Expression analysis of putative arcA, arcB and arcC genes partially cloned from Lactobacillus plantarum isolated from wine

AIMS: The aim of this paper was to study if homofermentative strains (Lacobacillus plantarum) capable of malolactic fermentation in wine can degrade arginine via the ADI pathway. METHODS AND RESULTS: Homofermentative lactic acid bacteria (LAB) isolated from a typical red wine were investigated for their ability to produce citrulline. Citrulline was formed suggesting that the arginine metabolism takes place via the arginine deiminase (ADI) pathway and not via the arginase/urease pathway. Ammonia was also detected with Nessler's reagent, and all the strains examined were able to produce ammonia. Identification of homofermentative LAB was performed using 16S ribosomal sequence analysis. The strains were further classified as belonging to L. plantarum species. Furthermore, the genes encoding for the three pathway enzymes (ADI, ornithine transcarbamylase, carbamate kinase) were partially cloned and gene expression was performed at two different pH values (3.6 and 4.5). CONCLUSIONS: The results suggest that citrulline production in wine, could be performed by homofermentative LAB. SIGNIFICANCE AND IMPACT OF THE STUDY: Homofermentative malolactic bacteria (L. plantarum) may degrade arginine through the ADI pathway.     

Bacteriocin production by lactic acid bacteria isolated from Rioja red wines

Forty-two lactic acid bacteria (LAB) of the genera Lactobacillus (32), Leuconostoc (6), Pediococcus (3) and Lactococcus (1), isolated from Rioja red wines, were tested for antimicrobial activity. All these strains, as well as 18 Leuconostoc oenos and 19 yeast strains were used as indicators. Only nine strains showed antimicrobial activity, and all were of the species Lactobacillus plantarum, which constitutes the predominant microflora in Rioja red wines after alcoholic fermentation. Lact. plantarum strain J-51 showed the widest range of action, inhibiting the growth of 31 strains of the four studied LAB genera. Lact. plantarum J-51 antimicrobial activity was lost after treatment with proteases, suggesting a proteinaceous nature for this activity. It was found to be stable between pH 3 and 9 and under strong heating conditions (100 degrees C for 60 min). Polymerase chain reaction (PCR) analysis of Lact. plantarum J-51 genome revealed the presence of the plnA gene that encodes the plantaricin precursor PlnA. A 366-bp fragment was sequenced and showed 95% identity with pln locus of Lact. plantarum C-11. The deduced precursor peptide sequence showed one mutation (Gly7 to Ser7) at the double glycine leader peptide, and the three putative 26-, 23- and 22-residue active peptides remain identical to those of Lact. plantarum C-11. Therefore, antimicrobial peptides constitute a potent adaptation advantage for those strains that dominate in a medium such as wine, and can play an important role in the ecology of wine microflora.     

In situ activity of a bacteriocin-producing Lactococcus lactis strain. Influence on the interactions between lactic acid bacteria during sourdough fermentation

AIMS: To biochemically characterize the bacteriocin produced by Lactococcus lactis ssp. lactis M30 and demonstrate its effect on lactic acid bacteria (LAB) during sourdough propagation. METHODS AND RESULTS: A two-peptide bacteriocin produced by L. lactis ssp. lactis M30 was purified by ion exchange, hydrophobic interaction and reversed phase chromatography. Mass spectrometry of the two peptides and sequence analysis of the ltnA2 gene showed that the bacteriocin was almost identical to lacticin 3147. During a 20-day period of sourdough propagation the stability of L. lactis M30 was demonstrated, with concomitant inhibition of the indicator strain Lactobacillus plantarum 20, as well as the non-interference with the growth of the starter strain Lact. sanfranciscensis CB1. CONCLUSIONS: In situ active bacteriocins influence the microbial consortium of sourdough LAB and can "support" the dominance of insensitive strains during sourdough fermentation. SIGNIFICANCE AND IMPACT OF THE STUDY: The in situ bacteriocinogenic activity of selected lactococci enables the persistence of insensitive Lact. sanfranciscensis strains, useful to confer good characteristics to the dough, at a higher cell concentration with respect to other LAB of the same ecosystem.     

