Studies on Milk Clotting Enzymes Produced by Basidiomycetes

In the course of screening tests of Basidiomycete proteolytic enzymes, it was observed that some strains produced milk clotting enzymes with fairly weak proteolytic activities.

When sucrose-polypeptone and sucrose-corn steep liquor media were used, only 6 strains out of 44 strains tested showed weak milk clotting activities. Cheddar cheese making with culture filtrates of these 6 strains revealed that the culture filtrates of 2 strains, Irpex lacteus Fr. and Fomitopsis pinicola (Fr.) Karst., were able to produce Cheddar cheese of good quality.

On the other hand, when sucrose-distillers solubles media were used, a lot of strains showed high proteolytic activity in addition to high milk clotting activity. The ratio of milk clotting to proteolytic activities (MCA/PA) was assumed to be an important index for the selection of organism, and F. pinicola and Coriolus consors (Berk.) Imaz. were selected as the strain with high MCA/PA ratio.

As the investigation on culture conditions of 3 strains mentioned above showed that F. pinicola and I. lacteus, were richly productive of milk clotting enzymes, the 2 strains except C. consors were used for further studies on cheese making.

Cheddar cheese making with crude enzymes revealed that cheese products produced by the enzyme of F. pinicola had a slightly bitter taste after 5 months’ ripening but that those produced by the enzyme of I. lacteus had good quality.     

Protease-producing psychrotrophic bacteria isolated from Antarctica

The extracellular protease production capacity of 840 bacterial strains isolated during the austral summers of 1989/90 and 1991/92 from different sources of the Antarctic ecosystem was analysed in skim-milk agar plates. Thirty-four psychrotrophic strains were selected, classified at genus level and tested from proteolytic activity by the azocasein method from the cell-free supernatant of submerged cultures. Thirty-two of the selected strains were Gram-negative bacteria and Pseudomonas was the predominant genus. Three Pseudomonas maltophilia strains showed the highest levels of proteolytic activity at 20°C. No correlation was observed between the proteolytic activity estimated by the ring of hydrolysis in skim-milk agar plates and the activity measured by the azocasein method. The results suggest that these psychrotrophic strains are potentially useful for developing a biotechnological process to produce proteases with high activity at moderate temperatures.     

A Newly Isolated Organic Solvent Tolerant Staphylococcus saprophyticus M36 Produced Organic Solvent-Stable Lipase

Thirty-eight high lipase activity strains were isolated from soil, seawater, and Brassica napus. Among them, a novel organic solvent tolerant bacterium (strain M36) was isolated from the seawater in Jiangsu, China. Isolate M36 was able to grow at high concentration of benzene or toluene up to 40% (vol/vol), and later identified as Staphylococcus saprophyticus by biochemical test and 16s ribosomal DNA sequence. No work on Staphylococcus producing lipase with organic solvent tolerance has been reported so far. The lipase of strain M36 whose activity in liquid medium was 42 U mL⁻¹ at 24-h incubation time was stable in the presence of 25% (vol/vol) p-xylene, benzene, toluene, and hexane.     

Interactions between Lactobacillus sakei and CNC (Staphylococcus xylosus and Kocuria varians) and their influence on proteolytic activity

Aims: To evaluate interactions between Lactobacillus sakei and coagulase negative cocci (CNC) (Staphylococcus xylosus and Kocuria varians) and to investigate the influence of these interactions on their own proteolytic activity. Methods and Results: Interactions occurring between strains of Lact. sakei and CNC were assessed by spectrophotometric analysis. The growth of 35 strains of Lact. sakei, used as indicators, was compared to that obtained combining the same strains with growing cells or cell‐free supernatants of 20 CNC (18 Staph. xylosus and 2 K. varians). The proteolytic activity expressed by single strains or by their combinations was assessed on sarcoplasmic protein extracts by sodium dodecyl sulfate‐polyacrylamide gel electrophoresis. The results evidenced that interactions are able to affect not only the growth but also the in vitro proteolytic activity of Lact. sakei and CNC used in combination. Conclusions: A relationship between the presence of interactions among useful strains and the strength of technological characteristics, such as proteolysis, was defined. Significance and Impact of the Study:  The study highlighted that CNC are able to stimulate the growth of some Lact. sakei strains. At the same time, this interaction positively influences the proteolytic activity of strains used in combination. Given the importance of proteolysis during the ripening of fermented meats, this phenomenon should be taken into account to select meat starter cultures.     

