Feng Xu

In vitro probiotic characteristics of Lactobacillus plantarum ZDY 2013 and its modulatory effect on gut microbiota of mice

Lactobacillus plantarum ZDY 2013, a novel strain isolated from Chinese traditional fermented acid beans, was systematically evaluated for its survival capacity under stress conditions (pH, bile salt, simulated gastrointestinal tract, and antibiotics), production of exopolysaccharide and antagonism against 8 pathogens. Its effect on mice gut microbiota was also investigated by quantitative PCR and PCR-denaturing gradient gel electrophoresis. The results showed that ZDY 2013 can grow at pH 3.5 and survive at pH 2.0 for 6 h and at 0.45% bile salt for 3 h. The exopolysaccharide yield was up to 204±7.68 mg/L. The survival rate of ZDY 2013 in a simulated gastrointestinal tract was as high as 65.84%. Antagonism test with a supernatant of ZDY 2013 showed maximum halo of 28 mm against Listeria monocytogenes. The inhibition order was as follows: Listeria monocytogenes, Salmonella typhimurium, Escherichia coli, Pseudomonas aeruginosa, Shigella sonnei, Enterobacter sakazakii, and Staphylococcus aureus. Lactobacillus plantarum ZDY 2013 was sensitive to some antibiotics (e.g., macrolide, sulfonamides, aminoglycoside, tetracyclines and β-lactams), whereas it was resistant to glycopeptides, quinolones, and cephalosporins antibiotics. Denaturing gradient gel electrophoresis profile demonstrated that ZDY 2013 administration altered the composition of the microbiota at various intestinal loci of the mice. Moreover, the quantitative PCR test showed that the administration of ZDY 2013 enhanced the populations of Bifidobacterium and Lactobacillus in either the colon or cecum, and reduced the potential enteropathogenic bacteria (e.g., Enterococcus, Enterobacterium, and Clostridium perfringens). Lactobacillus plantarum ZDY 2013 exhibited high resistance against low pH, bile salt, and gastrointestinal fluid, and possessed antibacterial and gut microbiota modulation properties with a potential application in the development of dairy food and nutraceuticals.

Safety Assessment and Probiotic Evaluation of Enterococcus Faecium YF5 Isolated from Sourdough

Enterococcus faecium YF5, a strain previously isolated from sourdough, was assessed for safety and probiotic potential. Its virulence and antibiotic resistant phenotypes (cytolysin and gelatinase production, antibiotic susceptibility) and genes (cylA, gelE, ace, agg, esp, and vanA) were surveyed. Results indicated that the tested virulence determinants were nontoxic. In addition, E. faecium YF5 was sensitive to 3 antibiotics such as amoxicillin, vancomycin, and chloramphenicol. Furthermore, results of in vivo animal acute oral toxicity of E. faecium YF5 studies were similar to the control group that indicated no abnormalities. In addition, E. faecium YF5 stably survived in low pH, bile salts, gastric, and intestinal fluids in vitro. Moreover, E. faecium YF5 was found to adhere to human colon cancer cell line HT-29 at 3.39 (±0.67) × 10(5) CFU/mL. When cocultured with pathogenic organisms (Enterobacter sakazakii CMCC45402, Escherichia coli CMCC44102, enterohemorrhage Escherichia coli O157: H7 CMCC44828, Salmonella Typhimurium CMCC50071, Shigella flexneri 301, and Shigella sonnei ATCC 29930) and 2 gram-positive strains (Listeria monocytogenes CMCC54001 and Staphylococcus aureus CMCC 26003), it inhibited these foodborne pathogens with exception of S. aureus. Therefore, E. faecium YF5 can be regarded as a safe strain and it may be used as a probiotic preparation or for microecologics.

The beneficial effect of exopolysaccharides from Bifidobacterium bifidum WBIN03 on microbial diversity in mouse intestine

BACKGROUND The structure of exopolysaccharides (EPS) produced by Bifidobacterium and their beneficial effects on human health have been fully studied, but only a few studies have investigated their influence on microbial diversity in the human/animal intestine. RESULTS The strain named Bifidobacterium bifidum WBIN03 with high growth rate and exopolysaccharide (EPS) yield was selected to study the effect of its EPS on modifying the intestinal microbiota of mice. The results indicated that EPS significantly increased the growth of lactobacilli and total anaerobic bacteria, and exerted their inhibition effect on the growth of enterobacteria, enterococci and Bacteroides fragilis. Denaturing gradient gel electrophoresis (DGGE) analysis indicated that the EPS significantly increased the diversity of total bacteria and lactobacilli, but significantly decreased the diversity of enterobacteria. When receiving a low dose of EPS, Bacteroidales sp./Lactobacillus sp. occupied the dominant position, and L. johnsonii, L. animalis and L. reuteri were identified as the dominant strains when receiving a high concentration of EPS. CONCLUSION The combination of viable cell count, DGGE and sequencing was used as an effective method to assess the microbial diversity in mouse intestine, and the benefit effect of EPS from B. bifidum WBIN03 on probiotics and antagonistic effect against pathogens would guaranteed the health of their hosts.

