Production of acidocin B, a bacteriocin of Lactobacillus acidophilus M46 is a plasmid-encoded trait: Plasmid curing, genetic marking by in vivo plasmid integration, and gene transfer

Abstract Plasmid-curing studies suggest that acidocin B production is encoded by the 14-kb plasmid pCV461 in Lactobacillus acidophilus M46. Loss of pCV461 from the original producer strain M46 did not coincide with loss of immunity to acidocin B. Bacteriocin activity determination after SDS-PAGE showed that a substance of 2.4 kDa, absent in the culture supernatant of the mutant strain M46A2, lacking pCV461, represented acidocin B activity. In order to introduce a positive selection criterion, pCV461 was marked in vivo by the erythromycin resistance marker of pE194, present on pUC19 containing a 1.4-kb Hin dIII fragment of pCV461, after plasmid integration. Introduction of this recombinant plasmid into the mutant strain M46A2 or Lactobacillus plantarum resulted in erythromycin-resistant, acidocin B-producing transformants, showing unambiguously that acidocin B is encoded by pCV461.     

Antimicrobial activity of lactobacilli: preliminary characterization and optimization of production of acidocin B, a novel bacteriocin produced by Lactobacillus acidophilus M46

Approximately 1000 lactobacillus strains were isolated and screened for the production of antimicrobial activity, using a target panel of spoilage organisms and pathogens. Only eight positive strains were found; two of these were studied in more detail. Lactobacillus salivarius M7 produces the new broad spectrum bacteriocin salivaricin B which inhibits the growth of Listeria monocytogenes, Bacillus cereus, Brochothrix thermosphacta, Enterococcus faecalis and many lactobacilli. A new atypical bacteriocin produced by Lact. acidophilus M46, acidocin B, combines the inhibition of Clostridium sporogenes with a very narrow activity spectrum within the genus Lactobacillus and was selected for further characterization. Acidocin B is sensitive to trypsin, heat-stable (80°C for 20 min) and can be extracted from the culture supernatant fluid with butanol. Native acidocin B occurs as a large molecular weight complex (100 kDa), while with SDS-PAGE the partly purified activity migrates as a peptide of 2·4 kDa. Optimization of the cultivation conditions resulted in an eightfold increase of the amount of acidocin B produced during growth. Growth is not necessary for acidocin B production; washed producer cells can synthesize the bacteriocin in a chemically defined production medium. The application potential of acidocin B is discussed.     

Genome sequence of Lactobacillus rhamnosus strain CASL, an efficient L-lactic acid producer from cheap substrate cassava

Lactobacillus rhamnosus is a type of probiotic bacteria with industrial potential for L-lactic acid production. We announce the draft genome sequence of L. rhamnosus CASL (2,855,156 bp with a G+C content of 46.6%), which is an efficient producer of L-lactic acid from cheap, nonfood substrate cassava with a high production titer.     

来自詹氏乳杆菌的耐热D-乳酸脱氢酶的酶学性质研究

【目的】D-乳酸脱氢酶是催化丙酮酸合成D-乳酸的关键酶。由于其不耐热,从而限制了D-乳酸高温发酵菌株的构建。本文从詹氏乳杆菌中克隆新型D-乳酸脱氢酶研究其酶学性质,为构建D-乳酸高温发酵菌株,进一步降低D-乳酸生产成本奠定基础。【方法】通过克隆詹氏乳杆菌的D-乳酸脱氢酶,将其进行体外表达,并与来自植物乳杆菌中的D-乳酸脱氢酶的最适温度、最适pH、动力学参数及热稳定性和热失活性相比较,研究詹氏乳杆菌D-乳酸脱氢酶的耐热性。【结果】詹氏乳杆菌的D-乳酸脱氢酶最适温度(45 °C)比植物乳杆菌中的D-乳酸脱氢酶的最适温度(30 °C)高很多,热失活的时间和温度均要比植物乳杆菌中D-乳酸脱氢酶高很多。同时其催化效率(kcat/Km)是植物乳杆菌D-乳酸脱氢酶的3倍左右。【结论】詹氏乳杆菌的D-乳酸脱氢酶具有更好的耐热性和更高的催化活力。     

