| PSP(Polysaccharidiopeptide) is a kind of polysaccharide peptide polymer, which is extracted from the deep layer cultivated mycelia of Cov-1strain of Coriolus versicolr. PSP can obviously improve the body’s immune ability and prevent the cancer as an auxiliary antitumor and immunostimulating agent. In this paper, a method of1-phenyl-3-methyl-5-pyrazolone (PMP) pre-column derivatization high performance liquid chromatographic (HPLC) analysis for monosaccharides of PSP was established, and on the basis of the standard quality detection methods of PSP, we developed the optimization of culture medium for increasing the production and reducing the cost of PSP.The effects of the chromatographic column and volume ratios of acetonitrile to phosphate buffer and pH of mobile phase on retention and separation of PMP derivatives of the seven monosaccharides were investigated. The results showed that the optimum chromatographic column was Luna C18(250X4.60mm,5μm, phenomenex), and the optimum volume ratio of acetonitrile to buffer was18:82and that the pH was at7.1. The other conditions were as follow:the column temperature was30℃, the detection wavelength was254nm, the flow rate was1.0mL/min and the injection volume was10μL. The developed method was successfully applied to the analyses of monosaccharide compositions in PSP. The analytical results showed that the monosaccharide compositions of PSP are mannose, rhamnose, glucose, xylose galactose, arabinose and fucose.A simple and sensitive HPLC method for simultaneous determination of seven kinds of monosaccharides by pre-column derivatization with PMP was developed. By studying the acid concentration, reaction time and temperature, the optimum method of polysaccharide hydrolysis was developed, and PSP was completely hydrolyzed in4mol/L trifluoroacetic acid for6hours at120℃.The result of the validation methodology has proved that phenol-sulfuric acid method has good linearity, high stability, high recoveries, and had no accessory material interference to the sample detection, and it is suitable for the detection of sugar content in PSP. Protein content detection, as well as measurement of molecular weight distribution of PSP have been verified and applied to analyse many batches of PSP products. By the established method of pre-column derivatization HPLC, we detected the monosaccharide compositions of ten batches of PSP raw material, ten batches of PSP capsules, and PSP extracted from different strains, different parts of Coriolus versicolor, and polysaccharide from the other different fungi. The results showed that the monosaccharide compositions for raw material and PSP capsules are stable relatively, and they are mainly consisted of mannose, glucosamine hydrochloride, rhamnose, glucose, xylose, galactose, arabinose, fucose with the molar ratio112.1±16.50:6.32±2.68:14.46±2.42:100:54.26±10.36:19.74±10.72:13.31±4.94:34.73±3.57and84.75±8.87:9.68±3.02:15.68±2.27:100:34.43±2.34:9.56±1.30:8.01±2.09:35.45±4.90. PSP from different strains of Corious versicolor, Polystictus Glycopeptide, and polysaccharide from the fruiting body of Coriolus versicolor, fruiting body of Polyporus umbellatus and the mycelium of Coprinus comatus possess the similar monosaccharide compositions with different molar ratio.The result has showed that the monosaccharide compositions and molar ratio of different products are various, and the established PMP pre-column derivatization HPLC analysis method can be used to the quality control for PSP.We studied the influence of the total amount of carbon and nitrogen, carbon and nitrogen proportion and different carbon sources on PSP production to optimize the fermentation culture medium. The results suggested that the optimum medium is: glucose4.8%, soybean powder1.9%, yeast powder0.2%, K2HPO40.05%, MgSO40.05%, with natural pH. The total carbon and nitrogen is6.9%, and the carbon nitrogen ratio is17:1, the production of the mycelia up to19.06g/L, the yield of PSP up to3.18g/L. Sugar content and protein content of the PSP is36.22%and28.54%respectively, the proportion of the polysaccharide peptide with the weight-average molecular weight great than or equal to40000Dalton is53.36%, and the monosaccharide compositions of the PSP are mannose, glucosamine hydrochlride, rhamnose, glucose, xylose, galactose, arabinose and fucose. The result of the investigation of carbon sources showed that lactose, corn flour and wheat flour can obviously improve the mycelia production and PSP yield, up to19.56g/L,21.69g/L and21.20g/L and2.95g/L,2.84g/L and2.91g/L respectively. Small molecular carbon source such as sucrose, maltose and maltodextrin are cheaper, which can replace glucose and ensure good production and quality of mycelia and PSP. In the study of carbon and nitrogen proportion, we found that with the increase of the carbon nitrogen ratio, the proportion of glucose in the PSP rise as well. So we can regulate the production of the mycelia and the monosaccharide compositions of PSP by changing the carbon and nitrogen proportion of the medium. We studied three proportion factors three level orthogonal tests on total amount of carbon and nitrogen, carbon and nitrogen ratio, and carbon source. The results show that the mycelia yield as the test index has significant difference. The optimum ratio of culture medium is as follow:total amount of carbon and nitrogen is8%, carbon and nitrogen ratio is13.5:1, and carbon source is corn flour. Under this condition, the mycelia yield is27.08g/L, the PSP yield PSP is3.73g/L, which is2.11and2.11times of the traditional method respectively. The sugar and protein content of the PSP is38.95%and31.04%respectively.
|
PDF Full Text Request