Modelling the growth of Weissella cibaria as a function of fermentation conditions

Aims:  To investigate the effect of pH, water activity ( a _w) and temperature on the growth of Weissella cibaria DBPZ1006, a lactic acid bacterium isolated from sourdoughs.
Methods and Results:  The kinetics of growth of W. cibaria DBPZ1006 was investigated during batch fermentations as a function of pH (4·0–8·0), a _w (0·935–0·994) and temperature (10–45°C) in a rich medium. The growth curve parameters (lag time, growth rate and asymptote) were estimated using the dynamic model of Baranyi and Roberts (1994. A dynamic approach to predicting bacterial growth in food. Int J Food Microbiol 23, 277–294). The effect of pH, a _w and temperature on maximum specific growth rate (μ_max) were estimated by fitting a cardinal model. μ_max under optimal conditions (pH = 6·6, a _w = 0·994, T  = 36·3°C) was estimated to be 0·93 h⁻¹. Minimum and maximum estimated pH and temperature for growth were 3·6 and 8·15, and 9·0°C and 47·8°C, respectively, while minimum a _w was 0·918 (equivalent to 12·2% w/v NaCl).
Conclusions:  Weissella cibaria DBPZ1006 is a fast-growing heterofermentative strain, which could be used in a mixed starter culture for making bread.
Significance and Impact of the Study:  This is the first study reporting the modelling of the growth of W. cibaria , a species that is increasingly being used as a starter in sourdough and vegetable fermentations.     

The characteristics of a new non-spore-forming cellulolytic mesophilic anaerobe strain CM126 isolated from municipal sewage sludge

A new mesophilic anaerobic cellulolytic bacterium, CM126, was isolated from an anaerobic sewage sludge digester. The organism was non-spore-forming, rod-shaped, Gram-negative and motile with peritrichous flagella. It fermented microcrystalline Avicel cellulose, xylan, Solka floc cellulose, filter paper, L-arabinose, D-xylose, β-methyl xyloside, D-glucose, cellobiose and xylitol and produced indole. The % G + C content was 36. Acetic acid, ethanol, lactic acid, pyruvic acid, carbon dioxide and hydrogen were produced as metabolic products. This strain could grow at 20–44·5°C and at pH values 5·2–7·4 with optimal growth at 37–41·5°C and pH 7. Both endoglucanase and xylanase were detected in the supernatant fluid of a culture grown on medium containing Avicel cellulose and cellobiose. Exoglucanase could not be found in either supernatant fluid or the cell lysate. When cellulose and cellobiose fermentation were compared, the enzyme production rate in cellobiose fermentation was higher than in cellulose fermentation. The optimum pH for both enzyme activities was 5·0, the optimum temperature was 40°C for the endoglucanase and 50°C for the xylanase. Both enzyme activities were inhibited at 70°C. Co-culture of this organism with a Methanosarcina sp. (A145) had no effect on cellulose degradation and both endoglucanase and xylanase were stable in the co-culture.     

Cultural conditions for the production of bacteriocin by a native isolate of Lactobacillus delbruecki ssp. bulgaricus CFR 2028 in milk medium

The effect of growth parameters and the molecular basis for antibacterial activity by a natural isolate of Lactobacillus delbruecki ssp. bulgaricus CFR 2028 was studied. The inhibition was tested against a toxigenic strain of Bacillus cereus F 4810. When grown in milk medium, the activity was highest at an incubation temperature of 37 °C in 48 h. The antibacterial activity appeared to be produced between late logarithmic and early stationary phases. The active principle was proteinaceous in nature (bacteriocin) and stable to low pH (3·8–5·0) and heat (75 °C for 30 min). There was also the possible role of hydrogen peroxide in bringing about inhibition. The strain of Lact. delbruecki ssp. bulgaricus CFR 2028 revealed the presence of plasmid DNA bands of 9·4 and 6·5 kbp, respectively, in agarose gel electrophoresis. The above strain has the potential to be used as a biopreservative in popular Indian fermented foods.     

