Large scale purification protocol for carnocin KZ 213 from Carnobacterium piscicola

Large scale purification of a class IIa bacteriocin has been developed to recover pure carnocin KZ 213 produced by Carnobacterium piscicola 213. Most previous protocols reported in the literature for the purification of small peptides have used reversed phase chromatography but scale-up is difficult. The first step of this new protocol is hydrophobic interaction chromatography, the second and third steps are cation exchange chromatography. The protocol leads to a complete recovery of carnocin KZ 213 with 95% purity and to a concentration factor of 83. From 10 l culture supernatant, 5.8 mg carnocin KZ 213 have been produced with a specific activity of 8,500 UA g⁻¹. The protocol is easy to implement for larger volumes.     

Properties of a bacteriocin produced by Carnobacterium piscicola CP5

Summary Carnobacterium piscicola CP5 produced a bacteriocin named carnocin CP5 that inhibited Carnobacterium, Enterococcus and Listeria spp. and among the Lactobacillus spp. only Lactobacillus delbrueckii ssp. Carnocin CP5 was stable 1h at 100°C at pH 7.0. It was inactivated by numerous proteolytic enzymes. Production of carnocin, CP5 occured in MRS broth regulated at pH 7.0. The apparent molecular weight of the bacteriocin in the crude extract was greater than 10 kDa, but around 5 kDa after action of SDS or urea. Novobiocin treatment led to non-producer variants.     

Influence of pH,temperature and culture media on the growth and bacteriocin production by vaginal Lactobacillus salivarius CRL 1328

AIMS: To study the influence of pH, temperature and culture medium on the growth and bacteriocin production by vaginal Lactobacillus salivarius subsp. salivarius CRL 1328. METHODS AND RESULTS: The study was performed using a complete factorial experimental design. Lactobacillus salivarius was cultivated in LAPTg and MRS broths, adjusted to specific initial pH, and at different temperatures of incubation. The growth, which was evaluated by the Gompertz model, was higher in MRS broth than in LAPTg broth. The initial pH of the culture medium and the temperature had a dramatic effect on the production of bacteriocin. The optimal conditions for bacteriocin production were different to those for optimal growth. The decrease in the pH of the culture medium was parallel to the growth; pH had similar final values in both the MRS and the LAPTg broths. CONCLUSIONS: The optimal growth conditions were recorded in MRS broth, with an initial pH of 6.5 and a temperature of 37 degrees C. The maximum bacteriocin activity was obtained in LAPTg after 6 h at 37 degrees C, and at an initial pH of 6.5 or 8.0. SIGNIFICANCE AND IMPACT OF THE STUDY: The application of a complete factorial design, and the evaluation of the growth parameters through the Gompertz model, enabled a rapid and simultaneous exploration of the influence of pH, temperature and growth medium on both growth and bacteriocin production by vaginal Lact. salivarius CRL 1328.     

Industrial Whey Utilization as a Medium Supplement for Biphasic Growth and Bacteriocin Production by Probiotic Lactobacillus casei LA-1

The ability of probiotic Lactobacillus casei LA-1 for bacteriocin production using industrial by-products, such as whey, as supplement in growth medium has been demonstrated for the first time. Whey was investigated as a sole carbon source in cooperation with other components to substitute expensive nutrients as MRS for economical bacteriocin production. Industrial whey-supplemented MRS medium was then selected as to determine the effect of four variables (temperature, initial pH, incubation time, and whey concentration) by response surface methodology on bacteriocin production. Statistical analysis of results showed that two variables have a significant effect on bacteriocin production. Response surface data showed maximum bacteriocin production of 6,132.33?AU/mL at an initial pH of 7.12, temperature 34.29?°C, and whey concentration 13.74?g/L. The production of bacteriocin started during the exponential growth phase, reaching maximum values at stationary phase, and a biphasic growth and production pattern was observed. Our current work demonstrates that this approach of utilization of whey as substitution in costly medium as MRS has great promise for cost reduction in industry for the production of novel biological metabolic product that can be utilized as a food preservative.     

