Antibacterial activity of Lactobacillus strains isolated from dry fermented sausages

One hundred strains of lactic acid bacteria isolated from dry cured sausages were tested for antagonistic activity against a set of test strains. Nine of 52 strains of Lactobacilus casei and three of 48 strains of Lact. plantarun produced inhibition zones against the indicator species. The substance excreted by Lact. casei CRL 705 was active against Lact. plantarum, Listeria monocytogenes, Staphylococcus aureus and a wide range of Gram-negative bacteria. The activity of the antibacterial compound from Lact. casei CRL 705 was destroyed by papain, trypsin and pepsin, but was resistant to heat (100°C for 20 min), lysozyme and catalase. The agent was produced during the growth cycle and when the concentrated and neutralized supernatant fluid was added to a fresh culture of sensitive cells it produced a rapid inactivation. A decrease in optical density (O.D.) over time, indicative of cell lysis, was also observed. These characteristics allowed us to identify the inhibitory compound as a bacteriocin which we termed Lactocin 705.     

Partial characterization of a bacteriocin produced by Lactobacillus helveticus

AIMS: To investigate the antimicrobial activity of a strain of Lactobacillus helveticus. METHODS AND RESULTS: The culture supernatant fluid Lact. helveticus G51 showed antimicrobial activity against thermophilic strains of Lactobacillus. Purification of the active compound was achieved after gel filtration and ion exchange chromatography. As revealed by SDS-PAGE, active fractions were relatively homogeneous, showing a protein with a molecular mass of 12.5 kDa. The antimicrobial compound was heat labile, inactivated by proteolytic enzymes and had a bactericidal mode of action. CONCLUSION: The antimicrobial activity expressed by Lact. helveticus G51 was correlated with the production of a bacteriocin with properties that were different to other helveticins. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has provided further data on Lact. helveticus bacteriocins. The strong activity of the bacteriocin towards various thermophilic lactobacilli warrants further investigation for its potential to obtain attenuated cultures for the enhancement of the cheese-ripening process.     

Antibacterial polypeptides of Lactobacillus species

Twelve of 79 strains of the genus Lactobacillus , mainly isolated from plants or fermenting material, were found to inhibit at least one of the nine indicator strains of the species Lact. brevis, Pediococcus damnosus and Leucanostoc oenos . The antimicrobial activities from Lact. brevis B 37 and Lact. casei B 80 were caused by polypeptides detectable in the culture liquids. They are bacteriocins with a narrow antimicrobial spectrum. Brevicin 37 from Lact. brevis B 37 was active against many lactic acid bacteria and Nocardia corallina , whereas caseicin 80 from Lact. casei B 80 inhibits only one other strain of Lact. casei . Brevicin 37 is stable at 121C, caseicin 80 is inactivated above 60C, and both are inactivated under alkaline conditions.     

Identification and characterization of helveticin V-1829, a bacteriocin produced by Lactobacillus helveticus 1829.

Lactobacillus helveticus 1829 produced an antimicrobial agent, designated helveticin V-1829, that demonstrated antagonistic activity against closely-related species. The agent was excreted into MRS agar, and was present in the supernatant fluids from both overnight broth and clotted milk cultures. It was heat labile (inactivated by 50 degrees C for 30 min) and was stable over the pH range 2.5 to 6.5. Production of the substance was pH-dependent and maximum yields were obtained in MRS broth cultures maintained at pH 5.5. Helveticin V-1829 was partially purified following growth of the producing strain in a semi-defined MRS medium and precipitating the cell-free filtrate with ammonium sulphate to 30% saturation. The cleared supernatant fluid was then brought to 60% saturation and the resulting precipitate pelleted and dialysed in 0.3 mol/l phosphate buffer. The partially purified inhibitor was sensitive to several proteolytic enzymes, and it was bactericidal in its mode of action against indicator cells of Lact. helveticus 1844 and Lact. delbrueckii subsp. bulgaricus 1489, indicating that it was a bacteriocin. A DNA probe specific for the helveticin J structural gene failed to hybridize to total genomic DNA of Lact. helveticus 1829, indicating that helveticin V-1829 is not significantly related to helveticin J.     

