Screening for potential probiotic from spontaneously fermented non-dairy foods based on in vitro probiotic and safety properties

The aim of this study was to screen potential probiotic lactic acid bacteria from Chinese spontaneously fermented non-dairy foods by evaluating their probiotic and safety properties. All lactic acid bacteria (LAB) strains were identified by 16S rRNA gene sequencing. The in vitro probiotic tests included survival under low pH and bile salts, cell surface hydrophobicity, auto-aggregation, co-aggregation, antibacterial activity, and adherence ability to cells. The safety properties were evaluated based on hemolytic activity and antibiotic resistance profile. The salt tolerance, growth in litmus milk, and acidification ability were examined on selected potential probiotic LAB strains to investigate their potential use in food fermentation. A total of 122 strains were isolated and identified at the species level by 16S rRNA gene sequencing and included 62 Lactobacillus plantarum, 40 Weissella cibaria, 12 Lactobacillus brevis, 6 Weissella confusa, and 2 Lactobacillus sakei strains. One W. cibaria and nine L. plantarum isolates were selected based on their tolerance to low pH and bile salts. The hydrophobicity, auto-aggregation, co-aggregation, and antagonistic activities of these isolates varied greatly. All of the 10 selected strains showed multiple antibiotic resistance phenotypes and no hemolytic activity. The highest adhesion capacity to SW480 cells was observed with L. plantarum SK1. The isolates L. plantarum SK1, CB9, and CB10 were the most similar strains to Lactobacillus rhamnosus GG and selected for their high salt tolerance and acidifying activity. The results revealed strain-specific probiotic properties were and potential probiotics that can be used in the food industry.     

In Vitro Characterization of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains with Inhibitory Activity on Enteropathogens for Use as Potential Animal Probiotics

The present study evaluates the probiotic properties of three Lactobacillus plantarum strains MJM60319, MJM60298, and MJM60399 possessing antimicrobial activity against animal enteric pathogens. The three strains did not show bioamine production, mucinolytic and hemolytic activity and were susceptible to common antibiotics. The L. plantarum strains survived well in the simulated orogastrointestinal transit condition and showed adherence to Caco-2 cells in vitro. The L. plantarum strains showed strong antimicrobial activity against enterotoxigenic Escherichia coli, Shiga toxin-producing E. coli, Salmonella enterica subsp. enterica serovar Typhimurium, Choleraesuis and Gallinarum compared to the commercial probiotic strain Lactobacillus rhamnosus GG. The mechanism of antimicrobial activity of the L. plantarum strains appeared to be by the production of lactic acid. Furthermore, the L. plantarum strains tolerated freeze-drying and maintained higher viability in the presence of cryoprotectants than without cryoprotectants. Finally, the three L. plantarum strains tolerated NaCl up to 8% and maintained >60% growth. These characteristics of the three L. plantarum strains indicate that they could be applied as animal probiotic after appropriate in vivo studies.     

Comparative evaluation of automated ribotyping and RAPD-PCR for typing of <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. occurring in dental caries

A group of 67 Lactobacillus spp. strains containing Lactobacillus casei/paracasei, Lactobacillus fermentum, Lactobacillus gasseri, Lactobacillus plantarum, Lactobacillus rhamnosus and Lactobacillus salivarius species isolated from early childhood caries and identified to the species level in a previous study (?vec et al., Folia Microbiol 54:53–58, 2009) was characterized by automated ribotyping performed by the RiboPrinter^® microbial characterization system and by randomly amplified polymorphic DNA fingerprinting (RAPD-PCR) with M13 primer to evaluate these techniques for characterization of lactobacilli associated with dental caries. Ribotyping revealed 55 riboprints among the analysed group. The automatic identification process performed by the RiboPrinter system identified 18 strains to the species level, however cluster analysis divided obtained ribotype patterns into individual clusters mostly corresponding to the species assignment of particular strains. RAPD-PCR fingerprints revealed by the individual Lactobacillus spp. showed higher variability than the ribotype patterns and the fingerprint profiles generated by the analysed species were distributed among one to four clusters. In conclusion, ribotyping is shown to be more convenient for the identification purposes while RAPD-PCR fingerprinting results indicate this method is a better tool for typing of Lactobacillus spp. strains occurring in dental caries.     