Rapid detection of Staphylococcus aureus in dairy and meat foods by combination of capture with silica-coated magnetic nanoparticles and thermophilic helicase-dependent isothermal amplification

Staphylococcus aureus is one of the main pathogens in dairy and meat products; therefore, developing a highly sensitive and rapid method for its detection is necessary. In this study, a quantitative detection method for Staph. aureus was developed using silica-coated magnetic nanoparticles and thermophilic helicase-dependent isothermal amplification. First, genomic DNA was extracted from lysed bacteria using silica-coated magnetic nanoparticles and amplified using thermophilic helicase-dependent isothermal amplification. After adding the nucleic-acid dye SYBR Green I to the amplicons, the fluorescence intensity was observed using a UV lamp or recorded using a fluorescence spectrophotometer. This detection system had a detection limit of 5×10(0) cfu/mL in pure culture and milk-powder samples and 5×10(1) cfu/mL in pork samples using a UV light in less than 2h. In addition, a good linear relationship was obtained between fluorescence intensity and bacterial concentrations ranging from 10(2) to 10(4) cfu/mL under optimal conditions. Furthermore, the results from contaminated milk powder and pork samples suggested that the detection system could be used for the quantitative analysis of Staph. aureus and applied potentially to the food industry for the detection of this pathogen.

Analysis of the intestinal microbial community structure of healthy and long-living elderly residents in Gaotian Village of Liuyang City

Gaotian, one typical conservative village in rural area of South China, is differentiated from other adjacent village for its longevity and health situation of residents. To ascertain the difference of intestinal microbial community between Gaotian and other region, high-throughput sequencing and systematical bioinformation analyses was adopted to compare 21 samples in long life group with 28 in control group. The α diversity showed that the diversity of species of intestinal flora of Gaotian villagers was higher than that of control group, while the β diversity showed that the similarity of intestinal flora for Gaotian residents was also much higher than that of control group. OTU cluster analysis and Venn diagram showed that the intestinal microbial community of Gaotian villagers is different from that of control group. To quantitatively compare the main flora constitution in all samples, real-time PCR was performed, and the results showed that the biomass of Enterococcus, Lactobacillus, Enterobacteriaceae, Clostridium perfringens, and Bacteroides of Gaotian villages is generally significantly higher than that of control group. Remarkably, some special species, i.e., Methanobacterium, Butyricimonas, Deinococcus, and Streptococcaceae, have been found in Gaotian villagers. Overall, this study lays a preparatory basis for exploration of the resources of special species from healthy and long-living elderly Gaotian villagers and for proposal of a hypothesis, namely, the diversity in intestinal flora of Gaotian might contribute to the longevity and health of local residents. Further study should be focused on screening and functional evaluation of the special species in the long-life residents.

Rapid detection of Campylobacter jejuni using fluorescent microspheres as label for immunochromatographic strip test

Campylobacter jejuni is a worldwide foodborne pathogen recognized as a leading cause of human gastrointestinal enteritis. A rapid, sensitive, and specific method is required to monitor food and water in cases of contamination by this pathogen. This report presents a novel immunochromatographic test (ICT) using fluorescent microspheres labeled with polyclonal antibodies of C. jejuni as the capture reagent dispensed onto the conjugate pad. Polyclonal antibodies against the outer membrane protein PEB1 of C. jejuni were used as the detective reagent at the test line, whereas the goat anti-rabbit IgG was used on the control line. PEB1 was obtained by gene cloning and expression to prepare its antibody. In this study, a simple and rapid ICT is reported for detecting C. jejuni for the first time with a detection limit of 106 CFU/ mL.

