Study On The Active Component That Enhancing Cyprinus Carpio Immunological Function Of A Thermophilic Bacterium

Immunostimulants, also known as immune enhancers, can enhance the immune response of host used alone or simultaneously with the antigen. Immunostimulants can improve animals' immune function and disease resistance. The resorce of the immunostimulants includs aimal, plant and microorganisms.The main purpose of this study is to screen the strain which's metabolisms can enhancing the immunity of cyprinus carpio and then isolated the activity agents, identified the chemistry structure. This research provides the basis for follow-up mechanism, synthesis and industrial production. Major test methods, procedures, results are as follows:1. Selected strain:XA-1 to XA-7 as the object. After fermentation, centrifugating, filtrating, concentrating, the fermentation broth was manufacturered as the feed of cyprinus carpio. The treatmental cycle is 28 days, assay the leucocyte phagocytic activity and phagocyt bactericidal activity every 7 days to investigate the 7 metabolites'effect on cyprinus carpio immune function. The result showed that these two indicaters of group XA-1, XA-4 have all improved by some extent and shown immune enhancement activity. Other groups of the broth in this experiment have no significant effect on immune function under the same conditions and feedings. So we selected the strain XA-1 as the further research object and identified its' classification through the methods consisited of morphology, physio-biochemistry experiment and 16SrRNA. The results suggested that the strain was belong to Anoxybacillus, the species was unidentified yet.2. Cultivate the XA-1 in large scale, treat the broth with centrifugation,cryoconcentration step by step and procured the extractum of metabolites. The cyprinus carpio were intraperitoneal injected with extracts of petroleum spirit, acetic ether, chloroform, methanol, then tested their lysozyme activity,leucocyt phagocytic activity,SOD activity and phagocyt bactericidal activity every 7 days to determine the active site of active component. The result showed that active component in metabolites of XA-1 was a high-polarity matter, the active site localized in methanol and the methanolic extract have a higher content in active component.3. In the 3rd experiment, The immunopotentiating compounds have been isolated from the metabolites of XA-1 under the bioassay-guided isolation and identification for its immunopotentiating effect and chemical structure. The isolation and purification of the compound were consisted of macroporous adsorptive resins, silicagel chromatographic column. At last, the compounds A (265 mg) and B (194 mg) had been isolated and purified. The results of experiment suggested that, the compounds A and B groups showd the immunoenhancing activity and the B had showd the strongest activity. Through the procedure consisiting of (Infrared Radiation), ESI-Mass Spectrum (ESI-MS), Nuclear Magnetic Resonance (NMR) and Element analysis, the possible structure of compound A had been identified as cyclic (proline-glycine) dipeptide (C7H10N2O2), compounds B had been identified as cyclic (Hydroxyproline-alanine) dipeptide (C8H12N2O3).From the above results, we concluded that the main active component enhancing the immunity of cyprinus carpio was cyclic (Pro-Gly) dipeptide and cyclic (Hyp-Ala) dipeptide in the metabolism of thermophilic bacterium.