Antagonistic Activity of Lactic Acid Bacteria <Emphasis Type="Italic">Lactobacillus</Emphasis> spp. against Clinical Isolates of <Emphasis Type="Italic">Klebsiella pneumoniae</Emphasis>

The screening of three strains of lactic acid bacteria identified as Lactobacillus rhamnosus, Lactobacillus reuteri, and Lactobacillus helveticus showed significant antagonistic activity against Klebsiella pneumoniae strains characterized by multiple antibiotic resistance. Lactobacilli cocultivated with the Klebsiella strains inhibited their growth 20 to 86% on the first and second days, respectively. Exoproteome analysis of L. rhamnosus cocultivated with K. pneumoniae revealed the induction of peptidoglycan hydrolases, including extracellular lytic transglycosylases, family II (MltA), and endopeptidases capable of disrupting the peptidoglycan bacterial cell wall.     

Assessment of the antifungal activity of <Emphasis Type="Italic">Lactobacillus</Emphasis> and <Emphasis Type="Italic">Pediococcus</Emphasis> spp. for use as bioprotective cultures in dairy products

Fungi are commonly involved in dairy product spoilage and the use of bioprotective cultures can be a complementary approach to reduce food waste and economic losses. In this study, the antifungal activity of 89 Lactobacillus and 23 Pediococcus spp. isolates against three spoilage species, e.g., Yarrowia lipolytica, Rhodotorula mucilaginosa and Penicillium brevicompactum, was first evaluated in milk agar. None of the tested pediococci showed antifungal activity while 3, 23 and 43 lactobacilli isolates showed strong antifungal activity or total inhibition against Y. lipolytica, R. mucilaginosa and P. brevicompactum, respectively. Then, the three most promising strains, Lactobacillus paracasei SYR90, Lactobacillus plantarum O_VI9 and Lactobacillus rhamnosus BIO_III28 at initial concentrations of 10⁵ and 10⁷ CFU/ml were tested as bioprotective cultures against the same fungal targets in a yogurt model during a 5-week storage period at 10 °C. While limited effects were observed at 10⁵ CFU/ml inoculum level, L. paracasei SYR90 and L. rhamnosus BIO_III28 at 10⁷ CFU/ml respectively retarded the growth of R. mucilaginosa and P. brevicompactum as compared to a control without selected cultures. In contrast, growth of Y. lipolytica was only slightly affected. In conclusion, these selected strains may be good candidates for bioprotection of fermented dairy products.     

Safety,probiotic and technological properties of <Emphasis Type="Italic">Lactobacilli</Emphasis> isolated from unpasteurised ovine and caprine cheeses

Eleven Lactobacillus plantarum from Slovak ovine and caprine lump and stored cheeses, and from four commercial probiotic and yogurt cultures (Lactobacillus plantarum, Lactobacillus reuteri, Lactobacillus acidophilus) identified using a Maldi-TOF MS analysis were screened in vitro for selected aspects correlated with safety (antibiotic susceptibility patterns, biochemical and haemolytic activity, presence of genes responsible for biogenic amines production), functional traits (including acid, bile tolerance and antimicrobial activity), ecological roles (ability to produce biofilms), and technological applications (acidification and milk coagulation capacity) for assurance of their quality and diversity. The antibiotic susceptibility showed two L. plantarum strains, 19l5 and 18l4, with the presence of the non-wild-type ECOFFs (epidemiological cut-off) for clindamycin and/or gentamicin. All these strains expressed a high acid tolerance at pH 2.5 after a 4 h exposure (bacteria viability varied between 60% and 91%), and bile resistance at 0.3% oxgall ranged from 60% to 99% with no haemolytic activity. Three wild L. plantarum strains, 17l1, 16l4, 18l2, had no harmful metabolic activities, and formed strong biofilms that were measured by a crystal violet assay. Simultaneously, the acid cell-free culture supernatant (ACFCS) from L. plantarum 18l2 had a marked inhibitory effect on the viability of the pathogens as evaluated by flow-cytometry, and also exhibited fast acidification and milk coagulation. As a result, we conclude that L. plantarum 18l2 can be included as part of the created lactobacilli collection that is useful as a starter, or starter adjunct, in the dairy industry, due to its desirable safety and probiotic characteristics, together with rapid acidification capacity compared with other investigated strains from commercially accessible products.     

