Heat-resistant Proteases Research Articles

Research on the quality deterioration of ultrahigh temperature milk products during shelf life: core microorganisms and related characteristics

Commercial sterility does not guarantee the sustained stability of ultrahigh temperature (UHT) milk over 6 months shelf life. We explore the microbiota presented in normal (SZ) and quality deteriorated UHT milk (QY and WY) products from the same brand. Based on high-throughput sequencing research results, 11 phyla and 54 genera were identified as dominant microbiota. Pseudomonas, Streptococcus, and Acinetobacter as core functional microbiota significantly influenced the UHT milk quality properties. Moreover, principal component analysis (PCA) and multivariate analyses were used to examine the quality characteristics, including 11 physicochemical parameters, 10 fatty acids, and 2 enzyme activities, in normal and quality deteriorated UHT milk. We found that the abundance of Pseudomonas increased in quality deteriorated milk (WY) and showed a significant positive correlation with heat-resistant protease content. Acinetobacter in quality deteriorated milk (QY) also considerably contributed to the content of heat-resistant lipase, which resulted in spoilage deterioration of UHT milk.

Biosynthesis, behavior and fate of volatile organic sulfide in Lentinus edodes (Berk.) upon hot-air drying treatment

This study elucidated the biosynthesis and changing behaviors of organic sulfide in shiitake mushrooms upon hot-air drying treatment. The changes of aw, moisture migration, contours of taste and flavor, organic sulfide, and 4 key enzyme activities were monitored throughout three drying procedures (CT/ST1/ST2). Results showed that drying rate was related to the moisture migration. Key enzymes of γ-GTase, ASFase and CS lyase were heat-resistant proteases, while C-Dase exhibited low thermal stability with the activity decreased during treatment. A total of 17 organic sulfides were identified and PLS analyses suggested 6 cyclic polysulfides were formed by C-Dase desulfurization, while 5 thioethers generation were related to the thermal cleavage of direct precursors (straight-chain di/tris/tetrasulfonyl esters) and Maillard reaction. These results indicated that ST2 drying procedures had a positive effect on the formation of cyclic polysulfides at the end of drying pried and the achievement of premium flavor qualities.

The effect of extracellular protease secreted by Pseudomonas fluorescens W3 on the quality of UHT milk

The heat-resistant protease produced by psychrotrophic bacteria is harmful to the quality of UHT milk, and the complex structure formed by fat and protein also affects the hydrolysis of protein by protease. The protease secreted by Pseudomonas fluorescens W3 was added to whole milk and skimmed milk before UHT treatment to analyze its effect on the quality of two kinds of UHT milk. During the preservation of UHT milk, increases in proteolysis, viscosity, hardness, sediment content, zeta potential, and particle size were observed. Extracellular protease reduced the whiteness, pH, and cohesiveness of UHT milk during the storage. The protease secreted by P. fluorescens W3 was easier to hydrolyze casein than whey protein, and κ-casein of casein was easily hydrolyzed by the protease. Milk fat accelerated the hydrolysis of casein by protease secreted by P. fluorescens W3, which led to a faster decline in the quality of whole UHT milk. Novelty impact statement Pseudomonas fluorescens W3 can secrete a thermostable protease AprX with a molecular weight of about 45 kDa. The protease AprX has stronger hydrolysis ability on κ-casein than other caseins. Fat can promote the activity of protease AprX, so whole milk is more susceptible to the damage of protease AprX.

Whey protein influences the production and activity of extracellular protease from Pseudomonas fluorescens W3

The heat-resistant protease secreted by Pseudomonas in raw milk is an important reason for reducing the quality of dairy foods. Protease from Pseudomonas fluorescens W3 (P. fluorescens W3) performed higher activity in milk than that in tryptic soy broth (TSB). In this study, the effects of different milk components on the activity of protease from P. fluorescens W3 were explored. The results showed that whey protein was the main reason that influenced the protease activity secreted by P. fluorescens W3, which exert positive correlation with the concentration of whey protein content (from 0.3% to 1%). The zymogram and mass spectrometry proved that the protease secreted by P. fluorescens W3 was AprX, which had a better ability to hydrolyze casein than whey protein. The AprX hydrolysised casein by the suquence as follows: κ-casein > β-casein > α-casein. The results of RT-qPCR showed that whey protein could increase the expression of gene aprX by P. fluorescens W3. The results could help us better understand how milk promoted the protease activity of Pseudomonas fluorescens. Destroying whey protein may become the key method to inhibit the activity of protease from psychrotrophic bacteria, and reduce the waste of dairy foods.

