【作者】 方俊；

【导师】 卢向阳； 邱业先； 李宗军；

【作者基本信息】 湖南农业大学 ， 植物学， 2006， 博士

【摘要】 我国每年屠宰生猪近5亿头，占世界生猪屠宰总量的45％，猪血资源十分丰富，但目前除少量用于制作血豆腐、血肠或直接食用外，基本未得到开发利用，不仅浪费了资源，同时又严重污染了环境。本课题以新鲜猪血为原料，利用自筛的A32菌株进行发酵，获得低分子量的猪血多肽。同时研究了猪血多肽的抗氧化、降血脂、增强免疫功能等生物活性，主要结果如下： 1．发酵猪血优良菌株的分离筛选 利用平板划线法，从农村个体屠宰场土壤中分离获得了菌株A32，该菌株在以猪血为惟一氮源的培养基上生长旺盛且蛋白酶活力强。利用该菌株发酵猪血粉，使猪血粉的蛋白质含量下降了28.7％，氨基酸态氮含量上升了4.20倍，可溶性蛋白质含量上升了60％，说明A32菌株是发酵猪血的优良菌株。 2．菌株鉴定 生理生化鉴定：革兰氏染色后为阳性，有芽孢，无伴孢晶体，该菌在细胞壁染色后菌体内呈浅色，外围有明显的紫色圈，初步鉴定为产芽孢细菌。 分子生物学鉴定：将A32菌株16s rDNA序列提交GenBank进行Blast比对，经过同源序列比较，最终鉴定A32为枯草芽孢杆菌。 3．A32菌株产蛋白酶条件研究 通过单因素试验以及正交试验确定了A32优化培养基配方：豆粕粉2％、玉米粉3％、0.4mmol／L Mn²⁺、Na₂HPO₄·12H₂O 0.4％、KH₂PO₄ 0.03％、Na₂CO₃ 0.01％、pH 7.2；同时还研究了培养时间、摇瓶转速、接种量、温度对产酶的影响，试验结果表明培养时间48h、接种量3％、温度30℃组合有利于蛋白酶的产生，摇瓶转速190r／min较为理想。 4．A32菌株发酵猪血条件的优化 在探讨了发酵时间、温度、接种量、起始pH值，装料量等单因素对猪血发酵影响的基础上，进行二次回归旋转组合设计，以接种量、发酵温度、发酵时间三个因素为自变量，以水解度DH为响应值，应用响应面分析法研究了发酵时间、发酵温度、接种量对肽产量的影响。试验结果表明，发酵温度与时间及接种量对猪血肽的生成有显著影响，发酵条件优化结果为接种量为4％、发酵温度为31℃、发酵时间为60h，根据数学模型预测值为37.97％，试验实际值为37.956％。 5．猪血多肽的分离及其成分分析更多还原

【Abstract】 About five hundred million swine were killed in our country every year. It was about 45% of the world’s. The blood of swine was very abundant. At present, it wasn’t utilized on the whole except that a little was used as soybean curd, blood bowels and food. It not only wasted the sources but also severe contamination surroundings. It was studied in this topic that fresh swine blood was fermented to get peptides by A32 strain. At the same time, it was studied that bioactivities of PSB such as antioxygen, norblood-fat and renforcing immune function. Main results were reported as follows:1. The filter and separation of fermentable blood meal strainsBy utilizing plate streaking, A32 was gained from soil of individual slaughter house in countryside. The strain could grow prosperity on medium with swine blood as only nitrogen source and the enzyme activity of its protease was high. Utilizing A32 to ferment swine blood, the protein of power from swine blood decreased 28.7%, the nitrogen of free amino acids increased 4.20 times and dissolving protein increased 60%. All these demonstrated that A32 was a fine strain to ferment swine blood.2. Appraisement of strainPhysiology and biochemistry appraisement: Gram staining was negatively and it had germ. Vivo of thalline was tint, the surrounding of thalline was purple circle. The strain was initial appraised as bacillus.Molecular biology: Refer 16s rRNA sequence to GenBank and contrasted in Blast, it was appraised as bacillus subtilis through homologization sequence comparison.3. The condition of toco-protease of A32 strainThe condition of toco-protease was studied on various factors and orthogonal experiments. The optimum fermentation medium containing 2% soybean powder, 3% cornmeal, Na₂HPO₄·12H₂O 0.4%, KH₂PO₄更多还原