Extracellular proteolytic activity ofCryptococcus neoformans

Eight strains ofCryptococcus neoformans var.neoformans isolated from AIDS patients in the Infectious Disease Institute, University of Turin, Italy, were examined for growth and extracellular proteolytic activity in culture with solid and liquid media. All of the strains grew well on Yeast Carbon Base (YCB) agar medium supplemented with both 0.1% (w/v) bovine serum albumin (BSA) and 0.01% (w/v) polypeptone (Pp), and produced a clear proteolytic zone around their colonies, whereas they exhibited less growth and proteolytic activity on YCB medium supplemented with BSA alone. Strain #8 with a strong proteolytic activity was cultured in three different liquid media. Its growth was limited in YCB medium supplemented with 0.1% BSA, but was moderate in that with 0.01% Pp. Enhanced growth was supported by the addition of both BSA and Pp to the YCB medium. The relative value of the final cellular yields obtained with the above YCB-0.1% BSA, YCB-0.01% Pp and YCB-0.1% BSA-0.01% Pp media was approximately 1:10:20. In the culture with YCB medium containing both BSA and Pp, a rapid decrease in the amount of BSA was demonstrated by a spectrophotometric assay and gel electrophoresis of the culture supernatant after the log-to-stationary phase. The proteolytic activity in the culture supernatant became detectable after the log phase when tested with skim milk agarose plates. These results allowed us to conclude thatCr. neoformans var.neoformans is able to secrete protease and to utilize protein as a source of nitrogen.     

Characterization of microaerophilic bacteria isolated from the coastal waters of Tokyo Bay, Japan

Abstract A total of 84 strains of microaerophiles were isolated from the Edo River mouth, Tokyo Bay, Japan, and their morphological, physiological and biochemical properties and mole percent guanine-plus-cytosine contents (mol%) of DNAs extracted from them were examined. Eighty-eight to 100% of the strains, collected in summer, grew at 15–37°C but only 5% of the strains grew at 5°C. Seventy-seven to 100% of the strains, collected in winter, were able to grow at 10–25°C but 79% of the strains were unable to grow at 37°C. The shift in growth-range of bacteria strongly suggests that the microflora of estuarine microaerophiles changes seasonally. All the strains could grow at 50% seawater while 70 and 42% of the strains could not grow at 0 and 100% seawater, respectively.
On the other hand, 72 strains, out of the 84 strains, clustered into 9 phena using numerical taxonomy methods. The strains belonging to phena 1 through 9 were all Gram-negative, non-spore-forming rod-shaped organisms which were negative for susceptibility to vibrostatic agent O/129, H₂S production and acid from dulcitol, and the G + C contents of DNAs ranged from 29.8 to 56.6 mol%. However, almost all strains could not be identified and are suggested to be new species.     

The Extracellular Proteases of the Phytopathogenic Bacterium Xanthomonas campestris

The culture liquids of three Xanthomonas campestris pv. campestris strains were found to possess proteolytic activity. The culture liquid of strain B611 with the highest proteolytic activity was fractionated by salting-out with ammonium sulfate, gel filtration, and ion-exchange chromatography. The electrophoretic analysis of active fractions showed the presence of two proteases in the culture liquid of strain B611, the major of which was serine protease. The treatment of cabbage seedlings with the proteases augmented the activity of peroxidase in the cabbage roots by 28%.     

Psychrotrophic amylolytic bacteria from deep sea sediment of Prydz Bay, Antarctic: diversity and characterization of amylases

Seventeen psychrotrophic bacteria with cold-adaptive amylolytic, lipolytic or proteolytic activity were isolated from deep sea sediment of Prydz Bay, Antarctic. They were affiliated with γ-Proteobacteria (12 strains) and gram-positive bacteria (5 strains) as determined by 16S rDNA sequencing. The amylase-producing strains belonged to genus Pseudomonas, Rhodococcus, and Nocardiopsis. Two Pseudomonas strains, 7193 and 7197, which showed highest amylolytic activity were chosen for further study. The optimal temperatures for their growth and amylase-producing were between 15 and 20°C. Both of the purified amylases showed highest activity at 40°C and pH 9.0, and retained 50% activity at 5°C. The SDS-PAGE and zymogram activity staining showed that the molecular mass of strain 7193 and 7197 amylases were about 60 and 50 kDa respectively. The Pseudomonas sp. 7193 amylase hydrolyzed soluble starch into glucose, maltose, maltotriose, and maltotetraose, indicating that it had both activities of α-amylase and glucoamylase. The product hydrolyzed by Pseudomonas sp. 7197 amylase was meltotetraose.     