Characterization of Lactobacillus plantarum from wine must by PCR species-specific and RAPD-PCR

AIMS: Physiological and molecular analysis such as PCR species-specific and randomly amplified polymorphic PCR (RAPD-PCR) have been used for typing of Lactobacillus plantarum strains from typical wine must. METHODS AND RESULTS: Phenotypic tests such as API 50CH and evaluation of D-L-lactate production from glucose were used to perform a preliminary characterization of lactobacilli. Furthermore, 18 strains of lactobacilli were analyzed by PCR species-specific oligonucleotides based on short sequences of the recA gene. CONCLUSIONS: Four strains were identified as belonging to the L. plantarum species and were further analysed by RAPD-PCR. The RAPD-PCR profiles were similar in all strains that had positive results for species-specific PCR, suggesting that the four L. plantarum strains were closely related. SIGNIFICANCE AND IMPACT OF THE STUDY: Using PCR species-specific as a preliminary screening test and then RAPD-PCR can be as considered the most reliable method of performing a rapid and correct typing of L. plantarum from wine must.     

The Lactobacillus plantarum strain ACA-DC287 isolated from a Greek cheese demonstrates antagonistic activity in vitro and in vivo against Salmonella enterica serovar Typhimurium

AIMS: The purpose of this study was to investigate the antibacterial activity of the Xynotyri cheese isolate Lactobacillus plantarum ACA-DC287 using a set of in vitro and in vivo assays. METHODS AND RESULTS: The co-culture of L. plantarum strain ACA-DC287 and Salmonella enterica serovar Typhimurium strain SL1344 results in the killing of the pathogen. The killing activity was produced mainly by non-lactic acid molecule(s) that were present in the cell-free culture supernatant of the L. plantarum strain ACA-DC287. The culture of the L. plantarum strain ACA-DC287 inhibited the penetration of S. typhimurium SL1344 into cultured human enterocyte-like Caco-2/TC7 cells. In conventional mice infected with S. typhimurium SL1344, the intake of L. plantarum strain ACA-DC287 results in a decrease in the levels of Salmonella associated with intestinal tissues or those present in the intestinal contents. In germ-free mice, the L. plantarum strain ACA-DC287 colonized the gastrointestinal tract. CONCLUSIONS: The L. plantarum strain ACA-DC287 strain exerts anti-Salmonella activity similar that of the established probiotic strains Lactobacillus rhamnosus GG, Lactobacillus casei Shirota YIT9029 and Lactobacillus johnsonii La1. SIGNIFICANCE AND IMPACT OF THE STUDY: The observation that a selected cheese Lactobacillus strain exerted antibacterial activity that was similar to those of probiotic Lactobacillus strains, is of interest for the use of this strain as an adjunct strain for the production of health-giving cheeses.     

Survival, growth and persistence under farm conditions of a Lactobacillus plantarum strain inoculated into liquid pig feed

AIMS: To investigate the survival and persistence of Lactobacillus plantarum REB1 in the fermentation process of liquid pig feed on a working farm in standard production conditions. METHODS AND RESULTS: Two feed types, a control diet [nonfermented liquid feed (NFLF)] and a fermented diet [fermented liquid feed (FLF)], were compared. A rifampicin-resistant mutant L. plantarum REB1-Rif was used to initiate the fermentation of the feed. Inoculation with the experimental strain was repeated one or two times per week throughout the three month growing period. Four microbial groups were followed using standard microbiological techniques, as well as pH. Lactic acid bacteria (LAB) counts were high already at the beginning in FLF, while it took nine days to reach the corresponding LAB levels in NFLF. Yeasts were stable in FLF, whereas in NFLF there were occasional high counts during the first week. The numbers of Enterobacteriaceae were low, although in NFLF they were variable. The average pH in NFLF was 4.5 and 4 in FLF. CONCLUSIONS: The inoculation of L. plantarum REB1-Rif provided a LAB population of log 9 colony forming units (CFU) ml(-1) from the first feeding day, stable numbers of yeast and pH, and a drastic reduction of Enterobacteriaceae. SIGNIFICANCE AND IMPACT OF THE STUDY: The inoculation by L. plantarum REB1-Rif offered a FLF microbiologically stable from the first week in actual production conditions.     