Application of antimicrobial-producing lactic acid bacteria to control pathogens in ready-to-use vegetables

Five psychrotrophic strains of lactic acid bacteria (Lactobacillus casei, Lact. plantarum and Pediococcus spp.) were isolated from 22 samples of commercial salads. These strains were shown to inhibit Aeromonas hydrophila, Listeria monocytogenes, Salmonella typhimurium and Staphylococcus aureus on MRS agar, in salads and in juice prepared from vegetable salads. Lactobacillus casei IMPCLC34 was most effective in reducing total mesophilic bacteria and the coliform group; Aer. hydrophila, Salm. typhimurium and Staph. aureus disappeared after 6 d of storage, while the counts for L. monocytogenes remained constant. The potential application of antimicrobial-producing lactic acid bacteria as biopreservatives of ready-to-use vegetables is suggested.     

Survivability of a probiotic Lactobacillus casei in the gastrointestinal tract of healthy human volunteers and its impact on the faecal microflora

AIM: The aim of this study was to measure the gastrointestinal survival of Lactobacillus casei and its impact on the gut microflora in healthy human volunteers. METHODS AND RESULTS: Twenty healthy volunteers took part in a double-blind placebo-controlled probiotic feeding study (10 fed probiotic, 10 fed placebo). The probiotic was delivered in two 65 ml aliquots of fermented milk drink (FMD) daily for 21 days at a dose of 8.6 +/- 0.1 Log(10)Lact. casei CFU ml(-1) FMD. Faecal samples were collected before, during and after FMD or placebo consumption, and important groups of faecal bacteria enumerated by fluorescent in situ hybridization (FISH) using oligonucleotide probes targeting the 16S rRNA. The fed Lact. casei was enumerated using selective nutrient agar and colony identity confirmed by pulsed field gel electrophoresis. Seven days after ingestion of FMD, the Lact. casei was recovered from faecal samples taken from the active treatment group at 7.1 +/- 0.4 Log(10) CFU g(-1) faeces (mean +/- SD, n = 9) and numbers were maintained at this level until day 21. Lact. casei persisted in six volunteers until day 28 at 5.0 +/- 0.9 Log(10) CFU g(-1) faeces (mean +/- SD, n = 6). Numbers of faecal lactobacilli increased significantly upon FMD ingestion. In addition, the numbers of bifidobacteria were higher on days 7 and 21 than on days 0 and 28 in both FMD fed and placebo fed groups. Consumption of Lact. casei had little discernible effect on other bacterial groups enumerated. CONCLUSIONS: Daily consumption of FMD enabled a probiotic Lact. casei strain to be maintained in the gastrointestinal tract of volunteers at a stable relatively high population level during the probiotic feeding period. SIGNIFICANCE AND IMPACT OF THE STUDY: The study has confirmed that this probiotic version of Lact. casei survives well within the human gastrointestinal tract.     

Characterization of pentocin TV35b, a bacteriocin-like peptide isolated from Lactobacillus pentosus with a fungistatic effect on Candida albicans

Lactobacillus pentosus TV35b, isolated from the posterior fornix secretions of the vagina of a prenatal patient, produced a bacteriocin-like peptide (pentocin TV35b), which is inhibitory to Clostridium sporogenes, Cl. tyrobutyricum, Lact. curvatus, Lact. fermentum, Lact. sake, Listeria innocua, Propionibacterium acidipropionici, Propionibacterium sp. and Candida albicans. The mechanism of activity of pentocin TV35b is bactericidal, as shown by a decrease in the viable cell numbers of Lact. sake from approximately 4 x 108 to less than 10 cfu ml - 1 over a period of 4 h. Pentocin TV35b added to the growth medium of C. albicans stimulated the formation of pseudohyphae during the first 36 h, followed by a slight repression in cell growth. Production of pentocin TV35b was at its maximum towards the end of the logarithmic growth phase of strain TV35b. The peptide was purified by ammonium sulphate precipitation, followed by SP-Sepharose cation exchange chromatography. The molecular size of pentocin TV35b was estimated to be between 2.35 and 3.4 kDa, according to tricine-SDS PAGE. However, results obtained by electrospray ionization mass spectroscopy indicated that the peptide is 3930.2 Da in size. Amino acid analysis performed by using the Pico-Tag(R) method and a Nova-Pak C18 HPLC column indicated that pentocin TV35b consists of 33 amino acids with a total mass of 3929.63 Da. Pentocin TV35b is inactivated when treated with papain and Proteinase K, but remains active after incubation at pH 1-10 for 2 h at 25 degrees C, and when heat-treated for 30 min at 100 degrees C.     

