Quantitative studies of heat-stable proteinase from Pseudomonas fluorescens P1 by the enzyme-linked immunosorbent assay

Pseudomonas fluorescens P1 is a psychrotrophic bacterium isolated from milk. Proteinase P1, the main extracellular heat-stable proteinase fraction of P. fluorescens P1, has been purified to homogeneity. A procedure with a sandwich enzyme-linked immunosorbent assay, using microplates and alkaline phosphatase conjugate was shown to detect 0.25 ng of proteinase P1 in 1 ml of reconstituted skim milk or defatted cream. The method offers the combination of sensitivity and specificity for the detection of these enzymes in milk and dairy products. In reconstituted skim milk cultures proteinase P1 was detectable when the CFU approached 10(7)/ml. Cultures in milk diluted 1:10, 1:30, or 1:100 with water showed detectable proteinase at population densities close to 10(6) CFU/ml. Aeration stimulated proteinase production; thus, a skim milk culture with shaking at 5 degrees C had a proteinase level of 36,000 ng/ml after 7 days as compared to 80 ng/ml in a stationary culture. The rate of inactivation of proteinase P1 at 150 and 55 degrees C as expressed by residual antigenic activity determined by the enzyme-linked immunosorbent assay was somewhat different from the rate determined on the basis of residual proteolytic activity. The specificity of the enzyme-linked immunosorbent assay with proteinase P1 antibodies was identical for proteinase P1 and for enzymes from six other strains of P. fluorescens, one Chromobacterium strain, and one Flavobacterium strain. Some psychrotrophic strains produced immunologically unrelated proteinase(s). These preliminary observations indicate that proteinase P1-related enzymes are common among psychrotrophs appearing as spoilage bacteria in milk.     

Quantitative studies of heat-stable proteinase from Pseudomonas fluorescens P1 by the enzyme-linked immunosorbent assay.

Pseudomonas fluorescens P1 is a psychrotrophic bacterium isolated from milk. Proteinase P1, the main extracellular heat-stable proteinase fraction of P. fluorescens P1, has been purified to homogeneity. A procedure with a sandwich enzyme-linked immunosorbent assay, using microplates and alkaline phosphatase conjugate was shown to detect 0.25 ng of proteinase P1 in 1 ml of reconstituted skim milk or defatted cream. The method offers the combination of sensitivity and specificity for the detection of these enzymes in milk and dairy products. In reconstituted skim milk cultures proteinase P1 was detectable when the CFU approached 10(7)/ml. Cultures in milk diluted 1:10, 1:30, or 1:100 with water showed detectable proteinase at population densities close to 10(6) CFU/ml. Aeration stimulated proteinase production; thus, a skim milk culture with shaking at 5 degrees C had a proteinase level of 36,000 ng/ml after 7 days as compared to 80 ng/ml in a stationary culture. The rate of inactivation of proteinase P1 at 150 and 55 degrees C as expressed by residual antigenic activity determined by the enzyme-linked immunosorbent assay was somewhat different from the rate determined on the basis of residual proteolytic activity. The specificity of the enzyme-linked immunosorbent assay with proteinase P1 antibodies was identical for proteinase P1 and for enzymes from six other strains of P. fluorescens, one Chromobacterium strain, and one Flavobacterium strain. Some psychrotrophic strains produced immunologically unrelated proteinase(s). These preliminary observations indicate that proteinase P1-related enzymes are common among psychrotrophs appearing as spoilage bacteria in milk.     

