Huazhi Xiao

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.

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.

Analysis of bacterial diversity of Chinese Luzhou-flavor liquor brewed in different seasons by Illumina Miseq sequencing

Luzhou-flavor liquor is one of the most popular liquors in China, produced by the distillation of Zaopei, a mixture of fermented and fresh cereals. The quality and flavor of Luzhou-flavor liquor are closely related to the multitudinous bacteria in Zaopei. Little is known about the effect of brewing seasons, a main operating parameter, on the bacterial diversity of Zaopei. In this study Illumina Miseq sequencing platform was first applied to study the bacterial succession in Zaopei during liquor fermentation processes in winter and summer. The research results showed that 10 phyla and 13 families were detected in Zaopei. Differences in bacterial communities of summer and winter Zaopei were apparent. Proteobacteria was the most abundant phylum in winter Zaopei. However, bacteria belonging to Firmicutes were most frequently detected in summer Zaopei. The diverse genera decreased sharply at the first half of the 40-day fermentation stage, and then bacterial population were mainly represented by Acetobacter and Lactobacillus both in winter and summer Zaopei. Interestingly, the summer Zaopei contained significantly higher proportions of LAB and lower proportions of Acetobacter than winter Zaopei. Clustering analysis also revealed that bacterial community structure was distinct between the Zaopei fermented in the two seasons. Thermoactinomycetaceae, Prevotella, Alcaligenes, and Gluconacetobacter not previously identified in Zaopei were revealed in this study by high-throughput sequencing. This study might be helpful in achieving better control over the quality of Luzhou-flavor liquor brewed in different seasons.

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.

Characterization of highly branched dextran produced by Leuconostoc citreum B-2 from pineapple fermented product

A strain Leuconostoc citreum B-2 was isolated from homemade fermentation product of pineapple and its polysaccharide yield was 28.3g/L after cultivating the strain in Man-Rogosa-Sharpe (MRS) medium with 75g/L sucrose. The exopolysaccharide (EPS) was characterized by gas chromatography (GC), Fourier-transform infrared (FT-IR) spectra, high-performance size-exclusion chromatography (HPSEC), nuclear magnetic resonance (NMR) spectroscopy and scanning electron microscope (SEM) analysis in present study. The monosaccharide composition of EPS was glucose and molecular weight was 3.77×106Da. FT-IR and NMR spectra revealed that the B-2 EPS was composed of 75% α-(1→6) linked d-glucopyranose units existing in the main chain with 19% α-(1→3) branching and only a few α-(1→2) branching. The SEM of the dried EPS appeared irregular sheets with glittering surface and compact structure. Water solubility index and water holding capacity of B-2 EPS were 80% and 450%, respectively. All the mentioned characteristics suggested that the EPS has a potential application in the food, cosmetic and pharmaceuticals industry.

Optimization and purification of glucansucrase produced by Leuconostoc mesenteroides DRP2-19 isolated from Chinese Sauerkraut

Abstract Strain DRP2-19 was detected to produce high yield of glucansucrase in MRS broth, which was identified to be Leuconostoc mesenteroides. In order for industrial glucansucrase production of L. mesenteroides DRP2-19, a one-factor test was conducted, then response surface method was applied to optimize its yield and discover the best production condition. Based on Plackett–Burman (PB) experiment, sucrose, Ca2+, and initial pH were found to be the most significant factors for glucansucrase production. Afterwards, effects of the three main factors on glucansucrase activity were further investigated by central composite design and the optimum composition was sucrose 35.87 g/L, Ca2+ 0.21 mmol/L, and initial pH 5.56. Optimum results showed that glucansucrase activity was increased to 3.94 ± 0.43 U/mL in 24 hr fermentation, 2.66-fold higher than before. In addition, the crude enzyme was purified using ammonium sulfate precipitation, ion-exchange chromatography, and gel filtration. The molecular weight of glucansucrase was determined as approximately 170 kDa by Sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The enzyme was purified 15.77-fold and showed a final specific activity of 338.56 U/mg protein.