In vitro growth control of selected pathogens by Lactobacillus acidophilus- and Lactobacillus casei-fermented milk

AIMS: Food-borne pathogen inhibition was tested in the presence of a mixture of Lactobacillus acidophilus and Lactobacillus casei during fermentation under controlled pH conditions. METHODS AND RESULTS: The growth of Escherichia coli O157:H7, Salmonella serotype Typhimurium, Staphylococcus aureus, Listeria innocua, Enterococcus faecium and Enterococcus faecalis was evaluated for 48 h at 37 degrees C. In the presence of the lactic acid bacteria (LAB), an increase of the generation time was observed for all the gram-positive bacteria evaluated. Staphylococcus aureus was the most sensitive strain showing an increase of the generation time by 210%. However, for all the gram-negative bacteria evaluated, no inhibition occurred after 8 h of fermentation. The soluble portion of Lact. acidophilus- and Lact. casei-fermented milk was recuperated and tested for its antimicrobial activity. Listeria innocua and Staph. aureus were the most sensitive to the presence of fermented milk supernatant showing an inhibition of 85.9% and 84.7%, respectively. This soluble fraction was neutralized to eliminate the antimicrobial effect of the organic acids produced; the most sensitive strains were L. innocua and E. coli O157:H7 showing an inhibition of 65.9% and 61.9%, respectively. Finally, the soluble fraction was neutralized and irradiated at 45 kGy using a (60)Co source to eliminate the possible antimicrobial effect of both organic acids and bacteriocin-like substances. Enterococcus faecalis, E. coli O157:H7 and Staph. aureus were the most affected bacteria by this fraction, showing 39.1, 32 and 31.2% inhibition, respectively. CONCLUSIONS: The results obtained in this study suggest the implication of both organic acids and bacteriocin-like inhibitory substances in the antimicrobial activity observed in the soluble fraction of the probiotic preparation. SIGNIFICANCE AND IMPACT OF THE STUDY: This study revealed the antimicrobial mechanisms of action of Lact. acidophilus- and Lact. casei-fermented milk used to prevent antibiotic-associated diarrhoea.     

Anti-bacterial activity of Lactobacillus plantarum strain SK1 against Listeria monocytogenes is due to lactic acid production

AIMS: The aim of this research was to investigate the potential of Lactobacillus plantarum strain SK1 for use as a biological control agent against Listeria monocytogenes and determine its mechanism of anti-listerial activity. METHODS AND RESULTS: Co-growth of Lact. plantarum SK1 and L. monocytogenes UMCC98 in MRS broth showed that anti-listerial activity of Lact. plantarum SK1 occurred during late log/early stationary phase of growth. This coincided with a reduction in broth pH to 4.26. Evidence obtained from the analysis of cell-free culture filtrates of strain SK1 grown in MRS broth using thin-layer chromatography and growth of L. monocytogenes in pH-adjusted culture filtrates suggested that the anti-listerial activity was due to lactic acid production alone. Trials of Lact. plantarum SK1 on radishes stored at 5 degrees C showed that it had statistically significant (P < 0.05) anti-listerial activity. CONCLUSIONS: The anti-listerial activity of Lact. plantarum SK1 was due to lactic acid production alone. A small-scale trial on radishes stored at 5 degrees C showed it to have significant anti-listerial activity in planta. SIGNIFICANCE AND IMPACT OF THE STUDY: This organism has potential as a biological control agent for L. monocytogenes.     