Detection and partial characterization of a bacteriocin produced by Carnobacterium piscicola 213

BLIS 213, is a bacteriocin-like inhibitory substance produced by Carnobacterium piscicola 213. It is active against Carnobacterium, Enterococcus and Listeria spp. No activity was observed against tested Lactobacillus, Lactococcus, Leuconostoc and Pediococcus strains, nor against Gram-negative bacteria. The BLIS 213 activity was inactivated by several proteolytic enzymes. It was heat resistant (121°C for 20 min), and stable over a pH range of 2–8. Activity was determined by a dilution micromethod; it was increased after SDS treatment. A mutant strain which lacks bacteriocin production was isolated and designated as Carnobacterium piscicola 213a. It had the same phenotypic and biochemical properties as the parent strain, and was not sensitive to bacteriocin activity. The apparent molecular weight of the bacteriocin in the crude extract was greater than 10 kDa. It was about 6 kDa after SDS-PAGE of a partially purified bacteriocin by adsorption on producer cells. The isoelectric point of the BLIS 213 was around 9.3. Received 21 January 1997/ Accepted in revised form 25 April 1997     

Influence of media and temperature on bacteriocin production by<Emphasis Type="Italic"> Bacillus cereus</Emphasis> 8A during batch cultivation

Cerein 8A is a bacteriocin produced by the soil bacterium Bacillus cereus 8A, isolated from native woodlands of Brazil. The influence of temperature and media on the growth of B. cereus 8A and the production of this bacteriocin was studied during batch cultivation. Maximum activity was detected by cultivation in brain/heart infusion broth, reaching 3200 activity units ml^–1. Bacteriocin was also produced in peptone, MRS, Mueller–Hinton and nutrient broth, while no activity was observed during cultivation in thioglycollate or tryptic soy broth. Temperature had a strong influence on bacteriocin production, which was higher at 30 °C than at 25 °C. An important decrease in bacteriocin activity was observed at 37 °C. The relationship between growth and specific production rates, as a function of the temperature, showed different kinetics of production and there were several peaks in the specific production rates during growth. Bacteriocin was produced at the stationary phase, indicating it is synthesized as a secondary metabolite.     

Optimization of culture conditions and medium composition for the production of micrococcin GO5 by Micrococcus sp. GO5

To enhance the production of micrococcin GO5, a bacteriocin produced by Micrococcus sp. GO5, cultivation conditions and medium composition were optimized. The optimal initial pH and temperature for bacteriocin production were 7.0-9.0 and 37 degrees C, respectively. Micrococcus sp. GO5 displayed the highest micrococcin GO5 activity when grown in modified MRS medium that contained lactose or sucrose, rather than glucose, as a carbon source. The maximum bacteriocin activity was obtained in modified MRS medium containing 0.5% tryptone and 1.0% yeast extract as nitrogen sources instead of the other nitrogen sources present in MRS medium. Bacteriocin production was greatly affected by the concentration of K(2)HPO(4); strain GO5 produced eight-fold more bacteriocin in medium containing 2.0-2.5% K(2)HPO(4) than in medium containing 0.2% K(2)HPO(4). The optimal concentration of MgSO(4).7H(2)O for bacteriocin production was 0.5%. The production of micrococcin GO5 was increased 32-fold in shake flask culture and 16-fold in a bioreactor using the optimized medium (TY medium), compared with culturing in MRS medium.     

Effect of medium components on bacteriocin production by Lactobacillus plantarum strains ST23LD and ST341LD, isolated from spoiled olive brine

Bacteriocin ST23LD levels of 2930AU/OD were recorded in MRS broth (pH of 6.5) and in the presence of tryptone and yeast extract as sole nitrogen sources. Growth in MRS broth at an initial pH of 6.0 yielded only 1460AU/OD bacteriocin ST23LD. Activities of 5861AU/OD were recorded with maltose (20, 30 and 40 g/l) as sole carbon source and 9036AU/OD with the addition of 2.0-10.0 g/l KH2PO4. Bacteriocin ST341LD levels of 2850 and 2841AU/OD were recorded in MRS broth at an initial pH of 6.0 or 5.5, respectively. Only 709AU/OD was recorded in the same medium with an initial pH of 6.5. Bacteriocin ST341LD production was stimulated by the presence of tryptone. However, glucose at 10 and 40 g/l, or the presence of 5.0 or 10.0 g/l K2HPO4, resulted in a 50% reduction of bacteriocin activity. Glycerol in the growth medium repressed bacteriocin production. No increased bacteriocin production was recorded in medium supplemented with vitamins.     