Intra-specific variation of Lactobacillus plantarum and Lactobacillus pentosus in sensitivity towards various bacteriocins

Fifty-two strains belonging to the Lactobacillus plantarum species group were identified and typed. They represented 32 clones of Lactobacillus plantarum and 7 clones of Lactobacillus pentosus. Sensitivity of all strains towards bacteriocins of four different producer strains was investigated using a deferred inhibition test (DIT). Substantial intra-specific variation in sensitivity of clones was observed towards bacteriocinogenic lactic acid bacteria producing nisin ( Lactococcus lactis ATCC 11454) or pediocin PA-1 ( Pediococcus acidilactici PAC-1.0), while none of the strains were sensitive towards the two remaining bacteriocin producers. The minimum inhibitory concentration (MIC) of nisin towards selected strains confirmed the DIT results. No correlation between the susceptibility of fourteen selected strains towards nisin and an array of antibiotics was found. The present study indicates that the variation in bacteriocin-sensitivity within target species might be a potential limitation for the application of bacteriocins as biopreservatives.     

Ribosomal resistance as a wide-spread self-defence mechanism in aminoglycoside-producing Micromonospora species

Abstract The mechanism of self-defence against their own product was studied in five aminoglycoside-producing Micromonospora (M.) species: M. grisea (verdamicin producer); M. inyoensis (sisomicin producer); M. sagamiensis (sagamicin producer); M. rhodorangea (antibiotic G-418 producer) and M. zionensis (antibiotic G-52 producer). Analysis of cell-free extracts of these organisms showed that they were devoid of modification enzymes specific for aminoglycosides. They contained, however, high-level resistant ribosomes. Mixed subunit exchange experiments of ribosomes from the producer strains and from a sensitive non-producing species ( M. melanosporea ) demonstrated that it is the 30S subunit which confers resistance.     

Genome sequence of the bacteriocin-producing Lactobacillus curvatus strain CRL705

Lactobacillus curvatus is one of the most prevalent lactic acid bacteria found in fermented meat products. Here, we present the draft genome sequence of Lactobacillus curvatus CRL705, a bacteriocin producer strain isolated from an Argentinean artisanal fermented sausage, which consists of 1,833,251 bp (GC content, 41.9%) and two circular plasmids of 12,342 bp (pRC12; GC, 43.9%) and 18,664 bp (pRC18; GC, 34.4%).     

Effect of Lactobacillus-protective cultures with bacteriocin-like inhibitory substances' producing ability on microbiological, chemical and sensory changes during storage of refrigerated vacuum-packaged sliced beef

AIMS: To investigate the effect of applying two different Lactobacillus-protective cultures, with bacteriocin-like inhibitory substances' (BLIS) producing ability, individually or in combination, on microbiological, chemical and sensory changes during storage of refrigerated vacuum-packaged sliced beef meat. METHODS AND RESULTS: Lactobacillus sakei CECT 4808 and Lactobacillus curvatus CECT 904(T), which were shown to be producers of BLIS, were inoculated individually or in combination on slices of beef M. semitendinosus. The samples were vacuum packaged and stored at 4 +/- 1 degrees C and were assessed during a 28-day storage period for microbiological [Enterobacteriaceae, Pseudomonas spp., lactic acid bacteria (LAB), Brochothrix thermosphacta and yeasts and moulds], chemical (pH, protein hydrolysis degree, lipid oxidation), sensory (abnormal odour) parameters and instrumental colour. Samples inoculated with the Lact. sakei strain and samples inoculated with the combination of the two strains had significantly (P < 0.05) lower spoilage microbial counts than those inoculated with the Lact. curvatus strain alone or the controls, while both chemical parameters (including lipid oxidation) and abnormal odour scores were also significantly (P < 0.05) improved by the former. Moreover, Lact. sakei alone showed a better preserving effect (P < 0.05) than the combination of both strains in the majority of the parameters tested. Instrumental colour measurements changed with storage time, but no treatment effects (P >or= 0.05) were observed during the whole 28-day storage period. CONCLUSIONS: The BLIS producer Lact. sakei CECT 4808 strain may be used for improving preservation of vacuum-packaged beef slices, as regards spoilage microbial counts and the chemical parameters tested in this study. SIGNIFICANCE AND IMPACT OF THE STUDY: Inoculation with the BLIS producer Lact. sakei CECT 4808 strain would provide an additional hurdle to improve storage life of refrigerated vacuum-packaged sliced beef. Furthermore, this strain demonstrated limited antioxidative ability, which could make a contribution to the prevention of lipid oxidation in meat and meat products.     