Succinate production and citrate catabolism by Cheddar cheese nonstarter lactobacilli

AIMS: To identify strains of Cheddar cheese nonstarter lactobacilli that synthesize succinate from common precursors and characterize the biochemical pathways utilized. METHODS AND RESULTS: Whole cell incubations of Lactobacillus plantarum, Lactobacillus casei, Lactobacillus zeae and Lactobacillus rhamnosus, were used to identify strains that accumulated succinate from citrate, l-lactate, aspartic acid or isocitrate. In vivo 13C-nuclear magnetic resonance spectroscopy (13C-NMR) identified the biochemical pathway involved at pH 7.0, and under conditions more representative of the cheese ripening environment (pH 5.1/4% NaCl/13 degrees C). Enzyme assays on cell-free extracts were used to support the pathway suggested by 13C-NMR. CONCLUSIONS: The Lact. plantarum strains studied synthesize succinate from citrate by the reductive tricarboxylic acid (TCA) cycle at either pH 7.0 or pH 5.1/4% NaCl/13 degrees C. Lactobacillus casei, Lact. zeae and Lact. rhamnosus strains lack one or more enzymatic activities present in this pathway, and do not accumulate succinate from any of the four precursors studied. SIGNIFICANCE AND IMPACT OF THE STUDY: The addition of Lact. plantarum strains to milk during cheese manufacture may increase the accumulation of the flavour enhancer succinate.     

Purification and Partial Characterization of Novel Bacteriocin L23 Produced by <Emphasis Type="Italic">Lactobacillus</Emphasis><Emphasis Type="Italic">fermentum L</Emphasis>23

Lactobacillus fermentum strain L23 produced a small bacteriocin, designated bacteriocin L23, with an estimated molecular mass of < 7000 Da. Isolation, purification, and partial characterization of bacteriocin L23 are described. It displayed a wide inhibitory spectrum including both Gram-negative and Gram-positive pathogenic strains and two species of Candida. The antibacterial activity of cell-free culture supernatant fluid was not affected by catalase or urease but was abolished by the proteolytic enzymes trypsin and protease VI. Bacteriocin L23 was heat stable (60 min at 100°C) and showed inhibitory activity over a wide pH range (4.0 to 7.0). The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin sample was prepared by a process of ammonium sulfate precipitation, gel filtration, thin-layer chromatography, bioautography, and reversed-phase HPLC.     

Bacteriocinogenic activity of lactobacilli from fermented sausages

During the screening of the inhibitory activity of 254 strains of lactobacilli isolated from fermented sausages at different times of ripening, 22% of the strains showed inhibition that was not related to acid or hydrogen peroxide, towards one or more indicator strains. Not all the strains were capable of secreting the inhibitory compound in the supernatant fluid. The characterization of the inhibitory compound from three strains showed that they were bacteriocins with a bactericidal mode of action and a molecular weight exceeding 10000 Da. Lactobacillus plantarum CTC 305, CTC 306 and Lact. sake CTC 372 inhibited Listeria monocytogenes. Lactobacillus sake CTC372 was cured of two plasmids of 84.8 kbp and 41.3 kbp, losing the production and the immunity of a bacteriocin as well as the ability to ferment lactose.     

Purification and characterization of a bacteriocin produced by Lactobacillus acidophilus IBB 801

Lactobacillus acidophilus IBB 801 produces a small bacteriocin, designated acidophilin 801, with an estimated molecular mass of less than 6.5 kDa. It displays a narrow inhibitory spectrum (only related lactobacilli but including the Gram-negative pathogenic bacteria Escherichia coli Row and Salmonella panama 1467) with a bactericidal activity. The antimicrobial activity of cell-free culture supernatant fluid was insensitive to catalase but sensitive to proteolytic enzymes such as trypsin, proteinase K and pronase, heat-stable (30 min at 121 degrees C), and maintained in a wide pH range. The proteinaceous compound was isolated from cell-free culture supernatant fluid and purified. Crude bacteriocin was isolated as a floating pellicle after ammonium sulphate precipitation (40% saturation) and partially purified by extraction/precipitation with chloroform/methanol (2/1, v/v). Further purification to homogeneity was performed by reversed phase Fast Performance Liquid Chromatography. The amino acid composition was determined. Amino acid sequencing revealed that the N-terminal end was blocked.     

Inhibitory effect of selected lactic acid bacteria on microflora associated with ready-to-use vegetables

The addition of selected lactic acid bacteria strains had a remarkable inhibitory effect on the growth dynamics of microflora associated with ready-to-use vegetables, during refrigerated storage. In particular, coliforms and enterococci were strongly reduced or eliminated from the products from the third day of storage. Lactobacillus casei strains proved more effective than pediococci. The use of lactic cultures able to produce bacteriocins and to grow at low temperatures could be a useful tool to preserve fresh vegetables and to ensure their microbiological safety.     

