Zhijiang Zhou

Isolation and identification of antifungal peptides from Bacillus BH072, a novel bacterium isolated from honey.

A bacterial strain BH072 isolated from a honey sample showed antifungal activity against mold. Based on morphological, biochemical, physiological tests, and analysis of 16S rDNA sequence, the strain was identified to be a new subspecies of Bacillus sp. It had a broad spectrum of antifungal activity against various mold, such as Aspergillus niger, Pythium, and Botrytis cinerea. Six pairs of antifungal genes primers were designed and synthesized, and ituA, hag, tasA genes were detected by PCR analysis. The remarkable antifungal activity could be associated with the co-production of these three peptides. One of them was purified by 30-40% ammonium sulfate precipitation, Sephadex G-75 gel filtration and anion exchange chromatography on D201 resin. The purified peptide was estimated to be 35.615 kDa and identified to be flagellin by micrOTOF-Q II. By using methanol extraction, another substance was isolated from fermentation liquor, and determined to be iturin with liquid chromatography-mass spectrometry (LC-MS) method. The third possible peptide encoded by tasA was not isolated in this study. The culture liquor displayed antifungal activity in a wide pH range (5.0-9.0) and at 40-100°C. The result of the present work suggested that Bacillus BH072 might be a bio-control bacterium of research value.

Optimization of antifungal lipopeptide production from Bacillus sp. BH072 by response surface methodology.

Antifungal lipopeptide produced by Bacillus sp. BH072 was extracted from fermentation liquor and determined as iturin A by liquid chromatography-mass spectrometry (LC-MS). For industrial-scale production, the yield of iturin A was improved by optimizing medium components and fermentation conditions. A one-factor test was conducted; fermentation conditions were then optimized by response surface methodology (RSM) to obtain the following: temperature, 29.5°C; pH 6.45; inoculation quantity, 6.7%; loading volume, 100 ml (in 500 ml flasks); and rotary speed, 150 rpm. Under these conditions, the mass concentration of iturin A was increased from 45.30 mg/ml to 47.87 mg/ml. The following components of the medium were determined: carbon sources (glucose, fructose, sucrose, xylose, rhamnose, and soluble starch); nitrogen sources (peptone, soybean meal, NH4Cl, urea, and ammonium citrate); and metal ions (Zn2+, Fe3+, Mg2+, Mn2+, Ca2+, and K+). The effects of these components on iturin A production were observed in LB medium. We selected sucrose, soybean meal, and Mg2+ for RSM to optimize the conditions because of several advantages, including maximum iturin A production, high antifungal activity, and low cost. The optimum concentrations of these components were 0.98% sucrose, 0.94% soybean meal, and 0.93% Mg2+. After iturin A production was optimized by RSM, the mass concentration reached 52.21 mg/ml. The antifungal specific activity was enhanced from 350.11 AU/mg to 513.92 AU/mg, which was 46.8% higher than the previous result. The present study provides an important experimental basis for the industrial-scale production of iturin A and the agricultural applications of Bacillus sp. BH072.

Isolation and characterization of dextran produced by Leuconostoc citreum NM105 from manchurian sauerkraut.

A water-soluble exopolysaccharide (EPS) was produced by Leuconostoc ctireum NM105 from homemade manchurian sauerkraut. After culturing the strain in Man-Rogosa-Sharpe medium containing 5% sucrose at 25°C for 48h, the EPS was purified and a yield of 23.5g/L was achieved. The EPS consisted exclusively of glucose and the weight-average molecular weight was 1.01×10(8)Da. The structural characterization of the purified EPS determined by FT-IR, (1)H, (13)C and two-dimensional NMR spectroscopy demonstrated that the glucan contained α-(1→6)-linked d-glucopyranose units, 2,6-linked d-glucopyranose units and terminal α-d-glucopyranose units at a ratio of 1:1:1. The microstructure of the dried dextran appeared a sheet-like smooth glittering and highly branched surface. The NM105 dextran showed high water solubility and excellent water retention. All the results suggested that the highly α-(1→2) branched dextran might have the potential to serve as valuable polymers applied in foods, cosmetics and other fields.