Behaviour of Listeria monocytogenes in meat and its control by a bacteriocin-producing strain of Lactobacillus sake

Lactobacillus sake Lb 706 can release a bacteriocin inhibitory to Listeria monocytogenes . In MRS broth, viable counts decreased rapidly when Lact. sake Lb 706 was added, whereas growth of the listerias was not affected by a bacteriocin-negative variant of the same Lactobacillus strain. Inhibition of L. monocytogenes was also observed in pasteurized minced meat inoculated with Lact. sake Lb 706. The bacteriocin produced is apparently effective in meat. However, the effect of the bacteriocin producer was less evident in minced meat than in broth. In comminuted cured raw pork filled into casings (German-type 'fresh Mettwurst'), L. monocytogenes was able to grow at a pH of 6.3, but addition of Lact. sake Lb 706 prevented the growth of listerias during the first few days after manufacture. At normal pH (5.7) L. monocytogenes did not multiply and addition of Lact. sake Lb 706 reduced viable counts of listerias by about one log cycle. Lactobacillus sake Lb 706 therefore may have some potential as a protective culture in meat products.     

Behaviour of Listeria monocytogenes in meat and its control by a bacteriocin-producing strain of Lactobacillus sake

Lactobacillus sake Lb 706 can release a bacteriocin inhibitory to Listeria monocytogenes. In MRS broth, viable counts decreased rapidly when Lact. sake Lb 706 was added, whereas growth of the listerias was not affected by a bacteriocin-negative variant of the same Lactobacillus strain. Inhibition of L. monocytogenes was also observed in pasteurized minced meat inoculated with Lact. sake Lb 706. The bacteriocin produced is apparently effective in meat. However, the effect of the bacteriocin producer was less evident in minced meat than in broth. In comminuted cured raw pork filled into casings (German-type 'fresh Mettwurst'), L. monocytogenes was able to grow at a pH of 6.3, but addition of Lact. sake Lb 706 prevented the growth of listerias during the first few days after manufacture. At normal pH (5.7) L. monocytogenes did not multiply and addition of Lact. sake Lb 706 reduced viable counts of listerias by about one log cycle. Lactobacillus sake Lb 706 therefore may have some potential as a protective culture in meat products.     

Intermediate chains of exopolysaccharides from Lactobacillus rhamnosus RW‐9595M increase IL‐10 production by macrophages

Aims: To evaluate the immunosuppressive properties of the exopolysaccharide (EPS) from high‐EPS producer Lactobacillus rhamnosus RW‐9595M on inflammatory cytokines produced by macrophages. Methods and Results: The conditioned media (CM) were produced by macrophages treated with parental Lact. rhamnosus ATCC 9595 and its isogenic variant, the high‐EPS producer Lact. rhamnosus RW‐9595M, and the levels of TNF‐α, IL‐6, IL‐10 and IL‐12 were evaluated. Results revealed that CM from parental Lact. rhamnosus induced higher levels of TNF‐α, IL‐6 and IL‐12 but inhibited IL‐10 production, whereas its mucous variant induced low or no TNF‐α and IL‐6. Addition of purified EPS to macrophages treated with parental Lact. rhamnosus decreased the inflammatory cytokines and inhibited the metabolic activity of lymphocytes. The intermediate polysaccharide chains (16–30 units) produced by time‐controlled hydrolysis of EPS increased the IL‐10 produced by macrophages. Conclusions: Polysaccharide chains of EPS induced immunosuppression by the production of macrophagic anti‐inflammatory IL‐10. Significance and impact of the Study: These results indicate that the EPS from Lact. rhamnosus RW‐9595M may be useful as a new immunosuppressive product in dairy food.     

