Spontaneous sauerkraut fermentation was performed at industrial scale in "Prehrana Inc.", Varaždin, in order to select autochthonous lactic acid bacteria (LAB) which were evaluated according probiotic criteria and tested for their capacity as probiotic starter cultures. At the end of the spontaneous sauerkraut fermentation, total LAB counts reached 9.0×10(5) CFU/ml. This underlines that the need for addition of the well characterised probiotic cultures, in appropriate viable cell counts, would be valuable in probiotic sauerkraut production. Phenotypic characterisation through API 50 CHL and SDS-PAGE of cell protein patterns revealed that Lactobacillus plantarum is predominant LAB strain in homofermentative phase of fermentation. Autochthonous LAB isolates SF1, SF2, SF4, SF9 and SF15 were selected based on the survival in in vitro gastrointestinal tract conditions. RAPD fingerprints indicated that the selected autochthonous LAB were distinct from one another. All of the strains efficiently inhibited the growth of indicator strains and satisfied technological properties such as acidification rate, tolerance to NaCl and viability during freeze-drying. Strains Lb. paraplantarum SF9 and Lb. brevis SF15, identified by AFLP DNA fingerprints, have shown the best properties to be applied as probiotic starter cultures, because of their highest adhesion to Caco-2 cells and expression of specific, protective S-layer proteins of 45 kDa in size. With addition of these strains, probiotic attribute of the sauerkraut will be achieved, including health promoting, nutritional, technological and economic advantages in large scale industrial sauerkraut production.
S-layers represent the simplest biological membranes developed during the evolution and are one of the most abundant biopolymers on Earth. Current fundamental and applied research aim to reveal the chemical structure, morphogenesis and function of S-layer proteins (Slps). This is the first paper that describes the Slps of certain Lactobacillus brevis strain isolated from sauerkraut. The whole genome sequence (WGS) analysis of the L. brevis SF9B strain uncovered three genes encoding the putative Slps, but merely one, identified as similar to the SlpB of L. brevis ATCC 14869, was expressed. Slp-expressing SF9B cells exhibited increased survival in simulated gastrointestinal
BackgroundAdhesiveness to intestinal epithelium, beneficial immunomodulating effects and the production of pathogen-inhibitory compounds are generally considered as beneficial characteristics of probiotic organisms. We showed the potential health-promoting properties and the mechanisms of probiotic action of seven swine intestinal Lactobacillus amylovorus isolates plus the type strain (DSM 20531T) by investigating their adherence to porcine intestinal epithelial cells (IPEC-1) and mucus as well as the capacities of the strains to i) inhibit the adherence of Escherichia coli to IPEC-1 cells, ii) to produce soluble inhibitors against intestinal pathogens and iii) to induce immune signaling in dendritic cells (DCs). Moreover, the role of the L. amylovorus surface (S) –layers - symmetric, porous arrays of identical protein subunits present as the outermost layer of the cell envelope - in adherence to IPEC-1 cells was assessed using a novel approach which utilized purified cell wall fragments of the strains as carriers for the recombinantly produced S-layer proteins.ResultsThree of the L. amylovorus strains studied adhered to IPEC-1 cells, while four strains inhibited the adherence of E. coli, indicating additional mechanisms other than competition for binding sites being involved in the inhibition. None of the strains bound to porcine mucus. The culture supernatants of all of the strains exerted inhibitory effects on the growth of E. coli, Salmonella, Listeria and Yersinia, and a variable, strain-dependent induction was observed of both pro- and anti-inflammatory cytokines in human DCs. L. amylovorus DSM 16698 was shown to carry two S-layer-like proteins on its surface in addition to the major S-layer protein SlpA. In contrast to expectations, none of the major S-layer proteins of the IPEC-1 -adhering strains mediated bacterial adherence.ConclusionsWe demonstrated adhesive and significant pathogen inhibitory efficacies among the swine intestinal L. amylovorus strains studied, pointing to their potential use as probiotic feed supplements, but no independent role could be demonstrated for the major S-layer proteins in adherence to epithelial cells. The results indicate that many intestinal bacteria may coexist with and confer benefits to the host by mechanisms not attributable to adhesion to epithelial cells or mucus.