Optimization, purification and structural characterization of a dextran produced by L. mesenteroides isolated from Chinese sauerkraut

A higher yield of dextran strain Leuconstoc mesenteroides TDS2-19 was isolated from Chinese sauerkraut juice. Effects of the three main factors on exopolysaccharide (EPS) yield were investigated by central composite design (CCD) and the optimum composition was sucrose 117.48g/L, sodium acetate 4.10g/L, and initial pH 6.88. Optimum results showed that EPS yield was increased to 71.23±2.25g/L in 48h fermentation, 31.24% higher than before. The molecular weight (Mw) of ESP was 8.79×107Da, as determined by high-performance size-exclusion chromatography (HPSEC). Fourier transform infrared spectra (FT-IR) and nuclear magnetic resonance spectra (NMR) showed that the polysaccharide synthesized by L. mesenteroides TDS2-19 in the MRS medium was dextran with a peak, a linear backbone composed of consecutive α-(1→6)-linked d-glucopyranose units. No branching was observed in the dextran structure. The present study suggested that L. mesenteroides TDS2-19 might be used for the industrial-scale production of linear dextran.

Characterization of the microbial communities along the gastrointestinal tract of sheep by 454 pyrosequencing analysis

Objective The gastrointestinal tract of sheep contain complex microbial communities that influence numerous aspects of the sheep’s health and development. The objective of this study was to analyze the composition and diversity of the microbiota in the gastrointestinal tract sections (rumen, reticulum, omasum, abomasum, duodenum, jejunum, ileum, cecum, colon, and rectum) of sheep. Methods This analysis was performed by 454 pyrosequencing using the V3-V6 region of the 16S rRNA genes. Samples were collected from five healthy, small tailed Han sheep aged 10 months, obtained at market. The bacterial composition of sheep gastrointestinal microbiota was investigated at the phylum, class, order, family, genus, and species levels. Results The dominant bacterial phyla in the entire gastrointestinal sections were Firmicutes, Bacteroidetes, and Proteobacteria. In the stomach, the three most dominant genera in the sheep were Prevotella, unclassified Lachnospiraceae, and Butyrivibrio. In the small intestine, the three most dominant genera in the sheep were Escherichia, unclassified Lachnospiraceae, and Ruminococcus. In the large intestine, the three most dominant genera in the sheep were Ruminococcus, unclassified Ruminococcaceae, and Prevotella. R. flavefaciens, B. fibrisolvens, and S. ruminantium were three most dominant species in the sheep gastrointestinal tract. Principal Coordinates Analysis showed that the microbial communities from each gastrointestinal section could be separated into three groups according to similarity of community composition: stomach (rumen, reticulum, omasum, and abomasum), small intestine (duodenum, jejunum, and ileum), and large intestine (cecum, colon, and rectum). Conclusion This is the first study to characterize the entire gastrointestinal microbiota in sheep by use of 16S rRNA gene amplicon pyrosequencing, expanding our knowledge of the gastrointestinal bacterial community of sheep.

Synthesis of magnetic framework composites for the discrimination of Escherichia coli at the strain level

Rapid and efficient characterization and identification of pathogens at the strain level is of key importance for epidemiologic investigations, which still remains a challenge. In this work, solvothermically Fe3O4-COOH@MIL-101 composites were fabricated by in situ crystallization approach. The composites combine the excellent properties of both chromium (III) terephthalate (MIL-101) and carboxylic-functionalized magnetite (Fe3O4-COOH) particles and possess the efficient peptides/proteins enrichment properties and magnetic responsiveness. Fe3O4-COOH@MIL-101 composites as magnetic solid phase extraction materials were used to increase the discriminatory power of MALDI-TOF MS profiles. BSA tryptic peptides at a low concentration of 0.25 fmol μL(-1) could be detected by MALDI-TOF MS. In addition, Fe3O4-COOH@MIL-101 composites were successfully applied in the selective enrichment of the protein biomarkers from bacterial cell lysates and discrimination of Escherichia coli at the strain level. This work provides the possibility for wide applications of magnetic MOFs to discriminate pathogens below the species level.

