Physiological studies of the Pediococcus pentosaceus biofilm.

Pediococcus pentosaceus, a bacterium recently used in human and animal probiotics, was used in combination with supports made from polylactic acid composite soybean meal was used to study biofilm formation, and it was found that dense biofilms developed by Day 1. Proteomic comparison between planktonic and biofilm cultures of P. pentosaceus showed distinct expression patterns of intracellular and extracellular proteins. Type I glyceraldehyde-3-phosphate dehydrogenase was upregulated in biofilm cultures and mediated cell adhesion and encouraged biofilm production. GMP synthase, which regulates GMP synthesis and acts as an intracellular signal molecule to control cell mechanisms and has been exploited in the development of new therapeutic agents, was also upregulated in the biofilm mode of growth. The present work serves as a basis for future studies examining the complex network of systems that regulate lactic acid bacterial (LAB) biofilm formation and can serve as a framework for studies of production of therapeutic agents from LAB.

Expression of coagulin A with low cytotoxic activity by Pediococcus pentosaceus ST65ACC isolated from raw milk cheese.

AIMS: We aimed to evaluate some specific conditions for growth of Pediococcus pentosaceus ST65ACC and its bacteriocin expression through ABC transporters; to purify the bacteriocin and determine its sequence; and to evaluate the cytotoxicity potential of the purified bacteriocin(s). METHODS AND RESULTS: The results presented for growth behaviour of P. pentosaceus ST65ACC showed that the bacterial growth was slightly influenced when cultured in MRS broth with different amounts of inoculum: 1, 2, 5 and 10%. The bacteriocin activity increased when 5 and 10% inocula were used. The carbon source (glucose) used in different amounts (1, 2, 3 or 4%) had no significant effect on growth and bacteriocin production. The studied strain P. pentosaceus ST65ACC was able to metabolize xylooligosaccharide (XOS) as the sole carbon source, resulting in the production of an antimicrobial peptide. The genes involved in the ABC transport system and sugar metabolism of P. pentosaceus ST65ACC were expressed at different levels. The bacteriocin produced by P. pentosaceus ST65ACC was partially purified by precipitation with ammonium sulphate (40% saturation), followed by reversed-phase liquid chromatography, resulting in the identification of an active bacteriocin. Tandem mass spectrometry was used to identify the partial sequence KYYGNGVTCGKHSCSVDWGK sharing high similarity to coagulin A. The semi-purified bacteriocin had low cytotoxicity based on estimated values for maximal nontoxic concentration (MNC) and cytotoxicity concentration (CC50 ). CONCLUSIONS: The bacteriocin produced by P. pentosaceus ST65ACC is similar to coagulin, with low cytotoxicity, strong antimicrobial activity and possible additional metabolite routes in the producer cell. In addition to MRS broth, bacteriocin was produced also in medium containing XOS (as the single carbon source). SIGNIFICANCE AND IMPACT OF THE STUDY: To the best of our knowledge, this is the first report of evaluation of the role of ABC transporters in the expression of bacteriocin by P. pentosaceus, cultured in MRS and XOS.

Optimized medium via statistical approach enhanced threonine production by Pediococcus pentosaceus TL-3 isolated from Malaysian food.

BACKGROUND: Threonine is an essential amino acid that is extensively used in livestock industry as feed supplement due to its pronounced effect in improving the growth performance of animals. Application of genetically engineered bacteria for amino acid production has its share of controversies after eosinophils myalgia syndrome outbreak in 1980s. This has urged for continuous search for a food grade producer as a safer alternative for industrial amino acid production. Lactic acid bacteria (LAB) appear as an exceptional candidate owing to their non-pathogenic nature and reputation of Generally Recognized as Safe (GRAS) status. Recently, we have identified a LAB, Pediococcus pentosaceus TL-3, isolated from Malaysian food as a potential threonine producer. Thus, the objective of this study was to enhance the threonine production by P. pentosaceus TL-3 via optimized medium developed by using Plackett-Burman design (PBD) and central composite design (CCD). RESULTS: Molasses, meat extract, (NH4)2SO4, and MnSO4 were identified as the main medium components for threonine production by P. pentosaceus TL-3. The optimum concentration of molasses, meat extract, (NH4)2SO4 and MnSO4 were found to be 30.79 g/L, 25.30 g/L, 8.59 g/L, and 0.098 g/L respectively based on model obtained in CCD with a predicted net threonine production of 123.07 mg/L. The net threonine production by P. pentosaceus TL-3 in the optimized medium was enhanced approximately 2 folds compared to the control. CONCLUSIONS: This study has revealed the potential of P. pentosaceus TL-3 as a safer alternative to produce threonine. Additionally, the current study has identified the key medium components affecting the production of threonine by P. pentosaceus TL-3, followed by optimization of their concentrations by means of statistical approach. The findings of this study could act as a guideline for the future exploration of amino acid production by LAB.