Inhibitory effect of oral Lactobacillus against oral pathogens

Aims: To determine the inhibitory effect of oral Lactobacillus against putative oral pathogens. Methods and Results: Total 357 strains comprising 10 species of oral Lactobacillus, Lactobacillus fermentum (195), Lactobacillus salivarius (53), Lactobacillus casei (20), Lactobacillus gasseri (18), Lactobacillus rhamnosus (14), Lactobacillus paracasei (12), Lactobacillus mucosae (12), Lactobacillus oris (12), Lactobacillus plantarum (11) and Lactobacillus vaginalis (10) were used as producer strains. Inhibitory effect against a panel of indicators, periodontitis‐ and caries‐related pathogens, was assessed. Most oral Lactobacillus was able to inhibit the growth of both periodontitis‐ and caries‐related pathogens. The strongest inhibitory activity was associated with Lact. paracasei, Lact. plantarum, Lact. rhamnosus, Lact. casei and Lact. salivarius. Lactobacillus SD1–SD6, representing the six species with the strong inhibitory effect, inhibited growth of Streptococcus mutans ATCC 25175 in the biofilm model. Also, it was demonstrated that growth of Strep. mutans was inhibited in a mixture with Lact. paracasei SD1. The inhibition was enhanced in acidic condition and 5% glucose. Conclusions: The results have shown that oral Lactobacillus SD1–SD6 showed a strong inhibitory effect against Strep. mutans and Streptococcus sobrinus, as well as, Gram‐negative periodontal pathogens Porphyromonas gingivalis and Aggregatibacter actinomycetemcomitans. Significance and Impact of the Study: The results indicated that Lactobacillus may be of benefit as probiotics for the prevention of oral diseases.     

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.     

Transformation of folate-consuming Lactobacillus gasseri into a folate producer

Five genes essential for folate biosynthesis in Lactococcus lactis were cloned on a broad-host-range lactococcal vector and were transferred to the folate auxotroph Lactobacillus gasseri. As a result L. gasseri changed from a folate consumer to a folate producer. This principle can be used to increase folate levels in many fermented food products.     

Modeling the batch bacteriocin production system by lactic acid bacteria by using modified three-dimensional Lotka–Volterra equations

Different batch cultures of Lactococcus lactis CECT 539, a nisin-producing strain, were carried out in culture media prepared with whey and mussel processing wastes. From these cultures, a reasonable system of differential equations, similar to the three-dimensional Lotka–Volterra two predators-one prey model, was set up to describe, for the first time, the relationship between the absolute rates of growth, pH drop and nisin production.Thus, the nisin production system was described as a three-species (pH, biomass and nisin) ecosystem. In this case, both nisin and biomass production were considered as two pH-dependent species that compete for the nitrogen source. Excellent agreement (R² values ≥0.9885) resulted between model predictions and the experimental data, and significant values for all the model parameters were obtained. The developed model was demonstrated (R² values ≥0.9874) for five batch cultivations of the strains L. lactis CECT 539 in MRS broth and Lactobacillus sakei LB 706 (sakacin A producer), Pediococcus acidilactici LB42-923 (pediocin AcH producer), L. lactis ATCC 11454 (nisin producer) and Leuconostoc carnosum Lm1 (leuconocin Lcm1 producer) in TGE broth. These results suggest that the batch bacteriocin production system in these culture media can be successfully described by using the Lotka–Volterra approach.     

Interferon induction in murine peritoneal macrophage by stimulation with Lactobacillus acidophilus.