Enzymes from isolates of Pseudomonas fluorescens involved in food spoilage

AIMS: Psychrotrophic Gram-negative bacteria, such as Pseudomonas species, pose a significant spoilage problem in refrigerated meat and dairy products due to secretion of hydrolytic enzymes, especially lipases and proteases. This study characterized the enzymes produced by strains of Pseudomonas fluorescens isolated from pasteurized milk. METHODS AND RESULTS: Thirty-seven isolates of Ps. fluorescens from skimmed, semiskimmed and whole milk were all shown to be proteolytic and lipolytic on casein and tributyrin agar, respectively. The highest level of protease production by one isolate, SMD 31, from skimmed milk was in minimal salts medium containing 1 mmol x l(-1) calcium chloride at 20 degrees C. The proteases belonged to the class of metallo-proteases, as there was no residual activity with 10 mmol x l(-1) EDTA. They were heat stable and retained activity even after treatment at 121 degrees C for 20 min. One protease of 45-48 kDa was detected in unconcentrated supernatant fluid samples but, in three isolates from different milk sources, five proteases with molecular masses between 28 and 48 kDa were detected on a 12% zymogram casein gel following ultrafiltration. Attempts to purify the lipases proved unsuccessful. CONCLUSIONS: The characteristics of the major protease of 45-48 kDa correspond to those of proteases described for other Pseudomonas species isolated from a range of environments. However, the smaller proteases have not been described previously. SIGNIFICANCE AND IMPACT OF THE STUDY: In the absence of ultrafiltration the presence of the minor protease species may be missed and they may act as contaminants of the major protease in unpurified or semipurified samples.     

Numerical taxonomy of proteolytic psychrotrophs from Queensland raw milks

Eighty-seven proteolytic psychrotrophic micro-organisms were isolated from 11 bulk milk supplies of two Queensland factories from different climatic regions, before and after storage at 4 degrees C for 7 d. These isolates together with 15 reference strains formed the basis of a numerical taxonomic study involving 81 attributes. All but six isolates were pseudomonads. The strains clustered into nine groups, of which one group consisted of four yeasts. One group, containing 39 isolates, was designated as Pseudomonas fluorescens biovar 1; three groups, containing 27 isolates, as Ps. fluorescens biovar 5; and one group, containing 10 isolates, as Ps. putida biovar A. This study showed that the proteolytic psychrotrophic microflora of the 11 milks supplying the two factories was substantially different and that the proteolytic flora of 7 d refrigerated milk could not be estimated by examining the flora before storage.     

Characterization of a bacteriocin produced by Enterococcus faecium GM-1 isolated from an infant

AIM: To partially characterize the bacteriocin produced by the GM-1 strain of Enterococcus faecium, isolated from the faeces of a newborn human infant. METHODS AND RESULTS: The bacteriocin produced by E. faecium GM-1 showed a broad spectrum of activity against indicator strains of Escherichia coli, Staphylococcus aureus, Vibrio spp., Salmonella typhimurium, Listeria monocytogenes, Lactobacillus acidophilus, and Streptococcus thermophilus. Treatment of the GM-1 bacteriocin with proteolytic enzymes reduced its inhibitory activities. The bacteriocin was stable at 100 degrees C for 20 min and displayed inhibitory activity at neutral pH. The optimal production of bacteriocin from E. faecium GM-1 was obtained when the culture conditions were pH 6.0-6.5 and 35-40 degrees C. The inhibitory activity of the bacteriocin was not substantially changed by the use of different carbon sources in the media, except when galactose was substituted for glucose. The use of a sole nitrogen source caused a decrease in inhibitory activity. A bacteriocin gene similar to enterocin P was identified from the total DNA of E. faecium GM-1 by PCR and direct sequencing methods. CONCLUSION: E. faecium GM-1, which was isolated from the faeces of a newborn baby, produces an enterocin P-like bacteriocin with inhibitory activity against Gram-positive and Gram-negative bacteria, including food-borne pathogens. SIGNIFICANCE AND IMPACT OF THE STUDY: E. faecium GM-1, isolated from infant faeces, produces a new bacteriocin that is similar to enterocin P. This bacteriocin is heat stable and has a broad antibacterial spectrum that includes both Gram-positive and Gram-negative bacteria.     

