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PubMed2006

Lactobacillus casei CRL 431 and Lactobacillus rhamnosus CRL 1224 as biological controls for Aspergillus flavus strains.

The effect of two species of lactobacilli, Lactobacillus casei CRL 431 and Lactobacillus rhamnosus CRL 1224, on growth of different Aspergillus flavus strains was determined. A. flavus strains (Ap, TR2, or CF80) were grown in LAPTg broth at 37 degrees C for 7 days as a single culture and in association with L. casei CRL 431 or L. rhamnosus CRL 1224 at initial inoculum ratios of 1:1, 1:10, and 1:100. In most cases, the mixed cultures had a lower fungal growth and a lower pH than the control cultures. Mycelial dry weight was reduced to 73 and 85% using L. casei CRL 431 and L. rhamnosus CRL 1224, respectively. The pH decrease in mixed cultures when the fungal mycelial dry weight is reduced may play an important role in inhibition. The number of viable bacteria was variably affected by fungal growth. These results indicate that L. casei CRL 431 and L. rhamnosus CRL 1224 may be useful as potential biocontrol agent against A. flavus.

Antibiosis↗
PubMed1977

Comparative studies of lactic acid dehydrogenases in lactic acid bacteria. I. Purification and kinetics of the allosteric L-lactic acid dehydrogenase from Lactobacillus casei ssp. casei and Lactobacillus curvatus.

The stability, pH-dependence and kinetic properties of the Mn2+ and FDP-activated NAD-dependent lactic acid dehydrogenases from Lactobacillus casei ssp. casei (ATCC 393) and L. curvatus (DSM 20010) were studied after the enzymes were purified to homogeneity by affinity chromatography. Both enzymes are virtually unidirectional, catalysing efficiency only the reduction of pyruvate. They are similar with respect to the effector requirement and pH-optimum. They differ, however, in their electrophoretic mobility, heat stability, pH-dependence of the Mn2+ requirement and several kinetic properties. It is suggested that most of these differences are caused by differences of the negative charges in the vicinity of the FDP-binding site or the site responsible for the interaction of the subunits of the enzymatically active oligomeres.

Allosteric Regulation↗
PubMed2005

Lactobacillus harbinensis sp. nov., consisted of strains isolated from traditional fermented vegetables 'Suan cai' in Harbin, Northeastern China and Lactobacillus perolens DSM 12745.

Taxonomical analysis of two genetically distinguished Lactobacillus strains isolated from traditional Chinese fermented vegetables 'Suan cai' was performed. They formed L-lactate from glucose, were facultatively heterofermentative, and had a DNA G+C content of 53-54mol%. They fermented D- and L-arabinose. They produced lactate, ethanol and acetate from gluconate at a molar ratio of 1.1:0.4:0.7. Phylogenetic analysis of 16S rDNA revealed that the two strains were closely related to L. perolens. DNA-DNA hybridization analysis revealed that the two strains were different from L. perolens type strain DSM 12744 and formed a separate cluster with L. perolens DSM 12745. G + C molar content of DNA of the former is 51%, whereas those of the latter strains were in the range of 53-54%. Based on the results, we propose that the new species be named L. harbinensis sp. nov. and that L. perolens DSM 12745 be reclassified as L. harbinensis DSM 12745. The type strain of L. harbinensis DSM 16991T (= AHU 1762T = SBT 10908T).

Acetates↗
PubMed2001

Effect of different cultivation conditions on Lactobacillus manihotivorans OND32T, an amylolytic lactobacillus isolated from sour starch cassava fermentation.

Study of the cassava sour starch fermentation has led to the isolation of a new homofermentative amylolytic lactic acid bacterium, Lactobacillus manihotivorans OND32T, whose nutritional requirements have been investigated in this work. The main effect of deleting one of the substrate components of the MRS-starch medium was to reduce the amylase production. When starch fermentation with nitrogen as a gas phase was compared to fermentation under aerobic conditions, both growth and amylase production were reduced whereas lactic acid formation was not affected. Addition of carbon dioxide (> or = 20% v/v) to the nitrogen gas phase restored growth and amylase production. The amylase production was high with starch, maltose or cellobiose contrary to glucose, fructose and sucrose. During mixed fermentation of glucose and maltose, a diauxic growth was observed. The maltose consumption and the amylase production started after the glucose depletion. The presence of maltose altered the carbon assimilation from glucose, whereas the energetic pathway was not affected. It is concluded that the elimination of soluble sugars by the wet extraction of starch during the processing of cassava, together with the expected in situ CO2 production, are conditions favouring the growth and the amylase synthesis. However, these are likely to be limited by the low nitrogen content in cassava.