Proteolytic activity from a thermophilic Streptomyces megasporus strain SDP4

Multiple proteases secreted by a thermophilic actinomycete Streptomyces megasporus SDP4 after 18 h of growth at 55 °C are reported. The enzyme preparation exhibited activity over a broad pH and temperature range of pH 6–12 and 25–85 °C, respectively. Optimum activity was observed at pH 8·0, pH 10·0 and 55 °C and was calcium independent. Thermostability was enhanced in the presence of 0·01 mol l⁻¹ calcium ions and half-life was 30 min at 85 °C. The enzyme was active in the presence of SDS. Both, EDTA and PMSF were partially inhibitory, indicating the presence of serine and metal requiring proteases. Three active zones in the range of 90–30 kDa were detected post-electrophoretically.     

Production and stability of pediocin N5p in grape juice medium

The production and stability of pediocin N5p from Pediococcus pentosaceus , isolated from wine, were examined in grape juice medium. Maximum growth and higher titre (4000 U ml^-1) were observed at a initial pH of 7·5 and 30°C. The activity of the inhibitory substance was stable between pH values from 2·0 to 5·0 at 4° and 30°C. At pH 10·0 it was completely inactivated. When submitted to 30 min at 80°, 100° and 115°C, maximal stability was observed at pH 2·0. Ethanol up to 10% did not affect pediocin activity at acid pH, nor did 40–80 mg 1^-1 SO₂, independently or combined with different ethanol concentrations, affect inhibitory activity.     

Purification and characterization of glucoamylase of Aspergillus terreus NA-170 mutant

A catabolite-derepressed mutant strain of Aspergillus terreus NA-170 was isolated by multi-step mutagenesis. The mutant produced an appreciable yield of glucoamylase in the culture medium. The enzyme was purified and was shown to be homogeneous. It hydrolysed successive glucose residues from the non-reducing end of starch molecules. The purified enzyme had an optimum pH of 5·0 and was stable over the pH range 3·0–7·0. It was highly active over a broad temperature range, 30–75°C, with optimal activity at 60°C. The molecular weight was 70 000 as determined by Sephadex G-200 filtration. The enzyme showed a decrease in K _m values with increasing chain length of the substrate molecule.     

Production, purification and characterization of an α-amylase produced by Saccharomycopsis fibuligera

Saccharomycopsis fibuligera ST 2 produced high levels of extracellular amylase during the stationary phase of growth. Glucose or other low molecular weight metabolizable sugars did not repress the synthesis of the amylase, indicating the lack of catabolite repression in this organism. Of the nitrogen sources examined, yeast extract and corn steep liquor stimulated the highest yield of amylase. Ammonium sulphate inhibited α-amylase synthesis. The enzyme was purified 118-fold from the culture supernatant fluid by isopropanol precipitation and DEAE-Sephadex A50 chromatography. The purified enzyme was characterized as an α-amylase. The α-amylase had the following properties: molecular weight, 40900 ± 500; optimum temperature, 60°C; activation energy, 1600 cal/mol; optimum pH, 4·8–6·0; range of pH stability, pH 4·0–9·4; K_m (50°C, pH 5·5) for soluble starch, 0·572 mg/ml; final products of starch hydrolysis—glucose, maltose, maltotriose and maltotetraose.     

The preparation of ampicillin dextrin agar for the enumeration of Aeromonas in water

Introduction of ampicillin dextrin agar (ADA) has revealed problems in details of the preparation. The final pH of the medium varied substantially between different laboratories. Measuring temperature has a pronounced effect on the pH (0·7 units lower at 50°C than at 6°C). Addition of agar during medium preparation resulted in a fall in pH of 0·5 units. If poured plates were stored in the refrigerator, the pH was reduced by 0·1–0·4 units, in particular during the first day. Recovery of Aeromonas from pure cultures and naturally polluted samples was unaffected by variation in pH between 7·1 and 8·3 but colony differentiation was optimal at a higher pH. The use of ADA at a final pH of 7·8 ± 0·2 (at 25°C) is recommended. Different types of dextrin differed in respect of solubility, fermentability and colony differentiation. Optimal results were obtained with Difco 161 and Merck 3006.     