Isolation of a psychrotrophic Azospirillum sp. and characterization of its extracellular protease

A novel psychrotrophic bacterium secreting a protease was isolated from a mountain soil in Korea. On the basis of a 16S rDNA sequence analysis and physiological properties, the isolate was identified as an Azospirillum sp. The protease purified from the culture supernatant was a monomer in its native form with an apparent molecular mass of 48.6 kDa on SDS-PAGE. The protease was active in a broad pH range around 8.5 and at temperatures up to 40 degrees C and stable at temperatures below 30 degrees C for 3 days. The proteolytic activity was inhibited by iodoacetamide and EDTA. The Mg2+ ion did not activate the enzyme much but reversed the inhibition by EDTA, suggesting that the protease belongs to a cysteine protease stabilized by the Mg2+ ion.     

Bacteriocin-like substance production by Bacillus licheniformis strain P40

AIMS: To investigate the production of bacteriocin-like compounds by Bacillus spp. isolated from the Amazon basin. METHODS AND RESULTS: An antimicrobial substance produced by Bacillus licheniformis strain P40 was inhibitory to a broad range of indicator strains, such as Listeria monocytogenes, Bacillus cereus and clinical isolates of Streptococcus spp. The compound was stable at 100 degrees C, but lost its activity when treated at 121 degrees C/103.5 kPa for 15 min. It was resistant to the proteolytic action of trypsin and papain but sensitive to pronase E and was stable within a wide range of pH (3-11). The substance was bactericidal and bacteriolytic to L. monocytogenes. CONCLUSIONS: An antibacterial peptide produced by Bacillus licheniformis was characterized, presenting a broad spectrum of activity against pathogenic and spoilage organisms. SIGNIFICANCE AND IMPACT OF THE STUDY: The identification of a substance active against important pathogens addresses an important aspect of food safety.     

Isolation of a Pseudomonas species from fish intestine that produces a protease active at low temperature

A psychrotrophic bacterium producing a protease active at low temperatures was isolated from fish intestine and identified as a Pseudomonas species. Optimum growth and protease-producing temperatures of this strain were 15°C and 10°C, respectively. The maximum temperature for proteolytic activity was 25°C, an unusually low temperature.     

Fish protein hydrolysis by a psychrotrophic marine bacterium isolated from the gut of hake (Merluccius hubbsi)

The aims of this study were to identify a psychrotrophic bacterium, strain CR41, producing a cold adapted protease during growth at low temperatures and to evaluate the ability of the cells to hydrolyze hake fish protein. The strain was isolated from the intestinal tract of hake collected from the San Jorge Gulf (Patagonia, Argentina) and it was identified as Pseudoalteromonas. Growth and fish protein hydrolysis were determined using an aerated simple mineral medium plus 10% fish protein concentrate. Proteolytic activity was measured at 7 and 22 degrees C during culture in the concentrate. Protease production started in the exponential growth phase and reached a maximum during stationary phase. Protease activity at 7 degrees C was lower than at 22 degrees C. After 8 h of incubation, the percentage of hydrolyzed protein was 84% at 7 degrees C and 95% at 22 degrees C. Electrophoresis detection showed that degradation of muscle hake proteins was complete at both temperatures, and in gelatin zymograms extracellular activity showed two proteolytic bands with apparent molecular masses of approximately 31.6 and 62 kDa.     

Extracellular protease activity of different Pseudomonas strains: dependence of proteolytic activity on culture conditions