Influence of Environmental Factors on Bacteriocin Production by Human Isolates of <Emphasis Type="Italic">Lactococcus lactis</Emphasis> MM19 and <Emphasis Type="Italic">Pediococcus acidilactici</Emphasis> MM33

The influence of temperature, initial pH, and carbon and nitrogen sources on bacteriocin secreted by Lactococcus lactis MM19 (MM19) and Pediococcus acidilactici MM33 (MM33) was evaluated. It was found that 30 and 45 °C were the growth temperatures for higher nisin and pediocin production by MM19 and MM33, respectively. The initial pH values for higher production of nisin and pediocin were 9 and 6, respectively. Glucose and wheat peptone E430 were found as suitable carbon and nitrogen sources, respectively, for highest nisin production by MM19 at 30 °C and initial pH of 9. In these conditions, nisin production could be increased by 6.7 times as compared to the control medium (de Man, Rogosa, and Sharpe—MRS broth). Similarly, fructose and pea peptone were suitable carbon and nitrogen sources, respectively, for highest production of pediocin by MM33 at 45 °C and initial pH of 6. In these conditions, pediocin production by MM33 was increased by three times as compared to the control medium (tryptone-glucose-yeast extract—TGE broth).     

Effect of physical factors on the production of bacteriocin from Pediococcus acidilactici ITV 26

The effect of pH, temperature and agitation on growth and bacteriocin production by Pediococcus acidilactici ITV 126 was investigated. Experiments were made in flasks containing MRS medium at 30 to 40°C, pH 5 to 7 and agitation 0 to 200 rpm. Factor levels were arranged in a 2³ factorial design with central and axial points. Anova and Tukey paired comparison tests showed that a temperature of 35°C favored bacteriocin production, whereas 40°C was best for cell growth. A statistical interaction of temperature and agitation was observed affecting microbial growth. pH 5 favored both cell growth and bacteriocin production. Journal of Industrial Microbiology & Biotechnology (2001) 26, 191–195. Received 30 October 1999/ Accepted in revised form 31 January 2001     

Growth optimization of Pediococcus damnosus NCFB 1832 and the influence of pH and nutrients on the production of pediocin PD-1

AIMS: Optimization of the growth of Pediococcus damnosus NCFB 1832 and the production of pediocin PD-1 by traditional fermentation methods. METHODS AND RESULTS: Fermentation studies were conducted in De Man Rogosa and Sharpe (MRS) broth (Oxoid), preadjusted to specific pH values, and in MRS broth supplemented with various nitrogen sources, MnSO4, MgSO4 and Tween 80. The production of pediocin PD-1 closely followed the growth curve of Ped. damnosus NCFB 1832. Maximum levels of bacteriocin activity (3249 AU ml(-1)/O.D.max) were recorded in MRS broth with an initial pH of 6.7. In media with an initial pH of 4.5 bacteriocin activity as low as 222 AU ml(-1)/O.D.max was recorded. The highest bacteriocin activity was recorded in growth conditions allowing the greatest pH variation (highest DeltapH). The addition of bacteriological peptone (1.7%, w/v), MnSO4 (0.014%, w/v) and Tween 80 (3%, v/v) to MRS and adjustment of the medium pH to 6.7 resulted in a further increase in activity (from 3249 to 5078 AU ml(-1)/O.D.max). The same medium, but with an initial pH of 6.2, resulted in an 82.5% decrease in bacteriocin activity. CONCLUSIONS: Pediocin PD-1 production is not only stimulated by the presence of specific growth factors (e.g., bacteriological peptone, MnSO4 or Tween 80), but may also be stimulated by the lowering in pH during growth (highest DeltapH), and thus also the amount of organic acids produced. SIGNIFICANCE AND IMPACT OF THE STUDY: The production of pediocin PD-1 by the wild-type producer strain was significantly improved by using a defined medium and traditional fermentation methods.     

Identification and production of a bacteriocin from Enterococcus mundtii QU 2 isolated from soybean

AIMS: Identification of the bacteriocin produced by Enterococcus mundtii QU 2 newly isolated from soybean and fermentative production of the bacteriocin. METHODS AND RESULTS: The bacteriocin produced by Ent. mundtii QU 2 inhibited the growth of various indicator strains, including Enterococcus, Lactobacillus, Leuconostoc, Pediococcus and Listeria. The bacteriocin activity was stable at wide pH range and against heat treatment, but completely abolished by proteolytic enzymes. The bacteriocin was purified from the culture supernatant by the three-step chromatographic procedure. Mass spectrometry, amino acid sequencing and DNA sequencing revealed that the bacteriocin was similar to class IIa bacteriocins produced by other Ent. mundtii strains. The bacteriocin production decreased in the absence of glucose, nitrogen sources, or Tween 80 in MRS medium. Additionally, it was strongly suppressed by addition of Ca(2+) (CaCO(3) or CaCl(2)). In pH-controlled fermentations, the highest bacteriocin production was achieved at pH 6.0, whereas the highest cell growth was obtained at pH 7.0. CONCLUSIONS: Ent. mundtii QU 2 produced a class IIa bacteriocin. Some growth factors (e.g. Ca(2+) and pH) influenced the bacteriocin production. SIGNIFICANCE AND IMPACT OF THE STUDY: A new soybean isolate, Ent. mundtii QU 2 was found to be a class IIa bacteriocin producer. Factors influencing the bacteriocin production described herein are valuable for applications of the bacteriocins from Ent. mundtii strains.     

