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1.
Bile salts play an important role in the digestion of lipids in vertebrates and are synthesized and conjugated to either glycine or taurine in the liver. Following secretion of bile salts into the small intestine, intestinal microbes are capable of deconjugating the glycine or taurine from the bile salts, using an enzyme called bile salt hydrolase (Bsh). Intestinal lactobacilli are regarded as major contributors to bile salt hydrolysis in vivo. Since the bile salt-hydrolyzing strain Lactobacillus plantarum WCFS1 was predicted to carry four bsh genes (bsh1, bsh2, bsh3, and bsh4), the functionality of these bsh genes was explored using Lactococcus lactis heterologous overexpression and multiple bsh deletion strains. Thus, Bsh1 was shown to be responsible for the majority of Bsh activity in L. plantarum WCFS1. In addition, bsh1 of L. plantarum WCFS1 was shown to be involved in conferring tolerance to specific bile salts (i.e., glycocholic acid). Northern blot analysis established that bsh1, bsh2, bsh3, and bsh4 are all expressed in L. plantarum WCFS1 during the exponential growth phase. Following biodiversity analysis, bsh1 appeared to be the only bsh homologue that was variable among L. plantarum strains; furthermore, the presence of bsh1 correlated with the presence of Bsh activity, suggesting that Bsh1 is commonly responsible for Bsh activity in L. plantarum strains. The fact that bsh2, bsh3, and bsh4 genes appeared to be conserved among L. plantarum strains suggests an important role of these genes in the physiology and lifestyle of the species L. plantarum. Analysis of these additional bsh-like genes in L. plantarum WCFS1 suggests that they might encode penicillin acylase rather than Bsh activity, indicating their implication in the conversion of substrates other than bile acids in the natural habitat.  相似文献   

2.
Genes coding for bile salt hydrolase of Lactobacillus plantarum CGMCC 8198, a novel probiotic strain isolated from silage, were identified, analyzed and cloned. L. plantarum strongly resisted the inhibitory effects of bile salts and also decreased serum cholesterol levels by 20 % in mice with hypercholesterolemia. Using RT-PCR analysis, bsh2, bsh3 and bsh4 were upregulated by bile salts in a dose-dependent manner. All three bsh genes had high similarity with those of other Lactobacillus strains. All three recombinant BSHs had high activities for the hydrolysis of glycodeoxycholic acids and taurodeoxycholic acids.  相似文献   

3.
4.
Bile salt hydrolase (Bsh) active probiotic strains hydrolyze bile acid amino conjugates in vivo, which triggers cholesterol consumption in liver to synthesize new bile leading to consequential cholesterol lowering. Hence, bile salt hydrolyzing potential was the criterion to select L. fermentum NCDO394 for this study and its gene encoding Bsh was identified and cloned. The resulting nucleotide sequence of bsh gene contained an open reading frame (ORF) of 978 nucleotides encoding a predicted protein of 325 amino acids with a theoretical pI of 6.39. Moreover, deduced Bsh protein had high similarity with the Bshs of L. fermentum only and also exhibited significant similarity to the Pencillin V amidases of other Lactobacillus spp. Five catalytically important amino acids were highly conserved in L. fermentum Bsh while four amino acid motifs around these active sites, were not as consistent as in other Bsh proteins. Furthermore, L. fermentum bsh gene was sub-cloned into pET-28b(+) vector, and its expression was induced with 0.05 mM isopropylthiogalactopyranoside (IPTG) in Escherichia coli BL21(DE3). The recombinant Bsh (rBsh) was purified with homogeneity using Ni+2-NTA column and characterized for substrate specificity, pH and temperature. The rBsh hydrolyzed six major human bile salts with a slight preference towards glycine-conjugated bile salts. The optimum pH of rBsh was six, and its enzymatic activity declined below pH 5 and above pH 7. The enzyme was stable and functional even at 65 °C while showed its maximum activity at 37 °C. In conclusion, L. fermentum NCDO394 may be a promising candidate probiotic which may affect cholesterol metabolism in vivo.  相似文献   