Induction of interferon for a kind of dairy lactic acid bacteria, Lactobacillus acidophilus (L. acidophilus), was investigated in murine peritoneal macrophage (M phi) cultures. Lactobacillus acidophilus JCM 1034, 1132T, 1229 and 2125 induced IFN (12-34 I.U./ml) in M phi cultures in vitro. Strain 1132T- and 2125-induced IFNs were characterized as IFN alpha/beta by treatment with anti-IFNs serum. The results indicate that the inducing activity of IFNs may be one of the available biological parameters for designating the dairy products containing L. acidophilus as "physiologically functional foods."     

Antimicrobial action of Lentinus edodes juice on human microflora

The action of the juice of Shiitake mushroom (L. edodes) on pathogenic and opportunistic microorganisms, detected in cases of considerable dysbiotic changes (Escherichia coli O-114, Staphylococcus aureus, Enterococcus faecalis, Candida albicans), as well as on some bacterial eubiotic producer strains (Escherichia coli M-17, Bifidobacterium spp., Lactobacillus spp.). The juice of this mushroom at a concentration of 5% from the volume of the nutrient medium was found to produce a pronounced antimicrobial effect with respect to C. albicans, S. aureus, E. faecalis, E. coli O-114 and to stimulate the growth of E. coli M-17. Bifidobacteria and lactobacteria exhibited resistance to the action of L. edodes juice.     

Purification and Production of Novel Angiotensin I-Converting Enzyme (ACE) Inhibitory Bioactive Peptides Derived from Fermented Goat Milk

International Journal of Peptide Research and Therapeutics - In the study, five different Lactobacillus cultures i.e. L. rhamnosus (NK2) (KR080695), L. casei (NK9) (KR732325), L. fermentum (M5)...     

Selection and identification of anaerobic lactobacilli producing inhibitory compounds against vaginal pathogens

Two strains of Lactobacillus crispatus (15L08 and 21L07) and one strain of Lactobacillus jensenii (5L08) were selected from amongst 100 isolates from the vaginas of healthy premenopausal women for properties relevant to mucosal colonization and the production of H2O2 and/or bacteriocin-like compound. All three strains self-aggregated and adhered to vaginal epithelial cells, displacing well-known vaginal pathogens, such as Gardnerella vaginalis and Candida albicans. Lactobacillus crispatus 15L08 was characterized as a potential H2O2 producer. A high level of bacteriocin-like compound was synthesized by L. jensenii 5L08, with a bactericidal mode of action for G. vaginalis, C. albicans and Escherichia coli. However, H2O2-dependent activity alone was not sufficient to inhibit the growth of C. albicans. Simultaneous actions of H2O2 and bacteriocin-like compound produced by lactobacilli may be important for antagonizing pathogenic bacteria. These strains of lactobacilli may be excellent candidates for eventual use as probiotics to restore the normal microbial communities in the vaginal ecosystem.     

Conjugated linoleic acid producing potential of lactobacilli isolated from the rumen of cattle

Lactobacilli isolated from the rumen of cattle were subjected to morphological and biochemical characterizations followed by PCR-based identification. Among isolates, Lactobacillus brevis was found to be the most prevalent species in the rumen. For in vitro conjugated linoleic acid (CLA) production, the two isolates of L. brevis and one each of Lactobacillus viridescens and Lactobacillus lactis were selected. The sunflower oil (i.e., 0.25, 0.5 and 1.0%; a rich source of linoleic acid) was added to skim milk as a substrate for CLA production by isolates at 37 °C/12 h. L. brevis 02 was found to be the most potential CLA producer (10.53 mg CLA/g fat) at 0.25% concentration of sunflower oil followed by L. brevis 01 (8.27 mg CLA/g fat). However, at higher level of sunflower oil (i.e., 1.0%), L. lactis was the highest CLA producer (9.22 mg/g fat) when compared to L. brevis and L. viridescens. The results indicated that L. brevis and/or CLA production was inhibited with increasing concentration of sunflower oil in skim milk. In contrast, L. lactis and L. viridescens could tolerate the increasing concentrations of sunflower oil and produced higher CLA. Overall, L. brevis extends a possibility to be used as a direct-fed microbial for ruminants to increase the CLA content in milk, however, in vivo trials are needed for validation of results obtained.