Electrophoresis separation and MS analysis of proteins in fermentation broth of Poria cocos / 中草药

Objective: In order to carry out the study on proteins from Poria cocos fermentation broth, the proteins in the fermentation broth were separated and identified. Methods: Proteins were obtained by organic acid precipitation from the fermentation broth and the protein concentration was determined by Bradford method. The obtained P. cocos secreted proteins were separated on SDS-PAGE electrophoresis, subjected to in-gel digestion, then identified by mass spectrometric analysis followed by database searching. Results: The protein concentration in the fermentation broth was around 74.01 μg/mL, with the apparent molecular weight ranged from 2.8 × 104 to 1.3 × 105. A total of 52 P. cocos secreted proteins were identified, including catalase, protein kinase, alkaline protease, glucoamylase, lysozyme, and so on. Conclusion: P. cocos fermentation broth has abundant proteins, which could be a good material for the study of P. cocos protein and also a potential healthy food and beverage.

Simultaneous determination of xylitol and L-xylulose in fermentation broth with high performance liquid chromatography / 中国生化药物杂志

Objective A high performance liquid chromatographic (HPLC) method was established for the simultaneous determination of xylitol and L-xylulose in fermentation broth.Methods The chromatographic conditions were as follows:C18 column (250 mm ×4.6 mm) with the temperature 35℃, acetonitrile-water (85∶15,v/v)as mobile phase with the flow rate of 0.8 mL/min.Xylitol was detected by refractive index (RI) detector at 33℃and L-xylulose was determined by ultraviolet ( UV) detector at 210 nm at room temperature.Results This method showed good linearity over the range from 0.50~30.00 g/L with a correlation coefficient of 0.9995 for xylitol and 0.30~30.00 g/L with a correlation coefficient of 0.9986 for L-xylulose. Moreover, the limit of quantification (LOQ) for xylitol and L-xylulose were 0.58 and 0.40,respectively.The limit of determination (LOD) for xylitol and L-xylulose were 0.18 and 0.15,respectively.The relative standard deviations (RSDs) of intraday and interday for xylitol were less than 0.64%and 0.80%,respectively.The intraday and interday RSDs for L-xylulose were less than 0.31%and 0.59%.The recoveries of xylitol and L-xylulose in fermentation broth were between 99.00%-101.00%.Conclusion There was no interference from other constitutes in the fermentation broth by this method.The methods were suitable for the simultaneous determination of the substrate xylitol and the product L-xylulose in fermentation process.

High-throughput screening of telomerase inhibitors in actinomycetes fermentation broth / 中国药理学通报

Aim To discover specific telomerase inhibitors by high-throughput screening in the more than 2 000 strains of actinomycetes fermentation broth.Methods After verifying the three kinds of screening models for telomerase inhibitor by yeast PCR,the three yeast strains were inoculated overnight into yeast liquid culture which was lacking in histidine and contained 3-AT.Then concentration of yeast strains in culture liquid and dosage of screening sample were regulated for better adjustment to the high-throughput screening.At last the adjusted liquid culture containing yeast strain piped to 96-well plates,drug samples and control article were also added,then the yeast was incubated in the thermostat oscillator.At the same time OD_(595nm) of the yeast liquid was measured every 12 h by using Multifunctional Microplate Analyzer,and the samples whose inhibitory efficiency was over 0.45 were selected for secondary screening.Results The high-throughput screening method related to yeast three-hybrid system was effective in screening samples that had potential telomerase inhibitory ability.The initial concentration of yeast liquid OD_(595nm) was 0.04 and the best dosage of samples was 5 ml.14 active samples,whose inhibitory efficiency was over 0.45,were founded after primary and secondary screening.Conclusions High-throughput screening method related to yeast three-hybrid system is simple and stable in discovering telomerase inhibitors,and 14 new antitumor compounds are identified,which lays the foundation in the development of new anti-tumor agents.

Optimization of Technique Conditions for Decolorization and Extract of Exopolysaccharide by Cordyceps jiangxiensis / 微生物学通报

The technique conditions of decolonization of fermentation broth were successively optimized using single factor assay and orthogonal layout method in Cordyceps jiangxiensis. The optimal condition of decolorization was investigated to be 3g/100mL active carbon, 5 min absorption time, pH5 of fermented broth and 25℃absorption temperature. Under the optimal condition, the maximum decolorization rate of fermented broth reached 89. 6% , simultaneously 10. 7% consuming rate of exopolysaccahride was minimum. Subsequently, the extract condition of exopolysaccharide of C. jiangxiensis was further optimized by orthogonal layout design. The maximum exopolysaccharide production was 0. 38 g/L under the optimal condition, i. e. firstly fermented filtrate decolorized and deproteined was concentrated to 1/7 of its total volume, secondly concentration broth was mixed with four times its volume of absolute ethanol and stirred vigorously, lastly precipitation of exopolysaccharide proceeded at 4℃for 16 hrs and the exopolysaccharide collected by centrifugal ion and dryness.