AIMS: This study investigated the effect of growth conditions on proteolytic activity of a Pseudomonas strain, named Pseudomonas sp. LBSA1, isolated from bulk raw milk. It was compared with three Pseudomonas chlororaphis and one Pseudomonas fluorescens strain from culture collections. METHODS AND RESULTS: Bacteriae were grown in a minimal salt medium. For all the strains, addition of 1% (v/v) skim milk to the growth medium was sufficient to induce protease production in 48-h culture. Addition of 1 mmol l(-1) calcium chloride permitted the detection of proteolytic activity of four strains in 48-h cultures but not for Pseudomonas sp. LBSA1. The five strains presented two patterns of proteolytic activity when grown in the minimal salt medium supplemented with 2% (v/v) skim milk at various temperatures for 48 h. Two electrophoretic protease patterns were also obtained from the zymogram of extracellular medium for the five strains. CONCLUSIONS: The growth conditions permitting protease production are variable and do not depend on the genus of the producing strain. SIGNIFICANCE AND IMPACT OF THE STUDY: For the first time a study on proteolytic activity of P. chlororaphis strains is reported. Among the tested criteria, zymograms of extracellular medium were the only ones that permitted distinguishing the P. chlororaphis strains from the P. fluorescens strain.     

Psychrotrophic bacterial flora of raw ewes' milk, with particular reference to gram negative rods

The microbial flora of 141 samples of raw ewes' milk was determined, before and after storage for 72 h at 4 degrees and 7 degrees C. Penicillin-resistant bacteria represented ca 61% of 1760 psychrotrophic isolates from refrigerated milk samples. Pseudomonas fluorescens and Pseudomonas fluorescent group-related strains predominated (ca 86%) in the Gram negative psychrotrophic microflora. Leuconostoc dextranicum was the most frequent Gram positive psychrotrophic species isolated.     

Proteolytic activity of a yeast cell wall lytic Arthrobacter species

AIMS: To investigate properties of the proteolytic activity of a yeast cell wall lytic soil bacterium identified as an Arthrobacter species. METHODS AND RESULTS: The organism was grown at pH 7.5 and 30 degrees C in shake flasks on media with different complex subtrates. Highest proteolytic activity assayed with azocaseine was detected in media with wheat gluten. In addition, l-leucine, l-alanine exopeptidase activity and esterase activity were found. The proteolytic activity showed stability up to pH 12, with a maximum at pH 11. The temperature optimum was at 55 degrees C, but there was a loss in enzyme activity of 50% within 2 h. The proteolytic activity was inhibited by 3,4-dichloroisocumarin, whereas there was little or no effect with EDTA, pepstatin A or E64. CONCLUSIONS: The proteolytic activity is highly alkaline stable. The formation of the enzyme can be induced by media with high protein content.     

Isolation of proteolytic psychrotrophic yeasts from fresh raw seafoods

A total of 103 cultures of yeasts were isolated from seven kinds of fresh raw seafoods. The isolates comprised six genera, Candida, Cryptococcus, Debaryomyces, Rhodotorula, Sterigmatomyces and Trichosporon , and included 21 different species. All the isolates were psychrotrophic yeasts. Proteolytic activities of 50 psychrotrophic strains were studied by use of skim milk within the temperature range of 0–42°C. All the strains showed various degrees of proteolysis. In particular, Candida lipolytica. Trichosporon pullulans and Candida scottii were active species at low temperatures. Sensory spoilage due to the proteolytic yeasts were observed in mackerel homogenates stored at 10°C. C. lipolytica -inoculated homogenates caused spoilage with ammoniacal odours after 1 week of storage. Values of total volatile basic nitrogen at 10°C were highest with C. lipolytica among 35 strains tested, followed by Tr. pullans. Trichosporon cutaneum, C. scottii, Rhodotorula glutinis and Cryptococcus luteolus. Proteolytic psychrotrophic yeasts were widely distributed in raw seafoods.     

Demonstration of a proteolytic activity on intracellular origin in Aeromonas hydrophila LP 50

Aeromonas hydrophila LP 50, isolated from packaged pasteurized milk, was grown in glucose-polypeptone medium at 30 degrees C. The proteolytic activity of A. hydrophila LP 50, optimum at the stationary phase of growth, is attributed to extracellular or membrane protease; no intracellular proteolytic activity was shown.     