Amylases↗
PubMed1990

Prevention of gastrointestinal infection using immunobiological methods with milk fermented with Lactobacillus casei and Lactobacillus acidophilus.

The protective effect of feeding milk fermented with a mixture of Lactobacillus casei sp. and Lb. acidophilus sp. against Salmonella typhimurium infection in mice was compared with that obtained feeding milks fermented with these microorganisms individually. The survival rate obtained after oral infection with Sal. typhimurium was 100% in mice pretreated by feeding during 8 d with the mixture of Lb. casei and Lb. acidophilus fermented milks. Similar treatments with the individual milks were ineffective. Moreover, mice became more susceptible to infection with Sal. typhimurium after such treatment. The colonization of liver and spleen with the pathogen was markedly inhibited by the pretreatment with the mixture of fermented milk, while such inhibition was not observed using the Lb. casei and Lb. acidophilus milks. The highest levels of anti-salmonellae antibodies in serum and in intestinal fluid were found in the group of mice fed with the mixture and with Lb. casei fermented milk respectively. However, this latter milk was not effective in protecting against Sal. typhimurium. When the mice were first infected with Sal. typhimurium and then fed with the mixture of fermented milks, pathogen colonization was not prevented. The results suggest that the augmentation of resistance to salmonellae caused by the treatment with Lb. casei- + Lb. acidophilus-fermented milk was due to the anti-salmonellae protective immunity mainly mediated by the mucosal tissue. Milk fermented with this mixture could be used as an immunobiological method to prevent gastrointestinal infection.

Animals↗
PubMed2001

Vector-free cloning of a bacterial endo-1,4-beta-glucanase in Lactobacillus plantarum and its effect on the acidifying activity in silage: use of recombinant cellulolytic Lactobacillus plantarum as silage inoculant.

In this research, the advantage of use of cellulolytic recombinant Lactobacillus plantarum as microbial inoculants for alfalfa silage fermentation was evaluated. To such purpose, two L. plantarum strains, one (L. plantarum Lp80) currently commercialised and the other (L. plantarum B41) suitable as silage microbial additive, were genetically modified by integration of celA gene, encoding an alkaline endo-1,4-beta-glucanase from Bacillus sp., in the chromosome, by means of a vector-free cloning technique. The heterologous gene was cloned in two fashions: preceded by two promoters (AC1 modification) or in translational coupling with a partial upstream ORF (AC2 modification). Therefore two different genetically modified organisms (GMOs) per each wild-type (WT), producing 43-59 U/l cellulase in 16 h, were examined. Thirty-five micro-ensiling experiments were carried out by inoculating the WT or the derived GMOs. L. plantarum B41AC1 cellulolytic clone exhibited significantly increased acidification capacity in silage samples incubated at 37 degrees C. No advantage of use was evident for the other GMOs.

Cellulase↗
PubMed2001

Lactobacillus casei and Lactobacillus plantarum initiate catabolism of methionine by transamination.

AIMS: To study the ability of Lactobacillus casei and Lact. plantarum strains to convert methonine to cheese flavour compounds. METHODS AND RESULTS: Strains were assayed for methionine aminotransferase and lyase activities, and amino acid decarboxylase activity. About 25% of the strains assayed showed methionine aminotransferase activity. The presence of glucose in the reaction mixture increased conversion of methionine to 4-methylthio-2-ketobutanoate (KMBA) and 4-methylthio-2-hydroxybutanoate (HMBA) in all strains. The methionine aminotransferase activity in Lact. plantarum and Lact. casei showed variable specificity for the amino group acceptors glyoxylate, ketoglutarate, oxaloacetate and pyruvate. None of the strains showed methionine lyase or glutamate and methionine decarboxylase activities. CONCLUSION: The presence of amino acid converting enzymes in lactobacilli is strain specific. SIGNIFICANCE AND IMPACT OF THE STUDY: The findings of this work suggest that lactobacilli can be used as adjuncts for flavour formation in cheese manufacture.