Influence of nutrients on growth and bacteriocin production by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442

The aim of this study was to investigate the effect of complex nutrients on microbial growth and bacteriocin production, in order to improve bacteriocin synthesis during the growth cycle of Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442. The fermentations were conducted at the optimum pH and temperature for bacteriocin production (pH 5.5+/-0.1 and temperature 25+/-0.1 degrees C). Because of their association with the final biomass, conditions favouring the increase of the produced biomass resulted in the increase of bacteriocin activity in the growth medium. Since the produced final biomass and the final concentration of the bacteriocins were associated with the amount of the carbon (glucose) and nitrogen source, better growth of the lactic acid bacterial strains favoured the increase of the specific bacteriocin production. Additionally, the bacteriocin production was influenced by carbon/nitrogen ratio.     

Thermoactive extracellular proteases of <Emphasis Type="Italic">Geobacillus caldoproteolyticus</Emphasis>, sp. nov., from sewage sludge

A proteolytic thermophilic bacterial strain, designated as strain SF03, was isolated from sewage sludge in Singapore. Strain SF03 is a strictly aerobic, Gram stain-positive, catalase-positive, oxidase-positive, and endospore-forming rod. It grows at temperatures ranging from 35 to 65°C, pH ranging from 6.0 to 9.0, and salinities ranging from 0 to 2.5%. Phylogenetic analyses revealed that strain SF03 was most similar to Saccharococcus thermophilus, Geobacillus caldoxylosilyticus, and G. thermoglucosidasius, with 16S rRNA gene sequence identities of 97.6, 97.5 and 97.2%, respectively. Based on taxonomic and 16S rRNA analyses, strain SF03 was named G. caldoproteolyticus sp. nov. Production of extracellular protease from strain SF03 was observed on a basal peptone medium supplemented with different carbon and nitrogen sources. Protease production was repressed by glucose, lactose, and casamino acids but was enhanced by sucrose and NH₄Cl. The cell growth and protease production were significantly improved when strain SF03 was cultivated on a 10% skim-milk culture medium, suggesting that the presence of protein induced the synthesis of protease. The protease produced by strain SF03 remained active over a pH range of 6.0–11.0 and a temperature range of 40–90°C, with an optimal pH of 8.0–9.0 and an optimal temperature of 70–80°C, respectively. The protease was stable over the temperature range of 40–70°C and retained 57 and 38% of its activity at 80 and 90°C, respectively, after 1 h.     

Characterisation of the bacteriocins produced by two probiotic Lactobacillus isolates from idli batter

Lactobacillus plantarum JJ18 and Lactobacillus plantarum subsp. plantarum JJ60, probiotics from idli batter, produce bacteriocins JJ18 and JJ60 having a wide spectrum of activity. After optimising the environmental conditions for bacteriocin production the effect of various media components was determined. Maximum bacteriocin production was observed in MRS broth, pH 6.4 at 37 °C after 36 h. Tryptone (as nitrogen source) and glucose (as carbon source) are required for optimal production of bacteriocins JJ18 and JJ60. Activity was not affected by surfactants like Triton X-100, Tween 80 and Tween 20 or by treatment with NaCl, urea and EDTA. Protease treatment resulted in complete loss of activity of the partially purified bacteriocins JJ18 and JJ60, while lipase and α-amylase had no effect, indicating that the bacteriocin is a simple protein. Tris tricine SDS-PAGE electrophoresis depicted a single band of less than 3.5 kDa. However, the strain Lactobacillus plantarum JJ18 was inhibited by bacteriocin JJ60 and Lactobacillus plantarum JJ60 by bacteriocin JJ18, whereas no inhibition was observed against the respective producer strains, indicating that the two bacteriocins are different. The bacteriocins remained active over a wide range of pH and temperature. The bacteriocins were able to adsorb onto producer and target cells, Lactobacillus plantarum and Listeria monocytogenes and differentially in the presence of various surfactants, salts and solvents. A bactericidal mode of action was observed against Listeria monocytogenes. Given their wide spectrum of activity against various pathogens, the bacteriocins JJ18 and JJ60 can be applied as bio-preservatives in the food industry.     