BACTERIA THAT INDUCE MORPHOGENESIS IN ULVA PERTUSA (CHLOROPHYTA) GROWN UNDER AXENIC CONDITIONS1

Marine foliaceous green macroalgae such as Ulva lose their typical morphology when cultured aseptically in defined synthetic media. However, after reinfection by certain marine bacteria (isolated from unialgal cultures of Ulva pertusa Kjellman), the organisms regain their typical foliaceous or tubular morphology. To investigate the morphogenesis (MG) induced in U. pertusa by bacteria, we isolated and identified bacteria with MG activity on U. pertusa and studied the distribution of such bacteria in seawater and on various marine macroalgae. We isolated 1555 bacterial strains from 18 species of marine macroalgae (six Chlorophyta, five Phaeophyta, and seven Rhodophyta), from seawater and from sediment collected at the beach at Omaezaki, Shizuoka Prefecture; Japan. Of these, 676 bacterial strains (43.5%) showed MG activity. They were classified into six bacterial groups, Flavobacterium, Vibrio, Pseudomonas, Deleya, Escherichia, and gram-positive cocci. These bacteria were ubiquitous among the samples and were not specific to U. pertusa. Several plant growth regulators had no MG activity. Filter-sterilized supernatants of culture media of MG-active bacteria strains did not induce MG. Cocultivation of Ulva with active bacterial strains is so far the only way to induce the MG effect, which suggests that for MG direct contact between Ulva and the bacterial strain is necessary.     

Toxin production by Clostridium botulinum in grass.

Investigations on farms where botulism has occurred in cows showed that proteolytic Clostridium botulinum type B was present in newly made grass silages. Experiments were undertaken to study growth and toxin production of C. botulinum in grass. Of the strains tested only proteolytic strains of C. botulinum types A and B were able to produce toxin with grass as a substrate. Proteolytic strains of type B produced both medium (12S) and large (16S) toxin forms. The minimal water activity (aw) for toxin production at pH 6.5 and 5.8 was 0.94. At pH 5.3, toxin was produced at an aw of 0.985. These results indicate that proteolytic strains of C. botulinum (if present) may multiply and produce toxin in wilted grass silages.     

Toxin production by Clostridium botulinum in grass.

Investigations on farms where botulism has occurred in cows showed that proteolytic Clostridium botulinum type B was present in newly made grass silages. Experiments were undertaken to study growth and toxin production of C. botulinum in grass. Of the strains tested only proteolytic strains of C. botulinum types A and B were able to produce toxin with grass as a substrate. Proteolytic strains of type B produced both medium (12S) and large (16S) toxin forms. The minimal water activity (aw) for toxin production at pH 6.5 and 5.8 was 0.94. At pH 5.3, toxin was produced at an aw of 0.985. These results indicate that proteolytic strains of C. botulinum (if present) may multiply and produce toxin in wilted grass silages.     

Characterisation and identification of spoilage psychotrophic Gram-negative bacteria originating from Tunisian fresh fish

The contamination of fresh fish by spoilage bacteria is undesirable, particularly when Gram-negative bacteria, which produce thermo-resistant protease and lipase, can grow. The purpose of the present work was, therefore, to isolate and identify psychrotrophic Gram-negative (Psy G(-)) bacteria, isolated from 80 samples of 12 species of wild and aquacultured fresh seafood, by biochemical and molecular methods using 16S rRNA gene sequencing. Twenty-eight identified strains were studied to evaluate their catalase, nitrate reductase, lipolytic and proteolytic activities, as well as growth ability, at different temperatures, pH and NaCl concentrations. Among 150 Psy G(-) strains, the most dominant species found were: Pseudomonas fluorescens, Aeromonas hydrophila, Pseudomonas putida and Photobacterium damselae. All strains of Psy G(-) had catalase activity and were able to reduce nitrates to nitrites. Proteolytic activity on milk and on gelatin agar was demonstrated for the majority of the isolates. However, extracellular proteolytic activity as assessed by the azocasein method wasn’t very high in all the strains. Lipolytic activity, as assessed by the agar method, showed that 92.9 % of strains could hydrolyse egg yolk, against 82.1 % and 57 % that could hydrolyse Tween 20 and Tween 80, respectively.     