Effects of pH and sugar supplements on bacteriocin-like inhibitory substance production by Pediococcus pentosaceus.

To improve bacteriocin-like inhibitory substance (BLIS) production by Pediococcus pentosaceus ATCC 43200, the influence of pH as well as the addition of sugars-either prebiotic (inulin) or not (sucrose)-on its metabolism were investigated. This strain was grown at pH 5.0 or 6.0 either in glucose-based MRS medium (control) or after addition of 0.5, 1.0 or 1.5% (w/w) sucrose and inulin (GSI-MRS) in the same percentages. In the control medium at pH 5.0, cell mass concentration after 48 h of fermentation (Xmax = 2.26 g/L), maximum specific growth rate (µmax = 0.180 h-1) and generation time (Tg = 3.84 h) were statistically coincident with those obtained in supplemented media. At pH 6.0 some variations occurred in these parameters between the control medium (Xmax = 2.68 g/L; µmax = 0.32 h-1; Tg = 2.17 h) and the above supplemented media (Xmax = 1.90, 2.52 and 1.86 g/L; µmax = 0.26, 0.33 and 0.32 h-1; Tg = 2.62, 2.06 and 2.11 h, respectively). Lactate production was remarkable at both pH values (13 and 16 g/L) and improved in all supplemented media, being 34 and 54% higher than in their respective control media, regardless of the concentration of these ingredients. Cell-free supernatant of the fermented control medium at pH 5.0 displayed an antimicrobial activity against Enterococcus 101 5.3% higher than that at pH 6.0 and even 20% higher than those of all supplemented media, regardless of the concentration of supplements. BLIS production was favored either at pH 5.0 or in the absence of any additional supplements, which were able, instead, to stimulate growth and lactate production by P. pentosaceus.

Novel lactic acid bacteria strains as inoculants on alfalfa silage fermentation.

The effects of new strains of lactic acid bacteria on alfalfa silage fermentation were evaluated. The experiment was performed using a completely randomized design (with three replicates) based on a 6 × 6 factorial assay with 6 inoculants (I): Control (CTRL), Commercial inoculant (CI), Lactobacillus pentosus 14.7SE (LPE), Lactobacillus plantarum 3.7E (LP), Pediococcus pentosaceus 14.15SE (PP), and Lactobacillus plantarum 3.7E + Pediococcus pentosaceus 14.15SE (LP + PP), and six fermentation periods (P): 1, 3, 7, 14, 28 and 56 days. Alfalfa was wilted for 6 h in the field, which increased the dry matter content to 368 g/kg as fed. The CP and yeast population decreased during the fermentation process. Silage inoculated with the PP strain had the lowest pH values beginning at 14 d of fermentation and the lowest acetic acid concentration on the last day of fermentation. New strains more efficiently regulated enterobacteria and mold populations at days 56 and 28, respectively. Silages inoculated with the PP strain had a higher coefficient of in vitro dry matter digestibility than LP silages. All of the tested novel strains resulted in positive effects on at least one chemical property of the silage during the fermentation process. However, the adding of P. pentosaceus can be indicated as the better for silage quality considering the tested treatments in the present study.

Prebiotic effects of bovine lactoferrin on specific probiotic bacteria.

Bovine lactoferrin (bLf) is a natural iron-binding protein and it has been suggested to be a prebiotic agent, but this finding remains inconclusive. This study explores the prebiotic potential of bLf in 14 probiotics. Initially, bLf (1-32 mg/mL) treatment showed occasional and slight prebiotic activity in several probiotics only during the late experimental period (48, 78 h) at 37 °C. We subsequently supposed that bLf exerts stronger prebiotic effects when probiotic growth has been temperately retarded. Therefore, we incubated the probiotics at different temperatures, namely 37 °C, 28 °C, room temperature (approximately 22-24 °C), and 22 °C, to retard or inhibit their growth. As expected, bLf showed more favorable prebiotic activity in several probiotics when their growth was partially retarded at room temperature. Furthermore, at 22 °C, the growth of Bifidobacterium breve, Lactobacillus coryniformis, L. delbrueckii, L. acidophilus, B. angulatum, B. catenulatum, and L. paraplantarum were completely blocked. Notably, these probiotics started regrowing in the presence of bLf (1-32 mg/mL) in a significant and dose-dependent manner. Accordingly, bLf significantly increased the growth of Pediococcus pentosaceus, L. rhamnosus, and L. paracasei (BCRC 17483; a locally isolated strain) when their growth was retarded by incubation at 22 °C. In conclusion, bLf showed inconsistent prebiotic activity in the 14 probiotics at 37 °C, but revealed strong prebiotic activity in 10 probiotic strains at 22 °C. Therefore, this study enables determining additional roles of Lf in probiotic strains, which can facilitate developing novel combinational approaches by simultaneously using Lf and specific probiotics.