Isolation and characterization of heat stable proteinases from Pseudomonas isolate AFT 21

Pseudomonas strain AFT 21 produced three heat stable extracellular proteinases in milk and nutrient broth at 7 or 21 degrees C, but the proportions depended on medium and cultivation temperature. The three proteinases were EDTA- and o-phenanthroline-sensitive metalloenzymes and were not inhibited by N-ethylmaleimide or phosphoramidon. Proteinases I and II showed maximum activity at pH 7-7.5 and proteinase III at pH 8.5. All three enzymes showed maximum activity at 45-47.5 degrees C, but had relatively high (19-27% of maximum) activity at 4 degrees C. They were unstable at 55 degrees C in phosphate buffer, pH 6.6, or synthetic milk ultrafiltrate (SMUF) containing 12 mmol Ca2+, but were stabilized by short preheating at 100 degrees C. They were extremely heat stable in both phosphate buffer and SMUF, pH 6.6, at 70-150 degrees C. Their D-values at 140 degrees C were 69, 54 and 80 s respectively. The Z-values for Pseudomonas AFT 21 proteinase III in phosphate buffer and SMUF were 29.7 and 30.3 degrees C respectively; the corresponding activation energies for inactivation were 8.7 x 10(4) J mol-1 and 9.2 X 10(4) J mol-1.     

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.     

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 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.     

Autolysis of the proteinase from Pseudomonas fluorescens.

The gene encoding the proteinase from Pseudomonas fluorescens was cloned and sequenced in an effort to identify the cleavage sites involved in its autolysis at 50 degrees C. A single open reading frame consisting of 1449 nucleotides, encoding a protein of 482 amino acids, was found. Analysis of the N-terminal amino acid sequence of the purified proteinase indicated the presence of a prosequence consisting of 13 amino acid residues. The molecular weight of the mature protein was calculated as 48,900 based on the deduced amino acid sequence, which was consistent with that of the purified proteinase as determined by sodium dodecylsulfate-PAGE. Greater than 90% loss of proteolytic activity was observed upon heating at 50 degrees C for 2 min compared with the unheated enzyme. Incubation of the proteinase at 50 degrees C led to disappearance of the intact enzyme, as shown by sodium dodecyl sulfate-PAGE, whereas it was stable in the presence of the protease inhibitor o-phenanthroline. Autolytic fragments were fractionated by reverse-phase HPLC and subjected to N-terminal amino acid sequence analysis in an effort to determine the cleavage sites. The cleavage profile was not definitive; however, amino acid residues with small side chain groups, such as glycine or alanine, were frequently found adjacent to the cleavage sites.     

Growth of an extracellular proteinase-deficient strain of Pseudomonas fluorescens on milk and milk proteins

An extracellular proteinase-and lipase-deficient mutant of a psychrotroph, Pseudomonas fluorescens strain 32A, has been isolated and the absence of the proteinase enzyme confirmed by growth on differential media, enzyme assay and polyacrylamide gel electrophoresis. Competition between the parent and the mutant was observed when equal numbers of the 2 strains were inoculated together into raw skim-milk at 6 degrees C. Bitterness was detected at 6 degrees C in pasteurized skim-milk inoculated with the parent cells concurrent with the detection of proteolytic activity. In the case of the mutant, slight bitterness which did not increase with increasing cell numbers was detected in the absence of proteolysis. Mutant cells failed to grow on Na caseinate as the sole source of carbon. It was concluded that the extracellular proteinase, while not essential for growth in milk, does provide a selective advantage to the producer organism. This enzyme is, however, essential for growth on milk proteins and contributes to the development of bitterness in pasteurized milk.     

