Influence of complex nutrient source on growth of and curvacin a production by sausage isolate Lactobacillus curvatus LTH 1174

Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of a complex nutrient source were investigated in vitro during laboratory fermentations with modified MRS medium. A modification of the nutrient depletion model was used to fit the data describing growth and bacteriocin production. Both cell growth and bacteriocin activity were influenced by changes in the complex nutrient source concentration. Standard MRS medium clearly limited the growth of L. curvatus LTH 1174. Higher nutrient concentrations, up to a certain degree, led to improved growth, a higher attainable biomass concentration, and a higher bacteriocin activity in the supernatant. A lower concentration of complex nutrient source caused severe growth inhibition, leading to a lower biomass concentration but a much higher specific bacteriocin production. When examining the separate components of the complex nutrient source, a stimulating effect of bacteriological peptone on growth was found without an adverse effect on bacteriocin production, resulting in increased curvacin A activity. Furthermore, specific depletion of the amino acids tyrosine, serine, and asparagine/aspartic acid was observed for this strain.     

Effects of different spices used in production of fermented sausages on growth of and curvacin A production by Lactobacillus curvatus LTH 1174

Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the listericidal bacteriocin curvacin A. The effect of different spices relevant for the production of fermented sausages was investigated in vitro through laboratory fermentations with a meat simulation medium and an imposed pH profile relevant for Belgian-type fermented sausages. The influence on the growth characteristics and especially on the kinetics of curvacin A production with L. curvatus LTH 1174 was evaluated. Pepper, nutmeg, rosemary, mace, and garlic all decreased the maximum specific growth rate, while paprika was the only spice that increased it. The effect on the lag phase was minor except for nutmeg and especially for garlic, which increased it, yet garlic was stimulatory for biomass production. The maximum attainable biomass concentration (X(max)) was severely decreased by the addition of 0.40% (wt/vol) nutmeg, while 0.35% (wt/vol) garlic or 0.80% (wt/vol) white pepper increased X(max). Nutmeg decreased both growth and bacteriocin production considerably. Garlic was the only spice enhancing specific bacteriocin production, resulting in higher bacteriocin activity in the cell-free culture supernatant. Finally, lactic acid production was stimulated by the addition of pepper, and this was not due to the manganese present because an amount of manganese that was not growth limiting was added to the growth medium. Addition of spices to the sausage mixture is clearly a factor that will influence the effectiveness of bacteriocinogenic starter cultures in fermented-sausage manufacturing.     

Sodium Chloride Reduces Production of Curvacin A, a Bacteriocin Produced by Lactobacillus curvatus Strain LTH 1174, Originating from Fermented Sausage

Lactobacillus curvatus LTH 1174, a strain originating in fermented sausage, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of salt (sodium chloride) were investigated in vitro during laboratory fermentations using modified MRS medium. A model was set up to describe the effects of different NaCl concentrations on microbial behavior. Both cell growth and bacteriocin activity were affected by changes in the salt concentration. Sodium chloride clearly slowed down the growth of L. curvatus LTH 1174, but more importantly, it had a detrimental effect on specific curvacin A production (k_B) and hence on overall bacteriocin activity. Even a low salt concentration (2%, wt/vol) decreased bacteriocin production, while growth was unaffected at this concentration. The inhibitory effect of NaCl was mainly due to its role as an a_w-lowering agent. Further, it was clear that salt interfered with bacteriocin induction. Additionally, when 6% (wt/vol) sodium chloride was added, the minimum biomass concentration necessary to start the production of curvacin A (X_B) was 0.90 g (cell dry mass) per liter. Addition of the cell-free culture supernatant or a protein solution as a source of induction factor resulted in a decrease in X_B, an increase in k_B, and hence an increase in the maximum attainable bacteriocin activity.     

Activity of plantaricin SA6, a bacteriocin produced by Lactobacillus plantarum SA6 isolated from fermented sausage

N. REKHIF, A ATRIH AND G. LEFEBVRE. 1995. Plantaricin SA6, a bacteriocin produced by Lactobacillus plantarum SA6, exhibited an inhibitory action against several mesophilic lactobacilli. It was stable at 90–100°C at pH 2–4 and it remained stable in the presence of several organic solvents, urea or β-mercaptoethanol. Plantaricin SA6 bound specifically to the cell surface of only plantaricin SA6-sensitive bacteria. The putative receptors are not destroyed by different hydrolytic enzymes added to the phosphate buffer. Plantaricin SA6 acted as a bactericidal agent lysing sensitive strains, that became more permeable to ortho-nitro-phenol-β-galactoside and lost their intracellular K⁺ ions and u.v.-absorbing materials. Both the adsorption and lethal action of plantaricin SA6 were maximal between pH 4 and 7, but the range of temperature tested (5–37βC) had no effect. Ions (of several salts such as MgCl₂) inhibited the binding of plantaricin SA6 and protected cells against bacteriocin action.     