Carbon-Sulfur Lyases↗
PubMed2003

Identification of Lactobacillus alimentarius and Lactobacillus farciminis with 16S-23S rDNA intergenic spacer region polymorphism and PCR amplification using species-specific oligonucleotide.

AIMS: The restriction fragment length polymorphism (RFLP) method was used to differentiate Lactobacillus species having closely related identities in the 16S-23S rDNA intergenic spacer region (ISR). Species-specific primers for Lact. farciminis and Lact. alimentarius were designed and allowed rapid identification of these species. METHODS AND RESULTS: The 16S-23S rDNA spacer region was amplified by primers tAla and 23S/p10, then digested by HinfI and TaqI enzymes and analysed by electrophoresis. Digestion by HinfI was not sufficient to differentiate Lact. sakei, Lact. curvatus, Lact. farciminis, Lact. alimentarius, Lact. plantarum and Lact. paraplantarum. In contrast, digestion carried out by TaqI revealed five different patterns allowing these species to be distinguished, except for Lact. plantarum from Lact. paraplantarum. The 16S-23S rDNA spacer region of Lact. farciminis and Lact. alimentarius were amplified and then cloned into vector pCR(R)2.1 and sequenced. The DNA sequences obtained were analysed and species-specific primers were designed from these sequences. The specificity of these primers was positively demonstrated as no response was obtained for 14 other species tested. RESULTS AND CONCLUSIONS: The species-specific primers for Lact. farciminis and Lact. alimentarius were shown to be useful for identifying these species among other lactobacilli. The RFLP profile obtained upon digestion with HinfI and TaqI enzymes can be used to discriminate Lact. farciminis, Lact. alimentarius, Lact. sakei, Lact. curvatus and Lact. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: In this paper, we have established the first species-specific primer for PCR identification of Lact. farciminis and Lact. alimentarius. Both species-specific primer and RFLP, could be used as tools for rapid identification of lactobacilli up to species level.

Bacterial Typing Techniques↗
PubMed2001

Differentiation of Lactobacillus delbrueckii subsp. bulgaricus and Lactobacillus delbrueckii subsp. lactis by SDS-PAGE of cell-wall proteins.

AIMS: In the present study, a method based on SDS-PAGE fingerprinting of surface layer proteins was developed to identify Lactobacillus delbrueckii subsp. bulgaricus and subsp. lactis dairy isolates. METHODS AND RESULTS: The two subspecies, identified by species-specific PCR, were characterized by different SDS-PAGE cell-wall protein profiles; subspecies bulgaricus showed one band of about 31 kDa which, in some cases, was observed at a doublet, and subspecies lactis showed one band of about 21 kDa or 18 kDa. CONCLUSION: The sensitivity of this procedure for discriminating between the two subspecies was very high. The different types of SDS-PAGE profile for cell-wall proteins of the strains studied in this work did not seem to be correlated to the different dairies of origin. SIGNIFICANCE AND IMPACT OF THE STUDY: The method appears to be an efficient taxonomic tool. It has the advantage of easy gel interpretation over fingerprinting of whole-cell protein extracts, and may be used as an alternative to established PCR-based techniques which, though rapid and safe, require expensive instruments and reagents.

Bacterial Proteins↗
PubMed1999

Genetic diversity within Lactobacillus sakei and Lactobacillus curvatus and design of PCR primers for its detection using randomly amplified polymorphic DNA.

