Zhengjun Wu

Partial characterization and immunostimulatory activity of exopolysaccharides from Lactobacillus rhamnosus KF5

Lactobacillus rhamnosus KF5, a strain newly isolated from the faeces of a healthy human volunteer, has been shown to produce the exopolysaccharides (EPS) in skim milk. Two EPS fractions were separated from the fermented skim milk by removing proteins, ethanol precipitation, anion-exchange and gel permeation chromatography. Fraction S1, with a low average molecular weight of 1.36 × 10(4)Da, was composed of glucose, arabinose, glucosamine, galactosamine and galactose in an approximate molar ratio of 2.03:1.29:1.25:0.72:0.61. Such monosaccharide composition of the exopolysaccharide by lactic acid bacteria has not been reported so far, and S1 is likely to be an unusual type of microbial EPS. Whilst Fraction S2, with a high average molecular weight of 1.23 × 10(6)Da, contained rhamnose, glucose and galactose in a molar ratio of approximately 1.73:1.47:1.00. Both of EPS fractions could significantly stimulate splenocyte proliferation in vitro, indicating two EPS fractions had the potential immunomodulatory activity.

Aggregation and adhesion properties of 22 Lactobacillus strains

In this paper, the autoaggregating, coaggregating, hydrophobicity, and adhering abilities of 22 Lactobacillus strains belonging to different species were assessed. No correlation existed between autoaggregation and adhesion of the strains belonging to different species, whereas a positive correlation existed between autoaggregation and adhesion of the strains belonging to the same species. After treating with guanidine HCl, the autoaggregating and adhering abilities of some Lactobacillus strains decreased, indicating that surface-bound proteins and other macromolecules played a role in the adhering and autoaggregating abilities. The strains Lactobacillus plantarum 20 and 66 had higher adhesion and coaggregation abilities and should be further studied for their probable probiotic properties. Aggregating, coaggregating, and adhering abilities of Lactobacillus strains could be used as the preliminary criteria for selecting strains having probiotic potential.

Dextran synthesized by Leuconostoc mesenteroides BD1710 in tomato juice supplemented with sucrose

The characteristics of the growth of Leuconostoc mesenteroides BD1710 and the synthesis of dextran in tomato juice supplemented with 15% sucrose were assayed. L. mesenteroides BD1710 could synthesize approximately 32 g L(-1) dextran in the tomato-juice-sucrose medium when cultured at 28 °C for 48 h, which was on the same level as the dextran yield in a chemically defined medium. The viscosity of the cultured tomato-juice-sucrose medium with various dextran contents was also measured. The results of the monosaccharide composition, molecular-weight distribution, Fourier transform infrared spectra (FTIR) and nuclear magnetic resonance spectra (NMR) showed that the polysaccharide synthesized by L. mesenteroides BD1710 in the tomato-juice-sucrose medium was dextran with a peak molecular weight of 6.35 × 10(5)Da, a linear backbone composed of consecutive α-(1 → 6)-linked d-glucopyranosyl units and approximately 6% α-(1 → 3) branches.

Molecular characteristics of an exopolysaccharide from Lactobacillus rhamnosus KF5 in solution

The molecular weight and chain conformation of an exopolysaccharide (EPS), S2, from Lactobacillus rhamnosus KF5 were determined by size-exclusion chromatography combined with multi-angle laser light scattering (SEC-MALLS), dynamic light scattering (DLS), viscometry and atomic force microscopy (AFM). The weight-average molecular weight (Mw), intrinsic viscosity [ƞ], radii of gyration (R(g)) and hydrodynamic radii (R(h)) were 7.5 × 10(5) Da, 2.331 dL/g, 44.3 nm and 29.4 nm, respectively. The conformational parameters were calculated from the above data according to the theory of dilute polymer solutions. The Mark-Houwink-Sakurada exponent α was found to be 0.687, and the value of the structure-sensitive parameter ρ (R(g)/R(h)) was 1.51. The results revealed that the EPS S2 existed as a random coil conformation in 0.1M NaNO3 aqueous solution. AFM further confirmed the random coil morphology of this molecule in aqueous solution. EPS S2 present extended chains (fibrous morphology), with circular side chains, dispersed in SDS solution.