Assessment of the production of Bacillus cereus protease and its effect on the quality of ultra-high temperature-sterilized whole milk

Bacillus cereus is one of the most important spoilage microorganisms in milk. The heat-resistant protease produced is the main factor that causes rotten, bitter off-flavors and age gelation during the shelf-life of milk. In this study, 55 strains of B. cereus were evaluated, of which 25 strains with protease production ability were used to investigate proteolytic activity and protease heat resistance. The results showed that B. cereus C58 had strong protease activity, and its protease also had the highest thermal stability after heat treatment of 70°C (30 min) and 100°C (10 min). The protease was identified as protease HhoA, with a molecular mass of 43.907 kDa. The protease activity of B. cereus C58 in UHT-sterilized whole milk (UHT milk) showed an increase with the growth of bacteria, especially during the logarithmic growth phase. In addition, the UHT milk incubated with protease from B. cereus C58 at 28°C (24 h) and 10°C (6 d) were used to evaluate the effects of protease on the quality of UHT milk, including protein hydrolysis and physical stability. The results showed that the hydrolysis of casein was κ-CN, β-CN, and αS-CN successively, whereas whey protein was not hydrolyzed. The degree of protein hydrolysis, viscosity, and particle size of the UHT milk increased. The changes in protein and fat contents indicated that fat globules floated at 28°C and settled at 10°C, respectively. Meanwhile, confocal laser scanning microscopy images revealed that the protease caused the stability of UHT milk to decrease, thus forming age gelation.

Identification of Pseudomonas jessenii and Pseudomonas gessardii as the most proteolytic Pseudomonas isolates in Iranian raw milk and their impact on stability of sterilized milk during storage.

Identification of the most proteolytic Pseudomonas strains that can produce heat-resistant proteases and contribute to the Ultra High Temperature (UHT) milk destabilization is of great interest. In the present study, among the 146 Pseudomonas isolates that encoded the aprX gene, five isolates with the highest proteolytic activity were selected and identified based on 16S rRNA, rpoD and gyrB gene sequences data. The identification results were confirmed by phylogenetic analysis based on multilocus sequence analysis and identified the representative isolates as P. jessenii (two isolates) and P. gessardii (three isolates). Casein zymography demonstrated the ability of these species to produce heat-resistant enzymes, AprX, with molecular mass of about 48 kDa during storage at 7° C for 72 h. In sterilized milk samples, the residual activity of AprX caused a considerable enhancement in the degree of protein hydrolysis, non-protein nitrogen and non-casein nitrogen contents of the samples during a two-month storage. This enhancement was slightly higher in samples containing enzyme produced by P. jessenii compared to P. gessardii ones, resulting in earlier onset of sterilized milk destabilization. Hence, this study revealed that P. jessenii and P. gessardii can play a considerable role in deterioration of Iranian commercial long-life milk.

Effect of a pre-milking teat foam and a liner disinfectant on the presence of mesophilic and (proteolytic) psychrotrophic bacteria prior to milking.

Contamination of raw milk by psychrotrophs can lead to the production of heat-resistant proteases and subsequent spoilage of UHT milk. Therefore, this research communication evaluated the effect of a pre-milking teat disinfectant (active components: L-(+)-lactic acid and salicylic acid) and a liner disinfectant (active components: peracetic acid and hydrogen peroxide) on the number of mesophilic and (proteolytic) psychrotrophic bacteria prior to milking. The teat orifices of 10 cows were sampled using a swabbing procedure before and after treatment with a pre-milking teat disinfectant on six subsequent days. On the teat orifices, there was a small but statistically significant decrease in the psychrotrophic bacterial counts between pre and post dipping. No differences were observed for the mesophilic bacterial counts and proteolytic active counts. Liners were also sampled using swabs pre and post disinfection. No statistically significant decrease in the bacterial counts was observed post liner disinfection, although there was a numerical decrease. Sixty-two percent of the proteolytic psychrotrophs were pseudomonads: 16.5% of which were P. fragi, 14.3% P. lundensis, 10.0% P. fluorescens and 2.9% P. putida. Trinitrobenzenesulfonic acid (TNBS) analysis revealed a wide variety in proteolytic activity (from 0 to 55 µmol glycine/ml milk) and the presence of high producers. It can be concluded that there was only a minor effect of teat and liner disinfection on the psychrotrophic bacterial counts indicating that the measures presented did not result in a reduction of the targeted bacteria on teat orifices and liners.