New Strains of Bacillus licheniformis and Bacillus coagulans producing Thermostable α-Amylase Active at Alkaline pH

Two species of Bacillus producing thermostable α-amylase with activity optima at alkaline pH are reported here. These organisms were isolated from soil and have been designated as Bacillus licheniformis CUMC 305 and B. coagulans CUMC 512. The enzymes released by these two species were partially purified up to about 81- and 72-fold respectively of the initial activity. The enzyme from B. licheniformis showed a wide temperature-range of activity, with optimum at 91°C. At this temperature it remained stable for 1 h. It retained 40–50% activity at 110°C and showed only 60% of its activity at 30°C. The enzyme showed a broad pH range of activity (4–10) retaining substantial activity on the alkaline side. The optimum pH was 9·5. The enzyme of B. coagulans showed activity up to 90°C, with optimum at 85°C and had a wide pH range with optimum at 7·5–8·5. The hydrolysis pattern of the substrate starch by these enzymes indicated that glucose, maltose, maltotriose and maltotetraose are the principal products rather than higher oligosaccharides.     

Effect of pH and NaCl on growth from spores of non-proteolytic Clostridium botulinum at chill temperature

The effect of combinations of temperature (2°, 3°, 4°, 5°, 8° and 10°C), pH (5·0–7·2) and NaCl (0·1–5·0% w/w) on growth from spores of non-proteolytic Clostridium botulinum types B, E and F was determined using a strictly anaerobic medium. Inoculated media were observed weekly for turbidity, and tests were made for the presence of toxin in conditions that approached the limits of growth. Growth and toxin production were detected at 3°C in 5 weeks, at 4°C in 3/4 weeks and at 5°C in 2/3 weeks. The resulting data define growth/no growth boundaries with respect to low temperature, pH, NaCl and incubation time. This is important in assessment of the risk of growth and toxin production by non-proteolytic Cl. botulinum in minimally processed chilled foods.     

Production of β-glucosidase (cellobiase) by Curvularia sp.

A Curvularia sp. isolated from soil was found to produce extracellular β-glucosidase activity when grown in yeast extract, peptone, carboxymethylcellulose (YPC) medium. An initial medium pH of 6·5 and cultivation temperature of 30°C were found to be most suitable for high enzyme productivity. The pH and temperature optima for the enzyme were 4·0 and 70°C, respectively. Under these conditions, the enzyme exhibited a K_m (0-nitrophenyl-β- d -glucoside) value of 0.20 mmol/l. Several divalent metal ions inhibited enzyme activity at high concentration. EDTA. also inhibited β-glucosidase activity.     

Influence of culture conditions on exopolysaccharide production by Lactobacillus rhamnosus strain C83

The influence of culture conditions on growth and exopolysaccharide (EPS) production by Lactobacillus rhamnosus strain C83 was investigated using fermentor batch cultures. A temperature shift (from 37 to 25 °C) at the beginning of the exponential growth phase (0–5 h) enhanced EPS production. Furthermore, an optimal environmental pH of 6·2–7·2 improved the performance of strain C83 and partially anaerobic culture conditions (pO₂ from 0 to 10%) triggered EPS over-production by the cells. The sugar composition of this polymer was independent of culture conditions, such as carbon source, medium composition, temperature, pH, pO₂ and growth phases. In all cases, galactose and glucose were the principal components.     

