Partial characterization and plasmid linkage of a non-proteinaceous antimicrobial compound in a Lactobacillus casei strain of vegetable origin

Lactobacillus casei IMPC LC34 of vegetable origin produces a non-proteinaceous inhibitory compound with a broad spectrum of activity towards Gram-positive and Gram-negative bacteria, including pathogens. The active substance, mainly produced in the stationary phase of growth, is insensitive to proteolytic enzymes, lipase and catalase, and is stable at 121 degrees C for 30 min. The inhibitory activity was detected either at 8 degrees C or at 37 degrees C. The active compound does not contain glucidic groups, is inactivated by Na-metaperiodate, and its molecular mass is between 2000 and 5000 Da. Plasmid curing experiments showed that both antimicrobial compound immunity and production determinants were encoded by an 8.8 kbp plasmid. The effectiveness of the active agent was verified on ready-to-use vegetables, using either the Lact. casei strain or its culture supernatant fluid as inoculant, compared with cured clone. The application potential of the Lact. casei strain or its culture supernatant fluid for assuring the microbiological safety of ready-to-use vegetables is discussed.     

Characterization and species recognition of Lactobacillus plantarum strains by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene

Twenty-one strains, labelled Lactobacillus plantarum or Lact. plantarum -like, and isolated from different natural sources, were characterized by restriction fragment length polymorphism (RFLP) of the 16S rRNA gene using Hin dIII and Eco RI cleaved chromosomal DNA, together with Lact. plantarum ATCC 14917^T, Lact. pentosus ATCC 8041^T, Lact. plantarum ATCC 10776 and Lact. plantarum ATCC 8014. The fermentation patterns on API 50CH were recorded at 30°C and 37°C for all strains. The phenotypes were heterogeneous, and the ability to ferment 17 of the 49 carbohydrates varied. The fermentation of some carbohydrates, for example D-raffinose and D-arabitol, was temperature-dependent. Strains having identical API profiles were separated by the plasmid profile. All strains but one (affiliated to Lact. casei ) had identical 16S ribosomal DNA sequences ( Lact. plantarum/Lact. pentosus ). The RFLP study resulted in identical ribopatterns for 17 of the strains, including the type strain of Lact. plantarum (pattern A1). Four strains had related fragment patterns to that of Lact. plantarum sensu stricto; three of these strains had more than 60% DNA: DNA homology to the type strain of Lact. plantarum , and one had less than 50% DNA: DNA homology to Lact. plantarum ATCC 14917^T. Two strains had fragment patterns similar to the type strain of Lact. pentosus , and they had more than 80% DNA: DNA homology to Lact. pentosus ATCC 8041^T. One of the Lact. pentosus strains shared one band with the A1 pattern. The ribopatterns of Lact. plantarum were homogeneous (identical for 85% of the strains), irrespective of phenotype and source of isolation. RFLP of the 16S rRNA genes using Eco RI and Hin dIII might be used for species recognition of Lact. plantarum , but seems less suitable for strain typing.     

Identification and characterization of homofermentative mesophilic Lactobacillusstrains isolated from artisan starter-free cheeses

Fifty-six strains of mesophilic lactobacilli from hand-made cheeses made without starters havebeen isolated, identified and characterized. Of these, 21 strains were classified as Lactobacillusplantarum , 18 as Lact. casei subsp. pseudoplantarum, 10 as Lact. curvatus , five as Lact. casei subsp. casei , and two remained unidentified. The numericalclassification of these strains, based on 80 different physiological and morphologicalcharacteristics, correlated well with the phenotypic classification. Most of the technologicallyimportant traits have been examined in these strains, which will allow the selection of some ofthem to be tested as adjunct cultures in the manufacture of dairy products.     

Antagonistic Action of Lactobacilli and Bifidobacteria in Relation to Staphylococcus aureus and Their Influence on the Immune Response in Cases of Intravaginal Staphylococcosis in Mice

The antibacterial activity of Lactobacillus casei IMV B-7280, Lact. acidophilus IMV B-7279, Bifidobacterium longum VK1, and B. bifidum VK2 strains or their various compositions in relation to Staphylococcus aureus in vitro and on models of experimental intravaginal staphylococcosis of mice was determined. It was found that under the influence of these strains and their various compositions, the in vitro growth of Staph. aureus was inhibited, and the number of colonies of Staph. aureus plated from the vagina of infected mice was significantly reduced. The antibacterial activity of these strains separately and in compositions correlated with their ability to improve the performance of the immune response. These strains were the most effective in the following compositions: Lact. casei IMV B-7280-B. longum VK1-B. bifidum VK2. Strains of Lact. casei IMV B-7280, Lact. acidophilus IMV B-7279, B. bifidum VK2, and B. longum VK1 are prospective components of future probiotic drugs efficient in treating staphylococcosis and for immunity correction.     