5.
Lactobacillus plantarum strains isolated and identified from naturally-fermented Chinese sauerkraut were examined in vitro for potential probiotic properties and in vivo for cholesterol-lowering effect in mice. Among 7 isolated L. plantarum strains, strains S2-5 and S4-1 were found to possess desirable probiotic properties including ability to survive at pH 2.0 for 60 min, tolerate pancreatin and bile salts, adhere to Caco-2 cells, produce high β-galactosidase activity and antimicrobial activity against Escherichia coli O157 and Shigella flexneri CMCC(B). In addition, strains S2-5 and S4-1 were susceptible to several antibiotics, and capable of reducing cholesterol level in MRS medium by assimilation of cholesterol at 20.39 and 22.28 μg ml?1, respectively. The in vivo study with L. plantarum S4-1 showed that feeding with fermented milk containing this strain was able to effectively reduce serum cholesterol level in mice, demonstrating its potential as an excellent probiotic candidate for applications in functional products.  相似文献   

6.
In this study, seven bacteriocinogenic and non-bacteriocinogenic LAB strains previously isolated from the intestines of Nile tilapia and common carp and that showed potent antibacterial activity against host-derived and non-host-derived fish pathogens were assayed for their probiotic and safety properties so as to select promising candidates for in vivo application as probiotic in aquaculture. All the strains were investigated for acid and bile tolerances, transit tolerance in simulated gastrointestinal conditions, for cell surface characteristics including hydrophobicity, co-aggregation and auto-aggregation, and for bile salt hydrolase activity. Moreover, haemolytic, gelatinase and biogenic amine-producing abilities were investigated for safety assessment. The strains were found to be tolerant at low pH (two strains at pH 2.0 and all the strains at pH 3.0). All of them could also survive in the presence of bile salts (0.3% oxgall) and in simulated gastric and intestinal juices conditions. Besides, three of them were found to harbour the gtf gene involved in pH and bile salt survival. The strains also showed remarkable cell surface characteristics, and 57.14% exhibited the ability to deconjugate bile salts. When assayed for their safety properties, the strains prove to be free from haemolytic activity, gelatinase activity and they could neither produce biogenic amines nor harbour the hdc gene. They did not also show antibiotic resistance, thus confirming to be safe for application as probiotics. Among them, Lactobacillus brevis 1BT and Lactobacillus plantarum 1KMT exhibited the best probiotic potentials, making them the most promising candidates.  相似文献   

7.
The search for probiotic candidates among lactic acid bacteria (LAB) isolated from food may uncover new strains with promising health and technological properties. Lactobacillus mucosae strains attracted recent research attention due to their ability to adhere to intestinal mucus and to inhibit pathogens in the gastrointestinal tract, both related to a probiotic potential. Properties of interest and safety aspects of three Lb. mucosae strains (CNPC006, CNPC007, and CNPC009) isolated from goat milk were investigated employing in vitro tests. The presence of genetic factors related to bile salt hydrolase production (bsh), intestinal adhesion properties (msa, map, mub, and ef-tu), virulence, and biogenic amine production were also verified. All strains exhibited the target map, mub, and ef-tu sequences; the msa gene was detected in CNPC006 and CNPC007 strains. Some of the searched sequences for virulence factors were detected, especially in the CNPC009 strain; all strains carried the hyl gene, related to the production of hyaluronidase. Lb. mucosae CNPC007 exhibited a high survival rate in simulated gastric and enteric conditions. Besides, all strains exhibited the bsh sequence, and CNPC006 and CNPC007 were able to deconjugate salts of glycodeoxycholic acid (GDC). Regarding technological properties for dairy product applications, a relatively higher milk acidification and clotting capacity, diacetyl production, and proteolytic activity were registered for CNPC007 in comparison to the other strains. Collectively, the results aim at Lb. mucosae CNPC007 as a promising probiotic candidate for application in dairy products, deserving further studies to confirm and explore its potential.  相似文献   