The presence of prtP proteinase gene in natural isolate Lactobacillus plantarum BGSJ3–18

Aims:  The study of proteolytic activity and examination of proteinase gene region organization in proteolytically active Lactobacillus plantarum strains from different natural sources.
Methods and Results:  A set of 37 lactobacilli was distinguished by using multiplex PCR assay. Results showed that 34 strains were Lact. plantarum and three of them were Lact. paraplantarum . The examination of proteolytic activity revealed that 28 Lact.   plantarum and two Lact.   paraplantarum hydrolyse β-casein. Further analyses of all proteolytically active Lact. plantarum with primers specific for different types of CEPs demonstrated that strain BGSJ3–18 has prtP catalytic domain as well as prtP – prtM intergenic region showing more than 95% sequence identity with the same regions present in Lact. paracasei , Lact. casei and L. lactis . No presence of prtB , prtH or prtR proteinase genes was detected in any of tested Lact. plantarum strains.
Conclusions:  One out of 28 analysed Lact. plantarum strains harbours the prtP -like gene. The other proteolytically active Lact. plantarum probably possesses a different type of extracellular proteinase(s).
Significance and Impact of the Study:  It is the first report about the presence of the prtP –like gene in Lact. plantarum , which illustrates the mobility of this gene and its presence in different species.     

Lactose metabolism and lactase gene sequence homologies amongst lactobacilli

A number of strains of Lactobacillus spp., including the thermophilic and mesophilic dairy species, were screened for the presence of β -galactosidase ( β -gal) and phospho- β -galactosidase (pbg) enzyme activities. The majority of lactose fermenting strains exhibited β -gal rather than pbg enzyme activity with the highest levels in the thermophilic dairy species.
Correlation between these enzymes and the presence of specific genetic determinants was sought using probes for β -gal and pbg genes from Lactobacillus casei ssp. casei strain 64H. Southern transfer and filter hybridization showed that the β-gal probe shared homology with one strain of Lact. casei ssp. casei only. Sequences homologous to the pbg gene were detected only in plasmid DNA from the same strain of Lact. casei ssp. casei and with plasmid DNA from an apparently unrelated strain of Lactobacillus which exhibited no pbg activity. Two other strains of Lact. casei ssp. casei appeared to show homology between their chromosomal DNA and the pbg gene probe. No other homologies were detected. Therefore, although lactase activity could be detected in many strains of Lactobacillus spp., the genetic determinants involved did not share extensive homology.     

Identification and characterization of helveticin V-1829, a bacteriocin produced by Lactobacillus helveticus 1829

Lactobacillus helveticus 1829 produced an antimicrobial agent, designated helveticin V-1829, that demonstrated antagonistic activity against closely-related species. The agent was excreted into MRS agar, and was present in the supernatant fluids from both overnight broth and clotted milk cultures. It was heat labile (inactivated by 50°C for 30 min) and was stable over the pH range 2.5 to 6.5. Production of the substance was pH-dependent and maximum yields were obtained in MRS broth cultures maintained at pH 5.5. Helveticin V-1829 was partially purified following growth of the producing strain in a semi-defined MRS medium and precipitating the cell-free filtrate with ammonium sulphate to 30% saturation. The cleared supernatant fluid was then brought to 60% saturation and the resulting precipitate pelleted and dialysed in 0.3 mol/l phosphate buffer. The partially purified inhibitor was sensitive to several proteolytic enzymes, and it was bactericidal in its mode of action against indicator cells of Lact. helveticus 1844 and Lact. delbrueckii subsp. bulgaricus 1489, indicating that it was a bacteriocin. A DNA probe specific for the helveticin J structural gene failed to hybridize to total genomic DNA of Lact. helveticus 1829, indicating that helveticin V-1829 is not significantly related to helveticin J.     

