Abstract:Abstract:Equol，a microbial metabolite of daidzein，has been hypothesized as clue to the effectiveness of soy and its isoflavones but only 33%－50% of human population can excrete equol．Recently studies indicated that hydrogen gas playsan important role in equol formation． However，researches focus on this area are just started．In this paper，the corresponding research results of previous studies have been reviewed，including the underlying mechanism of equol formation and bioavailability，factors influencing equol production and the role of hydrogen gas on microbial equol formation.

Abstract:Abstract:［Objective］To construct an efficient gene knock-out system for Paenibacillus polymyxa SC2.［Methods］Temperature sensitive plasmid pRN5101 was transformed into P．polymyxa SC2 by electrotransformation．A mutant SC2-E was obtained，in which pmxE was disrupted by homologous recombination． To confirm whether pmxE was knocked out，we used antibacterial activity assay and high performance liquid chromatography to analyze the ability of mutants synthesizing polymyxin.［Results］We developed an efficient gene knock-out system for P. polymyxa SC2. Plasmid of pRN5101 could replicate at 28℃ and suicide at 39℃ in SC2. Mutants lost the ability of synthesizing polymyxin，indicating that pmxE gene was successfully knocked out.［Conclusion］The constructed gene knock-out system for P. polymyxa provides a highefficiency tool to detect genes function for P. polymyxa．

Abstract:Abstract:［Objective］ To broaden the substrate spectrum including L-arabinose，Ralstonia eutropha W50，a mutant strain with high yield of poly-β-hydroxybutyrate (PHB)，was metabolically engineered by expressing the genes encoding L-arabinose catabolic enzymes and high-affinity L-arabinose transporter from Escherichia coli.［Methods］The promoter fragment of PHB synthase gene phaC1 (Ppha C1) from R．eutropha H16 and the araBAD genes from E.coli W3110 were cloned by PCR and inserted into expression vector pBBR1MCS． The resulting recombinant plasmid was transformed into W50 to generate W50-1．The araFGH gene from E.coli W3110 was introduced into W50-1 by plasmid system or homologous recombination，yielding W50-2 and W50-3 respectively． The fermentation characteristics of the three engineered strains were investigated.［Results］ The flask fermentation experiments of the engineered strains show that W50-1 carrying the arabinose catabolic genes under the control of Ppha C1 could grow in the fermentation medium containing 0.1 mol/L arabinose as the sole carbon source，but could not utilize low concentration arabinose (0.01 mol/L). However，W50-2 and W50-3 containing the gene of high-affinity arabinose transporter were able to utilize low concentration arabinose. In the fermentation medium containing 0.1 mol/L arabinose，the biomass of W50-3 was 2.5 fold higher than that of W50-1，and the PHB accumulation amount of W50-3 accounted for 38.6% of the cell dry weight.［Conclusion］ R.eutropha W50 was able to metabolize L-arabinose by the expression of araBAD genes，and the simultaneous expression of araFGH genes could further improve its ability of L-arabinose utilization. By using L-arabinose as the sole carbon source，the recombinant strain W50-3 can accumulate a noticeable level of PHB.

Abstract:Abstract:［Objective］Many tandem repeats exist in FLO1 gene of Saccharomyces cerevisiae，which might have great regulatory effect on the conformation and function of flocculation protein (flocculin)．In this study，we analyzed the effect of 3'-terminal tandem repeats B，C and D complete deletion on the function of flocculin.［Methods］We constructed the derived gene FLO1bcd with complete deletion of tandem repeats B，C and D of FLO1 by fusion PCR． We then constructed plasmid pYCF1bcd by insertion of FLO1bcd into YCp50，and transformed such plasmid，pYCF1 and YCp50 into S.cerevisiae YS58 separately to generate recombinant strains YSF1bcd，YSF1 and YSP50. We compared the flocculation ability and characteristics of these strains.［Result］Compared to YSF1，YSF1bcd displayed only a slight reduction (4%) in flocculation ability in optimal flocculation buffer (50 mmol /L NaAC，pH 4.5)．Moreover，the dependence of flocculation on Ca2+ ，sensitivity to metal ions and ethanol，and the specificity to different sugars showed no obvious difference between strains YSF1 and YSF1bcd．However，strain YSF1bcd displayed much higher flocculation levels than strain YSF1 under conditions with extreme pH，high temperature，or high concentration of mannose.［Conclusion］Combined deletion of tandem repeats B，C and D adjacent to the 3'-terminal of FLO1 increases the conformation stability of flocculin in response to changes of pH，temperature or concentration of mannose in environment，but does not influence the other characteristics of flocculation.