Potential probiotic properties of lactic acid bacteria isolated from the intestinal mucosa of healthy piglets

In the present study, the probiotic properties of 52 lactic acid bacteria strains, isolated from the intestinal mucosa of 60-day-old healthy piglets, were evaluated in vitro in order to acquire probiotics of potential application. Based on acidic and bile salt resistance, 11 lactic acid bacteria strains were selected, among which 1 was identified as Pediococcus acidilactici, 3 as Enterococcus faecium, 3 as Lactobacillus rhamnosus, 2 as Lactobacillus brevis, and 2 as Lactobacillus plantarum by 16S rRNA gene sequencing. All selected strains were further investigated for transit tolerance in simulated upper gastrointestinal tract, for adhesion capacity to swine intestinal epithelial cells J2 (IPEC-J2), for cell surface characteristics including hydrophobicity, co-aggregation and auto-aggregation, and for antimicrobial activities. Moreover, hemolytic, bile salt hydrolase and biogenic amine-producing abilities were investigated for safety assessment. Two E. faecium (WEI-9 and WEI-10) and one L. plantarum (WEI-51) exhibited good simulated upper gastrointestinal tract tolerance, and showed high auto-aggregation and co-aggregation with Escherichia coli 1570. The strains WEI-9 and WEI-10 demonstrated the highest adherence capacity. The 11 selected strains mentioned above exhibited strong antimicrobial activity against E. coli CVCC1570, Staphylococcus aureus CVCC1882 and Salmonella pullorum AS1.1859. None of the 11 selected strains, except WEI-9 and WEI-33, exhibited bile salt hydrolase, hemolytic or biogenic amine-producing abilities. This work showed that the E. faecium WEI-10 and L. plantarum WEI-51were found to have the probiotic properties required for use as potential probiotics in animal feed supplements.     

Antibacterial and Antibiofilm Activity of Lactic Acid Bacteria Isolated from Traditional Artisanal Milk Cheese from Northeast China Against Enteropathogenic Bacteria

The present study aims to investigate the probiotic properties of novel strains of lactic acid bacteria isolated from traditional artisanal milk cheese from Northeast China and to explore their antibacterial activity against enteropathogenic bacteria. Of the 321 isolates, 86 exhibited survival in low pH, resistance to pancreatin, and tolerance to bile salts; of these, 12 inhibited the growth of more than seven enteropathogenic bacteria and exhibited antibiofilm activities against Staphylococcus aureus CMCC26003 and/or Escherichia coli CVCC230. Based on 16S ribosomal RNA sequence analysis, the 12 isolates were assigned to Lactobacillus plantarum (7), Lactobacillus helveticus (3), Pediococcus acidilactici (1), and Enterococcus faecium (1) species. In addition, 5 of the 12 strains were susceptible to most of the tested antibiotics. Furthermore, four strains with sensitivity to antibiotics showed significantly high levels of hydrophobicity similar to or better than the reference strain Lactobacillus rhamnosus GG. Moreover, three strains were confirmed safe through non-hemolytic activities and bacterial translocation. Overall, the selected Lact. plantarum 27053 and 27172 and Lact. helveticus 27058 strains can be considered potential probiotic strains and candidates for further application in functional food and prevention or treatment of gastrointestinal diseases.     

