【作者】 张琛；

【导师】 樊美珍； 李增智； 李春如；

【作者基本信息】 安徽农业大学 ， 微生物学， 2005， 硕士

【摘要】 钒是人体必需的微量元素之一,大量研究表明,钒在动物和人体中起着重要的生理作用,另外,钒与细胞凋亡、癌症和营养代谢紧密相关,并作用于免疫系统。体内钒的缺乏会导致糖、脂代谢紊乱,从而诱发糖尿病和心血管疾病,而在人体中补充微量的钒,对这些疾病有明显的治疗作用。 本实验以啤酒酵母(Saccharomyces cerevisiae, Meyen ex Hansen)为载体,在培养基中添加偏钒酸铵,利用啤酒酵母可以富集微量元素的特性,将无机钒富集到细胞内。 对 5 株本室保藏的啤酒酵母进行了筛选,经过抗性筛选和摇瓶发酵培养获得了一株编号为 RCEF1023 的富钒菌株,作为研究菌株。 通过对液体培养基的筛选以及葡萄糖、麦芽汁、蛋白胨、酵母浸出粉四因子的正交实验,确定了 RCEF1023 菌株的最适发酵培养基配方为:葡萄糖 1% 、酵母浸出粉0.3%、 蛋白胨 0.5% 、11BX 麦芽汁 25%。 在液体摇瓶培养条件试验中还研究了钒浓度、培养温度、pH、培养时间、接种量和装液量等因素对啤酒酵母 RCEF1023 菌株的生物量及钒含量的影响,初步优化了RCEF1023 菌株的富钒培养条件,结果表明:钒加入量为 40μg/mL,接种量为 10%,pH 为 6.0,装液量为 50mL/500mL 三角瓶,培养温度为 28℃,48h 后,富钒酵母生物量为 0.663g/100mL,钒含量达到 587.6μg/g。同时发现培养基的钒浓度,PH 和装液量显著影响富钒酵母的钒含量和生物量。 为了测定富钒酵母中有机钒的含量,在实验中采用了浸出法使钒酵母中的无机钒和有机钒得以分离,从而测得了钒酵母中有机钒含量。有机钒含量可达到 474.9μg/g。 对富钒酵母自溶条件进行了研究,结果表明:pH 7.0, 55℃, 2 4h的条件下,自溶物中蛋白质含量最高,为最佳自溶条件,对影响酸—热破碎法的条件进行了研究,结果表明 :3mol/LHCl,浸泡 6min,热处理 3min,迅速冷却的条件下,蛋白质含量最高,为最佳破碎条件。将两种方法进行比较,发现破碎后内容物溢出量及钒含量非常接近,差别不明显。 此外,通过对普通酵母和钒酵母进行 200nm 到 600nm 全波长紫外光谱扫描分析,发现钒酵母在 311nm 处有一特征吸收峰;通过红外光谱分析,发现钒酵母在 1794cm-1处有特征吸收峰。 粗蛋白样品经 Sephadex G-100 色谱柱分离,得到二个主要组分 FractionⅠ和FractionⅡ,得率分别为 8.2%、16.0%。其钒含量分别为 FractionⅠ1.66μg/mg、FractionⅡ8.08μg/mg。更多还原

【Abstract】 The vanadium is one of the essential trace elements of human body. A large amount ofresearch shows that vanadium plays an important physiological function in animal and thehuman bodies. In addition, vanadium related to withering and death of cell, cancer as wellas nutrition metabolism by acting on the immune system. The shortage of the internalvanadium will cause the metabolism disorder of sugar and fat, and consequently result indiabetes and cardiovascular diseases, while the supply of micro-vanadium in the humanbody will obviously help to treat these diseases. Brewer’s yeast is able to assimilate a great number of minerals and trace element intoa nutritious product. In this work, brewer’s yeast was used to enrich vanadium by addingNH4VO3into culture media, making inorganic vanadium to be accumulated into cells. From 5 strains of brewer’s yeast of stock culture in this laboratory, Saccharo mycesceveisiae RCEF1023 which is suitable for V-rich yeasts fermentation, was selected byresistant screening and flask fermentation. Through liquid culture screening and the orthogonal design experiment ,the recipe offermentive medium was prescribed: 1% glucose, 0. 3% yeast extract, 0.5% peptone , 25%11BX wort. The concentration of vanadium, culture temperature , pH , culture time , inoculmsvolume, volume of medium etc factor were also studied and the primary optimization ofcultivation conditions was selected through fermentation test. The result showed that theoptimal condtions for the enrichment of vanadium into yeast was obtained after 48h at28℃, when inoculms volume was 10% (V/V) with 50mL of culture in 500mL shake flaskswhich was grown in culture fluid containing 40μg/mL V+5. The initial pH was adjusted 6.0.Under the optimal cultivation conditions, the biomass (dry weight) of 100mL fermentationreached 0.663g and enrichment quantity of vanadium was about 587.6μg/g. It was evidentthat the concentration of vanadium, pH and volume of medium greatly affected thebiomass and vanadium content of the brewer’s yeast. In order to test the content of the organic vanadium in vanadium-rich brewer’s yeast,adopting lixiviation process to separate inorganic vanadium and organic vanadium fromthe vanadium yeast , so the content of organic vanadium in the vanadium yeast could beaccurately determined. The content of organic vanadium in the test was 474.9μg/g.Study on the autolysis condition of vanadium-rich brewer’s yeast turned out that theoptimal conditions for antolysis were pH 7.0, 55℃and 24h for acid-heat shiver 3mol/LHCl, 6min soaking, and 3min.heating No significant difference was observed betweenthe two methods in their shiver effect. In addition, with ultraviolet spectrometry scanning within 200~600nm measurementof the ordinary yeast and vanadium-rich yeast, the vanadium-rich yeast has a characteristicabsorption in 311nm. With the determination of infrared spectrometry, the vanadium yeasthas a characteristic absorption in 1794cm-1. The raw protein was further separated into two fractions by Sephadex G-100 gelchromatography. The yield rates of FractionⅠand FractionⅡwere 8.2%,16.0%.Thevanadium content of FractionⅠand FractionⅡwas 1.66μg/mg and 8.08μg/mg. Analyzing of 16 kinds of amino acid, it turned out that the content of Met and Phe invanadium-rich yeast was higher than that in normal yeast. This Experiment has also compared the absorption and utilization betweeninorganic vanadium and vanadium yeast by using moina macrocpa. Taking moinamacrocpa as the animal’s model, studying the influence of vanadium yeast and inorganicvanadium from the pharmacodynamics aspect . The result of the toxic test shows thatvanadium-rich brewer’s yeast can effectively reduce the toxicity of the vanadium.更多还原