Potential applications of high pressure homogenisation in processing of liquid milk

Studies of the potential of high pressure homogenisation (HPH) for the combined pasteurisation/ homogenisation of raw bovine milk were undertaken. Raw milk was preheated to 45 degrees C and HPH-treated at 150, 200 or 250 MPa; milk outlet temperature at these pressures were 67, 76.8 and 83.6 degrees C, respectively, with a holding time of approximately 20 s. Raw and commercially pasteurized and homogenized (CPH) milk samples were analysed as controls. Fat globules in HPH samples were approximately half the size of those in CPH samples, although differences were not significant (P>0.05). beta-Lactoglobulin was denatured at pressures > or =150MPa, although little denaturation of alpha-lactalbumin was observed. Numbers of psychrotrophic bacteria in raw milk were reduced by 2.73 log cycles by HPH at 150 MPa and were uncountable following HPH at 200 or 250 MPa. Mesophilic bacterial counts were reduced by 1.30, 1.83 and 3.06 log cycles by HPH at 150, 200 or 250 MPa, respectively. No viable Staphylococcus aureus nor coliform cells remained in any HPH milk samples. HPH did not affect the colour of milk and HPH samples did not cream during refrigerated storage. The activities of plasmin, alkaline phosphatase and lactoperoxidase in milk were all greatly reduced by HPH. Pseudomonas fluorescens, inoculated into milk (approximately 10(6) cfu/ml), was reduced to undetectable levels by HPH at 200MPa (milk inlet temperature, approximately 10 degrees C); however, Ps. fluorescens proteinase was quite resistant to HPH under such conditions. Overall, owing to the significant increase in temperature and the possibility of varying the holding time, there may be potential applications for HPH as a novel liquid milk processing technique, combining many advantages of conventional homogenization and pasteurization of milk in a single process.     

High pressure inactivation of microorganisms inoculated into ovine milk of different fat contents

High hydrostatic pressure inactivation of Escherichia coli, Pseudomonas fluorescens, Listeria innocua, Staphylococcus aureus, and Lactobacillus helveticus were studied. These microorganisms were inoculated at a concentration between 10(7) and 10(8) cfu/ml in Ringer solution and in ovine milk adjusted to 0, 6, and 50% fat content to evaluate the baroprotective effect of fat content on inactivation of microorganisms. Treatments of pressurization consisted of combinations of pressure (100 to 500 MPa) and temperature (4, 25, and 50 degrees C) for 15 min. Gram-negative microorganisms were more sensitive than were Gram-positive ones (more destruction P. fluorescens > E. coli > or = List. innocua > Lb. helveticus > S. aureus). Pressurizations at low temperature (4 degrees C) produced greater inactivation on P. fluorescens, List. innocua, and Lb. helveticus than at room temperature (25 degrees C), whereas for E. coli and S. aureus the results were opposite. Ovine milk per se (0% fat) showed a baroprotective effect on all microorganisms, but percentage of fat (6 and 50%) did not show a progressive baroprotective effect in all pressurization conditions or for all microorganisms.     

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.     

Phenotypic and genotypic characterization of Pseudomonas fluorescens isolated from bulk tank milk.

Pseudomonas fluorescens isolates (n = 55) isolated from farm bulk tank milk (n = 55) from dairy herds in eastern South Dakota and western Minnesota were examined for phenotypic (biotype, proteolytic, and lipolytic profiles) and genotypic (plasmid profiles and 16S-23S PCR ribotypes) characteristics. The observed phenotypic and genotypic characteristics were used to conduct phylogenetic analysis. Pseudomonas fluorescens belonged to 28 API 20 NE biotypes and 14 proteolytic and lipolytic profiles. It was observed that 80, 91, and 58% of the isolates were proteolytic at 7, 22, and 32 degrees C, respectively. Only 7, 44, and 7% of the isolates were lipolytic at the same three temperatures. Pseudomonas fluorescens was more likely to produce proteinases at 7 and 22 degrees C and lipases at 22 degrees C. Only 9 of 55 isolates of P. fluorescens harbored plasmids. This small percentage of plasmid-bearing isolates provided insufficient data for inferences related to the distribution of plasmid-bearing clonal types. Based on 16S-23S PCR ribotyping, P. fluorescens belonged to 14 subtypes. The 16S-23S PCR ribotyping technique allowed differentiation between strains; however, it did not concur with the biotypes and proteolytic and lipolytic profiles. Use of biotypes in conjunction with proteolytic and lipolytic profiles might have practical value for conducting trace-back studies related to P. fluorescens. Based on phylogenetic analysis, it was inferred that for the given geographical area and time period, P. fluorescens isolated from farm bulk tank milk consists of a large heterogeneous group of organisms.     