Abstract:Abstract:［Objective］Antagonistic endophytic strains with strongly inhibitory activity to mulberry bacterial blight (P．syringae pv．mori) were isolated from mulberry endophytes．we identified the antagonistic endophyte and optimized the fermentation conditions．［Method］Streak plate method was used to separate the endophytes from healthy mulberry tissues after strict surface disinfection．Antagonistic endophytes were screened out through inhibition zone method．Strain SWg2 was identified by morphological，physiological and biochemical characteristics and 16S rDNA sequence analysis．The conditions of fermentation and medium composition were optimized through single factor and orthogonal experiment.［Result］In total 77 endophytic strains have been isolated from healthy mulberry. SWg2 showed strong and stable antagonistic activity to mulberry bacterial blight. The morphological，cultural， physiological，and biochemical characteristics assays indicated that SWg2 belongs to Pantoea sp．The 16S rDNA sequence phylogenetic analysis reveals that SWg2 appeared a sister lineage to P．agglomerans．The optimized culture conditions of strain SWg2 were liquid volume 20 mL in 100 mL flask，170 r /min at 28℃，inoculation size of 4% for 5 d with a medium of 2.0% glycerol，2.0% NH4NO3，0.1% KH2 PO4，0.15% MgSO4·7H2O at initial pH of 7.5． ［Conclusion］The antagonistic endophytic strain SWg2 to mulberry bacterial blight was identified as P．agglomerans. SWg2 strain shows stronger antagonistic action to mulberry bacterial blight under optimized fermentation conditions．

Abstract:Abstract:［Objective］To investigate the composition of culturable bacteria from soil of Grove Mountains，East Antarctica and analyze the production of extracellular hydrolytic enzymes and antibacterial and antifugal activities of these culturable bacteria.［Methods］We used spread plate method to obtain culturable bacteria.Phylogenetic relationship was analysed based on their 16S rRNA gene sequences. We used plate method and agar block method to detect the production of extracellular enzymes and antibacterial and antifungal activities of these bacteria，separately．［Results］We obtained a total of 39 isolates from all the soil samples. They belonged to 20 genera and grouped into Firmicutes，Proteobacteria，Actinobacteria，Bacteroidetes，Deinococcus-Thermus lineages，of which each group occupied 48.7%，25.6%，20.5%，2.6%，2.6% of the total，respectively．Bacillus was dominant．We isolated different strains from soil stored at different temperatures，and this may be explained as viable bacteria were different at diverse temperatures．Of the 39 isolates 33 showed the activity of producing at least one extracellular enzyme．Amylase-producing strains were the most (64.1%)，6 strains were able to inhibit the growth of at least one tested bacterium or fungus.［Conclusion］Strains with extracellular enzyme activity and antibacterial and antifungal activity would contribute to the exploration of cold-active enzymes and antibacterial (antifungal) material in Antarctica．