Influence of Complex Nutrient Source on Growth of and Curvacin A Production by Sausage Isolate Lactobacillus curvatus LTH 1174

Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the antilisterial bacteriocin curvacin A. Its biokinetics of cell growth and bacteriocin production as a function of various concentrations of a complex nutrient source were investigated in vitro during laboratory fermentations with modified MRS medium. A modification of the nutrient depletion model (Leroy and De Vuyst, Appl. Environ, Microbiol. 67:4470-4473, 2001) was used to fit the data describing growth and bacteriocin production. Both cell growth and bacteriocin activity were influenced by changes in the complex nutrient source concentration. Standard MRS medium clearly limited the growth of L. curvatus LTH 1174. Higher nutrient concentrations, up to a certain degree, led to improved growth, a higher attainable biomass concentration, and a higher bacteriocin activity in the supernatant. A lower concentration of complex nutrient source caused severe growth inhibition, leading to a lower biomass concentration but a much higher specific bacteriocin production. When examining the separate components of the complex nutrient source, a stimulating effect of bacteriological peptone on growth was found without an adverse effect on bacteriocin production, resulting in increased curvacin A activity. Furthermore, specific depletion of the amino acids tyrosine, serine, and asparagine/aspartic acid was observed for this strain.     

The curing agent sodium nitrite,used in the production of fermented sausages,is less inhibiting to the bacteriocin-producing meat starter culture Lactobacillus curvatus LTH 1174 under anaerobic conditions

Curvacin A is a listericidal bacteriocin produced by Lactobacillus curvatus LTH 1174, a strain isolated from fermented sausage. The response of this strain to an added curing agent (sodium nitrite) in terms of cell growth and bacteriocin production was investigated in vitro by laboratory fermentations with modified MRS broth. The strain was highly sensitive to nitrite; even a concentration of 10 ppm of curing agent inhibited its growth and both volumetric and specific bacteriocin production. A meat simulation medium containing 5 ppm of sodium nitrite was tested to investigate the influence of the gas phase on the growth and bacteriocin production of L. curvatus LTH 1174. Aerating the culture during growth had no effect on biomass formation, but the oxidative stress caused a higher level of specific bacteriocin production and led to a metabolic shift toward acetic acid production. Anaerobic conditions, on the other hand, led to an increased biomass concentration and less growth inhibition. Also, higher maximum volumetric bacteriocin activities and a higher level of specific bacteriocin production were obtained in the presence of sodium nitrite than in fermentations under aerobic conditions or standard conditions of air supply. These results indicate that the inhibitory effect of the curing agent is at least partially masked under anaerobic conditions.     

Purification and characterization of curvaticin L442, a bacteriocin produced by Lactobacillus curvatus L442

Lactobacillus curvatus L442, isolated from Greek traditional fermented sausage prepared without the addition of starters, produces a bacteriocin, curvaticin L442, which is active against the pathogen Listeria monocytogenes. The bacteriocin was purified by 50% ammonium sulphate precipitation, cation exchange, reverse phase and gel filtration chromatography. Partial N-terminal sequence analysis using Edman degradation revealed 30 amino acid residues, revealing high homology with the amino acid sequence of sakacin P. Curvaticin L442 is active at pH values between 4.0 and 9.0 and it retains activity even after incubation for 5 min at 121 °C with 1 atm of overpressure. Proteolytic enzymes and α-amylase inactivated this curvaticin, while the effect of lipase was not severe.     

Sakacin B, a bacteriocin produced by Lactobacillus sake isolated from Greek dry fermented sausages

When Lactobacillus sake 251, a strain isolated from naturally fermented Greek dry sausage was grown in MRS broth it excreted an antimicrobial factor that differed from organic acids and hydrogen peroxide. The substance was proteinaceous, heat stable and inhibitory towards various lactic acid bacteria of meat origin. This suggested that a narrow spectrum bacteriocin, designated sakacin B, was present in the broth. Sakacin B displayed a bactericidal mode of action on sensitive cells without causing cell lysis. It was secreted during late logarithmic phase and was stable within a pH range 2 to 9. In vitro production of sakacin B by the producer strain in a mixed culture caused a strong biocidal effect on growing indicator cells. Sakacin B was partially purified and found not to contain unusual amino acids. That it was a hydrophobic peptide was confirmed by SDS-PAGE electrophoresis. The molecular weight of sakacin B was estimated to be 6.3 kDa.     

