| To utilize clam resources with high-value, in this paper clam(Mactra chinensis) was fermented by Natto bacteria(Bacillus natto), the problems as follows were explored and solved.1. Free amino acid nitrogen content in the fermented product and natto kinase activity were used as the evaluation index to explore the best fermentation condition.Through single factor and orthogonal design test, the results showed that the best fermentation condition were as follows: 43 ℃, 0.6% inoculated quantity, fermentation time 48 h, ratio of water to material 2:1. Under this condition, free amino acid nitrogen content in the fermented products was the highest(conversion rate could be up to73.10%). When fermentation condition was 41 ℃, 0.8% inoculated quantity, fermentation time 48 h and ratio of water to material 3:1, natto kinase activity in fermented products were the highest.2. The extraction, purification, composition and biological activity of polysaccharides from the fermented products were studied successively. The traditional water extraction was used to obtain crude polysaccharide with extraction ratio of 6.5%).With the aid of DEAE Sepharose Fast Flow and Sephacry-400 column chromatography,three kinds of polysaccharides were achieved with purity of 93.92%, 89.91% and 83.73%(named XJW-1, XJW-2 and XJW-3, respectively). Then content of protein, sulfate and amino sugar in them was determined. Results showed that for XJW-1, contents of both protein and amino sugar were 1.03%. However, the content of sulfate was less and the yield was 0.20%. For XJW-2, protein content was 0.22%. It contained trace amounts of sulfate but no amino sugar, and the yield was 0.19%. For XJW-3, protein content was1.25%. However, it contained trace amounts of sulfate without amino sugar, and the yield was 0.23%. HPLC analysis results indicated that for XJW-1, the molecular weight was81.68 KD and its constitutional unit mainly was Glc. For XJW-2, its molecular weight was 31.73 KD and the constitutional units included Man, GlcN, Glc and Gal with the mole ratio of 1.095 : 1.164 : 96.430 : 1.312(Man : GlcN : Glc : Gal). For XJW-3, its molecular weight was 16.944 KD and the constitutional units included Man, GlcN, Glcand Gal with the mole ratio of 14.23 : 13.98 : 73.11 : 1.32(Man : GlcN : Glc : Gal).Results of antioxidant activity and antitumor activity showed that the three kinds of polysaccharides possessed obvious scavenging activity to hydroxyl radicals and superoxide anion free radicals. Furthermore, their scavenging activity to hydroxyl free radicals was strongger than that of the same concentration of Vc. In general, the order of antioxidant activity of 3 kinds of polysaccharides was XJW-3 > XJW-2 > XJW-1. Three kinds of polysaccharides all showed inhibitant effect to glioma cells, human cervical squamous cancer, neuroblastoma cells and cervix cancer cells and demonstrated time and dose dependent manner(positive correlation). Among them, three kinds of polysaccharides presented the strongest proliferation to cervical squamous cancer cell,secondly to astoma cells and glioma cells and the cervical cancer cells. Treated by three kinds of polysaccharides, four kinds of cancer cells changed clearly in cell morphology,and the number of the cells decreased or the close connection among cells interrupted, or clustered and even broke directly. The specific mechanism remains to be further studied.3. The fermentation product was extracted by petroleumether, ethyl acetate and n-butanol successively to get the extractums with different polarity accordingly. Results of the bacteriostatic activity to Staphylococcus aureus showed that the ethyl acetate phase was better than that of petroleum ether and n-butanol phase, so in the subsequent experiment, ethyl acetate phase was chose as experiment material with Staphylococcus aureus as indicator. By purification methods of silica gel column chromatography, gel column chromatography and HPLC, 4 kinds of compounds was purified from ethyl acetate phase. Through NMR and MS combined with the physical and chemical properties and literature comparison, contrast the identification of the compound structure,the two compounds was confirmed as follows: Ergosta-5,22,24(28)-trien-3β-ol,(20S)-cholest-5-en-3β-ol. |
PDF Full Text Request