Abstract:Abstract:［Objective］ To evaluate coal bed methane production potential and characterize the in situ microbial communities of coal bed.［Methods］ Coal samples were incubated under anaerobic conditions: mimicking coal bed condition，supplementing with methanogenic hydrocarbon degrading consortium，or adding with exogenetic substrate．Methane production was observed over time using gas chromatograph，and the in situ bacterial and archaeal communities were revealed using pyrosequencing.［Results］Enrichment incubation revealed that 3 of total 10 coal samples microcosms produced methane; bioaugmentation and substrate addition could enhance methane production of coal sample HF．Hydrogenotrophic Methanoculleus and acetoclastic Methanosaeta dominated the archaeal community of coal sample SL，while the bacterial domain was mainly composed of Firmicutes (54.4%)，Proteobacteria (30.9%)，uncultured bacteria (10.8%)，Caldiserica (1.5%) and Thermotogae (1.3%).［Conclusion］The methane production potential of coal bed samples with different maturity is different; the in situ coal bed microcosms are likely involved in hydrocarbons degradation and methane production.

Abstract:Abstract:［Objective］The work aimed to isolate and culture the acidophilic and moderately thermophilic microorganisms for leaching the sulfide ore.［Methods］We enriched and incubated iron- or sulfur-oxidizing strains from muddy water of acuric hot spring utilizing ferrous irons or elemental sulfur as substrates． Then，we identified the strains by their morphological，physiological，biochemical properties and phylogenetic positions，and estimated their bioleaching potential according to their oxidation rate of pyrite.［Results］Two acidophilic，aerobic and facultative heterotrophic bacterial strains，Costa C and Costa E，were isolated from the samples of sulfuric hot springs of Costa Rica. Cells of the two strains were gram-positive，spore-forming and rod-shaped [(0.4－0.6)μm×(2.5－4.0)μm and (0.4－0.7)μm×(2.4－4.9)μm，respectively].Strain Costa C grew at a temperature range of 30℃ －55℃ and a pH range of 1.2－5.0，optimally at 50oC and 2.8．Strain Costa E grew at a temperature range of 30℃－55℃ and at a pH range of 1.4－5.0， optimally at 40oC and 2.8. Two strains could autotrophically grow on inorganic substrates such as ferrous irons，element sulfur and K2 S4O6 and also could utilize organic substrates like yeast extract for heterotrophic growth．Phylogenetic analysis based on 16S rRNA gene sequences alignment demonstrated that the highest similarity between strain Costa C，Costa E and other species of the genus Sulfobacillus was above 99%.［Conclusion］Based on morphological，physiological and biochemical analysis，Costa C and Costa E can be affiliated to the genus Sulfobacillus，for which the names Sulfobacillus sp. strain Costa C and Sulfobacillus sp. strain Costa E were proposed． Both strains could oxidize pyrite，and the oxidation rates arrived 63.0 mg/L·d and 56.8 mg/L·d，respectively.

Abstract:Abstract:［Objective］In this study，we studied systematically the prevalence of Salmonella in foods from various parts of South China. Isolated strains were characterized by serotyping and enterobacterial repetitive intergenic consensus polymerase chain reaction (ERIC-PCR)，to provide data support for effectively tracing the source of Salmonella and controlling food contamination.［Methods］In total 400 unique food samples were collected from retail markets in South China，including meat product (74)，frozen product (56)，seafood (80)，mushroom (54)，ready-to-eat food (80)，vegetables (32)，dairy (24)．Food samples were examined by qualitative method according to national food safety standard (GB4789.4-2010) and most probable number (MPN) method． The predominant serotype of Salmonella was figured out when all isolated strains were characterized by serotyping using slide agglutination method． We also applied ERIC-PCR for genetic diversity analysis of Salmonella isolates.［Results］Of the total 400 food samples，75(18.8%) tested positive for Salmonella．The results showed that MPN value of 97.3% (73/75) positive samples below 10 MPN/g．Ninety-three Salmonella isolates were belonged to 9 groups and 29 different serovars． Salmonella Derby and Salmonella Typhimurium were the two predominant serovars. The isolated strains were further discriminated by ERIC-PCR．The results revealed 96 strains including 93 isolates，Salmonella Enteritidis CMCC 50335，Salmonella Typhi CMCC 50098 and S. Typhimurium ATCC 14028 were divided into 15 clustering groups and generated 56 genotypes according to the similarity coefficient of 0.75，which suggested various genotypes of Salmonella.［Conclusion］The overall prevalence of Salmonella was high，however lower contamination level in foods from cities of South China．S. derby and S. typhimurium were found to be predominant serovars．ERIC-PCR fingerprints database of Salmonella from food samples in South China were preliminarily established．Salmonella present in foods were phenotypically and genotypically diverse．