The genotypic and phenotypic diversity among isolates of the Lactobacillus curvatus/Lactobacillus graminis/Lactobacillus sakei group was evaluated by comparing RAPD data and results of biochemical tests, such as hydrolysis of arginine, D-lactate production, melibiose and xylose fermentation, and the presence of haem-dependent catalase. Analyses were applied to five type strains and to a collection of 165 isolates previously assigned to L. sakei or L. curvatus. Phenotypic and RAPD data were compared with each other and with previous DNA-DNA hybridization data. The phenotypic and genotypic separation between L. sakei, L. curvatus and L. graminis was clear, and new insights into the detailed structure within L. sakei and L. curvatus were obtained. Individual strains could be typed by RAPD and, after the elimination of similar or identical isolates, two sub-groups in both L. curvatus and L. sakei were defined. The presence or absence of catalase activity further distinguished the two L. curvatus sub-groups. By cloning and sequencing specific RAPD products, pairs of PCR primers were developed that can be used to specifically detect L. curvatus, L. sakei and each of the L. sakei sub-groups.

Animals↗
PubMed2001

Characterization of the Lactobacillus casei group and the Lactobacillus acidophilus group by automated ribotyping.

A total of 91 type and reference strains of the Lactobacillus casei group and the L acidophilus group were characterized by the automated ribotyping device Riboprinter microbial characterization system. The L. casei group was divided into five (C1-C5) genotypes by ribotyping. Among them, the strain of L. casei ATCC 334 was clustered to the same genotype group as most of L. paracasei strains and L casei JCM 1134T generated a riboprint pattern that was different from the type strain of L. zeae. These results supported the designation of L. casei ATCC 334 as the neotype strain, but were not consistent with the reclassification of L. casei JCM 1134T as L. zeae. The L. acidophilus group was also divided into 14 (A1-A11, B1-B3) genotypes by ribotyping. L. acidophilus, L. amylovorus, L. crispatus and L. gallinarum generated ribotype patterns that were distinct from the patterns produced by L. gasseri and L. johnsonii. This result confirmed previous data that the L. acidophilus group divided to two major clusters. Five strains of L. acidophilus and two strains of L. gasseri were correctly reidentified by ribotyping. Most strains belonging to the L. casei group and the L. acidophilus group were discriminated at the species level by automated ribotyping. Thus this RiboPrinter system yields rapid, accurate and reproducible genetic information for the identification of many strains.

Humans↗
PubMed2004

Thermal and chemical resistance of Lactobacillus casei and Lactobacillus paracasei bacteriophages.

AIMS: The survival of two collection Lactobacillus casei and L. paracasei bacteriophages when subjected to thermal and chemical treatments was investigated. METHODS AND RESULTS: Thermal resistance was evaluated by heating phage suspensions at 63, 72 and 90 degrees C in three different media [Tris-magnesium gelatin (TMG) buffer: 10 mmol l(-1) Tris-Cl, 10 mmol l(-1) MgSO(4) and 0.1% w/v gelatin; Man Rogosa Sharpe (MRS) broth and reconstituted nonfat dry skim milk (RSM)]. A marked heat sensitivity was evident in both phages, as 15 min at 72 degrees C was enough to completely inactivate (6 log(10) reduction) them. No clear influence was demonstrated by the suspension media. The phages also showed similar resistance to biocides. Peracetic acid and sodium hypochlorite (800 ppm) were the most effective ones, destroying the phages within 5 min. Concentrations of 75 and 100% ethanol were not suitable to inactivate phage particles even after 45 min. Isopropanol did not show an effect on phage viability. CONCLUSIONS: The data obtained in this work are important to design more effective control procedures in order to inactivate phages in dairy plants and laboratories. SIGNIFICANCE AND IMPACT OF THE STUDY: This work will contribute to enhance the background knowledge about phages of probiotic bacteria.

2-Propanol↗
PubMed1995

Characterization of genetic elements required for site-specific integration of Lactobacillus delbrueckii subsp. bulgaricus bacteriophage mv4 and construction of an integration-proficient vector for Lactobacillus plantarum.