In vitro inhibition of Helicobacter pylori NCTC 11637 by organic acids and lactic acid bacteria

In this Study the effects of both pH and organic acids on Helicobacter pylori NCTC 11637 were tested. Lactobacillus acidophilus, Lact. casei, Lact. bulgaricus, Pediococcus pentosaceus and Bifidobacterium bifidus were assayed for their lactic acid production, pH and inhibition of H. pylori growth. A standard antimicrobial plate well diffusion assay was employed to examine inhibitory effects. Lactic, acetic and hydrochloric acids demonstrated inhibition of H. pylori growth in a concentration-dependent manner with the lactic acid demonstrating the greatest inhibition. This inhibition was due both to the pH of the solution and its concentration. Six strains of Lact. acidophilus and one strain of Lact. casei subsp. rhamnosus inhibited H. pylori growth where as Bifidobacterium bifidus, Ped. pentosaceus and Lact. bulgaricus did not. Concentrations of lactic acid produced by these strains ranged from 50 to 156 mmol 1⁻¹ and correlated with H. pylori inhibition. The role of probiotic organisms and their metabolic by-products in the eradication of H. pylori in vivo remains to be determined.     

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.     

Genetic marker for differentiating beer-spoilage ability of Lactobacillus paracollinoides strains

AIMS: To determine whether the beer-spoilage ability is an intrinsic character of Lactobacillus paracollinoides and identify a genetic marker for differentiating the beer-spoilage ability of strains belonging to this species. METHODS AND RESULTS: The ribotype of a nonspoilage strain, Lact. brevis ATCC8291, was found to be identical with that of Lact. paracollinoides LA7. The 16S rDNA sequence analysis and DNA-DNA hybridization study indicates that nonspoilage ATCC8291 should belong to Lact. paracollinoides. We further isolated nonspoilage variants from Lact. paracollinoides LA2(T) and LA9 by incubating these strains at 30 degrees C. To identify a genetic marker for differentiating the beer-spoilage ability of Lact. paracollinoides, open reading frames 5 (ORF5), the previously reported genetic marker for Lact. brevis, was evaluated. As a result, ORF5 homologues were detected in all of the 12 beer-spoilage strains of Lact. paracollinoides, while this ORF was not found in ATCC8291 or the two nonspoilage variants obtained from LA2(T) and LA9. CONCLUSIONS: Lactobacillus paracollinoides is not an intrinsic beer-spoiler and the nonspoilage strain Lact. brevis ATCC8291 should be reclassified as Lact. paracollinoides. ORF5 was found to be useful for differentiating beer-spoilage ability of this species. SIGNIFICANCE AND IMPACT OF THE STUDY: The finding that Lact. paracollinoides includes nonspoilage strains necessitates brewers to use a genetic marker that is associated with the beer-spoilage ability of this 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.     

A survey of lactic acid bacteria in Italian silage

G razia , L. & S uzzi , G. 1984. A survey of lactic acid bacteria in Italian silage. Journal of Applied Bacteriology 56 , 373–379.
Lactic acid bacteria, isolated from Italian ensiled products, were represented by strains of the genera Lactobacillus and Leuconostoc . The predominant strains were heterofermentative lactobacilli, with Lactobacillus buchneri being the most frequent. Among homofermentative lactic acid bacteria, strains of Lact. plantarum and Lact. casei were recovered. Almost all strains utilized malic acid and showed good acid-tolerance, but only some of them were able to metabolize malic acid at extremely low pH; these were five homofermentative lactobacilli (4 Lact. plantarum and 1 Lacr. casei var. casei ) and two heterofermentative lactobacilli ( Lact. cellobiosus and Lactobacillus sp.).     

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.     