Fermenting Cucumber, a Potential Source for the Isolation of Pediocin-Like Bacteriocin Producers

Summary A strain of Pediococcus acidilactici CFR K7 isolated from cucumber, produced an antimicrobial peptide which acted against Leuconostoc mesenteroides, selected strains of Lactobacillus spp., Pediococcus spp. and Enterococcus spp. The partially purified bacteriocin had molecular weight of ~4.6 kDa, heat stability in a range of 40–121 °C and was active over a wide range of pH (2.0–9.0). This bacteriocin possessed strong antilisterial activity and was susceptible to proteolytic enzymes. Southern hybridization using the PCR-generated pedA probe established that the gene for the bacteriocin was plasmid-borne as in the case of pediocin PA-1. Nucleotide sequence of the pedAB gene indicated 100% homology to a pediocin AcH/PA-1. Certain bacteriocinogenic strains isolated from naturally fermented cucumber were tested by colony hybridization using the pedA gene probe. Nine out of twenty colonies reacted with the probe indicating their ability to produce the pediocin-like bacteriocin. These nine colonies were further tested for their antimicrobial spectrum, proteolytic inactivation and plasmid profile. It was found that a few of them were active against Bacillus cereus, Micrococcus luteus and Listeria monocytogenes. Their proteolytic inactivation showed that the antimicrobial compound was susceptible to proteinase K. Colony hybridization could thus enable rapid detection of pediocin and pediocin-like bacteriocin producers among a population of bacteriocinogenic strains.     

Phenotypic Variations and Molecular Identification of <Emphasis Type="Italic">Salmonella enterica</Emphasis> Serovar Typhimurium Cells Under Starvation in Seawater

In seawater, enteric bacteria evolve toward a stressed state that is difficult to identify because of major alterations of their phenotype. In this study, we incubated four reference strains of S. enterica serovar Typhimurium in seawater microcosms for 10 months and studied the modifications of their main phenotypic characters. All of the strains lost some key characters used for traditional identification of the Salmonella genus. They became able to produce acetoin, and tryptophane deaminase activity became positive, but they lost the capacity to use rhamnose. We were able to show some modifications of the level of enzymatic profile as well as in their antibiotic susceptibility. The atypical cells of S. enterica serovar Typhimurium were identified by polymerase chain reaction (PCR) methods using the internal transcribed spacer region, and they were confirmed by multiplex PCR after the simultaneous amplification of the phoP, Hin, and H-li genes.     

Technological characteristics of yeast strains and their potential as starter adjuncts in Greek-style black olive fermentation

The technological properties of Debaryomyces hansenii (15 strains) and Torulaspora delbrueckii (32 strains) isolated from Greek-style black olives under conditions typical for black olive fermentation were studied. Furthermore, the killer character of the strains was assessed as well as their antimicrobial action against food-borne pathogens. All strains could grow at 15°C and low pH (2.5), whereas the majority of the strains were able to grow at 10% (w/v) NaCl, assimilated d-galacturonic acid, and showed lipolytic activity. Only 33% of D. hansenii and 9% of T. delbrueckii strains could hydrolyse 1% (w/v) oleuropein. A large majority of the strains tolerated 0.3% (w/v) bile salts, which in correlation with acid resistance indicates probiotic potential. Cross-reactions between culture filtrates of D. hansenii and T. delbrueckii and 56 yeast strains isolated during spontaneous Greek-style black olive fermentation were conducted. Focusing on their lytic activity, 17 mycogenic strains were selected. Culture filtrates of the mycogenic strains inhibited strains of L. monocytogenes, B. cereus and S. typhimurium. The active substance was heat resistant (stable after heating at 100°C for 10 min) as well as stable over a pH range from 4.0 to 6.5. The possible inhibition of undesirable yeast contaminants and food-borne pathogens in situ on fermented olives as well as the probiotic potential of strains used as starter adjuncts would contribute to the improvement of quality of the fermented product.     

Alkaline protease from <Emphasis Type="Italic">Bacillus sp.</Emphasis> isolated from coffee bean grown on cheese whey

Two strains of Bacillus, one from a culture collection (B. subtilis ATCC 6633) and a wild type (Bacillus sp. UFLA 817CF) isolated during coffee fermentation in the south of Minas Gerais, Brazil, were evaluated in relation to secretion of alkaline proteases. The strains were grown on nutrient broth, nutrient broth with sodium caseinate and nutrient broth with three different concentrations of cheese whey powder for 72 h. Samples were collected at 24-h intervals to evaluate the proteolytic activity, protein content and cell population. Maximum protease activity was observed after 24-h growth for both the microorganisms, a period that coincided with the end of the exponential phase. The specific activity values were, respectively, 839.8 U/mg for B. subtilis ATCC 6633 and 975.9 U/mg for Bacillus sp. UFLA 817CF. The 60% saturation presented the best results for specific protease activity in all the growth culture media tested with B. sp. UFLA 817CF. Bacillus sp. UFLA 817CF showed highest enzymatic activity at pH 9.0 and 40°C in the three culture media tested. The protease obtained from culture of the wild Bacillus strain presented stability at pH 7.0 and considerable heat stability at 40°C and 50°C, and could be an alternative for the industry to utilize cheese whey to produce proteolytic enzymes.     