Numerical taxonomy of proteolytic psychrotrophs from Queensland raw milks

Eighty-seven proteolytic psychrotrophic micro-organisms were isolated from 11 bulk milk supplies of two Queensland factories from different climatic regions, before and after storage at 4°C for 7 d. These isolates together with 15 reference strains formed the basis of a numerical taxonomic study involving 81 attributes. All but six isolates were pseudomonads. The strains clustered into nine groups, of which one group consisted of four yeasts. One group, containing 39 isolates, was designated as Pseudomonas fluorescens biovar 1; three groups, containing 27 isolates, as Ps. fluorescens biovar 5; and one group, containing 10 isolates, as Ps. putida biovar A. This study showed that the proteolytic psychrotrophic microflora of the 11 milks supplying the two factories was substantially different and that the proteolytic flora of 7 d refrigerated milk could not be estimated by examining the flora before storage.     

Bacillus cereus is common in the environment but emetic toxin producing isolates are rare

AIMS: To determine the incidence of emetic toxin producing Bacillus cereus in soil, animal faeces and selected vegetable produce to compare the results with the previously reported high incidence in rice paddy fields. To examine whether the emetic toxin has antibiotic activity. METHODS AND RESULTS: The incidence of emetic toxin producing B. cereus was evaluated by plating on selective agar 271 samples of soils, animal faeces, raw and processed vegetables. Overall, 45.8% of samples were positive for B. cereus. One hundred and seventy-seven B. cereus isolates were recovered at 30 degrees C with the grand mean spore count being 2.6 +/- 1.7 log(10) CFU g(-1) and 148 B. cereus isolates were recovered at 7 degrees C with the grand mean spore count being 2.2 +/- 1.2 log(10) CFU g(-1) of the 177 B. cereus isolated at 30 degrees C, only 3 were positive for emetic toxin production at a titre of 1/64, 1/32, 1/16, respectively. Also, 1 of 148 B. cereus isolated at 7 degrees C was positive for emetic toxin production to a titre of 1/128. All positive isolates came from washed or unwashed potato skins, one was psychrotrophic as determined by PCR and growth at 7 degrees C on subculture. The emetic toxin was not shown to have any antibiotic effects in growth inhibition studies. CONCLUSIONS: While B. cereus was a common isolate, the incidence of the emetic strain was rare. This is in contrast to previous findings of the high incidence in rice paddy fields and the processing environment, which may suggest rice is a selective area for growth of the emetic strain of B. cereus. SIGNIFICANCE AND IMPACT OF STUDY: The finding that a psychrotrophic isolate of B. cereus can produce emetic toxin is the first ever such observation and suggests the possibility that psychrotrophic isolates could grow in refrigerated fresh foods and cause emesis. The incidence of emetic B. cereus strains in rice paddy fields now requires further study for comparison with the low incidence found in other soils. The emetic toxin failed to inhibit the growth of other bacterial, fungal and yeast species. Whether the toxin (which is similar in structure to the antibiotic valinomycin) plays a competitive role in the environment therefore remains unclear.     

Characterization of a bacteriocin produced by a newly isolated Bacillus sp. Strain 8 A

AIMS: The aim of this research was to investigate the production of bacteriocins by Bacillus spp. isolated from native soils of south of Brazil. METHODS AND RESULTS: A bacteriocin produced by the bacterium Bacillus cereus 8 A was identified. The antimicrobial activity was produced starting at the exponential growth phase, although maximum activity was at stationary growth phase. A crude bacteriocin obtained from culture supernatant fluid was inhibitory to a broad range of indicator strains, including Listeria monocytogenes, Clostridium perfringens, and several species of Bacillus. Clinically relevant bacteria such as Streptococcus bovis and Micrococcus luteus were also inhibited. Bacteriocin was stable at 80 degrees C, but the activity was lost when the temperature reached 87 degrees C. It was resistant to the proteolytic action of trypsin and papain, but sensitive to proteinase K and pronase E.Bacteriocin activity was observed in the pH range of 6.0-9.0. CONCLUSIONS: A bacteriocin produced by Bacillus cereus 8 A was characterized, presenting a broad spectrum of activity and potential for use as biopreservative in food. SIGNIFICANCE AND IMPACT OF STUDY: The identification of a bacteriocin with large activity spectrum, including pathogens and spoilage microorganisms, addresses an important aspect of food safety.     