Abstract:Abstract:［Objective］To screen for compounds against influenza A virus by using the viral promoter reporter cell line HeLa-IAV-Lucand investigate their anti-viral mechanism.［Methods］We screened the inhibitors of influenza A virus by infecting HeLa-IAV-Luc cells with influenza A virus WSN /H1N1 and treating it with the compounds isolated from microbial metabolites．The cell lysates were then subjected to the luciferase assay． We conducted the pesudovirus assay to analyze whether the compound affected the function of hemagglutinin (HA)．We carried out the influenza viral promoter reporter assay to examine whether the compound could inhibitinfluenza viral RNA polymerase (vRNP) activity． The effect of antiviral compoundon influenza viral RNA synthesis was measured by real-time fluorescence quantitative PCR．［Results］Colletodiol inhibited the replication of influenza A virus in HeLa-IAV-Luc cells by luciferase assay．It did not inhibit the function of HA protein based on the results of the time-of-addition experiment and pseudovirus assay．The influenza viral polymerase promoter reporter assay indicated that Colletodiol could inhibit the activity of vRNP dramatically．Due to the inhibition of vRNP，the influenza viral RNA synthesis decreased significantly.［Conclusion］Compound Colletodiol was an inhibitor of influenza A virus． It blocks the replication of influenza A virus by reducing the activity of vRNP.

Abstract:Abstract:［Objective］ Tuberculosis is a chronic infectious disease caused by Mycobacterium tuberculosis complex．Hence，novel vaccines against TB are urgently needed and important to the public health.［Methods］Immunobiologic characteristics of a recombinant attenuated Listeria monocytogenes strain LMΔhly::Ag85b-esat-6 was evaluated.［Results］LMΔhly::Ag85b-esat-6 had lost the hemolytic activity．It was completely cleared from the livers and spleens of mice 5 days after inoculation via intravenous route．Furthermore，the LD50 of the recombinant strain increased by 4 Logs comparing to that of the parent strain． Histopathology reveals no obvious pathological changes following administration of the recombinant strain to mice，indicating its safety．In addition，the potential protective immune response was evaluated on C57BL/6 mice via intravenous immunization route．The results indicate that the antigen delivered by the recombination LM could induce Th1 type immune response and elicit strong cytotoxic lymphocyte effect against Ag85B-ESAT-6． ［Conclusion］Thus，LMΔhly::Ag85b-esat-6 had high safety to mice，and could be used as a novel vaccines candidate for preventing tuberculosis．

Abstract:Abstract:［Objective］ A new method was introduced for precise determination of the live cell titer of mycoplasma culture，and would be a candidate to replace the commonly used CCU (color change unit) assay.［Methods］The CCU50 (50% color change unit) was modified according to the method of TCID50 (50% tissue culture infective dose) assay used for viral titer assessment，and adopted to estimate the live cell titer of mycoplasma． Sensitivity and reproducibility of the CCU50 assay were assessed，and adaptability was checked with M．hyopneumoniae and M．synoviae.［Results］The CCU50 assay showed better reproducibility，sensibility and adaptability than traditional CCU assessment approaches.［Conclusion］The method could be applied to accurate titration determination for mycoplasma，and might be considered as a useful tool for the research of high density fermentation of mycoplasma and development of vaccine.