A novel strain of Lactobacillus mucosae isolated from a Gaotian villager improves in vitro and in vivo antioxidant as well as biological properties in d-galactose-induced aging mice

Twelve isolates isolated from the gastrointestinal tracts of Gaotian villagers in China, who had a lifespan of 92 yr, were examined for their antioxidants using free radical scavenging activity and 2,2-diphenyl-1-picrylhydrazyl. Three strains (i.e., Lactobacillus mucosae LMU1001, and Lactobacillus plantarum LPL0902 and LPL0302) were selected as candidates to prepare yogurt for testing their antioxidants in a model of d-galactose-induced aging mice, with vitamin C as a positive control. The results showed that L. mucosae LMU1001 was the best strain, which had similar in vivo antioxidant activity as vitamin C. A significant increase was found in the activities of glutathione peroxidase in serum and total superoxide dismutase in the liver, and a decrease in the level of malondialdehyde in serum. Regarding mRNA expression level detected quantitatively by real-time PCR, we observed that L. mucosae LMU1001 significantly upregulated antioxidant genes (i.e., MT1A and MT1M in HT-29 and Caco-2) and those genes (i.e., MT1, MT2, GPx1, and GPx2) in the intestinal tract of the model mice. Hence, this strain could be considered as a potential probiotic lactic acid bacterium for improving antioxidant levels in functional foods.

Detection of Cronobacter species in powdered infant formula by probe-magnetic separation PCR.

Cronobacter species are opportunistic foodborne pathogens associated with serious infections in preterm neonates and infants. Based on the epidemiological research, infant formula products are considered to be the main source of infections from this organism. Therefore, accurate methods are required for detection of Cronobacter species. In this study, the specific probe and primers for detection of this organism were designed and verified. The probe-magnetic beads were prepared for sequence capture, followed by PCR assay to detect the target gene. This probe-magnetic separation PCR assay could detect as few as 10(3) cfu/mL of Cronobacter in artificially contaminated infant formulas in less than 4 h. The combination of magnetic beads and PCR showed the potential for the detection of Cronobacter in infant formulas and may have applications in the dairy industry.

Efficacy of oral Bifidobacterium bifidum ATCC 29521 on microflora and antioxidant in mice

This study aimed to examine whether Bifidobacterium bifidum ATCC 29521, a species of colonic microflora in humans, is involved in the intestinal tract of mice. This study was also conducted to determine the antioxidant activity of this species by evaluating different microbial populations and reactive oxygen species isolated from feces and intestinal contents for 28 days of oral administration. Microbial diversities were assessed through bacterial culture techniques, PCR-DGGE, and real-time PCR. This study showed that the intake of B. bifidum ATCC 29521 significantly (p < 0.05) improved the ecosystem of the intestinal tract of BALB/c mice by increasing the amount of probiotics (Lactobacillus intestinalis and Lactobacillus crispatus) and by reducing unwanted bacterial populations (Enterobacter, Escherichia coli). Antioxidative activities of incubated cell-free extracts were evaluated through various assays, including the scavenging ability of DPPH radical (64.5% and 67.54% (p < 0.05), respectively, at 21 days in nutrients and 28 days in MRS broth), superoxide anion, and hydroxyl radical (85% and 61.5% (p < 0.05), respectively, at intestinal contents in nutrients and 21 days in MRS broth). Total reducing power (231.5 μmol/L (p < 0.05), 14 days in MRS broth) and mRNA level of genes related to oxidative stress were also determined. Results indicated that B. bifidum ATCC 29521 elicits a beneficial effect on murine gut microbiota and antioxidant activities compared with the control samples. This species can be considered as a potential bioresource antioxidant to promote health. Bifidobacterium bifidum ATCC 29521 may also be used as a promising material in microbiological and food applications.

Mutual growth-promoting effect between Bifidobacterium bifidum WBBI03 and Listeria monocytogenes CMCC 54001

In this study, Bifidobacterium bifidum WBBI03 and Listeria monocytogenes CMCC 54001 were selected to detect the changes in their growth pattern after mutual interaction between them. The proteomic changes after the interaction between the 2 bacteria were detected by the isobaric tags for relative and absolute quantitation method. The proteins related to the biosynthesis and cell reproduction were selected, and their changes at the transcriptional level were monitored by fluorescent quantitative PCR. Also, 3 other types of probiotic organisms and opportunistic pathogens were used to verify the results mentioned above. The results showed that growing the 2 organisms together could promote the growth of each other, resulting in earlier entry into the logarithmic phase. The results also showed that the expression of these proteins mostly tended to be upregulated at the translational and transcriptional level. The increase in the expression of these proteins might help promote the growth and reproduction of B. bifidum WBBI03 and L. monocytogenes CMCC 54001. One aspect of the biological significance of their presence in the normal intestine may be that the opportunistic pathogens promote the growth of the probiotics.