Characterization of Putative Adhesion Genes in the Potentially Probiotic Strain Lactobacillus plantarum BFE 5092.

Two putative adhesion genes of the potentially probiotic strain Lactobacillus plantarum BFE 5092, i.e., a gene with similarity to an aggregation-promoting factor gene apf5092, and the mucin-binding protein gene mub5092, were investigated in this study. The gene encoding apf5092 encoded a protein bearing a predicted 26 amino acid signal peptide and a LysM domain putatively involved in binding to peptidoglycan. Moreover, the deduced protein also showed an amino acid sequence characteristic of an aggregation-promoting factor. The genes were tested for expression under different environmental conditions, and transcriptional studies on apf5092 showed that the expression could be influenced by low temperature and pH within 30 min. The aggregation behavior of the cells also changed at the low pH condition, but less noticeably at low temperature. To further investigate the role of apf5092 in aggregation, it was cloned and expressed in E. coli. The transformed strain showed higher co-aggregation ability with Gram-positive bacteria. Transcription studies on mub5092 revealed that it could be induced by mucin when added to the growth medium within 30 min. The data suggested that L. plantarum BFE 5092 can quickly adapt to changing environmental conditions and that enhanced aggregation may be important to survive low pH conditions, e.g., of the stomach or of fermented foods, and may thus be an important colonization factor.

The effect of bacteriocin-producing Lactobacillus plantarum strains on the intracellular pH of sessile and planktonic Listeria monocytogenes single cells.

A wide range of lactic acid bacteria (LAB) produce bacteriocins mainly active against other closely related LAB, but some bacteriocins are also active against the food-borne pathogen Listeria monocytogenes. With the aim of increasing food safety it has thus been considered to utilise bacteriocins and/or bacteriocin-producing LAB as "natural" food preservatives in foods such as cheese, meat and ready-to-eat products. Some strains of Lactobacillus plantarum produce bacteriocins termed plantaricins. Using a single-cell based approach, the effect on the intracellular pH as a measure of the physiological state of sessile and planktonic L. monocytogenes (strains EGDe and N53-1) during co-culturing with plantaricin-producing L. plantarum (strains BFE 5092 and PCS 20) was investigated using fluorescence ratio imaging microscopy (FRIM). Mono-cultures of L. monocytogenes were used as control. Expression levels of plantaricin-encoding genes by sessile and planktonic L. plantarum were determined using qRT-PCR. L.plantarum BFE 5092 possesses the genes for plantaricin EF, JK and N, while L. plantarum PCS 20 contains the genes for plantaricin EF, although determination of the nucleotide sequence of the PCS 20 plantaricin E gene showed that this peptide is probably non-functional. When cultured as mono-culture, both L. monocytogenes strains maintained pH(i) at a constant level around 7.2-7.6 throughout the experiment, independently of the matrix. On a solid surface, L. plantarum BFE 5092 strongly affected pH(i) of L. monocytogenes N53-1 with only 20% of the cells being able to maintain pH(i) in the physiological optimal range with pH>7 and 52% of the cells with pH(i) approximately pH(ex,) showing that the cells had no proton gradient towards the environment. The effect on L. monocytogenes EGDe was less pronounced, but still notable. L.plantarum PCS 20 left both strains of L. monocytogenes virtually unaffected when co-cultured on a solid surface. In liquid, both L. plantarum strains strongly affected the physiological state of L. monocytogenes EGDe as judged by pH(i), whereas L. monocytogenes N53-1 was left virtually unaffected after 5h of co-culturing and after 8h 50% of the cells still maintained pH(i)>or=7. Higher concentrations of lactic acid were produced in liquid compared to a solid surface, and the different response of EGDe and N53-1 to the activities of the two L. plantarum strains probably reflect higher susceptibility of L. monocytogenes EGDe to organic acids compared to L. monocytogenes N53-1. Taken together, our results may be explained by the difference in the range of plantaricins produced by the two L. plantarum strains and matrix- and strain-related differences in the susceptibility of L. monocytogenes to plantaricins and organic acids. In conclusion, the present study represents the first demonstration of the ability of a bacteriocin-producing LAB to dissipate the proton gradient of sessile and planktonic L. monocytogenes.

Genetic analysis of the plantaricin EFI locus of Lactobacillus plantarum PCS20 reveals an unusual plantaricin E gene sequence as a result of mutation.

