Antimicrobial compounds produced by probiotic Lactobacillus brevis isolated from dairy products

A total of 38 lactic acid bacteria, belonging to Lactobacillus, isolated from 24 samples of traditional Egyptian dairy products, were screened for antimicrobial activity against different Gram-positive and Gram-negative bacteria. A strain of Lactobacillus brevis showed the best inhibitory activity when tested by well diffusion assay. The antibacterial activity was pronounced between early logarithmic and early stationary phases. The strain produced a heat-stable antimicrobial compound showing no reduction in activity after heat treatment from 60 to 100°C for 15 and 30 min. Since it was inactivated by proteolytic enzymes, it is considered to be proteinaceous in nature and, therefore, referred to as a bacteriocin-like substance. This compound was also active over a wide pH range (pH 2–6). The antimicrobial compound was partially purified by 40% ammonium sulfate precipitation. Lactobacillus brevis was tested for its in vitro antibiotics susceptibility, tolerance to bile salts, resistance to low pH values, acidifying activity, proteolytic activity, and haemolytic activity. The results showed the potential of L. brevis strain as a probiotic culture, and hence it can be utilized in the manufacturing of pharmaceuticals and dietary supplements.     

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.     

Atopic dermatitis‐mitigating effects of new Lactobacillus strain,Lactobacillus sakei probio 65 isolated from Kimchi

Aims

Atopic dermatitis (AD) is an inflammatory skin disease. Probiotics have been reported to modulate immune responses and thus are now being suggested as potential treatments for allergies. In this study, we investigated the inhibitory effects of Lactobacillus sakei probio 65 isolated from Kimchi on artificially inducing AD in NC/Nga mice.

Methods and Results

Oral administration of viable or heat‐inactivated Lact. sakei probio 65 improved the condition of skin and reduced scratching frequency. Serum levels of IgE and cutaneous T‐cell‐attracting chemokine (CTACK) were significantly decreased by this therapy. Dead Lact. sakei probio 65 also decreased IL‐4 and IL‐6 serum concentrations. Moreover, both live and dead Lact. sakei probio 65 inhibited the expression of Thymus and activation‐regulated chemokine and CTACK in AD‐like skin lesions. The increased levels of Foxp3 expression in the lesional skin and ears were also suppressed by Lact. sakei probio 65. In addition, Lact. sakei probio 65 inhibited β‐hexosaminidase release and the secretion of IL‐4, TNF‐α and IL‐6 from RBL‐2H3 cells.

Conclusions

Oral treatment with both viable and heat‐inactivated Lact. sakei probio 65 inhibits skin inflammation and AD‐like skin lesions, as well as mast cell activation.

Significance and Impact of the Study

Lactobacillus sakei probio 65 has an inhibitory effect on atopic dermatitis‐like skin lesions and may represent an effective new anti‐inflammatory agent.     

Lacticin, a bacteriocin produced by Lactobacillus delbrueckii subsp. lactis

Twenty-one strains of Lactobacillus delbrueckii and L. helveticus were tested for bacteriocin production against each other. Lactobacillus delbrueckii subsp. lactis JCM 1106 and 1107 produced an inhibitory agent active against L. delbrueckii subsp. bulgaricus JCM 1002 and NIAI yB-62, L. delbrueckii subsp. lactis JCM 1248 and L. delbrueckii subsp. delbrueckii JCM 1012. Lactobacillus delbrueckii subsp. lactis JCM 1248 inhibited only the growth of L. delbrueckii subsp. bulgaricus NIAI yB-62. These agents were sensitive to proteolytic enzymes and heating (at 60°C for 10min). These agents were considered to be bacteriocins and designated lacticin A and B.     