Altering the composition of caseicins A and B as a means of determining the contribution of specific residues to antimicrobial activity

Caseicin A (IKHQGLPQE) and caseicin B (VLNENLLR) are antimicrobial peptides generated through the bacterial fermentation of sodium caseinate, and on the basis of this and previous studies, they are active against many Gram-negative pathogens (Cronobacter sakazakii, Cronobacter muytjensii, Salmonella enterica serovar Typhimurium, Escherichia coli, Klebsiella pneumoniae, and Pseudomonas fluorescens) as well as the Gram-positive organism Staphylococcus aureus. Here we describe further studies with the aim of establishing the importance of specific (charged and nonpolar aliphatic) residues within the caseicin peptides and the effects that they have on the bacteria listed above. In order to achieve our objective, we created four derivatives of each caseicin (A1 to A4 and B1 to B4) in which specific residues were altered, and results obtained with these derivatives were compared to wild-type caseicin activity. Although conversion of cationic residues to alanine in caseicins B1 (R8A change), A1 (K2A), A2 (H3A), and A3 (K2A-H3A) generally resulted in their activity against microbial targets being reduced or unaltered, C. sakazakii DPC6440 was unusual in that it displayed enhanced sensitivity to three peptides (caseicins A1, A3, and B2) in which positively charged residues had been eliminated. While the replacement of leucine with alanine in selected variants (B3 and B4) resulted in reduced activity against a number of strains of Cronobacter and, in some cases, S. Typhimurium, these changes enhanced the activities of these peptides against DPC6440 and a number of S. aureus strains. It is thus apparent that the importance of specific residues within the caseicin peptides is dependent on the strain being targeted.     

Specific enumeration of lactic acid bacteria in fermenting grape must and wine by colony hybridization with non-isotopic DNA probes

A. LONVAUD-FUNEL, A. JOYEUX AND O. LEDOUX. 1991. Total DNA extracted from lactic acid bacteria commonly found in musts and wines was randomly labelled with digoxigenin. It was assayed for the detection of several species by dot-blot hybridization. The method proved to be specific as there was no cross-hybridization between most of the species belonging to the genera Leuconostoc, Pediococcus and Lactobacillus , homofermentative and heterofermentative ( Lact. plantarum, Lact. casei, Leuc. mesenteroides, Leuc. oenos, Ped. damnosus, Ped. pentosaceus ). However, it failed for some Lact. brevis strains which strongly hybridized with Lact. hilgardii.
Colony hybridization was performed directly on plates soon after enumeration. Eight probes of the most common species were used; it was possible to follow the evolution of each species during the vinification of two red wines. According to the phase of alcoholic fermentation, then malolactic fermentation, the predominance or regression of bacilli and cocci could be established.     

Isolation, properties and behaviour of tyramine-producing lactic acid bacteria from wine

Wines containing high levels of biogenic amines were investigated for the presence of tyramine-producing strains. Two different Lactobacillus brevis (IOEB 9809 and IOEB 9901) able to produce the amine were isolated. None of the isolated strains identified as Oenococcus oeni formed tyramine. In addition, other Lact. brevis and Lact. hilgardii strains from our collection (IOEB) and the American Type Culture Collection (ATCC) were strong tyramine producers. Lactobacillus brevis IOEB 9809 and Lact. hilgardii IOEB 9649 were found to produce tyramine and phenylethylamine simultaneously. The conditions that can influence tyramine formation in wine were evaluated for three strains of Lact. brevis (IOEB 9809 and IOEB 9901) and Lact. hilgardii (IOEB 9649). Tyrosine was the major factor affecting tyramine formation and was enhanced by the presence of sugars, mainly glucose. Tyrosine decarboxylase (TDC) activity greatly depended on the presence of the precursor, which suggested that tyrosine induced the TDC system. These results indicate that Lactobacillus could be the lactic acid bacteria responsible for tyramine production in wine.     

The antimicrobial effect of organic acids, sour dough and nisin against Bacillus subtilis and B. licheniformis isolated from wheat bread

Prevention of growth in wheat bread for more than 6 d of approximately 10⁶ rope-producing Bacillus subtilis spores per gram of dough was achieved by addition of propionic or acetic acids at levels of 0·10% v/w (based on flour weight), or by addition of 15% sour dough fermented with Lactobacillus plantarum C11, Lact. brevis L62, Lact. plantarum ('vege-start 60'), Lact. plantarum (ch 20), Lact. maltaromicus (ch 15), or the commercial sour dough starter culture, Lact. sanfrancisco L99. These cultures resulted in an amount of total titratable acids above 10 in the sour dough and a pH value below 4·8 in the final bread. Bacteriocin-producing lactic acid bacteria added as starter cultures in wheat dough and nisin (Nisaplin) at levels up to 100 p.p.m. g⁻¹ flour had no effect against B. subtilis and B. licheniformis strains, despite the fact that nisin-producing strains of Lactococcus lactis ssp. lactis among 186 strains of lactic acid bacteria had demonstrated inhibitory activity against B. subtilis and B. licheniformis in an agar spot assay.     