Gsy, a novel glucansucrase from Leuconostoc mesenteroides , mediates the formation of cell aggregates in response to oxidative stress

Leuconostoc mesenteroides is a member of lactic acid bacteria (LAB) with wide applications in the food and medical industries. Species in the genus Leuconostoc are catalase-negative and generally regarded as facultative anaerobic or aerotolerant organisms. Despite their extensive use in industry, certain issues concerning the aerobic life of L. mesenteroides, e.g., the mechanism involved in the tolerance to oxygen, remain to be addressed. In this manuscript, a survival strategy employed by L. mesenteroides BD3749 in response to oxidative stress was elucidated. BD3749 cells cultivated in medium with sucrose available synthesized large amounts of exopolysaccharides, mostly consisting of insoluble EPS. When BD3749 cells were challenged with oxidative stress, the amount of insoluble EPS was greatly enhanced. The synthesized EPSs reduced the accumulation of reactive oxygen species (ROS) in bacterial cells and improved their survival during chronic oxidative stress. Another study showed that Gsy, a novel glucansucrase in the GH70 family that is induced by sucrose and up-regulated following exposure to oxygen, was responsible for the synthesis of insoluble EPS. Gsy was subsequently demonstrated to play pivotal roles in the formation of aggregates to alleviate the detrimental effects on BD3749 cells exerted by oxygen.

Characterization of the levan produced by Paenibacillus bovis sp. nov BD3526 and its immunological activity.

Paenibacillus bovis sp. nov BD3526 synthesizes a large amount of exopolysaccharides (EPSs) (36.25g/L) in a semi-defined chemical medium containing 20% (w/v) sucrose. The EPSs were extracted from the cultured broth by ethanol precipitation and purified via anion-exchange and gel permeation chromatography. The Fourier transform infrared (FT-IR) and nuclear magnetic resonance (NMR) spectra showed that the primary EPS fraction (F1) was a linear β (2→6)-linked levan. The peak molecular weight (Mp) of the levan exceeded 2.6×10(6)Da based on high-performance size-exclusion chromatography (HPSEC). The levan adopted a spherical conformation in aqueous solution as confirmed by transmission electron microscopy (TEM). The corresponding levansucrase was identified by SDS-PAGE analysis and in situ polymer synthesis. The in vitro assay demonstrated that the levan significantly stimulated the proliferation of spleen cells and induced the expression of TNF-α, indicating its potential as a natural immunomodulator.

High Milk-Clotting Activity Expressed by the Newly Isolated Paenibacillus spp. Strain BD3526

Paenibacillus spp. BD3526, a bacterium exhibiting a protein hydrolysis circle surrounded with an obvious precipitation zone on skim milk agar, was isolated from raw yak (Bos grunniens) milk collected in Tibet, China. Phylogenetic analysis based on 16S rRNA and whole genome sequence comparison indicated the isolate belong to the genus Paenibacillus. The strain BD3526 demonstrated strong ability to produce protease with milk clotting activity (MCA) in wheat bran broth. The protease with MCA was predominantly accumulated during the late-exponential phase of growth. The proteolytic activity (PA) of the BD3526 protease was 1.33-fold higher than that of the commercial R. miehei coagulant. A maximum MCA (6470 ± 281 SU mL−1) of the strain BD3526 was reached under optimal cultivation conditions. The protease with MCA was precipitated from the cultivated supernatant of wheat bran broth with ammonium sulfate and purified by anion-exchange chromatography. The molecular weight of the protease with MCA was determined as 35 kDa by sodium dodecyl sulfate-polyacrylamide gels electrophoresis (SDS-PAGE) and gelatin zymography. The cleavage site of the BD3526 protease with MCA in κ-casein was located at the Met106–Ala107 bond, as determined by mass spectrometry analysis.

Purification and characterization of a novel milk-clotting metalloproteinase from Paenibacillus spp. BD3526

In this study, a milk-clotting enzyme (MCE) isolated from Paenibacillus spp. BD3526 was purified and characterized. The MCE was purified 8.9-fold with a 10.11% recovery using ammonium sulfate precipitation and anion-exchange chromatography and the specific milk-clotting activity (MCA) reached 6791.73 SU/mg. The enzyme was characterized as a 35kDa metalloproteinase, and the zymogen of which was encoded by a 1671 bp gene named zinc metalloproteinase precursor (zmp) with a predicted molecular weight of 59.6 kDa. The optimal temperature for MCA and proteolytic activity (PA) was 65°C and 60°C, respectively. The enzyme was stable over a pH range of 5.0-9.0 and at temperatures below 50°C. The MCA was completely inactivated when the enzyme was heated at 60°C for 30 min, and the PA was totally inactivated for 20 and 10 min when the enzyme was heated at 55°C and 60°C, respectively. The BD3526 enzyme was preferentially active towards κ-casein (κ-CN) and β-casein (β-CN), as determined by sodium dodecyl sulfate-polyacrylamide gels (SDS-PAGE), whereas the hydrolysis of αs-casein (αs-CN) was slow and comparable to that caused by chymosin and asparatic acid proteinase from Rhizomucor miehei. The cleavage site of the metalloproteinase in κ-CN was located at the Met106-Ala107 bond, as determined by mass spectrometry analysis.