Effects of Different Spices Used in Production of Fermented Sausages on Growth of and Curvacin A Production by Lactobacillus curvatus LTH 1174

Lactobacillus curvatus LTH 1174, a fermented sausage isolate, produces the listericidal bacteriocin curvacin A. The effect of different spices relevant for the production of fermented sausages was investigated in vitro through laboratory fermentations with a meat simulation medium and an imposed pH profile relevant for Belgian-type fermented sausages. The influence on the growth characteristics and especially on the kinetics of curvacin A production with L. curvatus LTH 1174 was evaluated. Pepper, nutmeg, rosemary, mace, and garlic all decreased the maximum specific growth rate, while paprika was the only spice that increased it. The effect on the lag phase was minor except for nutmeg and especially for garlic, which increased it, yet garlic was stimulatory for biomass production. The maximum attainable biomass concentration (X_max) was severely decreased by the addition of 0.40% (wt/vol) nutmeg, while 0.35% (wt/vol) garlic or 0.80% (wt/vol) white pepper increased X_max. Nutmeg decreased both growth and bacteriocin production considerably. Garlic was the only spice enhancing specific bacteriocin production, resulting in higher bacteriocin activity in the cell-free culture supernatant. Finally, lactic acid production was stimulated by the addition of pepper, and this was not due to the manganese present because an amount of manganese that was not growth limiting was added to the growth medium. Addition of spices to the sausage mixture is clearly a factor that will influence the effectiveness of bacteriocinogenic starter cultures in fermented-sausage manufacturing.     

Sequencing and expression analysis of the sakacin P bacteriocin produced by a Lactobacillus sakei strain isolated from naturally fermented sausages

A Lactobacillus sakei strain, designated as I151 and isolated from naturally fermented sausages, was found to produce the sakacin P bacteriocin which is active against Listeria monocytogenes. In this study, we performed the sequencing of the gene cluster involved in the production of the sakacin P, and we followed the expression of the sppA gene, encoding for the bacteriocin, in vitro, using Rogosa–Sharpe medium, and in situ, inoculating the strain in fermented sausages as starter culture. The results obtained underlined the high similarity (>99%) of the entire sakacin P gene cluster from the L. sakei studied here with others present in strains of L. sakei already described. Moreover, from the expression experiments, it was shown that the gene is expressed during the exponential phase and that production procedures typical of fermented sausages are not turning off the expression of the gene encoding the bacteriocin. The capability of the strain studied to produce sakacin P during production is considered an advantage for its use as starter culture to improve the safety aspect of traditional fermented sausages produced in Italy.     

Characterization and purification of a new bacteriocin with a broad inhibitory spectrum produced by Lactobacillus plantarum lp 31 strain isolated from dry-fermented sausage

Aims:  Characterization and purification of a new bacteriocin produced by Lactobacillus plantarum LP 31 strain, isolated from Argentinian dry-fermented sausage.
Methods and Results:  Lactobacillus plantarum LP 31 strain produces an antimicrobial compound that inhibits the growth of food-borne pathogenic bacteria. It was inactivated by proteolytic enzymes, was stable to heat and catalase and exhibited maximum activity in the pH range from 5·0 to 6·0. Consequently, it was characterized as a bacteriocin. It was purified by RP (reverse-phase) solid-phase extraction, gel filtration chromatography and RP-HPLC. Plantaricin produced by Lact. plantarum LP 31 is a peptide with a molecular weight of 1558·85 Da as determined by Maldi-Tof mass spectrometry and contains 14 amino acid residues. It was shown to have a bactericidal effect against Pseudomonas sp., Staphylococcus aureus , Bacillus cereus and Listeria monocytogenes.
Conclusions:  The bacteriocin produced by Lact. plantarum LP 31 may be considered as a new plantaricin according to its low molecular weight and particular amino acid composition.
Significance and Impact of the Study:  In view of the interesting inhibitory spectrum of this bacteriocin and because of its good technological properties (resistance to heat and activity at acidic pH), this bacteriocin has potential applications as a biopreservative to prevent the growth of food-borne pathogens and food spoilage bacteria in certain food products.     