Some technological properties of Penicillium roqueforti strains isolated from a home-made blue cheese

Nine strains of Penicillium roqueforti isolated from a traditional Spanish blue cheese (Valdeón cheese) along with two commercial strains were investigated for their ability to grow at different concentrations of salt and at different temperatures as well as for their proteolytic and lipolytic activities. Low concentrations of salt (1-3%) were stimulating for all the strains, with 1% salt being the concentration with the highest stimulating effect in nearly all. The rate of growth at 10°C was 2-3 times lower than at 25°C, the optimum temperature for the species. None of the strains, including the commercial cultures, showed proteolytic activity on casein agar, while all of them were lipolytic on tributyrin agar.     

Properties of bacteriocins of Morganella morganii

Some properties of Morganella morganii bacteriocins were determined. For this purpose two strains (115 and 137) which after mitomycin C induction produced bacteriocins in high titer were chosen. The influence of several physical and chemical factors such as: heating and storage at various temperatures, a freezing and thawing, an influence of buffered fluid of different pH, and digestion by trypsin, papain and lysozyme were investigated. Range of bacteriocin activity against various microorganisms and the ability to diffuse in agar were also determined. It was found on the basis of the results obtained that two bacteriocins showed different features. Bacteriocin "115" was thermostable, sensitive to proteolytic enzymes, able to diffuse in 1.5% agar. Bacteriocin designated "137" was thermolabile, intensive to proteolytic digestion, and incapable to diffuse in 1.5% agar. Activity of both bacteriocins was reduced after freezing and thawing. They were both insensitive to lysozyme digestion. Storage at room temperature reduced their activity faster than storage at the temperature of refrigerator. Their activity was completely stopped at pH 3.03, and significantly at pH 5.08 while environment of pH ranged from 7.08 to 11.0 did not influence their activity. Both bacteriocins showed narrow range of activity limited to the growth inhibition of sensitive strains belonging to Morganella morganii genus.     

Screening ofBifidobacterium strains for bacteriocin production

Summary Thirteen strains of bifidobacteria (Bifidobacterium) were examined for bacteriocin production. One strain among these produced bacteriocin which is active against lactic streptococci strains and against other species ofBifidobacterium, Clostridium andLactobacillus, but not against Gram-negative bacteria such asPseudomonas, Klebsiella, Serratia andEscherichia coli.Bacteriocin activity was inhibited by proteolytic enzymes, but not by heating 15 min or30 min at 100°C, and it was resistant for 15 min at 121°C and active at pH between 2 to 10.     

Proteolytic Activities of the Starch-Fermenting Ruminal Bacterium, Streptococcus bovis

The objective of this study was to characterize the extracellular proteolytic activity of Streptococcus bovis. Strains KEG, JB1, NCFB 2476, and K11.21.09.6C produced very similar large molecular weight (160–200 kDa) extracellular proteases that were specifically inhibited by PMSF, a serine protease inhibitor. Further experiments with S. bovis KEG indicated that cultures grown with casein as the sole added N source produced the greatest level of proteolytic activity, and the level of proteolytic activity was independent of growth rate. Clarified ruminal fluid (CRF) decreased proteolytic activity by 54% compared with cultures grown with casein alone, and addition of exogenous peptides and carbohydrates (CHO) to the CRF further reduced the level of proteolytic activity by 44% and 52%, respectively. These results suggested that the proteolytic activity of S. bovis KEG was modulated by available N source and that the proteolytic activity was present for reasons other than providing N for growth. The role of S. bovis in ruminal proteolysis requires further definition, but phenotypic similarity among some ruminal strains would suggest a common niche in ruminal proteolysis. The uniformity of proteolytic activities could make S. bovis a prime candidate for manipulation in ruminal proteolysis control strategies. Received: 12 January 1999 / Accepted: 19 May 1999