Species-Level Discrimination of Psychrotrophic Pathogenic and Spoilage Gram-Negative Raw Milk Isolates Using a Combined MALDI-TOF MS Proteomics-Bioinformatics-based Approach.

Identification of psychrotrophic pathogenic and spoilage Gram-negative bacteria using rapid and reliable techniques is important in commercial milk processing, as these bacteria can produce heat-resistant proteases and act as postprocessing contaminants in pasteurized milk. Matrix-assisted laser desorption/ionization-time-of-flight mass spectrometry (MALDI-TOF MS) is a proven technology for identification of bacteria in food, however, may require optimization for identification of pathogenic and spoilage bacteria in milk and dairy products. The current study evaluated the effects of various culture conditions and sample preparation methods on assigning of raw milk isolates to the species level by MALDI-TOF MS. The results indicated that culture media, incubation conditions (temperature and time), and sample preparation significantly affected the identification rates of bacteria to the species level. Nevertheless, the development of spectral libraries of isolates grown on different media using a web tool for hierarchical clustering of peptide mass spectra (SPECLUST) followed by a ribosomal protein based bioinformatics approach significantly enhanced the assigning of bacteria, with at least one unique candidate biomarker peak identified for each species. Phyloproteomic relationships based on spectral profiles were compared to phylogenetic analysis using 16S rRNA gene sequences and demonstrated similar clustering patterns with significant discriminatory power. Thus, with appropriate optimization, MALDI-TOF MS is a valuable tool for species-level discrimination of pathogenic and milk spoilage bacteria.

Proteolysis of casein micelles by heat-stable protease secreted by Serratia liquefaciens leads to the destabilisation of UHT milk during its storage

Serratia liquefaciens is a psychrotrophic species, frequently found in raw milk, which secretes Ser2, a heat-resistant protease. Involvement of this species in UHT milk destabilisation was investigated in the present study. Microfiltered milk was inoculated independently with strains S. liquefaciens L53 or L64. Then, UHT treatment was performed and stability of the corresponding UHT milk was investigated during three months of storage. The residual proteolytic activity of strain L53 led to destabilisation of UHT milk, with sedimentation and formation of aggregates. Hydrolysis of casein micelles was confirmed by the increase in the content of non-casein nitrogen and the identification of numerous peptides coming from the four caseins using mass spectrometry. For strain L64, no visual and biochemical alteration were found. This study showed that Ser2 resists UHT treatment and could be a cause of UHT milk destabilisation; however, this destabilisation by S. liquefaciens was strain-dependent.

Identification and characterization of a heat-resistant protease from Serratia liquefaciens isolated from Brazilian cold raw milk

The cold storage of raw milk before heat treatment in dairy industry promotes the growth of psychrotrophic microorganisms, which are known for their ability to produce heat-resistant proteolytic enzymes. Although Pseudomonas is described as the main causative genus for high proteolytic spoilage potential in dairy products, Serratia liquefaciens secretes proteases and may be found in raw milk samples as well. However, at the present there is no information about the proteolytic spoilage potential of S. liquefaciens in milk after heat-treatment. The main aim of this research was to assess the proteolytic spoilage potential of S. liquefaciens isolated from Brazilian raw milk and to characterize the involved protease. S. liquefaciens was shown to secrete one heat-resistant spoilage metalloprotease of, approximately, 52kDa encoded by the ser2 gene. The heat-resistance of Ser2 was similar to the aprX encoded metalloprotease produced by Pseudomonas. Although the ser2 gene was detected in all S. liquefaciens isolates tested in this study, the proteolytic activity of the isolates in milk was highly heterogeneous. Since nucleotide and deduced amino acid sequences of ser2 of all tested isolates are identical, this heterogeneity may be attributed to differences in enzyme expression levels or post-translational modifications.