Effect of dissolved carbon dioxide on thermal inactivation of microorganisms in milk

Postpasteurization addition of CO2 inhibits growth of certain microorganisms in dairy products, but few workers have investigated the effect of CO2 on the thermal inactivation of microorganisms during pasteurization. Concentrations of CO2 ranging from 44 to 58 mM added to raw whole milk significantly (P < 0.05) reduced the number of surviving standard plate count (SPC) organisms in milk heated over the range of 67 to 93 degrees C. A decrease in thermal survival rates (D-values) for Pseudomonas fluorescens R1-232 and Bacillus cereus ATCC 14579 spores in milk was positively correlated with CO2 concentrations (1 to 36 mM). D(50 degrees C)-values for P. fluorescens significantly decreased (P < 0.05) in a linear fashion from 14.4 to 7.2 min. D(89 degrees C)-values for B. cereus spores were significantly (P < 0.05) decreased from 5.56 min in control milk to 5.29 min in milk containing 33 mM CO2. The Weibull function was used as a model to describe the thermal inactivation of P. fluorescens, B. cereus spores, and SPC organisms in raw milk. Nonlinear parameters for the Weibull function were estimated, and survival data fitted to this model had higher R2 values than when fitted to the linear model, further providing support that the thermal inactivation of bacteria does not always follow first-order reaction rate kinetics. These results suggest that CO2 could be used as a processing aid to enhance microbial inactivation during pasteurization.     

Effects of high intensity pulsed electric field and thermal treatments on a lipase from Pseudomonas fluorescens

Milk and dairy products may contain microorganisms capable of secreting lipases that cause sensory defects and technological problems in the dairy industry. In this study, the effects of thermal and high-intensity pulsed electric field (HIPEF) treatments on an extracellular lipase from Pseudomonas fluorescens, suspended in a simulated skim milk ultrafiltrate (SMUF) have been evaluated. Heat treatments applied were up to 30 min from 50 to 90 degrees C. HIPEF treatments were carried out using pilot plant facilities in a batch or continuous flow mode, where treatment chambers consisted of parallel and coaxial configuration, respectively. Samples were subjected to up to 80 pulses at electric field intensities ranging from 16.4 to 37.3 kV/cm. This resulted in a lipase that was quite resistant to heat and also to HIPEF. High (75 degrees C-15 s) and low pasteurization treatments (63 degrees C-30 min) led to inactivations of 5 and 20%, respectively. Using the batch-mode HIPEF equipment, a 62.1% maximum activity depletion was achieved after 80 pulses at 27.4 kV/cm. However, when HIPEF treatments were applied in the continuous flow mode, an inactivation rate of just 13% was achieved, after applying 80 pulses at 37.3 kV/cm and 3.5 Hz. The results of both heat and HIPEF treatments on enzyme inactivation were adjusted with good agreement to a first-order kinetic model (R2 > 62.3%).     

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.     

Effects of high pressure homogenisation of raw bovine milk on alkaline phosphatase and microbial inactivation. A comparison with continuous short-time thermal treatments

Raw whole milk of high microbial quality (58 degrees C), but markedly decreased above 200 MPa when Tin=24 degrees C (T2>60 degrees C). In contrast to inactivation induced by continuous short-time thermal treatments, ALP inactivation induced by HP homogenisation was clearly due to mechanical forces (shear, cavitation and/or impact) in the HP valve and not to the short (<1 s) residence time at temperature T2 in the same valve. Inactivation of the three exogenous microorganisms led to similar conclusions. Homogenisation at 250 MPa or 300 MPa (Tin=24 degrees C) induced a 2-3 log cycle reduction of the total endogenous milk flora and a 1.5-1.8 log cycle reduction of inoculated List. innocua. Higher reduction ratios (2-4 log cycles) were obtained for the two other microorganisms. The highest levels of ALP inactivation corresponded to the highest extents of microbial reduction. Running the milk twice or three times through the homogeniser (recycling), keeping temperature T1 approximately 29 degrees C and pressure=200 MPa, increased homogenisation efficiency.     