Isolation and partial characterization of a bacteriocin produced by Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product

Lactobacillus plantarum BM‐1 isolated from a traditionally fermented Chinese meat product was found to produce a novel bacteriocin that is active against a wide range of gram‐positive and gram‐negative bacteria. Production of the bacteriocin BM‐1 started early in the exponential phase and its maximum activity (5120 AU/mL) was recorded early during the stationary phase (16 hr). Bacteriocin BM‐1 is sensitive to proteolytic enzymes but stable in the pH range of 2.0–10.0 and heat‐resistant (15 min at 121°C). This bacteriocin was purified through pH‐mediated cell adsorption–desorption and cation‐exchange chromatography on an SP Sepharose Fast Flow column. The molecular weight of the purified bacteriocin BM‐1 was determined to be 4638.142 Da by electrospray ionization Fourier transform mass spectrometry. Furthermore, the N‐terminal amino acid sequence was obtained through automated Edman degradation and found to comprise the following 15 amino acid residues: H₂N‐Lys‐Tyr‐Tyr‐Gly‐Asn‐Gly‐Val‐Tyr‐Val‐Gly‐Lys‐His‐Ser‐Cys‐Ser. Comparison of this sequence with that of other bacteriocins revealed that bacteriocin BM‐1 contains the consensus YGNGV amino acid motif near the N‐terminus. Based on its physicochemical characteristics, molecular weight, and N‐terminal amino acid sequence, plantaricin BM‐1 is a novel class IIa bacteriocin.     

Antimicrobial compounds produced by Actinomadura sp. AC104 isolated from an Algerian Saharan soil

During a search for nonpolyenic antifungal antibiotics, an actinomycete designated AC104 was isolated from a Saharan soil sample by a dilution agar plating method using a chitin - vitamins B medium supplemented with rifampicin. Isolate AC104 presented the morphological and the chemical characteristics of the genus Actinomadura. On the basis of 76 physiological tests and 16S rDNA analysis, this isolate was determined to be quite different from the known species of Actinomadura. It is active against filamentous fungi and both Gram-positive and Gram-negative bacteria. The production of antibiotic substances was investigated using several culture media. The highest antimicrobial activities were obtained on ISP2 medium. The benzenic extract contained five bioactive spots detected on thin layer chromatography plates. Among these antibiotics, a complex called 104A, which showed the more interesting antifungal activity, was selected and purified by reverse-phase high-pressure liquid chromatography. This complex is composed of four compounds. Ultraviolet-visible, infrared, mass, and 1H nuclear magnetic resonance spectroscopy studies showed that these molecules contain an aromatic ring substituted by aliphatic chains. These compounds differ from the known antibiotics produced by Actinomadura species.     

Antibacterial activity of Lactobacillus sake isolated from meat.

A total of 221 strains of Lactobacillus isolated from meat and meat products were screened for antagonistic activities under conditions that eliminated the effects of organic acids and hydrogen peroxide. Nineteen strains of Lactobacillus sake, three strains of Lactobacillus plantarum, and one strain of Lactobacillus curvatus were shown to inhibit the growth of some other lactobacilli in an agar spot test; and cell-free supernatants from 6 of the 19 strains of L. sake exhibited inhibitory activity against indicator organisms. Comparison of the antimicrobial spectra of the supernatants suggested that the inhibitory compounds were not identical. One of the six strains, L. sake Lb 706, was chosen for further study. The compound excreted by L. sake Lb 706 was active against various lactic acid bacteria and Listeria monocytogenes. Its proteinaceous nature, narrow inhibitory spectrum, and bactericidal mode of action indicated that this substance is a bacteriocin, which we designated sakacin A. Curing experiments with two bacteriocin-producing strains of L. sake resulted in mutants that lacked both bacteriocin activity and immunity to the bacteriocin. Plasmid profile analysis of L. sake Lb 706 and two bacteriocin-negative variants of this strain indicated that a plasmid of about 18 megadaltons may be involved in the formation of bacteriocin and immunity to this antibacterial compound. In mixed culture, the bacteriocin-sensitive organisms were killed after the bacteriocin-producing strain reached maximal cell density, whereas there was no decrease in cell number in the presence of the bacteriocin-negative variant.     

Arginine catabolism in Lactobacillus sake isolated from meat.