Heat Resistant Proteases Isolated from Medicinally Significant Spices: Partial Purification and Biochemical Characterization

AbstractProteases or proteinases are essential constituents for all the existing live forms. They act as important industrial enzymes occupying about 60% of total enzyme market. In the present study proteases extracted from nine medicinally important spices; Carum copticum, Syzygium aromaticum, Cuminum cyminum, Nigella sativa, Cinnamomum verum, Foeniculum vulgare, Zingiber officinale, Cinnamomum tamala and Curcuma longa; used in our food and as household medicines on regular basis, have been investigated. Amongst these spices, the specific activity of the isolated protease enzyme was found to be significantly high in Nigella sativa (204 units/mg) and Curcuma longa (124 units/mg) and therefore, their extract was further partially purified and biochemically characterized. The crude extract of the two spices on being subjected to salt precipitation using (NH4)2SO4 as neutral salt, yielded three fractions at 0-30%, 30-60% and 60-90% saturation level. The specific activity of protease enzyme was found to be highest in 0-30% fraction, obtained from both the sources. The values of specific activity and purification-fold of enzyme from Nigella sativa and Curcuma longa were found to be 409 U/mg, 590 U/mg and 2.0-, 4.8-, respectively. The enzyme showed maximum activity at pH and temperature conditions of 5.0 and 40°C, respectively, at 20 min of incubation time, in both the cases. The pH stability values of protease from Nigella sativa ranged from 5.0 to 9.0 whereas in Curcuma longa values were from 4.0 to 8.0. The enzyme from Nigella sativa and Curcuma longa exhibited resistance against heat treatment upto 60°C and 50°C, respectively, increasing the industrial feasibility. The present work hereby, indicates that Nigella sativa and Curcuma longa may serve as good source of thermostable protease; an enzyme of great relevance in various chemical and bio-industries along with pharma sector.

Heat Resistant Proteases Isolated from Medicinally Significant Spices: Partial Purification and Biochemical Characterization

Heat Resistant Proteases Isolated from Medicinally Significant Spices: Partial Purification and Biochemical Characterization

Comparison of identification systems for psychrotrophic bacteria isolated from raw bovine milk

Psychrotrophic bacteria in raw milk produce heat-resistant extracellular proteases, resulting in spoilage and shelf-life reduction of ultrahigh temperature treated milk and milk products. Controlling of these spoilage microbes requires rapid and reliable identification systems for screening of raw milk. This study aimed to compare commercial bacterial identification systems with a genetic method (considered as the ‘gold standard’ method) for the identification of heat-resistant protease producing bacteria in raw milk. Five bacterial identification systems were compared based on typability, discrimination power (i.e. Simpson's Index of Diversity), reproducibility and speed of analysis. The accuracy of 16S rRNA gene sequencing, Biolog, MALDI-TOF MS, API, and Microbact for the identification of Gram negative bacilli at the species level was 100.0%, 86.8%, 63.2%, 60.5% and 57.9%, respectively. The Gram positive bacilli were identified by 16S rRNA gene sequencing, Biolog, MALDI-TOF MS, and API with accuracies at the species level of 100.0%, 85.0%, 95.0% and 90.0%, respectively. The 16S rRNA gene sequencing and phylogenetic analysis discriminated Pseudomonas fluorescens, Pseudomonas syringae, Hafnia alvei, Bacillus cereus, Bacillus pumilus and Bacillus licheniformis to the subspecies level. The Simpson's Index of Diversity scores were 0.966, 0.711, 0.496, 0.472, and 0.140, for 16S rRNA gene sequencing, Biolog, MALDI-TOF MS, API and Microbact, respectively. Limited reference profiles in the databases of Biolog, MALDI-TOF MS, API and Microbact systems reduced their accuracy in bacterial identification, compared to 16S rRNA gene sequencing. The rapidity of each assay is in the following order; MALDI-TOF MS>16S rRNA gene sequencing>Biolog>Microbact>API. The reproducibility of the assays is in the order of 16S rRNA gene sequencing>API>Microbact>MALDI-TOF MS>Biolog. Thus, 16S rRNA gene sequencing appears to be the most reliable and robust system for the identification of dairy spoilage bacteria. The Biolog system is suitable for the identification of Gram negative spoilage bacteria, while MALDI-TOF MS and API systems are suitable for the identification of Gram positive spoilage bacteria isolated from raw milk. The commercial systems used in this study have been developed and extensively used for the identification of clinical microbes but only a limited number of studies used those systems to identify the environmental microorganisms that often contaminate raw milk. Therefore, comparison of those systems for the identification of spoilage microbes in raw milk would provide better understanding of their suitability for routine dairy microbiology and more extensive dairy research.