Temperate phage mv4 integrates its DNA into the chromosome of Lactobacillus delbrueckii subsp. bulgaricus strains via site-specific recombination. Nucleotide sequencing of a 2.2-kb attP-containing phage fragment revealed the presence of four open reading frames. The larger open reading frame, close to the attP site, encoded a 427-amino-acid polypeptide with similarity in its C-terminal domain to site-specific recombinases of the integrase family. Comparison of the sequences of attP, bacterial attachment site attB, and host-phage junctions attL and attR identified a 17-bp common core sequence, where strand exchange occurs during recombination. Analysis of the attB sequence indicated that the core region overlaps the 3' end of a tRNA(Ser) gene. Phage mv4 DNA integration into the tRNA(Ser) gene preserved an intact tRNA(Ser) gene at the attL site. An integration vector based on the mv4 attP site and int gene was constructed. This vector transforms a heterologous host, L. plantarum, through site-specific integration into the tRNA(Ser) gene of the genome and will be useful for development of an efficient integration system for a number of additional bacterial species in which an identical tRNA gene is present.

Amino Acid Sequence↗
PubMed2000

Expression of cbsA encoding the collagen-binding S-protein of Lactobacillus crispatus JCM5810 in Lactobacillus casei ATCC 393(T).

The cbsA gene encoding the collagen-binding S-layer protein of Lactobacillus crispatus JCM5810 was expressed in L. casei ATCC 393(T). The S-protein was not retained on the surface of the recombinant bacteria but was secreted into the medium. By translational fusion of CbsA to the cell wall sorting signal of the proteinase, PrtP, of L. casei, CbsA was presented at the surface, rendering the transformants able to bind to immobilized collagens.

Bacterial Proteins↗
PubMed2000

The effect of Lactobacillus buchneri, Lactobacillus plantarum, or a chemical preservative on the fermentation and aerobic stability of corn silage.

Several microorganisms and one chemical preservative were tested for their effects on the fermentation and aerobic stability of corn silage. Whole-plant corn (one-half milk line, 31.3% dry matter) was ensiled in quadruplicate 20-L laboratory silos untreated or after the following treatments: Lactobacillus buchneri at 1 x 10(5) and 1 x 10(6) cfu/g of fresh forage; two different strains of L. plantarum, each at 1 x 10(6) cfu/g; and a buffered propionic acid-based product at 0.1% of fresh forage weight. After 100 d of ensiling, silage treated with L. buchneri (1 x 10(6) cfu/g) had a lower concentration of lactic acid compared with the untreated silage, but was similar to other treated silages. The silage treated with the high (1 x 10(6) cfu/g), but not the moderate rate (1 x 10(5) cfu/g) of L. buchneri also had a greater concentration of acetic acid (3.60%) and less yeasts (2.01 log cfu/g) when compared with other treatments (average of 1.88% acetic acid and 5.85 log cfu of yeasts/g). Silages treated with L. plantarums, the moderate rate of L. buchneri, and the chemical preservative took longer to heat than untreated silage when exposed to air, but improvements were numerically small (6.3 to 10.5 h). In contrast, silage treated with the high rate of L. buchneri never heated throughout a 900-h period of monitoring. Inoculating corn silage with 1 x 10(6) cfu/g of L. buchneri resulted in a more heterolactic fermentation and dramatically improved the aerobic stability of corn silage.

Acetic Acid↗
PubMed1996

The induction of cardioangitis by Lactobacillus casei cell wall in mice. I. The cytokine production from murine macrophages by Lactobacillus casei cell wall extract.

A single intraperitoneal injection of the water-soluble cell wall extract from Lactobacillus casei (LCWE) in mice can induce acute inflammatory heart involvement such as cardiac arteritis, similar to the heart lesions in Kawasaki disease. To clarify whether inflammatory cytokines participate in pathogenic mechanisms of LCWE-induced cardioangitis in mice, we studied in vitro cytokine production (IL-1, TNF alpha, and IL-6) from peritoneal macrophages (M phi) stimulated with LCWE in comparison with that of lipopolysaccharide (LPS). LCWE significantly activated M phi of BALB/c in production of IL-1 and TNF alpha compared with LPS. However, the magnitude of IL-6 synthesis by LCWE was approximately similar to that of LPS. These results demonstrate that the pathogenesis of LCWE- induced cardiac lesions may be associated with increased production of inflammatory cytokines.

Animals↗