Factors affecting production of an antilisterial bacteriocin by Carnobacterium piscicola strain A9b in laboratory media and model fish systems

AIMS: To investigate factors influencing bacteriocin production and bacteriocin stability of the bioprotective culture Carnobacterium piscicola strain A9b. METHODS AND RESULTS: Maximum activity was obtained in MRS7 broth (MRS adjusted to pH 7.2), with or without glucose. No bacteriocin was produced in APT broth when a low inoculum level (0.001%) was used. In contrast, inoculum level did not influence bacteriocin production in BHI and MRS7 without glucose. Bacteriocin production in APT was induced by the presence of an extracellular compound present in the sterile, filtered, cell-free supernatant fluid of a stationary-phase culture. Increasing concentrations of NaCl (2-7%) reduced bacteriocin production and maximum cell density of C. piscicola A9b when grown in cooked fish juice at 4 degrees C. CONCLUSION: Media composition, inoculum level and sodium chloride concentration affected production. SIGNIFICANCE AND IMPACT OF THE STUDY: The influence of NaCl on bacteriocin production may negate the inhibitory effect of C. piscicola A9b against Listeria monocytogenes in salty foods.     

Plantaricins S and T, Two New Bacteriocins Produced by Lactobacillus plantarum LPCO10 Isolated from a Green Olive Fermentation

Twenty-six strains of Lactobacillus plantarum isolated from green olive fermentations were tested for cross-antagonistic activities in an agar drop diffusion test. Cell-free supernatants from four of these strains were shown to inhibit the growth of at least one of the L. plantarum indicator strains. L. plantarum LPCO10 provided the broadest spectrum of activity and was selected for further studies. The inhibitory compound from this strain was active against some gram-positive bacteria, including clostridia and propionibacteria as well as natural competitors of L. plantarum in olive fermentation brines. In contrast, no activity against gram-negative bacteria was detected. Inhibition due to the effect of organic acids, hydrogen peroxide, or bacteriophages was excluded. Since the inhibitory activity of the active supernatant was lost after treatment with various proteolytic enzymes, this substance could be classified as a bacteriocin, designated plantaricin S. Plantaricin S was also sensitive to glycolytic and lipolytic enzymes, suggesting that it was a glycolipoprotein. It exhibited a bactericidal and nonbacteriolytic mode of action against indicator cells. This bacteriocin was heat stable (60 min at 100 degrees C), active in a pH range of 3.0 to 7.0, and also stable in crude culture supernatants during storage. Ultrafiltration studies indicated that plantaricin S occurred as multimolecular aggregates and that the size of the smallest active form is between 3 and 10 kDa. In sodium dodecyl sulfate-polyacrylamide gels, plantaricin S migrated as a peptide of ca. 2.5 kDa. Maximum production of plantaricin S was obtained in a fermentor system in unregulated pH and log-phase cultures of L. plantarum LPCO10 in MRS broth plus 4% NaCl. In these culture conditions, a second bacteriocin (designated plantaricin T) was produced in late-stationary-phase cultures of L. plantarum LPCO10. On the basis of its biological activity, its sensitivity to various enzymes, and its molecular weight (lower than that of plantaricin S) as assessed in sodium dodecyl sulfate-polyacrylamide gel electrophoresis, plantaricin T appeared different from plantaricin S. Curing experiments with L. plantarum LPCO10 resulted in the appearance of variants that no longer produced either of the two bacteriocins but that were still immune to both of them.     

Purification and characterization of a novel type of fimbriae from the oral anaerobe Bacteroides gingivalis.

Fimbriae and their constituent protein (fimbrilin) were purified to homogeneity from the bacterial wash fluid and cell lysate fraction, respectively, of Bacteroides gingivalis 381. Fimbriae, observed by negative staining, were curly, single-stranded filaments with a diameter of ca. 5 nm. The apparent molecular weight of the fimbrilin was 43,000. Fimbriae were resistant to sodium dodecyl sulfate denaturation at 70 degrees C. Heating at 100 degrees C in sodium dodecyl sulfate was needed to completely dissociate them to monomers of fimbrilin. Different sets of antigenic determinants seemed to be exposed on the surfaces of fimbriae and sodium dodecyl sulfate-denatured fimbrilin. Purified fimbriae did not show either hemagglutinating activity or hemagglutination inhibitory activity, although it has been inferred on the basis of circumstantial evidence that fimbriae are correlated to hemagglutinating activity of the organism. Hemagglutinin activity, however, was detected in culture supernatant, and this observation suggests that fimbriae of a different type or a lectin-like protein may be acting as hemagglutinin in B. gingivalis.