The Curing Agent Sodium Nitrite, Used in the Production of Fermented Sausages, Is Less Inhibiting to the Bacteriocin-Producing Meat Starter Culture Lactobacillus curvatus LTH 1174 under Anaerobic Conditions

Curvacin A is a listericidal bacteriocin produced by Lactobacillus curvatus LTH 1174, a strain isolated from fermented sausage. The response of this strain to an added curing agent (sodium nitrite) in terms of cell growth and bacteriocin production was investigated in vitro by laboratory fermentations with modified MRS broth. The strain was highly sensitive to nitrite; even a concentration of 10 ppm of curing agent inhibited its growth and both volumetric and specific bacteriocin production. A meat simulation medium containing 5 ppm of sodium nitrite was tested to investigate the influence of the gas phase on the growth and bacteriocin production of L. curvatus LTH 1174. Aerating the culture during growth had no effect on biomass formation, but the oxidative stress caused a higher level of specific bacteriocin production and led to a metabolic shift toward acetic acid production. Anaerobic conditions, on the other hand, led to an increased biomass concentration and less growth inhibition. Also, higher maximum volumetric bacteriocin activities and a higher level of specific bacteriocin production were obtained in the presence of sodium nitrite than in fermentations under aerobic conditions or standard conditions of air supply. These results indicate that the inhibitory effect of the curing agent is at least partially masked under anaerobic conditions.     

The identification of a low molecular mass bacteriocin,rhamnosin A,produced by Lactobacillus rhamnosus strain 68

Aims: This study focuses on the isolation and characterization of a peptide with bacteriocin‐like properties isolated from Lactobacillus rhamnosus strain 68, previously identified by 16S rRNA gene sequencing and originating from human gastrointestinal flora. Methods and Results: The peptide was isolated from a supernatant of bacteria maintained under restrictive conditions by a combination of ethanol precipitation and reversed‐phase chromatography. The molecular mass of the peptide as assessed by mass spectrometry was 6433·8 Da. An isoelectric point of 9·8 was determined by 2D‐PAGE. The peptide designated rhamnosin A inhibited Micrococcus lysodeikticus ATCC 4698 but did not inhibit Lactobacillus plantarum 8014 or Lact. plantarum 39268. Inhibitory activity against M. lysodeikticus at concentrations used in this study was shown to be bacteriostatic rather than bacteriolytic or bactericidal. Rhamnosin A retained biological activity after heat treatment (95°C, 30 min) but was sensitive to proteolytic activity of pepsin and trypsin. Conclusions: The N‐terminal sequence of rhamnosin A, as determined by Edman degradation and in more detail by blast analysis, did not show identity with any currently available Lact. rhamnosus HN001‐translated protein sequences, nor any significant similarity with other sequences in the nonredundant protein sequence database. Being a small, heat‐stable, nonlanthionine‐containing peptide, rhamnosin A should be categorized as a class II bacteriocin. Significance and Impact of the Study: This study describes a partial bacteriocin sequence isolated from Lact. rhamnosus 68 and broadens our understanding of bacteriocins.     

植物乳杆菌R260产细菌素发酵条件的研究

目的 获取植物乳杆菌R260产细菌素的最佳发酵条件.方法用琼脂扩散法测定发酵液对苏云金芽胞杆菌的抑菌效价.结果 产细菌素的最佳培养基是MRS培养基,最适起始Ph为6.5,最适接种量和接种种龄分别为3%和12 h,产细菌素最适发酵温度和时间分别为30℃和20 h：细菌素在对数期开始产生,稳定期产量达到最大值.结论 通过优化发酵条件提高了细菌素的产量,达1656 IU/ml.     

细菌素Paracin 1.7的纯化与性质研究

摘要：【目的】分离纯化（Lactobacillus paracasei）HD1.7所产生的细菌素并分析其特性。【方法】细菌素Paracin 1.7的纯化采用色谱技术,其分子量检测采用十二烷基磺酸钠－聚丙烯酰胺凝胶电泳（SDS-PAGE）,利用琼脂扩散法测定细菌素活力。【结果】Paracin 1.7分离于我国传统发酵食品酸菜发酵液中,其产生菌为副干酪乳杆菌。 Paracin 1.7可以抑制其它微生物的生长,为细菌素。该菌在稳定期可产生大量Paracin 1.7。经过阳离子交换层析、凝胶过滤层析以及高效液相色谱（HPLC）,对该细菌素进行了初步纯化,并经Tricine-SDS-PAGE检测其分子量大约为11 kDa。Paracin 1.7抑菌谱较广,其抑菌范围包括Proteus, Bacillus, Enterobacter, Staphylococcus, Escherichia, Lactobacillus, Microccus, Pseudomonas, Salmonella, Saccharomyces,其中有些为食品源致病菌。该细菌素在酸性及高温下稳定,对几种蛋白质酶敏感。该细菌素对敏感菌株的作用方式为抑菌。在4oC保存4个月后,Paracin 1.7的抑菌活性保持稳定。【结论】基于细菌素Paracin 1.7的性质,该细菌素可用作食品防腐剂。     