Purification and characterization of extracellular alkaline serine protease from Stenotrophomonas maltophilia strain S-1

AIMS: The present study was conducted by screening soil bacteria in an attempt to isolate a bacterium that produced extracellular alkaline protease, and for purification and characterization of the protease. METHODS AND RESULTS: Soil bacteria were screened by growth on casein as the sole carbon source. Characterization of a strain isolated from soil of Abashiri, Japan indicated a taxonomic affiliation to Stenotrophomonas maltophilia, and was named S-1 strain. The purified S-1 protease, designed S. maltophilia Protease-1 (SmP-1), exhibited an optimal pH of 12.0, optimal reaction temperature of 50 degrees C and a molecular mass of approximately 40 kDa by sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE). The cleavage sites of the oxidized-insulin B chain by SmP-1 were identified as Leu6-Cys7, Cys7-Gly8, Tyr16-Leu17 and Leu17-Val18. The N-terminal amino acid sequence of the purified alkaline protease was determined as NH2-SASAPMVSGVAALVLE. CONCLUSION: A novel extracellular alkaline serine protease was isolated from S. maltophilia strain S-1. The optimal pH of the proteolytic activity was pH 12.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The extremely high optimal pH and heat stability of the alkaline serine protease SmP-1 might make it widely applicable to food and other industries.     

Formation of guaiacol in chocolate milk by the psychrotrophic bacterium Rahnella aquatilis

AIMS: The aim of this study was to identify the causative agent of a smoky/phenolic taint in refrigerated full cream chocolate milk. METHODS AND RESULTS: Microbiological examination of spoiled and unspoiled milk samples from the same processor showed high numbers of the psychrotrophic coliform Rahnella aquatilis in the spoiled samples only. Gas chromatography/mass spectrometry (GC/MS) was used to identify and quantify the taint compound as guaiacol (2-methoxyphenol) in the spoiled milk. Challenge studies in UHT chocolate and white milks inoculated with the isolate and incubated at 4-5 degrees C and 8-9 degrees C for 6 d showed the production of guaiacol in chocolate milk only, which was confirmed and quantified by GC/MS. CONCLUSIONS: The results indicate that if present in refrigerated chocolate milk, Rah. aquatilis can produce guaiacol within the expected shelf-life of the product, even without temperature abuse. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first report that the coliform Rah. aquatilis can produce guaiacol in refrigerated chocolate milk products.     

Isolation and characterization of psychrophiles producing cold-active beta-galactosidase

AIMS: The present study was conducted to screen for psychrophilic micro-organisms that are able to hydrolyse lactose at low temperature, and to examine the cold-active beta-galactosidase produced by the isolated psychrophilic micro-organisms. METHODS AND RESULTS: Psychrophilic bacteria, which grow on lactose as a sole carbon source, were isolated from soil from Hokkaido, Japan. The phenotype and sequence of 16S rDNA of the isolated strains indicated a taxonomic affiliation to Arthrobacter psychrolactophilus. The isolated A. psychrolactophilus strains were able to grow on lactose at below 5 degrees C, and showed cold-active beta-galactosidase activity, which was highly specific at even 0 degrees C. CONCLUSIONS: Facts in this study may indicate the possibility that the isolated strains produce novel beta-galactosidases that are able to hydrolyse lactose at low temperature, although some strains have isozymes. SIGNIFICANCE AND IMPACT OF THE STUDY: It may be possible that the cold active beta-galactosidases from the isolated strains can be applied to the food industry, e.g. processing of milk and whey below 5 degrees C.