Improvement of Bacillus thuringiensis Bacteriocin Production Through Culture Conditions Optimization

Abstract

BUPM4 is a Bacillus thuringiensis subsp. kurstaki strain, isolated from Tunisian soil, producing an original bacteriocin named Bacthuricin F4. The optimization of the latter production conditions was carried out under several physicochemical conditions. It was found that the highest bacteriocin activity was reached at low aeration while bacteriocin synthesis yields were strongly reduced at higher ones. A balance between growth and bacteriocin synthesis, both highly dependent on aeration, was taken into account for the overproduction of bacteriocin. Both glucose and glycerol were shown to be necessary for Bacthuricin F4 maximal synthesis. In addition, the optimal carbon/nitrogen ratio for bacteriocin production is 9. In such optimal conditions, more than 4-fold greater bacteriocin production was obtained than when using TSB medium.     

Studies on cellulase production by a Bacillus subtilis

Bacillus subtilis AU-1 was found to produce carboxymethylcellulase (CMCase) and Avicelase activities in the culture supernatant when grown on a variety of carbohydrates as major carbon source. Maximum CMCase production was obtained in a liquid medium containing 0.2% D (+) raffinose as inducer, 0.5% each of yeast extract, casamino acids and proteose peptone at 50 °C and at an initial pH of 6.0. CMCase activity was detected at early log phase of growth, and reached the maximum level at early stationary phase of growth which occurred at the 10th hour of cultivation. The optimal temperature for CMCase activity was 65 °C, and the enzyme was highly stable up to 60 °C. CMCase synthesis was subjected to catabolite repression by glucose and cellobiose.     

Temperature and pH conditions that prevail during fermentation of sausages are optimal for production of the antilisterial bacteriocin sakacin K

Sakacin K is an antilisterial bacteriocin produced by Lactobacillus sake CTC 494, a strain isolated from Spanish dry fermented sausages. The biokinetics of cell growth and bacteriocin production of L. sake CTC 494 in vitro during laboratory fermentations were investigated by making use of MRS broth. The data obtained from the fermentations was used to set up a predictive model to describe the influence of the physical factors temperature and pH on microbial behavior. The model was validated successfully for all components. However, the specific bacteriocin production rate seemed to have an upper limit. Both cell growth and bacteriocin activity were very much influenced by changes in temperature and pH. The production of biomass was closely related to bacteriocin activity, indicating primary metabolite kinetics, but was not the only factor of importance. Acidity dramatically influenced both the production and the inactivation of sakacin K; the optimal pH for cell growth did not correspond to the pH for maximal sakacin K activity. Furthermore, cells grew well at 35 degrees C but no bacteriocin production could be detected at this temperature. L. sake CTC 494 shows special promise for implementation as a novel bacteriocin-producing sausage starter culture with antilisterial properties, considering the fact that the temperature and acidity conditions that prevail during the fermentation process of dry fermented sausages are optimal for the production of sakacin K.     

Influence of pH on the production of enterocin 1146 during batch fermentation

The production of enterocin 1146, a bacteriocin from Enterococcus faecium DPC1146, was studied during batch fermentation at pH 5, 5.5, 6 and 6.5. The bacteriocin was produced throughout the growth of the micro-organism, showing primary metabolite kinetics. Bacteriocin production stopped at the end of growth and was followed by a decrease in activity due primarily to adsorption on the cells of the producer. The optimal pH for enterocin 1146 production was 5.5, because of higher bacteriocin yield per unit of biomass and slower adsorption/degradation, while optimal pH for growth was between 6.0 and 6.5.     

Optimization of enterocin P production by batch fermentation of Enterococcus faecium P13 at constant pH

The influence of pH on growth, enterocin P production and glucose consumption by Enterococcus faecium P13 was studied during anaerobic batch fermentation in MRS broth at 32 degrees C in a fermentor. Growth and glucose consumption were maximal at pH 7.0. Enterocin P production displayed primary metabolite kinetics and was strongly dependent on pH. A maximum antimicrobial activity of 1,949 bacteriocin units (BU) ml(-1) was obtained at pH 6.0, which represented a four-fold increase compared with the antimicrobial activity obtained without pH regulation. The pH exerted a marked effect on the decrease in bacteriocin activity, with the decrease being maximal at pH 7.0. In this report, we propose models for the growth of E. faecium P13 as well as enterocin P production and inactivation. Enterocin P production decreased when potentially stress-inducing compounds (NaCl or ethanol) were included in the growth medium.