Dynamics and biodiversity of populations of lactic acid bacteria and acetic acid bacteria involved in spontaneous heap fermentation of cocoa beans in Ghana

The Ghanaian cocoa bean heap fermentation process was studied through a multiphasic approach, encompassing both microbiological and metabolite target analyses. A culture-dependent (plating and incubation, followed by repetitive-sequence-based PCR analyses of picked-up colonies) and culture-independent (denaturing gradient gel electrophoresis [DGGE] of 16S rRNA gene amplicons, PCR-DGGE) approach revealed a limited biodiversity and targeted population dynamics of both lactic acid bacteria (LAB) and acetic acid bacteria (AAB) during fermentation. Four main clusters were identified among the LAB isolated: Lactobacillus plantarum, Lactobacillus fermentum, Leuconostoc pseudomesenteroides, and Enterococcus casseliflavus. Other taxa encompassed, for instance, Weissella. Only four clusters were found among the AAB identified: Acetobacter pasteurianus, Acetobacter syzygii-like bacteria, and two small clusters of Acetobacter tropicalis-like bacteria. Particular strains of L. plantarum, L. fermentum, and A. pasteurianus, originating from the environment, were well adapted to the environmental conditions prevailing during Ghanaian cocoa bean heap fermentation and apparently played a significant role in the cocoa bean fermentation process. Yeasts produced ethanol from sugars, and LAB produced lactic acid, acetic acid, ethanol, and mannitol from sugars and/or citrate. Whereas L. plantarum strains were abundant in the beginning of the fermentation, L. fermentum strains converted fructose into mannitol upon prolonged fermentation. A. pasteurianus grew on ethanol, mannitol, and lactate and converted ethanol into acetic acid. A newly proposed Weissella sp., referred to as "Weissella ghanaensis," was detected through PCR-DGGE analysis in some of the fermentations and was only occasionally picked up through culture-based isolation. Two new species of Acetobacter were found as well, namely, the species tentatively named "Acetobacter senegalensis" (A. tropicalis-like) and "Acetobacter ghanaensis" (A. syzygii-like).     

Tyrosine-containing peptides are precursors of tyramine produced by lactobacillus plantarum strain IR BL0076 isolated from wine

ABSTRACT: BACKGROUND: Biogenic amines are molecules with allergenic properties. They are found in fermented products and are synthesized by lactic acid bacteria through the decarboxylation of amino acids present in the food matrix. The concentration of biogenic amines in fermented foodstuffs is influenced by many environmental factors, and in particular, biogenic amine accumulation depends on the quantity of available precursors. Enological practices which lead to an enrichment in nitrogen compounds therefore favor biogenic amine production in wine. Free amino acids are the only known precursors for the synthesis of biogenic amines, and no direct link has previously been demonstrated between the use of peptides by lactic acid bacteria and biogenic amine synthesis. RESULTS: Here we demonstrate for the first time that a Lactobacillus plantarum strain isolated from a red wine can produce the biogenic amine tyramine from peptides containing tyrosine. In our conditions, most of the tyramine was produced during the late exponential growth phase, coinciding with the expression of the tyrDC and tyrP genes. The DNA sequences of tyrDC and tyrP in this strain share 98% identity with those in Lactobacillus brevis consistent with horizontal gene transfer from L. brevis to L. plantarum. CONCLUSION: Peptides amino acids are precursors of biogenic amines for Lactobacillus plantarum strain IR BL0076.     

Characterization of lactobacilli isolated from caper berry fermentations

AIMS: To determine the metabolic and functional properties of lactobacilli isolated from caper fermentation. METHODS AND RESULTS: A collection of 58 lactobacilli from fermentation of caper berries (including species of Lactobacillus plantarum, Lactobacillus paraplantarum, Lactobacillus pentosus, Lactobacillus brevis and Lactobacillus fermentum) was studied. Strains were classified in different clusters according to sugar fermentation patterns. Most strains of L. plantarum (the predominant species in the fermentation) clustered in a single group. Analysis of enzymatic activities revealed a high incidence of leucine aminopeptidase, acid phosphatase, beta-galactosidase and beta-glucosidase among the different strains of lactobacilli. A high number of strains were able to degrade raffinose and stachyose. Phytase activity and bile salt hydrolase activity were only detected in certain strains of L. plantarum. CONCLUSIONS: Lactobacilli from caper fermentation are metabolically diverse, and some strains display functional properties of interest. SIGNIFICANCE AND IMPACT OF THE STUDY: Strains of lactobacilli with selected functional properties could be good candidates for future development of commercial starters for industrial caper fermentation.     