8.
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.  相似文献   

9.
Three probiotic Lactobacillus strains, Lactobacillus acidophilus, Lactobacillus plantarum, and Lactobacillus delbrueckii, were tested for their ability to assimilate and metabolize glycerol. Biodiesel-derived glycerol was used as the main carbon and energy source in batch microaerobic growth. Here, we show that the tested strains were able to assimilate glycerol, consuming between 38 and 48 % in approximately 24 h. L. acidophilus and L. delbrueckii showed a similar growth, higher than L. plantarum. The highest biomass reached was 2.11 g?L?1 for L. acidophilus, with a cell mass yield (Y X/S) of 0.37 g?g?1. L. delbrueckii and L. plantarum reached a biomass of 2.06 and 1.36 g?L?1. All strains catabolize glycerol mainly through glycerol kinase (EC 2.7.1.30). For these lactobacillus species, kinetic parameters for glycerol kinase showed Michaelis–Menten constant (K m) ranging from 1.2 to 3.8 mM. The specific activities for glycerol kinase in these strains were in the range of 0.18 to 0.58 U?mg?protein?1, with L. acidophilus ATCC 4356 showing the maximum specific activity after 24 h of cultivation. Glycerol dehydrogenase activity was also detected in all strains studied but only for the reduction of glyceraldehyde with NADPH (K m for DL-glyceraldehyde ranging from 12.8 to 32.3 mM). This enzyme shows a very low oxidative activity with glycerol and NADP+ and, most likely, under physiological conditions, the oxidative reaction does not occur, supporting the assumption that the main metabolic flux concerning glycerol metabolism is through the glycerol kinase pathway.  相似文献   

10.
Sixteen Lactobacillus plantarum strains, isolated from fermented table olives, were studied for the presence and expression of genes involved in the production of bacteriocins, pheromones and other peptides. The presence of 13 genes that belong to pln locus was monitored, while for the study of gene expression, producer strains were cultured in growth medium with variant salinity (0, 4, 6, and 8 % NaCl) and pH (3.5, 4.0, 4.5, and 6.4). The effect of producer strain on the growth of indicator microorganisms was evaluated using a well diffusion assay. In parallel, Real-Time PCR was employed to monitor the genetic expression of plnE/F and plnJ/K genes for strains that revealed the highest antimicrobial activity. The well diffusion assay showed that the growth of Lactobacillus pentosus was inhibited by six L. plantarum strains when cultured on control medium (0 % NaCl, pH 6.4). Moreover, when the same growth medium was supplemented with 4 and 6 % NaCl, the growth of L. pentosus was inhibited by three and two L. plantarum strains, respectively. Growth of L. pentosus was favoured when L. plantarum strains were cultured on a growth medium with lowered pH (3.5, 4.0, and 4.5). No inhibition of pathogens was observed, but in a few cases, inhibition of Aureobasidium pullulans was detected. The Real-Time PCR assay revealed that the expression of genes was dependent on producer strains and growth phase, whereas inhibition of indicator strains was enhanced in earlier stages of the growth curve in the presence of NaCl, although similar counts were obtained.  相似文献   

11.
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.  相似文献   

12.
Kefir—a traditional beverage whose consumption has been associated with health benefits—is a logical natural product to investigate for new probiotic strains. The aim of the present work was to isolate and identify kefir yeasts and select those with acid and bile tolerance to study their adhesion to epithelial cells and their transit through mouse gut. From 4 milky and 3 sugary kefir grains, 34 yeast strains were isolated and identified by means of classical microbiological and molecular-genetic methods (whole-cell protein pattern, internal-transcribed-spacer amplification, and analysis of restriction-fragment–length polymorphisms). We identified 4 species belonging to 3 genera—Saccharomyces cerevisiae (15 strains), Saccharomyces unisporus (6 strains), Issatchenkia occidentalis (4 strains), and Kluyveromyces marxianus (9 strains)—and selected 13 strains on the basis of resistance to low pH and bile salts. Among the strains selected, Kluyveromyces marxianus CIDCA 8154 and Saccharomyces cerevisiae CIDCA 8112 were further studied. Both strains evidenced the capacity to adhere to epithelial intestine-derived cells in vitro and to survive passage through the gastrointestinal tract of BALB/c mice. The investigation of the potential probiotic features of these kefir-yeast strains should be useful for the development of novel functional foods.  相似文献   

13.
Strains of Lactobacillus spp., isolated from sourdough and olive brines (seven strains), and three human isolates were screened for their antagonistic activity in coculture against the ureolytic pathogen Yersinia enterocolitica. A general reduction in the pathogen population was observed after 6 h when each Lactobacillus strain was cocultured with the pathogen at a ratio of 100:1 cfu ml−1, causing an almost complete inhibition of urease activity. Strains were also screened for their performances in in vitro tests such as adhesion ability to Caco-2 cells, tolerance to low pH, bile salts, and simulated digestion, which enabled the differences between strains to be highlighted. Three strains, L. paracasei IMPC2.1 and L. plantarum ITM21B and ITM5BG, met the main criteria for selecting effective probiotics: the ability to inhibit the pathogen Y. enterocolitica and, consequently, its urease activity (ITM21B); survival of simulated digestion (ITM21B and IMPC2.1); strong adhesion ability to enterocytes and good survival at low pH and in the presence of bile salts (ITM5BG and IMPC2.1).  相似文献   

