D.什特紫青霉降解沙棘籽原花色素的研究

【作者】 刘继伟；

【导师】 康传红；

【作者基本信息】 黑龙江大学 ， 生物化学与分子生物学， 2013， 硕士

【摘要】 沙棘籽中含有多种营养成分，原花色素作为其中一种被人们重点关注，它具有极强的抗氧化性使得其在食品、保健品、医药、工业等领域具有很好的应用价值。原花色素有低聚体与高聚体之分，相对于高聚体原花色素低聚体原花色素有着更好的生物活性，所以将原花色素高聚体降解为低聚体被广泛研究，主要的降解方法有化学法降解、微生物降解等。本实验室通过筛选找到能够降解沙棘原花色素的菌株，初步鉴定为D.什特紫青霉，本论文利用D.什特紫青霉对沙棘籽原花色素进行了降解。实验首先对D.什特紫青霉降解沙棘籽原花色素的培养基及培养条件进行了优化，通过对培养基及培养条件的单因素实验及均匀设计法进行回归分析获得D.什特紫青霉降解原花色素的最佳培养基为：麦芽糖含量为4.5g、硝酸钠含量为3.7g、硫酸镁含量为0.04g、硫化钾含量为0g。最佳培养条件为：pH值为6、降解时间为4天，在此条件下沙棘原花色素平均聚合度由6.057降解为1.983。在确定最佳降解条件后，本实验对比分析了降解前后原花色素的抗氧化能力，实验结果表明，当原花色素添加量为2.5mL时降解后原花色素对超氧阴离子的清除率达到95%以上，而降解前原花色素对超氧阴离子的清除率不到80%；当原花色素添加量为0.5mL时降解后原花色素对亚硝酸根离子的清除率达到90%以上，而降解前原花色素对亚硝酸根离子的清除率仅为44%，由此可见降解后原花色素具有更强的抗氧化能力。本实验还对D.什特紫青霉降解沙棘籽原花色素的降解机理进行了初步探索，首先研究了碳源对降解过程的影响，结果表明在缺少碳源的条件下D.什特紫青霉对沙棘籽原花色素无降解作用。同时又研究了D.什特紫青霉发酵液对沙棘原花色素的降解作用，结果表明发酵液对沙棘籽原花色素具有降解作用，而细胞破碎液对沙棘籽原花色素没有降解作用，初步推断微生物对对原花色素的降解作用为菌体分泌到细胞外的酶或其他物质对原花色素有降解作用。最后实验通过HPLC、ESI-质谱、红外光谱等方法对沙棘籽原花色素降解产物进行了分析，结果表明沙棘籽原花色素被D.什特紫青霉降解后低聚体含量明显增加，并且得到两种原花色素单体衍生物，根据质谱测定结果得到其相对分子量分别为343、311，根据红外测定结果发现其在结构中含有-OH、C=O、C-O、CH2等基团，表明降解后原花色素的基本结构没有发生变化，并根据原花色素单体结构信息，对这两种衍生物结构进行了初步判断。更多还原

【Abstract】 Sea-buckthorn seed contains a variety of nutrients, as one of these,proanthocyanidin is well known，it has a good application value in food, health products,pharmaceutical, industrial and other fields because it has a very strong antioxidantactivity. Proanthocyanidin contains oligomer and high polymer. Proanthocyanidinoligomer has a better biological activity respect than the high polymer proanthocyanidin,therefore making the high polymer proanthocyanidin into oligomer has been extensivelystudied. The main degradation method is thiolase, chemical degradation, microbialdegradation, and so on.My laboratory had found that the strains are able to degrade the sea buckthorn seedproanthocyanidins, identified as Penicillium purpurogenum straj. In this thesis,proanthocyanidin was degraded by Penicillium purpurogenum straj. Firstly, Theexperiment did an optimized on the conditions of media and culture Penicilliumdegradation proanthocyanidin. We obtain the experimental results by means of singlefactor experiment on the conditions of media and culture timely according to theuniform design method regression Analysis. The best medium is: Maltose4.5g,NaNO33.7g, MgSO40.04g, Kcl0g; the best culture conditions is pH value6, thedegradation time is4d. On this condition, the average polymerization degree ofseabuckthorn seed proanthocyanidin would be from6.057down to1.983.When the degradation conditions are determined, this experiment analysis thecapacity of antioxidant on proanthocyanidin out of degradation and ascorbic acid. It isfound that proanthocyanidin has a better capacity of antioxidant which was degradatedby Penicillium purpurogenum straj. The clearance of superoxide ions reaches95%when the adding amount of proanthocyanidin by degradated is2.5mL; The clearance ofnitrite ion reaches90%when the adding amount of proanthocyanidin by degradated is0.5mL.This study had also carried on the preliminary exploration on the degradationmechanism of proanthocyanidin degradated by Penicillium purpurogenum straj. Firstiy,the influence of degradation process by carbon source was studied,results showed thatseabuckthorn seed proanthocyanidin was not degradated by Penicillium purpurogenum straj. on the condition of lack of carbon source; On the meanwhile, the influence ofdegradation process by the fermented liquid of Penicillium purpurogenum straj wasstudied, results showed that seabuckthorn seed proanthocyanidin could be degradatedby fermented liquid, however it could not be degradated by cell lysates.At last, we made an analysis on degradation products by HPLC, ESI-MS, IRspectroscopy and other methods. Results showed that the oligomer proanthocyanidincontent increased obviously after degradation, and obtained two monomerproanthocyanidin derivatives whose relative molecular weight was343,311,and contain–OH,C=O,C-O,CH2group in these structures. According to the structure information ofmonomer proanthocyanidin, we made a preliminary judgment on the structures ofproanthocyanidin derivatives.更多还原