Lactobacillus sake isolated from meat can hydrolyze arginine via the arginine deiminase pathway. Two enzymes, arginine deiminase and ornithine transcarbamylase, have been revealed by detection of their reaction products, citrulline and ornithine, respectively. The production of citrulline depends on the concentration of glucose in a synthetic medium; it does not occur when the concentration of glucose is 27.5 mM or higher.     

Effect of bacteriocinogenic Lactobacillus spp. on the shelf life of fufu, a traditional fermented cassava product

Quantitative determination of antimicrobial compounds produced by the Lactobacillus strains under test was carried out. L. plantarum F1 produced the highest quantity of lactic acid (16.4 g/l) while the lowest amount (0.3 g/l) was produced by L. jensenii F9. All the test organisms produced hydrogen peroxide, with L. brevis OG1 having the highest yield of 0.037 g/l. Diacetyl was also produced by all the organisms, with L. plantarum F1 and L. brevis OG1 having the highest yield of 1.7 g/l, while the lowest producer was L. jensenii F9 (0.86 g/l). Determination of bacteriocin activity was carried out. L. plantarum F1 exhibited 6400 AU/ml bacteriocin activity, while L. brevis OG1 had the lowest activity of 3200 AU/ml using E. coli NCTC10418 as indicator organism. However, L. fermentum F5 and L. jensenii F9 did not produce any detectable bacteriocin. The pH value in the culture supernatant of L. plantarum F1 reached 3.1 within 48 h of incubation, while that of L. jensenii F9 was 5.2. Fufu was prepared using both bacteriocin-producing (BP) L. plantarum F1 and L. brevis OG1, and non-bacteriocin producing L. fermentum F5 and L. jensenii F9. No viable cells of Salmonella typhimurium ATCC13311 and Shigella flexneri AP23498 were detected after 12 h in the cassava products fermented with mixed starter culture of L. plantarum F1 and L. brevis OG1. The rate of survival of enteropathogens in cassava fermented with mixed starter cultures of L. plantarum F1 and L. brevis OG1 was much lower when compared to cassava fermented with mixed starter culture of L. fermentum F5 and L. jensenii F9. At 12 h, the viable count of E. coli NCTC10418 in cassava fermented with mixed starter cultures of L. plantarum F1 and L. brevis OG1 was 1.1 log₁₀ c.f.u./g whereas in cassava fermented with mixed starter cultures of L. fermentum F5 and L. jensenii F9, 8.5 log₁₀ c.f.u./g was obtained The study revealed that fufu produced with BP mixed starter cultures had a better shelf life and kept for 13 days before spoilage occurred, relative to 5 days observed for fufu produced using non-bacteriocin-producing starter cultures, and 6 days for the traditional fermented fufu.     

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.     

Maltose-fructose co-fermentation by Lactobacillus brevis subsp. lindneri CB1 fructose-negative strain

The Lactobacillus brevis subsp. lindneri CB1 fructose-negative strain utilized fructose in co-fermentation with maltose or glucose. Compared to the maltose (17 g/l) fermentation, the simultaneous fermentation of maltose (10 g/l) and fructose (7 g/l) increased cell yield (A ₆₂₀from 2.6 to 3.3) and the concentrations of lactic acid and especially of acetic acid (from 2.45 g/l to 3.90 g/l), produced mannitol (1.95 g/l) and caused a decrease in the amount of ethanol (from 0.46 g/l to 0.08 g/l). The utilization of fructose depended on the continuous presence of maltose in the growth medium and the two carbohydrates were consumed in a molar ratio of about 2:1. The presence of tagatose (a fructose stereoisomer) partially inhibited fructose consumption and consequently caused a decrease of the end products of the co-metabolism. Since maltose was naturally present during sourdough fermentation, the addition of only 6 g fructose/kg wheat dough enabled the co-fermentation of maltose and fructose by L. brevis subsp. lindneri CB1. A higher titratable acidity and acetic acid concentration, and a reduced quotient of fermentation (2.7) were obtained by co-fermentation compared with normal sourdough fermentation. Some interpretations of the maltose-fructose co-fermentation are given.     