Inactivation of Escherichia coli in milk by high-hydrostatic-pressure treatment in combination with antimicrobial peptides

We studied the inactivation in milk of four Escherichia coli strains (MG1655 and three pressure-resistant mutants isolated from MG1655) by high hydrostatic pressure, alone or in combination with the natural antimicrobial peptides lysozyme and nisin and at different temperatures (10 to 50 degrees C). Compared with that of phosphate buffer, the complex physicochemical environment of milk exerted a strong protective effect on E. coli MG1655 against high-hydrostatic-pressure inactivation, reducing inactivation from 7 logs at 400 MPa to only 3 logs at 700 MPa in 15 min at 20 degrees C. An increase in lethality was achieved by addition of high concentrations of lysozyme (400 microg/ml) and nisin (400 IU/ml) to the milk before pressure treatment. The additional reduction amounted maximally to 3 logs in skim milk at 550 MPa but was strain dependent and significantly reduced in 1.55% fat and whole milk. An increase of the process temperature to 50 degrees C also enhanced inactivation, particularly for the parental strain, but even in the presence of lysozyme and nisin, a 15-min treatment at 550 MPa and 50 degrees C in skim milk allowed decimal reductions of only 4.5 to 6.9 for the pressure-resistant mutants. A substantial improvement of inactivation efficiency at ambient temperature was achieved by application of consecutive, short pressure treatments interrupted by brief decompressions. Interestingly, this pulsed-pressure treatment enhanced the sensitivity of the cells not only to high pressure but also to the action of lysozyme and nisin.     

The formation of gamma-caseins during cooling of raw milk.

It has been shown that there is a time-dependent transfer of beta-casein and the milk serine proteinase system from micelles to milk serum with change of temperature from 38 to 4 degrees C. It has been established that the gamma-caseins can be formed by proteolytic degradation of beta-casein. By a simple extraction technique, the very hydrophobic gamma-casein fraction was separated from stored milks (26 and 4 degrees C) and estimated quantitatively. The results showed that the proteolytic degradation of beta-casein is faster at 4 degrees C than at room temperature and this can be explained by the immobilization of enzyme and substrate at the micelle surface at higher temperatures (26 degrees C). The results indicate that irreversible changes during cooling for short periods do not cause problems in milk processing, but the formation of gamma-caseins and phosphopeptides may influence the technological properties of raw milk stored for more than 48 h.     

Buffalo-milk enzyme levels, their sensitivity to heat inactivation, and their possible use as markers for pasteurization

The activities and rates of inactivation of four enzymes in raw buffalo milk were measured in relation to the process of heating to determine the value of these enzymes as markers for the evaluation of milk pasteurization. The activities of the enzymes alkaline phosphatase (ALP), lactic dehydrogenase (LDH), gamma-glutamyltransferase (GGT), and aspartate aminotransferase (AST) were measured before and after heating at 50, 60, 70, and 80 degrees C for 1, 3, 5, 10, 20, and 30 min. The enzyme GGT showed the highest activity (712 +/- 601 IU/liter), followed by LDH (386 +/- 183 IU/liter), ALP (295 +/- 164 IU/liter), and AST (18 +/- 4 IU/liter). Heating the milk at 50 degrees C for 1 to 30 min resulted in no effect on the activity of any of the enzymes. At 60 degrees C, ALP showed the highest sensitivity to heat inactivation, whereas all other enzymes showed resistance. At 70 degrees C, ALP activity was abolished completely after 1 min, whereas GGT and LDH lost most activity after 10 min, and AST still maintained 50% activity even after 30 min. At 80 degrees C, the activities of LDH and GGT were lost, whereas AST still retained some of its activity. The results suggest that in addition to ALP, LDH and GGT, but not AST, are potential markers for heat denaturation in buffalo milk, with GGT having the advantage that its concentration is the highest.     

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 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.