Proteolysis of casein micelles by Pseudomonas fluorescens CNRZ 798 contributes to the destabilisation of UHT milk during its storage

Among the incriminating factors in the destabilisation of ultra-high-temperature (UHT) milk during storage, the heat-resistant proteases of Pseudomonas are considered to play a role. The objective of this work was to study the consequences of contamination of raw skim milk with Pseudomonas fluorescens CNRZ 798 on the stability of the corresponding UHT milk during storage. After 92 days, milk destabilisation was determined by the presence of a gelled sediment and low value to phosphate test (2.4 mL for control against 0 mL of phosphate solution for milk contaminated before UHT treatment) and the presence of aggregates. For the UHT control milk and UHT milk manufactured from raw skim milk contaminated with P. fluorescens, an increase in the size of the casein micelles (205 and 332 nm, respectively), a decrease in the zeta potential (−16.6 and −14.0 mV, respectively) and decrease in the level of hydration (2.00 and 1.55 g of water per gramme of dried pellet, respectively) were detected. The increase in pH 4.6-soluble nitrogen and trichloroacetic acid-soluble nitrogen content for UHT milk previously contaminated were ten- and fivefold higher than those for control milk, respectively. The trichloroacetic acid-soluble nitrogen fraction of the milk contaminated before treatment contained 118, 22, 4 and 9 peptides from β-, αs1-, αs2- and κ-caseins in comparison to only 22, 19, 6 and 4 peptides for the control milk. This study showed that destabilisation of UHT milk was due to proteolysis of casein micelles.

Heterogeneity of heat-resistant proteases from milk Pseudomonas species

Pseudomonas fragi, Pseudomonas lundensis and members of the Pseudomonas fluorescens group may spoil Ultra High Temperature (UHT) treated milk and dairy products, due to the production of heat-stable proteases in the cold chain of raw milk. Since the aprX gene codes for a heat-resistant protease in P. fluorescens, the presence of this gene has also been investigated in other members of the genus. For this purpose an aprX-screening PCR test has been developed. Twenty-nine representatives of important milk Pseudomonas species and thirty-five reference strains were screened. In 42 out of 55 investigated Pseudomonas strains, the aprX gene was detected, which proves the potential of the aprX-PCR test as a screening tool for potentially proteolytic Pseudomonas strains in milk samples. An extensive study of the obtained aprX-sequences on the DNA and the amino acid level, however, revealed a large heterogeneity within the investigated milk isolates. Although this heterogeneity sets limitations to a general detection method for all proteolytic Pseudomonas strains in milk, it offers a great potential for the development of a multiplex PCR screening test targeting individual aprX-genes. Furthermore, our data illustrated the potential use of the aprX gene as a taxonomic marker, which may help in resolving the current taxonomic deadlock in the P. fluorescens group.

Seasonal influence on heat‐resistant proteolytic capacity of Pseudomonas lundensis and Pseudomonas fragi, predominant milk spoilers isolated from Belgian raw milk samples

Psychrotolerant bacteria and their heat-resistant proteases play a major role in the spoilage of UHT-processed dairy products. Summer and winter raw milk samples were screened for the presence of such bacteria. One hundred and three proteolytic psychrotolerant bacteria were isolated, characterized by API tests, rep-PCR fingerprint analysis and evaluated for heat-resistant protease production. Twenty-nine strains (representing 79% of the complete collection) were further identified by 16S rRNA gene sequencing, rpoB gene sequencing and DNA-DNA hybridizations. A seasonal inter- and intra-species influence on milk spoilage capacity (e.g. growth rate and/or protease production) was demonstrated. Moreover, this polyphasic approach led to the identification of Pseudomonas fragi and Pseudomonas lundensis (representing 53% of all isolates) as predominant producers of heat-resistant proteases in raw milk. The role of Pseudomonas fluorescens, historically reported as important milk spoiler, could not unequivocally be established. The use of more reliable identification techniques and further revision of the taxonomy of P. fluorescens will probably result in a different perspective on its role in the milk spoilage issue.