Purification and molecular characterization of antibacterial compounds produced by Lactobacillus murinus strain L1

AIMS: The aim of this work was to purify and characterize antibacterial compounds produced by Lactobacillus murinus strain L1. METHODS AND RESULTS: Antagonistic activity was observed in a deferred agar-spot assay against spoilage and pathogenic bacteria, but not against lactobacilli. The inhibitory activity occurred between pH 3.0 and 5.0, and was heat stable. The active compounds were purified by gel filtration chromatography and two peaks of antibacterial activity were observed using Bacillus cereus ATCC 11778 and Shigella sonnei ATCC 11060 as indicator strains. Two active low molecular weight compounds were responsible for this phenomenon and UV spectroscopy, gas chromatography and mass spectrometry were used to characterize them. One of them is lactic acid, while the other is a mono-substituted aromatic ring apparently constituted by group residues of m/z 192 linked in tandem to phenylalanine. CONCLUSIONS: Lactobacillus murinus produces at least two low molecular weight compounds active against B. cereus and Sh. sonnei. SIGNIFICANCE AND IMPACT OF THE STUDY: This is the first purification of a new broad-spectrum antibacterial compound from Lact. murinus which inhibits various pathogenic and food spoilage bacteria without acting on other lactobacilli. Using it as a biotechnological control agent of bacterial spoilage may be a promising possibility for the food industry.     

Metabolic profiling and biological activities of bioactive compounds produced by Pseudomonas sp. strain ICTB-745 isolated from Ladakh, India

In an ongoing survey of the bioactive potential of microorganisms from Ladakh, India, the culture medium of a bacterial strain of a new Pseudomonas sp., strain ICTB-745, isolated from an alkaline soil sample collected from Leh, Ladakh, India, was found to contain metabolites that exhibited broad-spectrum antimicrobial and biosurfactant activities. Bioactivity-guided purification resulted in the isolation of four bioactive compounds. Their chemical structures were elucidated by (1)H and (13)C NMR, 2D-NMR (HMBC, HSQC, (1)H,(1)H-COSY, and DEPT- 135), FT-IR, and mass spectroscopic methods, and were identified as 1-hydroxyphenazine, phenazine-1-carboxylic acid (PCA), rhamnolipid-1 (RL-1), and rhamnolipid-2 (RL-2). These metabolites exhibited various biological activities like antimicrobial and efficient cytotoxic potencies against different human tumor cell lines such as HeLa, HepG2, A549, and MDA MB 231. RL-1 and RL-2 exhibited a dose-dependent antifeedant activity against Spodoptera litura, producing about 82.06% and 73.66% antifeedant activity, whereas PCA showed a moderate antifeedant activity (63.67%) at 60 microgram/cm2 area of castor leaf. Furthermore, PCA, RL-1, and RL-2 exhibited about 65%, 52%, and 47% mortality, respectively, against Rhyzopertha dominica at 20 microgram/ml. This is the first report of rhamnolipids as antifeedant metabolites against Spodoptera litura and as insecticidal metabolites against Rhyzopertha dominica. The metabolites from Pseudomonas sp. strain ICTB-745 have interesting potential for use as a biopesticide in pest control programs.     

Characterization of a bacteriocin-like substance produced by Bacillus amyloliquefaciens isolated from the Brazilian Atlantic forest.

A Bacillus strain producing a bacteriocin-like substance was characterized by biochemical profiling and 16S rDNA sequencing. Phylogenetic analysis indicated that the strain has high sequence similarity with Bacillus amyloliquefaciens. The antimicrobial substance was inhibitory to pathogenic and food-spoilage bacteria, such as Listeria monocytogenes, Bacillus cereus, Serratia marcescens, and Pasteurella haemolytica. It was stable over a wide temperature range, but lost activity when the temperature reached 121 degrees C/15 min. Maximum activity was observed at acidic and neutral pH values, but not at alkaline pH. The antimicrobial substance was sensitive to the proteolytic action of trypsin, papain, proteinase K, and pronase E. Except for iturins, other antimicrobial peptides have not been described for B. amyloliquefaciens. The identification of a bacteriocin-like inhibitory substance active against L. monocytogenes addresses an important aspect of food protection.     