14.
The effect of two putative probiotic strains, Lactobacillus rhamnosus BFE5264 and Lactobacillus plantarum NR74, on the control of cholesterol efflux in enterocytes was assessed by focusing on the promotion of ATP-binding cassette sub-family G members 5 and 8 (ABCG5 and ABCG8). Differentiated Caco-2 enterocytes were treated with live bacteria, heat-killed bacteria, a bacterial cell wall fraction, and metabolites and were subjected to cholesterol uptake assay, mRNA analysis, and protein analyses. Following LXR-transfection by incubation with CHO-K1 cells in DNA-lipofectin added media, the luciferase assay was conducted for LXR analysis. Treatment of Caco-2 cells with L. rhamnosus BFE5264 (isolated from traditional fermented Maasai milk) and L. plantarum NR74 (isolated from Korean kimchi) resulted in the up-regulation of LXR, concomitantly with the elevated expression of ABCG5 and ABCG8. This was associated with the promotion of cholesterol efflux at significantly higher levels compared to the positive control strain L. rhamnosus GG (LGG). The experiment with CHO-K1 cells confirmed up-regulation of LXR-beta by the test strains, and treatment with the live L. rhamnosus BFE5264 and L. plantarum NR74 strains significantly increased cholesterol efflux. Heat-killed cells and cell wall fractions of both LAB strains induced the upregulation of ABCG5/8 through LXR activation. By contrast, LAB metabolites did not show any effect on ABCG5/8 and LXR expression. Data from this study suggest that LAB strains, such as L. rhamnosus BFE5264 and L. plantarum NR74, may promote cholesterol efflux in enterocytes, and thus potentially contribute to the prevention of hypercholesterolemia and atherosclerosis.  相似文献   

15.
16.
Estimation of bile tolerance, endurance to gastric and intestinal environment and adhesion potential to intestinal cells are significant selection criteria for probiotic lactic acid bacteria (LAB). In this paper, the probiotic potential of native bacteriocin-producing LAB isolated previously from indigenous source has been determined through quantitative approaches. Among fifteen anti-listerial bacteriocin-producing native LAB, ten strains were found to be bile tolerant. The presence of bile salt hydrolase (bsh) gene in native Lactobacillus plantarum strains was detected by PCR and confirmed by nucleic acid sequencing of a representative amplicon. Interestingly, three native LAB strains exhibited significant viability in simulated gastric fluid, analogous to the standard LAB Lactobacillus rhamnosus GG, while an overwhelming majority of the native LAB strains demonstrated the ability to survive and remain viable in simulated intestinal fluid. Quantitative adhesion assays based on conventional plating method and a fluorescence-based method revealed that the LAB isolates obtained from dried fish displayed significant in vitro adhesion potential to human adenocarcinoma HT-29 cells, and the adhesion level was comparable to some of the standard probiotic LAB strains. The present study unravels putative probiotic attributes in certain bacteriocin-producing LAB strains of non-human origin, which on further in vivo characterization could find specific applications in probiotic food formulations targeted for health benefits.  相似文献   

17.

Background

Probiotic microorganisms favorably alter the intestinal microflora balance, promote intestinal integrity and mobility, inhibit the growth of harmful bacteria and increase resistance to infection. Probiotics are increasingly used in nutraceuticals, functional foods or in microbial interference treatment. However, the effectiveness of probiotic organism is considered to be population-specific due to variation in gut microflora, food habits and specific host-microbial interactions. Most of the probiotic strains available in the market are of western or European origin, and a strong need for exploring new indigenous probiotic organisms is felt.

