共轭亚油酸论文：产共轭亚油酸丙酸菌的筛选及发酵条件研究.doc

共轭亚油酸论文：产共轭亚油酸丙酸菌的筛选及发酵条件研究【中文摘要】亚油酸(Linoleic Acid; LA)是人体必需的三大不饱和脂肪酸之一,作为亚油酸异构体的共轭亚油酸(Conjugated Linoleic Acid; CLA)因其具有多种重要生理功能而备受关注。目前CLA的生产主要是采用化学法,而利用生物转化法生产CLA也日渐成为研究的热点。本论文通过建立简单的CLA含量测定方法,从食品中分离筛选出产CLA量较高的丙酸菌菌株,并对筛选所得菌株的培养基和发酵转化条件进行优化以提高其产量,本论文的主要研究内容和结果如下：1.自奶酪中筛选获得具有产CLA能力的菌株共13株,其中菌株PF-3的相对最好,其产CLA能力为38.41gmL-1。利用气相色谱-质谱联用(GC-MS)对发酵产物进行检测,进一步确认菌株PF-3具备产CLA的能力。通过常规鉴定手段对菌株PF-3进行鉴定,鉴定结果显示该菌株为费氏丙酸杆菌(Propionibacterium freudenreichii)。2.对不同的培养基进行了初步优化,确定了MRS培养基作为发酵培养基。通过正交实验和响应面法(RSM)优化研究了丙酸杆菌PF-3发酵生产CLA的培养基成分和发酵条件。得到的最优培养基成分为：蛋白胨(2%)、牛肉膏(1%)、酵母膏(0.5%)、葡萄糖(2.5%)、乙酸钠(0.5%)、柠檬酸铵(0.3%)、磷酸氢二钾(0.2%)、硫酸锰(0.02%)、硫酸镁(0.02%)。最佳发酵条件为：菌体接种量10.2%(v/v),LA添加量0.075%,pH6.75,反应温度31, Tween-80作为乳化剂(LA/Tween-80为1：20)的条件下,厌氧振荡培养36h。菌株PF-3在优化后的条件下,其CLA产量为63.58g-mL-1。【英文摘要】Linoleic acid(LA) is one of the three essential unsaturated fatty acids, the conjugated linoleic acid(CLA) which is the isomer of linoleic acid, has recently attracted much attention because of its important variety of physiological functions. Currently, the mainly method to producing CLA is by chemical approaches, and the biological transformation method to producing CLA is becoming a research hotspot.In this thesis, we screened Propionibacterium strains with high CLA producing capacity from food, then optimized the fermentation medium and fermentation conditions based on the fast determination method of CLA, the main contents and results of this paper are as follows:1. There are 13 strains which producing CLA were screened from cheese. Strain PF-3, has the relatively highest CLA producing ability, the CLA productivity is up to 38.41gmL-1. The fermentation products were detected by Gas Chromatography-Mass Spectrometry(GC-MS), and the strain PF-3 has the ability to produce CLA by futher confirmed. According to the analysis of the cell and colony morphology and physio-biochemical characteristics, the bacterium strain PF-3 was identified as Propionibacterium freudenreichii finally.2. The different fermentation mediums were optimized preliminary, and the MRS medium was defined as culture medium. Fermentation conditions to produce CLA of strain PF-3 were studied by using response surface analytic method and orthogonal experiment. The optimum medium consisted of peptone 2%, beef extract 1%, yeast extract 0.5%, glucose 2.5%, sodium acetate 0.5%, ammonium Citrate 0.3%, dipotassium hydrogen phosphate 0.2%, manganese sulfate 0.02%, magnesium sulfate 0.02%; The optimum fermentation conditions to produce CLA were:cell inoculum 10.2%, LA 0.075%, pH 6.75, temperature 31, and Tween-80 as emulsifier(LA/Tween-801:20), the best fermentation time was 36h under anaerobic shaking condition. In the optimized conditions, the CLA productivity of the strain PF-3 is up to 63.58gmL-1.