Structure of the extracellular polysaccharide produced by Lactobacillus delbrueckii subsp. bulgaricus 291

The lactic acid bacterium Lactobacillus delbrueckii subsp. bulgaricus 291, when grown in skimmed milk, produced 80 mg/L exopolysaccharide with an average molecular mass of 1.4 x 10(3) kDa. Monosaccharide analysis, methylation analysis, MS, and 1D/2D NMR (1H and 13C) studies performed on the native polysaccharide, and on oligosaccharides obtained from a mild acid hydrolysate of the native polysaccharide, showed the polysaccharide to consist of branched pentasaccharide repeating units with the following structure: [structure: see text].     

Detection and characterization of a bacteriocin produced by Lactococcus lactis subsp. cremoris R isolated from radish

Bacteria isolated from radish were identified as Lactococcus lactis subsp. cremoris R and their bacteriocin was designated lactococcin R. Lactococcin R was sensitive to some proteolytic enzymes (proteinase-K, pronase-E, proteases, pepsin, α-chymotrypsin) but was resistant to trypsin, papain, catalase, lysozyme and lipase, organic solvents, or heating at 90 °C for 15, 30 and 60 min, or 121 °C for 15 min. Lactococcin R remained active after storage at −20 and −70 °C for 3 months and after exposure to a pH of 2–9. The molecular weight of lactococcin R was about 2·5 kDa. Lactococcin R was active against many food-borne pathogenic and food spoilage bacteria such as Clostridium, Staphylococcus, Listeria, Bacillus, Micrococcus, Enterococcus, Lactobacillus, Leuconostoc, Streptococcus and Pediococcus spp., but was not active against any Gram-negative bacteria. Lactococcin R was produced during log phase and reached a maximum activity (1600 AU ml⁻¹) at early stationary phase. The highest lactococcin R production was obtained in MRS broth with 0·5% glucose, at 6·5–7·0 initial pH values, 30 °C temperature and 18–24-h incubation times. Lactococcin R adsorbed maximally to its heat-killed producing cells at pH 6–7 (95%). Crude lactococcin R at 1280 AU ml⁻¹ was bactericidal, reducing colony counts of Listeria monocytogenes by 99·98% in 3 h. Lactococcin R should be useful as a biopreservative to prevent growth of food-borne pathogenic and food spoilage bacteria in ready-to-eat, dairy, meat, poultry and other food products. Lactococcin R differs from nisin in having a lower molecular weight, 2·5 kDa vs 3·4 kDa, and in being sensitive to pepsin and α-chymotrypsin to which nisin is resistant.     

口腔乳酸杆菌的分离及其益生特性

[目的]从口腔环境中筛选具有潜在益生特性的乳酸杆菌,用于防治口腔疾病的益生菌疗法.[方法]利用选择性培养基从健康志愿者的唾液和牙菌斑样品中筛选得到乳酸杆菌,然后验证他们对龋齿致病菌变异链球菌生长的抑制作用.同时考察分离得到的微生物是否具有可以定植或在口腔环境中生存的特性.[结果]本研究从牙菌斑样品中分离得到一株发酵乳杆菌Y29.该菌能够抑制变异链球菌的生长,并有自聚集和与其他口腔微生物共聚集形成生物膜的能力.此外,发酵乳杆菌Y29可耐受1.0 mg/mL溶菌酶和140μg/g过氧化氢,有利于其在可能含有多种抑菌物质的口腔动态环境中生存.[结论]发酵乳杆菌Y29在防治龋齿和保证口腔健康方面具有潜在的益生特性.