higher in milk than in buffer. The double exponential model may indicate differing heat stabilities of isoforms of the enzyme, or stabilization of the enzyme by some milk constituent. It is clear that the enzyme can survive, at least partially, processes such as heating at 55 degrees C for 30 min during manufacture of high-cook cheese varieties (45% survival), and HTST pasteurization (8% survival), and thus may contribute to proteolysis in a range of dairy products.     

The influence of nisin on the thermal resistance of Bacillus cereus

Decimal reduction times (D-values) at cooking and autoclaving temperatures (80 to 120 degrees C) of spores of Bacillus cereus ATCC 1479-8 in rice and milk (13% wt/vol) supplemented with nisin (25 microg/ml) were evaluated. The mean D-values at 97.8 degrees C in cooked white rice, phosphate buffer (pH 7.0), and rice water (pH 6.7) were 3.62, 1,99, and 1.34 min, respectively. From 80 to 100 degrees C, the mean reduction in D-values due to the addition of nisin to milk was 40%. The D-value at 110 degrees C was approximately 0.86 min for milk (control) and milk with nisin. The z-values ranged from 7.32 degrees C (phosphate buffer) to 10.37 degrees C (milk control).     

Inhibition by chelating agents of the formation of active extracellular proteinase by Pseudomonas fluorescens 32A

The effect of chelating agents on extracellular proteinase production by Pseudomonas fluorescens 32A was examined. Increasing concentrations of orthophosphate slightly stimulated growth while inhibiting proteinase synthesis. Fifty per cent inhibition was found at 35 and 28 mM-orthophosphate at 5 and 20 degrees C respectively. Extracellular protein concentration was reduced by 30% when cells were grown with 100 mM-orthophosphate. Polyacrylamide gel electrophoresis of the cell-free supernatants suggested that reduced enzyme synthesis had taken place as evidenced by the decrease in staining intensity of the protein band corresponding to the proteinase. Other phosphate compounds could replace orthophosphate as an inhibitor. Extent of inhibition was related to chain length; polyphosphates with 4-6 or 13-18 phosphorus atoms were the most effective inhibitors. EDTA (0.5 mM) completely inhibited proteinase synthesis. This inhibition could be partly reversed by Ca2+ and, to a lesser extent, Mn2+. Proteinase production at 5 degrees C in skim milk was completely inhibited by phosphate glass (P13-P18). Control experiments showed that loss of activity with chelators was not due to inhibition of preformed enzyme. The results suggest a possible role for polyphosphates in controlling proteinase production in stored milk.     

Effect of added proteinases and level of starter culture on the formation of biogenic amines in raw milk Manchego cheese

The influence of two proteinases (Bacillus subtilis neutral proteinase and Micrococcus sp. cysteine proteinase) and two starter culture levels (0.1% and 1%) on biogenic amine formation has been studied in raw ewes' milk Manchego cheese. Amino acid decarboxylating micro-organisms were determined on tyrosine enriched selective media. Biogenic amines were analysed by capillary electrophoresis in citrate buffer at pH 3.6. Addition of proteinases and level of starter culture did not influence the population of micro-organisms with amino acid decarboxylating activity, which represented on average 1% of the bacterial population in 30-day-old cheeses. Tyramine and histamine were detected in all batches of cheese from day 30. Concentrations of tyramine and histamine were higher in cheeses made from milk with neutral proteinase (up to 356 and 284 mg kg(-1), respectively, after 90 days) than in cheeses made from milk with cysteine proteinase (up to 269 and 189 mg kg(-1), respectively) or with no proteinase added (up to 305 and 226 mg kg(-1), respectively). Formation of tyramine and histamine was also favoured in cheeses made with 1% starter culture with respect to cheeses made with only 0.1% starter culture, probably due to the higher pH values of the former cheeses. After 90 days of ripening, concentrations of 10-20 mg kg(-1) phenylethylamine were observed in 9 of the 12 batches, and levels < 10 mg kg(-1) tryptamine were only detected in 3 batches, with no significant relationship between the concentration of these amines and proteinase addition or level of starter culture.     

Proteolytic activity of proteinases on macropeptide isolated from kappa-casein.

Proteolytic activities of chymosin, bovine pepsin, Mucor miehei rennet, Cryphonectria parasitica (formerly Endothia parasitica) rennet, trypsin, and chymotrypsin on kappa-casein macropeptide were measured. Macropeptide solutions (10 mg/ml of .05 M, pH 6.6 phosphate buffer) were incubated with the enzymes at 37 degrees C for various times, and their reactions were stopped by adding .025 ml of pepstatin (1 mg/ml of methanol). Peptides released from kappa-casein macropeptide were then fractionated using reverse-phase HPLC. At the pH of milk (pH 6.6), kappa-casein macropeptide was resistant to enzymic action by chymosin, bovine pepsin, and M. miehei and C. parasitica rennets. Bovine pepsin hydrolyzed kappa-casein macropeptide at pH 3. kappa-Casein macropeptide was readily hydrolyzed at pH 6.6 by trypsin and chymotrypsin. Possible physiological functions of the kappa-casein macropeptide are discussed in light of these findings.     

