【作者】 张中义；

【导师】 孙君社；

【作者基本信息】 中国农业大学 ， 农产品加工及贮藏工程， 2005， 博士

【摘要】 共轭亚油酸（CLA）有很好的抗癌、减肥、抗动脉硬化等生理活性。目前,CLA工业化生产是由化学异构法制得,其产物中异构体组成复杂,不利于在食品和医药方面使用。亚油酸生物异构化生成CLA,产物中异构体组成单一,乳酸菌异构化亚油酸生成CLA有很好的应用前景。 本课题对Lactobacillus plantarum转化亚油酸生成CLA过程进行了研究,内容包括菌种分离、筛选、鉴定,菌种诱变选育,CLA发酵过程动力学,洗涤细胞转化亚油酸生成CLA,亚油酸异构酶分离纯化,酶学性质研究和提高发酵牛乳CLA含量实验研究。论文主要内容如下: 1) 从酸菜中分离出一株较高CLA产量的乳酸菌L₂₉,鉴定为植物乳杆菌（Lactobacillusplanmrum置信度96.1%）。 2) 以L.plantarum L₂₉为出发菌株,通过紫外线照射和硫酸二乙酯（DES）诱变处理,选育出突变株L.plantarum LT2-6,CLA产量提高18.6%。 3) 对L.planmrum LT2-6在5 L罐规模的分批发酵过程进行了实验分析研究,获得了分批发酵过程动力学曲线,建立了分批发酵过程菌体生长、产物CLA生成和底物（葡萄糖）代谢数学模型。 4) Lplanmrum LT2-6洗涤细胞转化亚油酸生成CLA优化条件为:30℃,pH 7.0,亚油酸浓度1.5%（w/v）,细胞浓度20%（w/v）。反应64 h,CLA产量达到最大（7.86g/L）,亚油酸转化率为52.4%。经脂肪酸萃取和甲酯化,气相色谱检测显示,土要产物为cis-,trans-9,11.CLA。 5) L.plantarum LT2-6亚油酸异构酶分子量约为48.8 KDa,最适pH 6.0,最适温度30℃,不耐热,温度高于50℃时,稳定性下降很快。金属离子对此酶活力影响顺序为:Mg²⁺>Ca²⁺>Mn²⁺>Fe²⁺>Zn²⁺>Na⁺。酶动力学参数为:K_m=12.46 μmol/L,V_m=1.933×10^-1/μmol/h.mg。 6) 以L.plantarum LT2-6为牛乳发酵剂,转化亚油酸生成CLA的适宜温度为37℃。牛乳中添加葡萄糖和小分子氮源,可促进Lplantarum LT2-6生长和提高CLA产量。牛乳中亚油酸添加量超过0.075%时,对菌体生长和CLA生成均产生抑制。菌体生长对数期末尾和稳定期,CLA生成活跃。发酵24 h,CLA产量最大,生成的产物主要为cis-,trans-9,11-CLA。牛乳中加入0.075%的亚油酸,37℃,发酵24 h,亚油酸转化率为50%。脱脂牛乳添加亚油酸经L.plantarum LT2-6发酵后,CLA含量比普通酸乳提高5～6倍。更多还原

【Abstract】 Conjugated linoleic acid (CLA) has attracted much attention as a novel type of biologically beneficial functional lipid. For example, CLA reduces carcinogenesis, atherosclerosis, and body fat. Presently, CLA is produced through chemical isomerization of linoleic acid (LA), but by-productions of unexpected isomers are produced, which is not beneficial for the use of CLA in food and medicine. However, CLA isomers are much simpler through biology isomerization of LA than through chemical. And it will bring a bright promise for application of CLA, which is isomerized by lactic acid bacteria.The process of CLA production by Lactobacillus plantarum was studied. The research included bacteria isolation, screening, identification and induced mutation, also fermentation dynamics, CLA production from washed cells, LA isomerase purification and its properties were studied, in the end the CLA concentration in milk was increased by fermentation. Results as the follows:1) Lactic acid bacteria L29, with a high ability to produce CLA, was isolated from pickle. L29 was identified as a strain ofLactobacillus plantarum (confidence level 96.1%) .2) Lplantarum L29 was induced by ultraviolet radiation and DES, then a mutation strain LT2-6 was screened out, by which CLA production could increase 18.6 %.3) Fermentation dynamics curve was obtained by the study of the Lplantarum LT2-6 fermentation in batches in 5 L fermentor. A mathematics model was established based on the cells growth of L. plantarum LT2-6, production CLA accumulation and glucose metabolism.4) Washed cells of Lplantarum LT2-6 as the catalyst, the optimal reaction condition for CLA production was: temperature 30℃, pH 7.0, LA concentration 1.5% (w/v) , cells concentration 20%(w/v). After reacted for 64 h, CLA concentration was up to the maximal (7.86 g/L), and 52.4 % of LA was conversed to CLA. Then fatty acid was extracted, methylated and determined by gas chromatography analysis, the majority of resultants was cis-,trans-9,ll-CLA.5) Molecular weight of the LA isomerase of Lplantarum LT2-6 was about 48.8 KDa. The optimal reaction condition for the LA isomerase was pH 6.0, 30℃. This enzyme was non-resistant to heat relatively, if temperature was up to 50℃, stabilization ability of the enzyme declined soon. Kinds of metal ion had different effects on the enzyme activity, lowering sequence was Mg2+ > Ca2+ > Mn2+ > Fe2+ > Zn2+ > Na+. Km and V,,,, dynamics parameters of LA isomerase, were respectively 12.46 mol/L, 1.933x10-1moVh.mg.6) In the process of fermentation of skim milk by Lplantarum LT2-6, 37℃ was suitable for fermentation. The growth of cells was accelerated significantly supplementing with glucose in milk, so production of CLA was increased. Also small molecular nitrogen source materials supplemented in milk had the same effects on CLA production. However, when LA concentration supplemented in milk was more than 0.075 %, the growth of cells and CLA production were restrained. Production of CLA was active during the end of log phase and stationary phase. Fermentation for 24 h, CLA production was themaximum. Results showed that the majority of CLA in resultants was cis-,trans-9,ll-CLA. 0.075 % of LA supplemented in milk, and 50% of LA was bio-conversed into CLA in 37℃, fermentation for 24 h. After fermentation by Lplantarum LT2-6, CLA production was higher 5-6 times with skim milk supplemented LA than with market yoghourt without LA.更多还原