Identification and analysis of the function of surface layer proteins from three <Emphasis Type="Italic">Lactobacillus</Emphasis> strains

To identify and investigate the role of surface layer proteins (SLPs) on the probiotic properties of Lactobacillus strains, SLPs were extracted from Lactobacillus bulgaricus fb04, L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus NCFM by 5 mol/L lithium chloride. The molecular masses of the four SLPs were approximately 45–47 kDa as analyzed by SDS-PAGE. Hydrophobic amino acids were the main components of the four SLPs. The secondary structure content of the four SLPs showed extensive variability among different strains. After the SLPs were removed from the cell surface, the autoaggregation ability, coaggregation ability, and gastrointestinal tolerability of the four lactobacilli were significantly reduced as compared with the intact cells (P?<?0.05). When exposed to bile salt stress, L. rhamnosus fb06, L. gasseri fb07, and L. acidophilus NCFM expressed more SLPs as determined by Bradford method. In conclusion, the four lactobacilli all possessed functional SLPs, which had positive contributions to the probiotic properties of the four Lactobacillus strains. This research could reveal the biological contributions of SLPs from Lactobacillus strains and offer a theoretical basis for the application of lactobacilli and their SLPs in food and pharmaceutical industries.     

Biofilm formation and antimicrobial sensitivity of lactobacilli contaminants from sugarcane-based fuel ethanol fermentation

Industrial ethanol fermentation is subject to bacterial contamination that causes significant economic losses in ethanol fuel plants. Chronic contamination has been associated with biofilms that are normally more resistant to antimicrobials and cleaning efforts than planktonic cells. In this study, contaminant species of Lactobacillus isolated from biofilms (source of sessile cells) and wine (source of planktonic cells) from industrial and pilot-scale fermentations were compared regarding their ability to form biofilms and their sensitivity to different antimicrobials. Fifty lactobacilli were isolated and the most abundant species were Lactobacillus casei, Lactobacillus fermentum and Lactobacillus plantarum. The majority of the isolates (87.8%) were able to produce biofilms in pure culture. The capability to form biofilms and sensitivity to virginiamycin, monensin and beta-acids from hops, showed inter- and intra-specific variability. In the pilot-scale fermentation, Lactobacillus brevis, L. casei and the majority of L. plantarum isolates were less sensitive to beta-acids than their counterparts from wine; L. brevis isolates from biofilms were also less sensitive to monensin when compared to the wine isolates. Biofilm formation and sensitivity to beta-acids showed a positive and negative correlation for L. casei and L. plantarum, respectively.     

Selection and evaluation of functional characteristics of autochthonous lactic acid bacteria isolated from traditional fermented stinky bean (<Emphasis Type="Italic">Sataw-Dong</Emphasis>)

The aim of this study was to evaluate the technological and functional potential of lactic acid bacteria (LAB) isolated from fermented stinky bean (Sataw-Dong). Of the 114 LAB colonies isolated from spontaneously fermented stinky bean which showed inhibitory activity against two food-borne pathogens (Staphylococcus aureus DMST 4480 and Escherichia coli DMST 4212), the five isolates (KJ03, KJ15, KJ17, KJ22, KJ23) exhibiting excellent antagonistic activity were subjected to further study. These five strains showed titratable acidity as lactic acid in the range of 1.47–1.55 %, with strains KJ03 and KJ23 additionally exhibiting a high NaCl tolerance of >7 % (w/v). Using 16S rRNA gene sequence analysis, strains KJ03 and KJ23 were identified as Lactobacillus plantarum and L. fermentum, respectively, and further investigated for their functional properties in vitro. Both strains survived well in a simulated gastrointestinal tract environment with <1 log cell decrease over 8 h (>8 log CFU/ml). Lactobacillus plantarum KJ03 showed the best performance with respect to cholesterol removal (53 %), while L. fermentum KJ23 showed the highest cell-surface hydrophobicity (39.5 %). Neither of the two strains showed any hemolysis activity. Both strains hydrolyzed glycodeoxycholic and taurodeoxycholic acids. In terms of antibiotic susceptibility, L. fermentum KJ23 was not sensitive to tetracycline. Taking all of the results into account, L. plantarum KJ03 possessed desirable in vitro functional properties. This strain is therefore a good candidate for further investigation for use in Sataw-Dong fermentation to assess its technological performance as a potential probiotic starter.     