Characterization of a dextran produced by Leuconostoc pseudomesenteroides XG5 from homemade wine

A water-souble exopolysaccharide (EPS) produced by Leuconostoc pseudomesenteroides XG5 from homemade wine was investigated. The EPS yield of 35.5g/L was achieved at 30°C for 48h in De Man-Rogosa-Sharpe (MRS) medium containing 12.5% sucrose. The EPS was a dextran composed exclusively of glucose and the molecular weight was 2.6×106Da. Fourier transform infrared spectra and nuclear magnetic resonance spectra revealed that the EPS was a dextran containing D-glucose residues in a linear chain with consecutive α-(1→6) linkages. Scanning electron microscopy of the EPS appeared a highly branched and porous structure. Rheological studies showed that the EPS had higher viscosity in 0.1M KCl solution, at lower temperature, or at acidic pH. Thermal gravimetric analysis and differential scanning calorimetric indicated that the EPS had excellent thermal stability with a degradation temperature of 313.80°C and melting point at 274.14°C. Water solubility index and water holding capacity of XG5 dextran were 90.2% and 412% respectively. The results suggest that L. pseudomesenteroides XG5 might be widely used in the production of linear dextran which has potential to serve as natural agent applied in food and other fields.

Production and characterization of bacterial cellulose produced by Gluconacetobacter xylinus isolated from Chinese persimmon vinegar

This study aimed to characterize the structural and physico-mechanical properties of bacterial cellulose (BC) produced by Gluconoacetobacter xylinus TJU-S8 which was isolated from Chinese persimmon vinegar. Thermogravimetric analysis (TGA) showed that BC exhibited a good thermal stability. Solid-state nuclear magnetic resonance (NMR), fourier transform infrared spectroscopy (FT-IR) and x-ray diffraction (XRD) analysis revealed that BC had a typical crystalline form of the cellulose I. The BC membrane had typical characteristics such as nanodimensional network and microfibrils obtained by scanning electron microscopy (SEM). Moreover, the bacterial cellulose chitosan (BC-C) membrane and bacterial cellulose carboxymethyl chitosan (BC-CC) membrane were synthesized which showed significant inhibition against the growth of both Escherichia coli and Staphylococcus aureus. These results indicated superior properties of BC that advocated its effectiveness for various applications.

Encoded protein from ycbR gene of enterohemorrhagic Escherichia coli O157:H7 associated with adherence to HEp-2 cells

Adhesion is one of the significant virulence factors in enterohemorrhagic Escherichia coli (EHEC) O157:H7 pathogenesis. It is regulated by specific loci in the genome sequence. This study mainly focused on investigating the influence of ycbR gene and its encoded YCBR protein on the adhesion of EHEC O157:H7 to HEp-2 cells. In the first part, mutants of EHEC O157:H7 were constructed through TnphoA mutagenesis and assayed for adherent ability. Six mutant strains with lost adherence to HEp-2 cells were isolated and then sequenced using a primer that hybridized to phoA sequence downstream of the fusion joint. The sequencing results indicated that the gene of eae and ycbR, between the initiation codon and the -10 sequence of Z4182, yciI, ARAC-type regulator protein, and high-affinity gluconate transporter of EHEC were all possibly related to adhesion. Of the six genes, the ycbR gene was cloned to the pET28a vector to analyze its function further. Recombinant YCBR protein fused to a His tag (YCBR-His) was expressed under IPTG induction and purified by Ni-NTA column. The purified protein was subcutaneously injected to rabbits to prepare antisera. The results of an adherence assay in the presence of anti-YCBR-His antibodies indicated that antibodies blocked the adherence of EHEC O157:H7 to HEp-2 cells. These observations suggested that ycbR encoded a novel adherence-associated determinant of EHEC O157:H7, which could contribute to the adhesive capacity of the bacteria.

Optimization, chain conformation and characterization of exopolysaccharide isolated from Leuconostoc mesenteroides DRP105

Response surface methodology (RSM) was used to optimize the fermentation condition of exopolysaccharide (EPS) producing strain Leuconostoc mesenteroides DRP105. Result showed that the optimum condition was sucrose 86.83g/L, tryptone 15.47g/L, initial pH7.18 and maximum yield was 53.79±0.78g/L in 36h fermentation. Chain conformation was characterized by Congo red test, β-elimination and circular dichroism (CD), which indicated that the EPS was O-linkage and exhibited random coil structure in aqueous solution. CD results concluded hydrogen-bond interaction and chirality might be connected with concentration. Purified EPS has a higher degradation temperature of 279.42°C, suggesting high thermal stability of the EPS. The absolute value of zeta potential and particle size were enhanced with increasing concentration. Crude EPS and its purified fraction were found to have moderate DPPH, hydroxyl, superoxide anion radicals scavenging activities and reducing power. This study provided a high yield EPS with unique characteristics for industrial applications.