Methods and Findings

An indigenous isolate Lp9 identified as Lactobacillus plantarum by molecular-typing methods was studied extensively for its functional and probiotic attributes, viz., acid and bile salt tolerance, cell surface hydrophobicity, autoaggregation and Caco-2 cell-binding as well as antibacterial and antioxidative activities. Lp9 isolate could survive 2 h incubation at pH 1.5–2.0 and toxicity of 1.5–2.0% oxgall bile. Lp9 could deconjugate major bile salts like glycocholate and deoxytaurocholate, indicating its potential to cause hypocholesterolemia. The isolate exhibited cell-surface hydrophobicity of ∼37% and autoaggregation of ∼31%. Presence of putative probiotic marker genes like mucus-binding protein (mub), fibronectin-binding protein (fbp) and bile salt hydrolase (bsh) were confirmed by PCR. Presence of these genes suggested the possibility of specific interaction and colonization potential of Lp9 isolate in the gut, which was also suggested by a good adhesion ratio of 7.4±1.3% with Caco-2 cell line. The isolate demonstrated higher free radical scavenging activity than standard probiotics L. johnsonii LA1 and L. acidophilus LA7. Lp9 also exhibited antibacterial activity against E. coli, L. monocytogenes, S. typhi, S. aureus and B. cereus.

Conclusion

The indigenous Lactobacillus plantarum Lp9 exhibited high resistance against low pH and bile and possessed antibacterial, antioxidative and cholesterol lowering properties with a potential for exploitation in the development of indigenous functional food or nutraceuticals.  相似文献   

18.
This study used SNaPshot minisequencing for species identification within the Lactobacillus plantarum group. A SNaPshot minisequencing assay using dnaK as a target gene was developed, and five SNP primers were designed by analysing the conserved regions of the dnaK sequences. The specificity of the minisequencing assay was evaluated using 35 strains of L. plantarum group species. The results showed that the SNaPshot minisequencing assay was able to unambiguously and simultaneously discriminate strains belonging to the species L. plantarum subsp. plantarum, L. plantarum subsp. argentoratensis, Lactobacillus paraplantarum, Lactobacillus pentosus and Lactobacillus fabifermentans. In conclusion, a rapid, accurate and cost-effective assay was successfully developed for species identification of the members of the L. plantarum group.  相似文献   

19.
In a changing scenario of food habits being associated with wellness factors through the concepts of probiotics and prebiotics, an attempt has been made to characterize on molecular basis, the desirable benefits associated with natural isolates of lactic acid bacteria, bifidobacteria, and yeasts. From a diverse range of foods and related samples, based on conventional microbiological protocols, three well-characterized natural isolates of Lactobacillus plantarum MTCC 5422, Bifidobacterium adolescentis MTCC 5423 and Saccharomyces cerevisiae MTCC 5421 were selected. The cultures of L. plantarum and B. adolescentis showed positive polymerase chain reaction (PCR) amplification with oligonucleotide primers targeting genus-specific 16 S rRNA for Lactobacillus and fructose-6-phosphate phosphoketolase for Bifidobacterium. Similarly, species-specific positive amplification in PCR was observed with primers of phytase (acid phosphatase) in S. cerevisiae and α-d-galactosidase and bile salt hydrolase in L. plantarum and B. adolescentis. The cultures of L. plantarum and B. adolescentis exhibited a broad spectrum antibacterial activity against selected foodborne pathogenic bacterial species and tolerance to acid and bile. Gene sequence of respective PCR-amplified products confirmed the genetic identity of the isolated cultures as L. plantarum and B. adolescentis showing 99% homology with the documented sequence of established gene bank.  相似文献   

20.
Samples of fermented herbs were used to isolate lactic acid bacteria (LAB). Of a total of 19 isolates, eight were resistant both to gastric acid and bile salts (glycocholic acid, GCA; taurocholic acid, TCA; glycodeoxycholic acid, GDCA; and taurodeoxycholic acid, TDCA). Most isolates exhibited a pH-dependent surface hydrophobicity: a pH of 4 conferred a greater hydrophobicity compared to a pH of 7. Based on the hydrophobicity characteristics, the LAB isolate WU-P19 from the traditional fermented herb Oroxylum indicum was selected for further study. WU-P19 was identified as Lactobacillus plantarum WU-P19. The presence of bile salts GCA and GDCA in the culture medium induced production of the relevant bile salt hydrolase. Relative to controls, the presence of the bile salts in the culture medium affected the carbon and nitrogen contents of the cells and their hydrophobicity. Cells grown in a medium free of bile salts were morphologically different to cells grown in the presence of GCA and GDCA. WU-P19 was resistant to several antibiotics. It produced β-galactosidase and inhibited growth of the tested pathogenic bacteria at various levels. In vitro, L. plantarum WU-P19 adapted well to conditions typical of the various zones of the human gastrointestinal tract. In view of the promising results, in vivo evaluations are planned for the isolate WU-P19.  相似文献   

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