Aciduric Strains of <Emphasis Type="Italic">Lactobacillus reuteri</Emphasis> and <Emphasis Type="Italic">Lactobacillus rhamnosus</Emphasis>, Isolated from Human Feces,Have Strong Adhesion and Aggregation Properties

Human feces were streaked onto MRS Agar adjusted to pH 2.5, 3.0, and 6.4, respectively, and medium supplemented with 1.0% (w/v) bile salts. Two aciduric strains, identified as Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 (based on 16S rDNA and recA sequences), were non-hemolytic and did not hydrolyze mucin. The surface of Lactobacillus reuteri HFI-LD5 cells has a weak negative charge, whereas Lactobacillus rhamnosus HFI-K2 has acidic and basic properties, and produces exopolysaccharides (EPS). None of the strains produce bacteriocins. Both strains are resistant to several antibiotics, including sulfamethoxazole-trimethoprim and sulphonamides. The ability of Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 to grow at pH 2.5 suggests that they will survive passage through the stomach. EPS production may assist in binding to intestinal mucus, especially in the small intestinal tract, protect epithelial cells, and stimulate the immune system. Lactobacillus reuteri HFI-LD5 and Lactobacillus rhamnosus HFI-K2 may be used as probiotics, especially in the treatment of small intestinal bacterial overgrowth (SIBO).     

Genotypic characterization of Lactic acid bacteria in gut microbiome of freshwater fish

The present study focused on identification and genotypic characterization of Lactic acid bacteria (LAB) in the intestine of freshwater fish. 76 strains of LAB were isolated and identified by 16S rRNA gene sequences and hsp60 gene sequences as different strains of Lactobacillus plantarum, Lactobacillus pentosus, Lactobacillus fermentum, Lactobacillus delbrueckii subsp. bulgaricus, Lactobacillus brevis, Lactobacillus reuteri, Lactobacillus salivarius, Pediococcus pentosaceus, Pediococcus acidilactici, Weissella paramesenteroides, Weissella cibaria, Enterococcus faecium, and Enterococcus durans. The hsp60 gene showed a higher level of sequence variation among the isolates examined, with lower interspecies sequence similarity providing more resolutions at the species level than the 16S rRNA gene. Phylogenetic tree derived from hsp60 gene sequences with higher bootstrap values at the nodal branches was more consistent as compared to phylogenetic tree constructed from 16S rRNA gene sequences. Closely related species L. plantarum and L. pentosus as well as species L. delbrueckii subsp. bulgaricus and L. fermentum were segregated in different cluster in hsp60 phylogenetic tree whereas such a distribution was not apparent in 16S rRNA phylogenetic tree. In silico restriction analysis revealed a high level of polymorphism within hsp60 gene sequences. Restriction pattern with enzymes AgsI and MseI in hsp60 gene sequences allowed differentiation of all the species including closely related species L. plantarum and L. pentosus, E. faecium and E. durans. In general, hsp60 gene with higher evolutionary divergence proved to be a better phylogenetic marker for the group LAB.     

<Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> as a Probiotic Potential from Kouzeh Cheese (Traditional Iranian Cheese) and Its Antimicrobial Activity

The aim of this study is to isolate and identify Lactobacillus plantarum isolates from traditional cheese, Kouzeh, and evaluate their antimicrobial activity against some food pathogens. In total, 56 lactic acid bacteria were isolated by morphological and biochemical methods, 12 of which were identified as Lactobacillus plantarum by biochemical method and 11 were confirmed by molecular method. For analyzing the antimicrobial activity of these isolates properly, diffusion method was performed. The isolates were identified by 318 bp band dedicated for L. plantarum. The isolated L. plantarum represented an inhibitory activity against four of the pathogenic bacteria and showed different inhibition halos against each other. The larger halos were observed against Staphylococcus aureus and Staphylococcus epidermidis (15 ± 0.3 and 14.8 ± 0.7 mm, respectively). The inhibition halo of Escherichia coli was smaller than that of other pathogen and some L. plantarum did not show any inhibitory activity against E. coli, which were resistant to antimicrobial compounds produced by L. plantarum. The isolated L. plantarum isolates with the antimicrobial activity in this study had strong probiotic properties. These results indicated the nutritional value of Kouzeh cheese and usage of the isolated isolates as probiotic strains.     