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.     

The effect of chlorogenic, gallic and quinic acids on the growth of spoilage strains of Lactobacillus collinoides and Lactobacillus brevis

The naturally occurring complex organic acids, chlorogenic acid, gallic acid and quinic acid, at concentrations of 100, 500 and 1000 mg l^-1 were evaluated for effects on the growth of three spoilage strains of Lactobacillus collinoides and one of Lact. brevis in acid tomato broth containing 5% (v/v) ethanol at pH 4.8. During early stages of growth, all the complex acids at each concentration stimulated growth of Lact. collinoides but not of Lact. brevis. During stationary phase, chlorogenic and gallic acids produced greater cell densities of all strains, whereas quinic acid generally had less effect. The presence of these complex acids in fruit products may increase the requirement for added preservative in order to prevent spoilage by certain strains of lactic acid bacteria.     

Arabinose fermentation by Lactobacillus plantarum in sourdough with added pentosans and alphaalpha-L-arabinofuranosidase: a tool to increase the production of acetic acid

Sixty-five strains of obligately and facultatively heterofermentative sourdough lactic acid bacteria were screened for their capacity to grow optimally in the presence of arabinose, ribose and xylose as carbon sources. Lactobacillus alimentarius 15F, Lact. brevis 10A, Lact. fermentum 1F and Lact. plantarum 20B showed higher growth rate, cell yield, acidification rate and production of acetic acid when some pentoses instead of maltose were added to the SDB medium. Lactobacillus plantarum 20B used arabinose also in a synthetic medium where complex growth factors such as yeast extract were omitted. Other Lact. plantarum strains did not show the same property. Pentosan extract was treated with alpha-L-arabinofuranosidase from Aspergillus niger or endo-xylanase from Bacillus subtilis to produce hydrolysates containing mainly arabinose and xylose, respectively. In particular, the hydrolysate containing arabinose substantiated the growth and the production of lactic acid and, especially, of acetic acid by Lact. plantarum 20B. Sourdough fermentation by Lact. plantarum 20B with addition of pentosan extract and alpha-L-arabinofuranosidase increased the acidification rate, titratable acidity and acetic acid content compared with traditional sourdough. A facultatively heterofermentative strain, Lact. plantarum 20B, also produced a sourdough with an optimal fermentation quotient.     

Rapid identification of Lactobacillus brevis using the polymerase chain reaction

AIMS: Species-specific PCR was applied to identify Lactobacillus brevis and the sensitivity and the specificity of the protocol were determined. METHODS AND RESULTS: Strains of Lact. brevis obtained from foods, particularly dairy products, and various strain collections, were identified by PCR using primers which amplified a 1340 bp fragment within the 16S rRNA gene. The PCR product was obtained after amplification of all the Lact. brevis strains tested; the size of the amplicon was as expected. No PCR products were observed after amplification from DNA of several lactic acid bacteria (LAB) species. CONCLUSIONS: A PCR method was optimized to identify Lact. brevis. The protocol was highly efficient and sensitive. SIGNIFICANCE AND IMPACT OF THE STUDY: Conventional phenotypic methods often lead to ambiguous identification of LAB species belonging to Lact. brevis. The proposed protocol is sensitive, specific, and can be applied to total DNA extracted by use of chelating matrix with loss of neither sensitivity nor specificity.     

The effect of hydroxycinnamic acids on the growth of wine-spoilage lactic acid bacteria

Hydroxycinnamic acids and their derivatives occur naturally in grape juice and wine. To assess their potential as natural preservatives the effect of caffeic, coumaric and ferulic acids on the growth of three wine-spoilage strains of Lactobacillus collinoides and one of Lact. brevis was studied in acid tomato broth containing 5% ethanol at pH 4.8. At concentrations of 500 and 1000 mg l^-1, all three compounds markedly inhibited growth; coumaric and ferulic acids were more effective than caffeic acid. At a concentration of 100 mg l^-1, all compounds stimulated growth. In general, the strains of Lact. collinoides were more susceptible both to inhibition and stimulation by the hydroxycinnamic acids than was the strain of Lact. brevis. The possible influence of hydroxycinnamic acids on the malolactic fermentation of wine is discussed.     

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.