Production of alkaline xylanase by an alkaliphilic Bacillus sp. isolated from an alkalinesoda lake

An alkaline xylanase-producing alkaliphilic Bacillus sp. AR-009 was isolated from analkaline soda lake in Ethiopia. The enzyme was optimally active at pH 9 and was stable over abroad pH range. The optimum temperature for xylanase activity, assayed at pH 9, was60°–65°C. Measured at pH 8 and 9, the enzyme had good stability at 55° and60°C. At both pH values, over 80% of its original activity was retained after heating for2·5 h at 55°C. At 60°C, the enzyme maintained 63% of its original activity after2·5 h incubation while at pH 9 it retained 54% of its original activity after 1 h heating. Theseproperties qualify the enzyme to be novel and potentially important for application in someindustrial processes.     

Isolation and growth physiology of novel thermoactinomycetes

A total of 34 thermophilic isolates identified as members of the genus Thermoactinomyces by a range of chemotaxonomic, microscopic and determinativebiochemical tests, were isolated from two acid soils. Growth studies in shake flask and fermenteridentified the isolates to be moderately acidophilic with growth occurring between pH4·5 and 6·0 with optima at pH 5·0. The isolates differed considerablyfrom known Thermoactinomyces cultures in their pH profile, colony morphology andin several biochemical tests.Extracellular enzyme activities are identified and partiallycharacterized in termsof their thermostability, pH and temperature profiles from crude supernatantfluid samples. Optimal protease, amylase and pullulanase activity was observed at pH5·0–5·5 and 75–80 °C with each showing T ₍₅₀₎ values of 10, 30 and 30 min, respectively. A highly thermotolerant extracellularesterase was also identified which retained 50% activity after 8 h at 90°C . This is the firstreport of an acidophilic member of the genus Thermoactinomyces.     

Factors affecting the production of hydrogen sulphide by Lactobacillus sake L13 growing on vacuum-packaged beef

Lactobacillus sake L13 produced hydrogen sulphide during growth at 0°C on vacuum-packaged beef of normal pH (5·6–5·8) when the packaging films used had oxygen permeabilities as high as 200 ml/m²/24 h/atm (measured at 25°C and 98% relative humidity. No hydrogen sulphide was detected when the film permeability was 300 ml/m²/24 h/atm. Sulphmyoglobin was formed whenever hydrogen sulphide was present except when the film permeability was very low (1 ml of oxygen/m²/24 h/atm). Lactobacillus sake L13 also produced hydrogen sulphide when grown on beef under anaerobic conditions at 5°C. When meat pH was high (6·4–6·6) hydrogen sulphide was first detected after incubation for 9 d. When 250 μg of glucose was added to each g of high pH meat, or when meat pH was normal (5·6–5·8), hydrogen sulphide was first detected after incubation for 18 d. The spoilage of beef by hydrogen sulphide-producing lactobacilli is more rapid when the pH of the meat is high because high-pH meat contains less glucose. Sulphmyoglobin formation and greening can be prevented by the use of packaging films of very low oxygen permeability.     

Comparison of the killer toxin of several yeasts and the purification of a toxin of type K2

A total of 13 killer toxin producing strains belonging to the genera Saccharomyces, Candida and Pichia were tested against each other and against a sensitive yeast strain. Based on the activity of the toxins 4 different toxins of Saccharomyces cerevisiae, 2 different toxins of Pichia and one toxin of Candida were recognized. The culture filtrate of Pichia and Candida showed a much smaller activity than the strains of Saccharomyces. Extracellular killer toxins of 3 types of Saccharomyces were concentrated and partially purified. The pH optimum and the isoelectric point were determined. The killer toxins of S. cerevisiae strain NCYC 738, strain 399 and strain 28 were glycoproteins and had a molecular weight of M_r=16,000. The amino acid composition of the toxin type K₂ of S. cerevisiae strain 399 was determined and compared with the composition of two other toxins.     