Viability and Stress Response of Putative Probiotic <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Strains in Honey Environment

Due to problem of preservation of dairy products which serve as a matrix for probiotics, it is challenging to use these probiotics as food supplements in many developing countries. To determine the suitability of the Lactobacillus strains for exploitation as probiotics in honey, we investigated the effect of their storage on the viability, functionality, and the mechanism associated with their protective effect. Three isolates obtained from our laboratory collection were identified through amplification of the 16S rRNA gene. The viability of the strains in honey at different storage conditions was studied. Three genes (hdc, gtf, and clpL) responsible for the resistance of bacteria in acidic environments were screened. SDS-PAGE analysis of total protein was performed to observe protein profile changes of the strains after exposure to honey. All the three isolates, namely, GGU, GLA51, and GLP56, were identified as Lactobacillus plantarum strains. After 28 days of storage in honey at 4 °C, viable cell concentrations of the three strains were higher than 2.04?×?10⁶ CFU/ml. During the same period at room temperature, only the Lactobacillus plantarum GLP56 strain remained viable with a cell concentration of 1.86?×?10⁴ CFU/ml. The clpL gene coding for ATPase was detected in all the three strains. The protein of molecular weight ~?50 kDa was absent in the protein profile of Lactobacillus plantarum GGU after 60 days of storage in honey at 4 °C. The Lactobacillus plantarum GLP56, Lactobacillus plantarum GLA51, and Lactobacillus plantarum GGU strains exposed to honey can withstand acidic environmental stress but their viability declines over time.     

Survey on proteolytic activity and diversity of proteinase genes in mesophilic lactobacilli

Lactocepins or CEPs are large cell wall bound extracellular proteinases of lactic acid bacteria, involved in protein breakdown and utilization. They are responsible for many health-promoting traits of food products fermented with these organisms, but also essential for probiotic effects of certain strains. Different mesophilic strains selected within the species Lactobacillus zeae, Lb. casei, Lb. rhamnosus, and Lb. plantarum were analyzed for their proteolytic activity towards main fractions of milk proteins—caseins and whey proteins. The strains showing excellent proteolytic features were further examined for presence of corresponding proteinase gene(s). It was found that Lb. zeae LMG17315 possessed catalytic domains of three distinct proteinase genes, unique feature in Lb. casei group, which are similar but not identical to previously characterized prtP and prtR genes. Lb. casei neotype strain ATCC393 was also analysed and based on obtained results its reclassification in taxon Lb. zeae is supported. In addition, we report catalytic domain of prtR-type gene in Lb. plantarum LMG9208, which is first such report in this species, and it is first time that this gene is reported outside Lb. casei group.     

Effect of <Emphasis Type="Italic">Lactobacillus plantarum</Emphasis> Tennozu-SU2 on <Emphasis Type="Italic">Salmonella</Emphasis> Typhimurium Infection in Human Enterocyte-Like HT-29-Luc Cells and BALB/c Mice

The probiotic properties and inhibitory effect on Salmonella Typhimurium adhesion on human enterocyte-like HT-29-Luc cells of three Lactobacillus plantarum strains isolated from fermented fish, beach sand and a coastal plant were determined. Compared with the type strain L. plantarum NBRC 15891^T, which was isolated from pickled cabbage, L. plantarum Tennozu-SU2 isolated from the acorn of a coastal tree showed high autoaggregation in de Man, Rogosa and Sharpe (MRS) broth and an antagonistic effect against S. Typhimurium in brain heart infusion (BHI) broth. Furthermore, heat-killed L. plantarum Tennozu-SU2 cells inhibited S. Typhimurium adhesion on HT-29-Luc cells. Both live and heat-killed L. plantarum Tennozu-SU2 cells showed an inhibitory effect on gut colonisation in BALB/c mice, as assessed by viable Salmonella count in faecal samples and by invasion into liver and spleen tissues. The properties shown in this study suggest that L. plantarum Tennozu-SU2 is useful as a starter and probiotic bacteria in functional food material.     