Use of bacteriocin promoters for gene expression in Lactobacillus plantarum C11

AIMS: To exploit promoters involved in production of the bacteriocin sakacin P for regulated overexpression of genes in Lactobacillus plantarum C11. METHODS AND RESULTS: Production of sakacin P by Lact. sakei LTH673 is controlled by a peptide-based quorum sensing system that drives strong, regulated promoters. One of these promoters (PorfX) was used to establish regulated overexpression of genes encoding chloramphenicol acetyltransferase from Bacillus pumilus, aminopeptidase N from Lactococcus lactis or chitinase B from Serratia marcescens in Lact. plantarum C11, a strain that naturally possesses the regulatory machinery that is necessary for promoter activation. The expression levels obtained were highly dependent on which gene was used and on how the promoter was coupled to this gene. The highest expression levels (14% of total cellular protein) were obtained with the aminopeptidase N gene translationally fused to the regulated promoter. CONCLUSIONS: Sakacin promoters permit regulated expression of a variety of genes in Lact. plantarum C11. SIGNIFICANCE AND IMPACT OF THE STUDY: This study shows the usefulness of regulated bacteriocin promoters for developing new gene expression systems for lactic acid bacteria, in particular lactobacilli.     

Identification and characterization of lactic acid bacteria isolated from traditional pickles in Sichuan, China

The pickle, a traditional fermented vegetable product, is popular in Sichuan Province of China. The objective of this study was to investigate the diversity of dominant lactic acid bacteria (LAB) in pickles by analyzing 36 samples collected from 6 different regions in Sichuan Province. The LAB counts in these samples varied from 3.90 to 8.40 log cfu ml^-1. In total, 185 presumptive LAB with Gram-positive and catalase-negative properties were obtained from these samples using MRS agar, and those strains were identified at the species level by physiological tests, 16S rRNA gene sequencing and multiplex PCR assay. The results revealed that all isolates were accurately identified as Enterococcus thailandicus (2 strains), Lactobacillus alimentarius (16 strains), L. brevis (24 strains), L. paracasei (9 strains), L. plantarum (81 strains), L. pentosus (38 strains), L. sakei (8 strains), L. spicheri (1 strain), Leuconostoc lactis (1 strain) and Pediococcus ethanolidurans (5 strains). The predominant LAB in Sichuan pickle was L. plantarum, which were isolated from most samples. The results also demonstrated that different regions in Sichuan Province have complex compositions of LAB species, and such a rich resource of LAB strains provides raw data for further studies involving probiotic strain selection.     

High-level gene expression in Lactobacillus plantarum using a pheromone-regulated bacteriocin promoter

AIMS: To use promoters and regulatory genes involved in the production of the bacteriocin sakacin P to obtain high-level regulated gene expression in Lactobacillus plantarum. METHODS AND RESULTS: In a plasmid containing all three operons naturally involved in sakacin P production, the genes encoding sakacin P and its immunity protein were replaced by the aminopeptidase N gene from Lactococcus lactis (pepN) or the beta-glucuronidase gene from Escherichia coli (gusA). The new genes were precisely fused to the start codon of the sakacin P gene and the stop codon of the immunity gene. This set-up permitted regulated (external pheromone controlled) overexpression of both reporter genes in L. plantarum NC8. For PepN, production levels amounted to as much as 40% of total cellular protein. CONCLUSIONS: Promoters and regulatory genes involved in production of sakacin P are suitable for establishing inducible high-level gene expression in L. plantarum. SIGNIFICANCE AND IMPACT OF THE STUDY: This study describes a system for controllable gene expression in lactobacilli, giving some of the highest expression levels reported so far in this genus.     

Genotypic and phenotypic characterization of garlic-fermenting lactic acid bacteria isolated from som-fak, a Thai low-salt fermented fish product

AIMS: To evaluate the importance of garlic for fermentation of a Thai fish product, and to differentiate among garlic-/inulin-fermenting lactic acid bacteria (LAB) at strain level. METHODS AND RESULTS: Som-fak was prepared by fermentation of a mixture of fish, salt, rice, sucrose and garlic. pH decreased to 4.5 in 2 days, but omitting garlic resulted in a lack of acidification. LAB were predominant and approximately one third of 234 isolated strains fermented garlic and inulin (the carbohydrate reserve in garlic). These strains were identified as Lactobacillus pentosus and Lact. plantarum. Randomly Amplified Polymorphic DNA (RAPD) analysis revealed one major RAPD type (29 strains) isolated from all stages of fermentation. CONCLUSION: Garlic was essential for acidification of som-fak and garlic-fermenting strains constituted a significant, homogeneous part of the LAB flora. SIGNIFICANCE AND IMPACT OF THE STUDY: The present study indicates the role of fructans (garlic/inulin) as carbohydrate sources for LAB. Fructan fermenters may have several biotechnological applications, for example, as probiotics.     