Keratinolytic activity from the broth of a feather-degrading thermophilic Streptomyces thermoviolaceus strain SD8

Keratinolytic activity was detected in the culture broth of feather-degrading thermophilic Streptomyces thermoviolaceus SD8. The crude enzyme was concentrated by precipitation with 80% saturation of ammonium sulphate and desalted by SephadexG-10–120 gel chromatography followed by lyophilization. The specific activity of the enzyme was enhanced 50-fold. PAGE analysis indicated a monomeric form with a molecular weight of 40 kDa. The optimum pH and temperature for production of the enzyme were 8 and 55 °C, respectively. The enzyme was stable at a pH range of 6·5–8·5 and up to 65 °C. The enzyme could hydrolyse fibrin, muscle, collagen, nail and hair and could produce leucine, threonine and tyrosine from feather. It could be a useful enzyme for waste treatment by promoting hydrolysis of the above substances in the sewage, or it could be used for animal feed preparation.     

The combined effect of sub-optimal temperature and sub-optimal pH on growth and toxin formation from spores of Clostridium botulinum

Low-acid foods (pH ≥ 4.5) are not sufficiently acidic to prevent growth of Clostridium botulinum in otherwise optimal conditions. The combination of sub-optimal pH and sub-optimal temperature may, however, result in a very significant reduction in the risk of growth of this bacterium compared with the risk in optimal conditions. The combined effect of incubation temperatures of 12° and 16°C and pH values between 5·2 and 5·5 on growth and toxin production from spores of Cl. botulinum during incubation for 28 d has been investigated. Growth and formation of toxin (type B) were detected only in medium at pH 5·5 and incubated at 16°C, corresponding to a probability of growth from a single spore within 14 d of 1·6 × 10^-5. The probability of growth in 28 d in the remaining conditions was <9 × 10^-6. After transfer of inoculated media from 12° to 30°C growth occurred at pH 5·2–5·5 within 19 d. After transfer of inoculated media from 12° to 20°C growth occurred at pH 5·5 and 5·4 but not at pH 5·3 or 5·2 in 40 d. Growth at pH 5·2–5·5 was accompanied by formation of toxin, in most cases of types A or B. In addition to the effect of sub-optimal temperature and pH, chelation of divalent metal ions by citrate may have contributed to inhibition.     

Bioemulsifier production by Bacillus stearothermophilus VR-8 isolate

Bacillus stearothermophilus produced an extracellular bioemulsifier during growth in a medium containing 4% crude oil. Over the temperature range of 45° to 70°C, maximum recovery (0·6 g 1^-1) occurred at 50°C. The emulsifier had its greatest activity on benzene, among the hydrocarbons tested. Acetone precipitated, dialysed emulsifier contained 46% protein, 16% carbohydrate and 10% lipid. The emulsification activity was stable over a broad range of temperature (50–80°C), pH (2–8) and salt concentration (5% NaCl, 5% CaCl₂ and 1% MgCl₂). Thus, this emulsifier was found to be better than liposan (showing emulsifying activity between pH 2–5 and stable up to 70°C) in terms of pH and temperature stability. Additionally, it was also salt tolerant, suggesting its potential use in crude oil tank clean-up and enhanced oil recovery.     

Effects of essential oil from mint (Mentha piperita) on Salmonella enteritidis and Listeria monocytogenes in model food systems at 4° and 10°C

The effect of mint ( Mentha piperita ) essential oil (0·5, 1·0, 1·5 and 2·0%, v/w) on Salmonella enteritidis and Listeria monocytogenes in a culture medium and three model foods; tzatziki (pH 4·5), taramosalata (pH 5·0) and pâté (pH 6·8), inoculated at 10⁷ cfu g^-1, at 4° and 10°C for ca 1 week was studied. In the culture medium supplemented with the essential oil, no growth was observed over 2 d at 30°C determined by a conductance method with a Malthus 2000 growth analyser. Salmonella enteritidis died in tzatziki in all treatments and declined in the other foods except for pâté at 10°C as judged with viable counts. Listeria monocytogenes populations showed a declining trend towards the end of the storage period but was increased in pâté. Mint essential oil antibacterial action depended mainly on its concentration, food pH, composition, storage temperature and the nature of the micro-organism.