Isolation of <Emphasis Type="Italic">Lactobacillus</Emphasis> strains from shellfish for their potential use as probiotics

Microorganisms intended for use as probiotics in aquaculture should exert antimicrobial activity and be regarded as safe not only for their aquatic hosts but also for their surrounding environments and humans. The objective of this work was to investigate antimicrobial activity against various pathogens, bile salt tolerance, and acid tolerance of 65 presumptive Lactobacillus spp. isolated from shellfish samples. Four strains (HL1, HL12, HL20, and JL28) were selected after qualitatively identifying high levels of antimicrobial activity against bacteria including Staphylococcus aureus, Salmonella typhimurium, Salmonella enteritidis, Escherichia coli O157:H7, Vibrio ichthyoenteri, Edwardsiella tarda, Streptococcus iniae, and V. parahaemolyticus. The sequence analysis of their 16S rRNA genes revealed that the four strains belong to the Lactobacillus plantarum species. In addition, their survivability was tested in bile salt and acidic conditions to show their potential use as probiotics in the gastrointestinal tract.     

Human milk and mucosal lacto- and galacto-<Emphasis Type="Italic">N</Emphasis>-biose synthesis by transgalactosylation and their prebiotic potential in <Emphasis Type="Italic">Lactobacillus</Emphasis> species

Lacto-N-biose (LNB) and galacto-N-biose (GNB) are major building blocks of free oligosaccharides and glycan moieties of glyco-complexes present in human milk and gastrointestinal mucosa. We have previously characterized the phospho-β-galactosidase GnbG from Lactobacillus casei BL23 that is involved in the metabolism of LNB and GNB. GnbG has been used here in transglycosylation reactions, and it showed the production of LNB and GNB with N-acetylglucosamine and N-acetylgalactosamine as acceptors, respectively. The reaction kinetics demonstrated that GnbG can convert 69 ± 4 and 71 ± 1 % of o-nitrophenyl-β-d-galactopyranoside into LNB and GNB, respectively. Those reactions were performed in a semi-preparative scale, and the synthesized disaccharides were purified. The maximum yield obtained for LNB was 10.7 ± 0.2 g/l and for GNB was 10.8 ± 0.3 g/l. NMR spectroscopy confirmed the molecular structures of both carbohydrates and the absence of reaction byproducts, which also supports that GnbG is specific for β1,3-glycosidic linkages. The purified sugars were subsequently tested for their potential prebiotic properties using Lactobacillus species. The results showed that LNB and GNB were fermented by the tested strains of L. casei, Lactobacillus rhamnosus (except L. rhamnosus strain ATCC 53103), Lactobacillus zeae, Lactobacillus gasseri, and Lactobacillus johnsonii. DNA hybridization experiments suggested that the metabolism of those disaccharides in 9 out of 10 L. casei strains, all L. rhamnosus strains and all L. zeae strains tested relies upon a phospho-β-galactosidase homologous to GnbG. The results presented here support the putative role of human milk oligosaccharides for selective enrichment of beneficial intestinal microbiota in breast-fed infants.     

Improvement of the quality of maize grain silage by a synergistic action of selected lactobacilli strains

As silage is one of the most important feed sources for dairy cattle it is recommended for farmers to preserve silage by fermentation. Interaction of the five strains of Lactobacillus genera [Lactobacillus buchneri A KKP 2047 p (LB), L. reuteri M KKP 2048 p (LR), L. plantarum K KKP 593 p (LPk), L. plantarum S KKP 2021 p (LPs), L. fermentum N KKP 2020 p (LF)] has been shown aiming to increase the safety of corn grain silage fodder. Experiments were conducted in polyethylene microsilos for 48 days and on production scale in an experimental farm for 3 years. Synergistic activity of the studied bacterial strains in terms of reducing aflatoxin B₁ and ochratoxin A levels was clear in these experimental variants wherein to the inoculants of the LB?+?LR strains subsequent bacterial strains LPk, LPs and LF were sequentially added. Silages inoculated with five bacterial strains were free from pathogens and showed the lowest yeast and mold count values among all experimental variants. As a result of employing the preparation starter culture for ensiling corn grain there were obtained silages characterized by high stability, microbiological and chemical purity, thus safe in feeding livestock.