【关键词】共轭亚油酸 丙酸菌 筛选 鉴定 发酵条件【英文关键词】Propionibacterium freudenreichii conjugated linoleic acid screening identification fermentation conditions【目录】产共轭亚油酸丙酸菌的筛选及发酵条件研究摘要3-4ABSTRACT4第1章 绪论11-261.1 共轭亚油酸的研究概况11-121.2 共轭亚油酸的生理学功能及应用12-171.2.1 抗癌作用及机理12-141.2.2 抗动脉粥样硬化作用14-151.2.3 降低脂肪沉积作用151.2.4 增强免疫作用15-161.2.5 改善骨组织代谢161.2.6 在食品工业中的应用16-171.2.7 在禽畜业中的应用171.3 CLA的化学合成17-191.3.1 碱性异构法181.3.2 金属催化异构法181.3.3 羟基脂肪酸脱水法18-191.4 CLA的生物合成19-211.4.1 瘤胃菌19-201.4.2 乳酸菌20-211.4.3 丙酸菌211.5 CLA的纯化方法21-231.5.1 尿素包合法21-221.5.2 低温结晶法221.5.3 吸附分离法221.5.4 脂肪酶浓缩法22-231.5.5 超临界流体萃取法231.6 CLA的检测方法23-251.6.1 紫外光谱法(UV)23-241.6.2 红外光谱法(IR)241.6.3 高效液相色谱法(HPLC)241.6.4 气相色谱法(GC)241.6.5 气-质联用法(GC-MS)24-251.7 本课题的研究目的及内容25-26第2章 产CLA丙酸菌的筛选与鉴定26-392.1 材料与设备26-282.1.1 样品262.1.2 主要试剂和仪器26-282.1.3 培养基282.2 试验方法28-322.2.1 菌种的分离筛选282.2.2 样品处理及筛选方法28-292.2.2.1 样品处理28-292.2.2.2 溴甲酚紫平板厌氧培养292.2.2.3 平板划线初筛292.2.2.4 复筛292.2.3 紫外分光光度法检测CLA29-302.2.3.1 LA和CLA吸收峰的测定292.2.3.2 CLA紫外吸收标准曲线的绘制29-302.2.4 发酵培养液中脂肪酸的萃取与紫外检测302.2.5 GC-MS分析发酵液脂肪酸产物302.2.5.1 脂肪酸的皂化和甲酯化302.2.5.2 气相色谱条件302.2.5.3 质谱条件302.2.6 革兰氏染色、显微形态和培养特征观察30-312.2.7 理化特性鉴定31-322.2.7.1 单糖发酵试验312.2.7.2 明胶液化试验312.2.7.3 七叶苷试验312.2.7.4 淀粉水解试验312.2.7.5 V-P试验312.2.7.6 甲基红试验312.2.7.7 硝酸盐还原试验31-322.2.7.8 H_2S试验322.2.7.9 石蕊牛乳试验322.2.7.10 吲哚试验(靛基质试验)322.2.7.11 运动性试验322.3 结果与分析32-382.3.1 LA和CLA特征吸收峰的确定32-332.3.2 CLA标准曲线的绘制33-342.3.3 产CLA菌株的筛选34-362.3.3.1 紫外检测结果34-352.3.3.2 GC-MS分析35-362.3.4 菌株PF-3的鉴定结果36-382.3.4.1 革兰氏染色、显微形态和培养特征36-372.3.4.2 生理生化试验结果37-382.4 本章小结38-39第3章 费氏丙酸杆菌生产CLA发酵条件的研究39-603.1 材料与设备39-403.1.1 菌种393.1.2 主要试剂和仪器39-403.1.3 培养基40(1) 玉米浆培养基40(2) 脱脂乳培养基40(3) MRS培养基40(4) 乳酸钠培养基40(5) LB培养基403.2 试验方法40-433.2.1 发酵培养基的优化40-413.2.1.1 不同培养基的优化40-413.2.1.2 碳源的优化413.2.1.3 氮源的优化413.2.1.4 氮源比例的确定413.2.1.5 无机盐的优化413.2.1.6 无机盐比例的确定413.2.2 发酵条件的优化41-433.2.2.1 培养方式对CLA产量的影响41-423.2.2.2 培养时间对CLA产量的影响423.2.2.3 LA添加量对CLA产量的影响423.2.2.4 培养温度对CLA产量的影响423.2.2.5 接种量对CLA产量的影响423.2.2.6 pH对CLA产量的影响423.2.2.7 乳化剂对CLA产量的影响42-433.2.2.8 响应面法(RSM)优化菌株发酵条件433.3 结果与分析43-583.3.1 培养基的优化结果43-483.3.1.1 不同培养基的影响43-443.3.1.2 碳源的优化结果44-453.3.1.3 氮源的优化结果45-463.3.1.4 无机盐比