Lactobacillus plantarum strains produce a variety of chromosomally encoded bacteriocins and often multiple bacteriocins are encoded by a single strain. In this study, the genetic loci for bacteriocin production of L. plantarum strains BFE 5092 and PCS20 were studied. These strains were investigated for their possible application as protective cultures in food preservation. The bacteriocin locus of strain BFE 5092 showed remarkable similarity to the plantaricin loci previously described for L. plantarum strains C11 and WCFS1. However, the locus of the L. plantarum PCS20 strain was unusual in that it showed an interesting mutation as a result of deletions within the plnE gene. These deletions led to a hypothetically produced peptide which is 2 amino acids shorter than plantaricin E. Furthermore, it differs by 24 amino acids, while it shares 30 identical amino acids i.e., 15 at the amino end and 15 at the carboxyl end of the hypothetical peptide. As a consequence, the amino acid sequence is changed such that a double-glycine-type leader peptide would not be encoded. This raises the question whether a functional peptide is being produced, even though RT-PCR studies showed that the plnE gene is obviously expressed. Furthermore, a transposase gene was located upstream of the plnEFI gene cluster and was inserted into a bacteriocin regulatory gene, the histidine protein kinase gene. Taken together, these facts indicate a loss of plantaricin gene function in L. plantarum PCS20 as a result of transposition and mutation.

Investigation into the Potential of Bacteriocinogenic Lactobacillus plantarum BFE 5092 for Biopreservation of Raw Turkey Meat.

The bacteriocin-producing Lactobacillus plantarum BFE 5092 was assessed for its potential as a protective culture in the biopreservation of aerobically stored turkey meat. This strain produces three bacteriocins, i.e. plantaricins EF, JK and N. The absolute expression of Lactobacillus plantarum BFE 5092 16S rRNA housekeeping gene, as well as l-ldh, plnEF and plnG genes as determined by quantitative, real-time-PCR, revealed that these genes were expressed to similar levels when the strain was grown at 8 and 30 °C in MRS broth. On turkey meat, Lactobacillus plantarum BFE 5092 did not grow but survived, as indicated by similar viable cell numbers during a 9-day storage period at 8 °C. When inoculated at 1 × 10(7) CFU/g on the turkey meat and subsequently stored at 10 °C, the culture did again not show good growth. Lactobacillus plantarum BFE 5092 could not inhibit the growth of naturally occurring listeriae or Gram-negative bacteria on the turkey meat at 10 °C, or that of Listeria monocytogenes when it was co-inoculated at a level of 1 × 10(5) CFU/g. Gene expression analyses showed that the bacteriocin genes were expressed on turkey meat stored at 10 °C. Moreover, the investigation into the absolute expression of the three plantaricin genes of Lactobacillus plantarum BFE 5092 in co-culture with Listeria monocytogenes on turkey meat by qRT-PCR showed that the plantaricin genes were indeed expressed during the low-temperature storage condition. The Lactobacillus plantarum BFE 5092 strain overall could not effectively inhibit L. monocytogenes and therefore it would not make a suitable protective culture for biopreservation of turkey meat stored aerobically at low temperature.

Use of Lactobacillus strains to start cassava fermentations for Gari production.

Two Lactobacillus strains, Lactobacillus plantarum BFE 6710 and Lactobacillus fermentum BFE 6620, were used to start cassava fermentations in a pilot study under field production conditions in Kenya, to determine their potential to establish themselves as predominant lactobacilli during the fermentation. Predominant strains from three fermentations were isolated throughout the 48 h fermentation period. The use of these strains in high numbers clearly resulted in 1 to 2 log higher lactic acid bacteria (LAB) counts over the course of the fermentation when compared to the uninoculated control. 178 predominant LAB isolates were grouped based on their phenotypic characteristics, and were characterised to strain level by RAPD-PCR, followed by PFGE strain typing. Overall, L. plantarum strains represented the majority of the isolates, followed by Weissella confusa and Lactococcus garvieae strains. The results of RAPD-PCR and PFGE strain typing techniques indicated that L. plantarum BFE 6710 was successful in asserting itself as a predominant strain. In contrast, L. fermentum BFE 6620 failed to establish itself as a predominant organism in the fermentation. The success of the L. plantarum strains to predominate in the cassava fermentation demonstrates the potential for development of Lactobacillus starter cultures to industrialise the Gari production process.