Autolysis and the endogenous proteinases characterised in beardless barb (Anematichthys apogon) muscle

Beardless barb is a common fish species used in fermentation of fish paste Ka-pi-plaa. Autolytic profile of beardless barb muscle showed the maximum autolysis was at 50 °C, at both acidic and alkaline pH values. With augmentation concentration of NaCl, autolytic activity slightly decreased. Endogenous proteinases isolated from fish muscle in crude extract forms were also characterised. The acidic proteinases had optimum activity at pH 3.0 and 50°C, and they showed higher proteolytic activity than the alkaline proteinases which were optimally active at pH 9.0 and 50 °C. Proteinases in peak at pH 3.0 were inhibited by pepstatin A, but those in peak at pH 9.0 were highly inhibited by PMSF, TLCK and soybean trypsin inhibitor, suggesting that both aspartic and serine proteinases were existed in beardless barb muscle. The proteinases were stable in pH range of 2.0-5.0 but unstable at the temperatures higher than 40 °C. NaCl suppressed the proteolytic activity, ATP activated the proteinase activity, while CaCl₂, MgCl₂ and CoCl₂ exhibited no influence on the activity. The results implied that cathepsin D is the predominant proteinase responsible for autolysis in beardless barb. The findings were useful to improve the processing and qualities of Ka-pi-plaa product using beardless barb as raw material.     

The cell-bound proteinase system of Lactobacillus casei--purification and characterization

The cell-wall crude extract from Lactobacillus casei NCDO 151 was partially purified by DEAE-Sephacel chromatography. Three active fractions were eluted. Two major peaks (eluted with 0.05 M and 0.27 M phosphate buffer) were further investigated. Peak I represented enzymatic activity with an optimum temperature of 40 degrees C, an optimum pH of 7.0 and was strongly inhibited by the serine proteinase inhibitor phenylmethylsulfonylfluoride. Peak II represented an enzymatic activity with an optimum temperature of 45 degrees C, an optimum pH of 7.5 and was totally inhibited by p-hydroxymercuribenzoate. None of the enzymes was affected by the metal chelator ethylenediaminetetraacetic acid at a concentration up to 1 x 10(-2).     

Purification and characterization of the extracellular proteinase of Pseudomonas fluorescens NCDO 2085

Pseudomonas fluorescens NCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 degrees C and pH 7.0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3.5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5.40 +/- 0.05 and a mol. wt of 40 200 +/- 2100. It is heat-stable having D-values at 74 and 140 degrees C of 1.6 and 1.0 min respectively; 40 and 70% of the original activity remained after HTST (74 degrees C/17 s) and ultra high temperature (140 degrees C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from other Pseudomonas spp.     

CYSTEINE PROTEINASE ACTIVITY IN JUMBO SQUID (DOSIDICUS GIGAS) HEPATOPANCREAS EXTRACTS

Jumbo squid specimens were captured, dissected, and the hepatopancreas was freeze dried for the extraction of proteolytic enzymes. An autolysis experiment conducted at 25C showed two peaks with maximum proteolysis at pH 3 and 5. The proteinase activity of the extract was measured using azocasein, BANA, Z‐PAAFC and Z‐AAAFC substrates. Activity of the extract with azocasein (pH 5) had a maximum at 55C and increased threefold with the inclusion of cysteine or DTT. The proteinase activity remained at least at 60% of the original after 45 h at 4C in the pH range of 3–8. Activity was inhibited 70–85% when extracts were treated with cysteine proteinase specific inhibitors. The proteinases extracted from jumbo squid hepatopancreas are mainly of the cysteine type and have significant activity towards a cathepsin L specific substrate. The understanding of proteinases from this tissue could have implications for quality control of jumbo squid food products.     

Properties of proteases from milk somatic cells and blood leukocytes

Proteolytic activity of proteases associated with somatic cells isolated from high SCC milk and proteases associated with leukocytes isolated from bovine blood was assayed at pH 6.6 and 5.2 in a model system consisting of a beta-casein (.96%) substrate in Jenness/Koops buffer. Intact beta-casein and casein proteolysis products were measured by densitometric analysis of SDS-PAGE gels. Relative proteolytic activity was expressed as percentage degradation of beta-casein after 24 h at 37 degrees C per 1 x 10(6) cells/ml. Proteolytic activity associated with somatic cells isolated from bovine milk was 27.5 and 13.6% at pH 6.6 and 5.2, respectively. Proteolytic activity associated with leukocytes isolated from bovine blood was 16.0 and 8.4% at pH 6.6 and 5.2, respectively. Proteolytic activity at pH 6.6 was significantly higher for the somatic cells isolated from milk than for leukocytes isolated from blood. The reason for the difference in proteolytic activity of leukocytes isolated from blood of a healthy cow versus somatic cells isolated from milk produced by a cow with mastitis is not known. Further work is needed to determine whether the difference may be caused by a higher proportion of activated macrophages in somatic cells isolated from milk produced by cows with mastitis than in the leukocytes isolated from blood of healthy cows.     

Breakdown of caseins by proteinases in bovine milks with high somatic cell counts arising from mastitis or infusion with bacterial endotoxin

Milk obtained from cows which were either infected by clinical mastitis or had been subjected to intramammary infusion of Escherichia coli endotoxin possessed high counts of somatic cells and very high levels of proteinase activity which hydrolysed the caseins almost completely in a few hours at 37 degrees C. The rate of hydrolysis of beta-casein was slightly greater than that of alpha S1-casein, but in both cases hydrolysis was enhanced by 6 cycles of freezing and thawing to disrupt somatic cell membranes. A study of the relationship between proteinase activity and cell count suggested that only some of the proteinase activity originated in the somatic cells and also that the identity of the cells making up the total cellular population was important. Maximum proteolysis occurred at 50-60 degrees C, but the temperature-activity curve was a broad peak. Likewise the pH versus activity plot was very broad and was almost flat over the pH range 6-9. Experiments with a number of inhibitors of proteinases failed to give a clear cut pattern of inhibition. All evidence obtained was consistent with the view that several different enzymes with different pH and temperature optima and different specificities contributed to the overall hydrolysis of caseins in these milks. From electrophoretic band patterns one of these enzymes was clearly plasmin, but in high cell count milks other proteinases also became significant.