Biodiversity of Lactobacillus sanfranciscensis strains isolated from five sourdoughs

AIMS: Five different sourdoughs were investigated for the composition of lactic acid bacteria (LAB) and the biodiversity of Lactobacillus sanfranciscensis strains. METHODS AND RESULTS: A total of 57 strains were isolated from five sourdoughs. Isolated strains were all identified by the 16S rDNA sequence and species-specific primers for L. sanfranciscensis. Results of identification showed that LAB strains were L. sanfranciscensis, Lactobacillus plantarum, Lactobacillus paralimentarius, Lactobacillus fermentum, Lactobacillus pontis, Lactobacillus casei, Weisella confusa and Pediococcus pentosaceus. A total of 21 strains were identified as L. sanfranciscensis and these isolates were detected in all five sourdoughs. Ribotyping was applied to investigate the relationship between intraspecies diversity of L. sanfranciscensis and sourdough. A total of 22 strains of L. sanfranciscensis including L. sanfranciscensis JCM 5668T were compared by ribotyping. The dendrogram of 21 ribotyping patterns showed four clusters, and L. sanfranciscensis JCM 5668T was independent of the others. The different biotypes of L. sanfranciscensis were present in two sourdoughs compared with other three sourdoughs. CONCLUSIONS: The LAB compositions of five sourdoughs were different and the relationship between intraspecies diversity of L. sanfranciscensis strains and five sourdoughs was shown by ribotyping. SIGNIFICANCE AND IMPACT OF THE STUDY: This study demonstrated that ribotyping was useful for distinguishing L. sanfranciscensis strains. A further important result is that the intra-species diversity of L. sanfranciscensis strains seems to be related to the sourdough preparation.     

Production of a heterologous nonheme catalase by Lactobacillus casei: an efficient tool for removal of H2O2 and protection of Lactobacillus bulgaricus from oxidative stress in milk

Lactic acid bacteria (LAB) are generally sensitive to H2O2, a compound that they can paradoxically produce themselves, as is the case for Lactobacillus bulgaricus. Lactobacillus plantarum ATCC 14431 is one of the very few LAB strains able to degrade H2O2 through the action of a nonheme, manganese-dependent catalase (hereafter called MnKat). The MnKat gene was expressed in three catalase-deficient LAB species: L. bulgaricus ATCC 11842, Lactobacillus casei BL23, and Lactococcus lactis MG1363. While the protein could be detected in all heterologous hosts, enzyme activity was observed only in L. casei. This is probably due to the differences in the Mn contents of the cells, which are reportedly similar in L. plantarum and L. casei but at least 10- and 100-fold lower in Lactococcus lactis and L. bulgaricus, respectively. The expression of the MnKat gene in L. casei conferred enhanced oxidative stress resistance, as measured by an increase in the survival rate after exposure to H2O2, and improved long-term survival in aerated cultures. In mixtures of L. casei producing MnKat and L. bulgaricus, L. casei can eliminate H2O2 from the culture medium, thereby protecting both L. casei and L. bulgaricus from its deleterious effects.     

Kinetic analysis of strains of lactic acid bacteria and acetic acid bacteria in cocoa pulp simulation media toward development of a starter culture for cocoa bean fermentation

The composition of cocoa pulp simulation media (PSM) was optimized with species-specific strains of lactic acid bacteria (PSM-LAB) and acetic acid bacteria (PSM-AAB). Also, laboratory fermentations were carried out in PSM to investigate growth and metabolite production of strains of Lactobacillus plantarum and Lactobacillus fermentum and of Acetobacter pasteurianus isolated from Ghanaian cocoa bean heap fermentations, in view of the development of a defined starter culture. In a first step, a selection of strains was made out of a pool of strains of these LAB and AAB species, obtained from previous studies, based on their fermentation kinetics in PSM. Also, various concentrations of citric acid in the presence of glucose and/or fructose (PSM-LAB) and of lactic acid in the presence of ethanol (PSM-AAB) were tested. These data could explain the competitiveness of particular cocoa-specific strains, namely, L. plantarum 80 (homolactic and acid tolerant), L. fermentum 222 (heterolactic, citric acid fermenting, mannitol producing, and less acid tolerant), and A. pasteurianus 386B (ethanol and lactic acid oxidizing, acetic acid overoxidizing, acid tolerant, and moderately heat tolerant), during the natural cocoa bean fermentation process. For instance, it turned out that the capacity to use citric acid, which was exhibited by L. fermentum 222, is of the utmost importance. Also, the formation of mannitol was dependent not only on the LAB strain but also on environmental conditions. A mixture of L. plantarum 80, L. fermentum 222, and A. pasteurianus 386B can now be considered a mixed-strain starter culture for better controlled and more reliable cocoa bean fermentation processes.