Purification and properties of a heat-stable enzyme of Pseudomonas fluorescens Rm12 from raw milk

Pseudomonas fluorescens Rm12 is a kind of Psychrotrophic bacteria growing in cold raw milk. It produced an extracellular heat resistant protease with an estimated molecular weight of 45 kDa by size exclusion chromatography and SDS-PAGE under both reducing and non-reducing conditions. The enzyme, designated Ht13, was purified to electrophoretic homogeneity from the culture supernatant by sequentially using ammonium sulfate precipitation, ion-exchange chromatography, hydrophobic chromatography and size exclusion chromatography. The specific activity of the enzyme increased 115.5-folds. The optimum pH value and temperature of Ht13 were 7.5 and 40 °C, respectively. Based on its biochemical characteristics, Ht13 can be included in the group of metalloproteases, which was inhibited by 1, 10-phenanthroline and EDTA but not by pepstatin A, chymostatin, STI, E-64, BBI, PMSF and pAPMSF. Mn2+ has positive effect on activity and can increased the heat resistance capability, while Ca2+ had a negligible effect. For the hydrolysis of azocasein, the Km was 0.012 mg mL−1. The enzyme showed typical heat-stable behavior. After treatment of 160 °C 20 s, the residual activity was 9%. The half-life of the enzyme at 160 °C in buffer with Mn2+ was approximately 12 s. Among several main milk proteins, Ht13 can cleave αs-casein, β-casein and κ-casein. The sequence of 1st–16th amino acids of N-terminal was MSKVKDKAIVSAAQAS, which was same as those proteases excreted from some other P. fluorescens. However, their molecular weights, the activation ion and amino acid composition were different, suggesting Ht13 from P. fluorescens Rm12 is a novel protease.

The relationship between quality criteria of goat milk, its technological properties and the quality of the final products

This paper presents the main criteria that enable the control of milk quality criteria that are included in a payment system based on goat milk quality, with regard to hygienic quality and the technological and sensorial impacts on the final products. Somatic cells, often linked to mastitis and thus to pathogens when the count is high, induce changes of the biochemical composition of milk. However, somatic cell count (SCC) does not seem to impair either cheese-making or sensorial characteristics of the cheeses for SSC close to 1,800,000 cells/ml. Some problems could, nevertheless, occur for the production of high heat-treated milks. The presence of colostrum (IgG) could impair heat-treated fluid milk production, while inhibiting substances could impair the fermentation process. Taking into account the great variability of natural inhibiting substances contained in goat milk, the bovine test applied to goat milk should be adapted. Bacteria count gives a good estimation of the hygienic quality of milk and although pathogens are also included in payment systems, these are not discussed. Nevertheless, different classes of bacteria should be distinguished due to the specific character or change each may cause, for example, psychrotrophic bacteria, which may produce heat resistant protease and lipase and induce organoleptic defects in goat milk products. The fat and protein contents in goat milk have been improved through genetics and feeding programmes. Protein percentage, however, is not a precise criterion to predict and ensure cheese yield. Casein percentage would be a more precise criterion to ensure cheese yield. The lipolysis of goat milk can be a useful optional criterion to ensure the quality of non-ripened lactic cheeses.

Manothermosonication of heat-resistant lipase and protease from Pseudomonas fluorescens: effect of pH and sonication parameters.

The effect of different parameters (pH, ultrasonic amplitude and pressure) on the resistance to heat and manothermosonication (MTS) treatments of heat resistant lipase and protease produced by Pseudomonas fluorescens B52 and NCDO 2085, respectively, were studied. Lipase B52 thermoresistance decreases with an increase of pH. However, inactivation by MTS seems to be pH independent. There were only slight increases in the MTS efficiency when increasing pressure at UHT temperatures and the effect of amplitude was different depending on treatment temperature. Protease NCDO 2085, which was very resistant to MTS at 30 degrees C. was very sensitive to MTS at 76 degrees C. Increases in applied pressure had no effect on MTS efficiency at 140 degrees C and its inactivation by MTS was almost temperature independent between 76-109 degrees C. Data obtained are compared with previous published data and inactivation mechanisms are discussed.

Detection of Heat-Resistant Proteases Produced by Psychrotrophs in Refrigerated Milk

Proteases from four species of Pseudomonas were added to pasteurized milk at .18, 1.8, and 18 units of protease activity per milliliter followed by incubation at 5C for 18 days. During this period milks were analyzed every 2 days for proteolysis by methods of Hull, ninhydrin, and absorption at 280nm. Sensory analysis was done twice weekly for 18 days. There were no significant differences noted for protease activity when ninhydrin and absorption at 280nm were used; however, only 18 units protease/ml were detected by the Hull test. Sensory evaluation correlated with the Hull test data. When 50 and 500 cells/ml were added to milk, a curvilinear increase in proteolysis was noted by 11 and 13 days, respectively, for three of the four Pseudomonas species as detected by the Hull method. Heat inactivation of the proteases at 62.8°C for 30min was 0 to 73.1% and 10 to 36% as measured by the Hull and agar diffusion methods, respectively.