| This paper is aim to give out the toxicological characteristics and the effect on improving immunity by mice experiment. Screening the high yield strain of polysaccharides by UV mutation method, at the same time ,we optimized the liquid ferment condition, studied the main factors of extraction rate and the degree of purification of polysaccharides. Under series of experiments, the physicochemical characteristics, molecular weight, molecular structure and composition of polysaccharides have been given.Above all we studied the bevel culture medium , found the wort saccharifying technology , and the bevel culture medium is: the sugar degree of wort is 6-8.5 Baume, pH 6-7, agar 2%.After 21 days we weighed the avoirdupois of mice normally poured stomach by polysaccharides, and weighed the liver, spleen , kidney, heart, lung and recorded the index of the organs respectively. The result shows that the experiment the lower concentration polysaccharides can increase the liver quality, spleen quality, but high concentration polysaccharides could reduce the spleen quality and there is no bad effect on the other organs. Seen from the content of SGPT, polysaccharides could lower the content of SGPT obviously, but no apparent effect on the content of blood-sugar and SGOT. The conclusion is that polysaccharides has no apparent toxic effect on the mice.Through the DTH experiment, after pouring the polysaccharides into stomach the mice's feet swollen degree has obvious changes compared with the reference team. The experiment illuminated that polysaccharides could improve the mice cellular immunity. In the experiment of chloroform fine poisoning ,Compared with reference team the SGPT content of the experimented team has been lowered which illustrated polysaccharides can protect liver. In the experiment of carbon granule clearance rate , polysaccharides can improve abdominal cavity macrophage phagocytize ability, prompt the formation of periphery blood E- rose loop, increase the transformation rate of vivo lymphocyte and strengthen nonspecific immunity by pouring polysaccharides. By the swimming experiment and resistance to hypoxia experiment, we knew that the effect of polysaccharides is not apparent on improving resistance to hypoxia capability and viability at anoxic condition , which needs us study in furtherstep.ZH-100 is the variant strain of Monascus AS 3.4701 through Ultra-violet mutations radiation treatments, the optimal mutation dose is 254nm, 2.0min. After ZH-100 has been cultured 3 generations, the yield of polysaccharides is 4.26g/L, 4.69g/L, 4.10g/L, and the average is 4.35g/L. This result proved that ZH-100 have steady transmissibility and it is higher 286% than Monascus sp.3.4071.Still another three experiments. 1. The orthogonal experiment on fermentation condition. 2. parallel experiment on the inorganic element. 3. Test of the optimal experiment condition. Conclusion can be drawn that: rice plasm as the culture medium (solid 10%), NaNO3 0.15%,MgSO4 ? 7H2O 0.15%,K2HPO4 0.25%, temperature 35 °C, 4000r/min, time 96h. The yield of polysaccharides is 5.53 g/L higher 27% than the initial.By the orthogonal experiment the optimal extraction condition is : temperature is 90 °C; the rate of material to liquor is 1:3; time is 30 min. The yield of polysaccharides is 6.66 g/L higher 20% than the initial.The total sugar content is 78%, measured through carbolic acid- sulfuric acid method. Reducing sugar content is 12.5%, measured by 3.5 dinitrosalicylic acid , polysaccharides content is 65.5%. the specimen soluble protein content is 5.2%, measured by Coomassie brilliant blue methods.The purification technology of Monascus raw polysaccharides has been testified by experiment, we got rid of sundry protein with Sevage method, took out of salt and small molecular substance with dialyses, separated polysaccharides with Sephadex G - 200 column chromatography. At last, we got the pure polysaccharides through collecting and evaporating separated polysaccharides.There was only one aquamarine spot on paper chromatography. Result shows that the polysaccharides we collected is a pure substance. And confirm that the developer is normal butyl alcohol-ethanol-water (12: 3: 5 ). Sephadex G-200 column chromatography had a single eluting peak that demonstrated the polysaccharide was homogeneity.Through a chain of biochemical experiment, we drew the conclusion that polysaccharides crude product is a light red powder, virgin is a kind of white powder and easy to dissolve in water. The water solution was thickness, pH was about 6. That is of achromatism and insipidity, theviscosity of polysaccharides water solution ( 1 %) was 460mPa.s, can not submit blue reacted with iodine reagent. The reaction of H2SO4-Phenol and polysaccharides shows nacarat; the reaction of Anthracene-ketone reagent shows cyan; specific rotatory power [ a ] D = 1.66. It is cannot be dissolved into high concentration ethanol, aether, acetone, hexyl acetate etc.The essay adopts the method of two-way thin layer chromatography. Polysaccharides was hydrolyzed by 2mol / L vitriol, developer chloroform: methanol: water =16: 9: 2. By measuring polysaccharides components, we knew that polysaccharides contains mannitose, glucose and galactose.Processed gel filtration chromatography by Sephadex G-200, we got the volume on the chromatography is 5mg/lml, O.lmol/LNaCl elution solution. The standard curve is drawn, according to the given molecular weight. The molecular weight of polysaccharides is 316KD.Infra-red spectra analysis: Porphyrize 200mg pure KBr and 2mg pure polysaccharides, blend uniformity and pressure by oil press into transparence flake. IR analysed by TJ270 - 30 infrared spectrophotometer, scanned area 4000 - 400cm"1, we drew the conclusion that polysaccharides was p - pyranoid form polysaccharide.Generally speaking, these experiments illustrates that polysaccharides had no toxic effect on biology organism, but it could improve the immunity of body greatly. By structure and compose studying, we know that polysaccharides is a kind of heteroglycan which is composed with mannitose, glucose and galactose. Polysaccharides is p - pyrane configuration which proves the conclusion of improving immunity of body. The predominance strain of high-yielding polysaccharide has been screened through substantive mutation. Then we optimized the liquid fermentation condition, abstraction condition and purification condition. And the theory could instruct production and exploit kinds of health food and beverage, which aims at improve the immunity level of sub- health crowd. |
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