The presence of salt and a curing agent reduces bacteriocin production by Lactobacillus sakei CTC 494, a potential starter culture for sausage fermentation

The specific conditions in the batter of raw fermented sausages may reduce the efficiency of bacteriocin-producing starter cultures. In this work, using in vitro fermentation, we found that sodium chloride and sodium nitrite interfere with the growth of Lactobacillus sakei CTC 494, an organism which produces the antilisterial bacteriocin sakacin K. Because sakacin K production follows primary metabolite kinetics, a decrease in cell formation resulted in a decrease in sakacin K production as well. Sodium chloride dramatically influenced bacteriocin production by decreasing both biomass production and specific bacteriocin production. Sodium nitrite, however, had no effect on specific bacteriocin production and decreased bacteriocin production only because of its effect on cell growth. Moreover, sodium nitrite enhanced the toxic effect of lactic acid on bacterial growth.     

Optimization of bacteriocin production by Lactobacillus plantarum ST13BR, a strain isolated from barley beer

The cell-free supernatant containing bacteriocin ST13BR, produced by Lactobacillus plantarum ST13BR, inhibits the growth of L. casei, Pseudomonas aeruginosa, Enterococcus faecalis, Klebsiella pneumoniae and Escherichia coli. Based on tricine-SDS-PAGE, bacteriocin ST13BR is 10 kDa in size. Complete inactivation or significant reduction in bacteriocin activity was observed after treatment with Proteinase K, trypsin and pronase, but not with catalase or alpha-amylase. Low bacteriocin activity (200 AU/ml) was recorded in BHI medium, M17 broth, 10% (w/v) soy milk, and 2% and 10% (w/v) molasses, despite good growth. Maximal bacteriocin activity (6,400 AU/ml) was recorded after 23 h in MRS broth, but only at 30 degrees C. Tween 80 in MRS broth increased bacteriocin production by more than 50%. Meat extract or yeast extract as sole nitrogen source, or a combination of the two (1 : 1) in MRS broth, stimulated bacteriocin production (6,400 AU/ml). Only 50% activity (3,200 AU/ml) was recorded with tryptone as sole nitrogen source, whereas a combination of tryptone, meat extract and yeast extract yielded 6,400 AU/ml. Bacteriocin production was not stimulated by the addition of glucose at 2.0% w/v (3,200 AU/ml), nor 2% (w/v) fructose, sucrose, lactose or mannose, respectively (800 AU/ml). Activity levels less than 200 AU/ml were recorded in the presence of 0.05% to 0.5% (w/v) maltose. Maximal bacteriocin production (6,400 AU/ml) was recorded in the presence of 2% (w/v) maltose. Maltose at 4.0% (w/v) led to a 50% reduction of bacteriocin activity. The presence of 1.0% (w/v) and higher KH(2)PO(4), or glycerol at 0.2% (w/v) suppressed bacteriocin production.     

Purification and partial amino acid sequence of curvaticin FS47, a heat-stable bacteriocin produced by Lactobacillus curvatus FS47.

Curvaticin FS47, a bacteriocin produced by Lactobacillus curvatus FS47, is inhibitory to Listeria monocytogenes, as well as Lactobacillus, Pediococcus, Enterococcus, and Bacillus spp. The bacteriocin was purified by 40% ammonium sulfate precipitation, solid-phase extraction, and reversed-phase high-pressure liquid chromatography. Purified curvaticin FS47 was determined to be 4.07 kDa by mass spectrometry and was partially sequenced. Thirty-one N-terminal amino acids were identified; the curvaticin FS47 protein sequence did not show homology to the pediocin-like group of bacteriocins.     

Characterization of two bacteriocins produced by Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages

Leuconostoc mesenteroides L124 and Lactobacillus curvatus L442, isolated from dry fermented sausages, produce bacteriocins antagonistic towards closely related species and pathogens, such as Listeria monocytogenes. The bacteriocins were inactivated by proteolytic enzymes and lipase but not by catalase and lysozyme. They were also heat stable, retaining activity after heating at 100 °C for 60 min. The bacteriocins were stable at pH values ranging from 2.0 to 8.0. Bacteriocin production was observed at low temperatures (10 and 4 °C) and in meat juice. The maximum bacteriocin activity was observed at the end of the exponential growth phase. The bacteriocins were produced in media with initial pH values ranging from 5.0 to 7.5, but not in media with a pH lower than 5.0 (weak bacteriocin activity of the antibacterial compound produced by Ln. mesenteroides L124 was observed at pH 4.5). Both bacteriocins exhibited strong bactericidal activity following cell/bacteriocin contact.