Background: We evaluated the functional capacity of plantaricin-producing Lactobacillus plantarum SF9C and S-layer-carrying Lactobacillus brevis SF9B to withstand gastrointestinal transit and to compete among the gut microbiota in vivo. Considering the probiotic potential of Lb. brevis SF9B, this study aims to investigate the antibacterial activity of Lb. plantarum SF9C and their potential for in vivo colonisation in rats, which could be the basis for the investigation of their synergistic functionality. Results: A plantaricin-encoding cluster was identified in Lb. plantarum SF9C, a strain which efficiently inhibited the growth of Listeria monocytogenes ATCC ® 19111 ™ and Staphylococcus aureus 3048. Homology-based three-dimensional (3D) structures of SF9C plantaricins PlnJK and PlnEF were predicted using SWISS-MODEL workspace and the helical wheel representations of the plantaricin peptide helices were generated by HELIQUEST. Contrary to the plantaricin-producing SF9C strain, the S-layer-carrying SF9B strain excluded Escherichia coli 3014 and Salmonella enterica serovar Typhimurium FP1 from the adhesion to Caco-2 cells. Finally, PCR-DGGE analysis of the V2-V3 regions of the 16S rRNA gene confirmed the transit of the two selected lactobacilli through the gastrointestinal tract (GIT). Microbiome profiling via the Illumina MiSeq platform revealed the prevalence of Lactobacillus spp. in the gut microbiota of the Lactobacillus-treated rats, even on the 10th day after the Lactobacillus application, compared to the microbiota of the healthy and AlCl 3-exposed rats before Lactobacillus treatment. Conclusion: The combined application of Lb. plantarum SF9C and Lb. brevis SF9B was able to influence the intestinal microbiota composition in rats, which was reflected in the increased abundance of Lactobacillus genus, but also in the altered abundances of other bacterial genera, either in the model of healthy or aberrant gut microbiota of rats. The antibacterial activity and capacity to withstand in GIT conditions contributed to the functional aspects of SF9C and SF9B strains that could be incorporated in the probiotic-containing functional foods with a possibility to positively modulate the gut microbiota composition.
Aim:The aim of this study was to investigate the in vitro probiotic potential of dairy yeast isolates from artisanal cheeses manufactured in Serbia and Croatia.Materials and Methods:Twelve yeast strains isolated from artisanal fresh soft and white brined cheeses manufactured in Serbia and Croatia were used in the study. Survival in chemically-simulated gastrointestinal conditions, adherence to epithelial intestinal cells and proliferation of gut-associated lymphoid tissue (GALT) cells were evaluated.Results:The results revealed that two strains of Kluyvereomyces lactis ZIM 2408 and ZIM 2453 grew above one log unit (Δ log CFU/ml) in the complex colonic medium during 24 h of cultivation, while Torulaspora delbrueckii ZIM 2460 was the most resistant isolate in chemically-simulated conditions of gastric juice and upper intestinal tract. It was demonstrated that the strains K. lactis ZIM 2408 and ZIM2441 and Saccharomyces cerevisiae ZIM 2415 were highly adhesive to Caco-2 cells, while strains K. lactis ZIM 2408 and Debaryomyces hansenii ZIM 2415 exhibit the highest adhesion percentage to HT29-MTX cells. All strains significantly (P < 0.0001) decreased the proliferation of GALT cells, suggesting the possible strain-specific immunomodulatory potential of the isolates.Conclusion:The dairy yeast isolates exhibit strain-specific probiotic properties, particularly the strain K. lactis ZIM 2408, which appears to be the best probiotic candidate in terms of all three criteria. Taking into account their immunomodulatory potential, the yeast isolates could be further tested for specific probiotic applications and eventually included in functional food formulated for patients suffering from diseases associated with an increased inflammatory status.
Background: Reliable high-throughput microbial pathogen identification in human urine samples is crucial for patients with cystitis symptoms. Currently employed methods are time-consuming and could lead to unnecessary or inadequate antibiotic treatment. Purpose of this study was to assess the potential of mass spectrometry for uropathogen identification from a native urine sample. Methods: In total, 16 urine samples having more than 10 5 CFU/mL were collected from clinical outpatients. These samples were analysed using standard urine culture methods, followed by 16S rRNA gene sequencing serving as control and here described culture-independent MALDI-TOF/TOF MS method being tested. Results: Here we present advantages and disadvantages of bottom-up proteomics, using MALDI-TOF/TOF tandem mass spectrometry, for culture-independent identification of uropathogens (e.g. directly from urine samples). The direct approach provided reliable identification of bacteria at the genus level in monobacterial samples. Taxonomic identifications obtained by proteomics were compared both to standard urine culture test used in clinics and genomic test based on 16S rRNA sequencing. Conclusions: Our findings indicate that mass spectrometry has great potential as a reliable high-throughput tool for microbial pathogen identification in human urine samples. In this case, the MALDI-TOF/TOF, was used as an analytical tool for the determination of bacteria in urine samples, and the results obtained emphasize high importance of storage conditions and sample preparation method impacting reliability of MS2 data analysis. The proposed method is simple enough to be utilized in existing clinical settings and is highly suitable for suspected single organism infectious etiologies. Further research is required in order to identify pathogens in polymicrobial urine samples.
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