盐胁迫下乳酸菌的高密度培养及冻干保护的研究.pdf

国内图书分类号 TS252 54 学校代码 10213 国际图书分类号 663 密级 公开 工学硕士学位论文 工学硕士学位论文 盐胁迫下乳酸菌的高密度培养及冻干保护 的研究 硕 士 研 究 生 王璐 导师 张兰威 教授 申请学位级别 工学硕士 学 科 专 业 食品科学与工程 所 在 单 位 食品科学与工程学院 答 辩 日 期 2010 年 6 月 26 日 授予学位单位 哈尔滨工业大学 Classified Index TS252 54 U D C 663 Dissertation for the Master Degree in Engineering RESEARCH ON HIGH CELL DENSITY CULTURE OF LACTIC ACID BACTERIA UNDER SALT STRESS AND ACTIVITY PROTECTION DURING FREEZE DRYING PROCESS Candidate Supervisor Academic Degree Applied for Specialty Affiliation Date of Defence Degree Conferring Institutio Wang Lu Prof Zhang Lanwei Master of Engineering Food Science School of Food Science and Engineering June 26th 2010 Harbin Institute of Technology 哈尔滨工业大学工学硕士学位论文 I 摘 要 摘 要 近年来 我国乳制品工业迅猛发展 尤其是酸奶发酵剂的生产 为了提高国产 酸奶发酵剂的市场竞争力 就需要开发出高活性 高品质的产品来满足日益增长的 消费需求 嗜热链球菌及保加利亚乳杆菌是生产酸奶发酵剂的主要菌种 本研究从 几个关键点入手 包括培养过程中乳酸中和剂的选择 乳酸菌耐盐驯化 乳酸菌营 养需求以及乳酸菌冻干损伤等 为高活性酸奶发酵剂的生产提供一定的理论基础 本研究分别对嗜热链球菌St sp1 1 及保加利亚乳杆菌Lb 34 5 在脱脂乳培养基与 配制培养基中的生长进行对比 确定两种菌均在 12 脱脂乳中生长状态良好 最大 活菌数分别为在配制培养基中的 2 8 倍及 2 6 倍 以脱脂乳为基础培养基 利用发 酵罐对乳酸菌的最佳乳酸中和剂进行选择 结果表明 对于两种菌使用 20 的 Na2CO3作为中和剂 其所能达到的活菌数最高 且最大比生长速率最大 实验证明 乳酸盐胁迫对菌体生长有很大的抑制作用 驯化乳酸菌对盐的耐受性可以进一步提 高菌体密度 球菌活菌数由 4 85 109 cfu mL提高到 5 83 109 cfu mL 杆菌活菌数由 1 28 109 cfu mL提高到 1 75 109 cfu mL 对增菌物质进行选择 确定腺嘌呤 甲酸钠 L 半胱氨酸盐酸盐有明显增菌作 用 50 mol L 腺嘌呤对球菌及杆菌促生长作用明显 5 mmol L 甲酸钠对球菌促生 长作用明显 10 mmol L 甲酸钠对杆菌促生长作用明显 50 mg L L 半胱氨酸盐酸盐 对球菌 杆菌有促生长作用 当浓度达 250 mg L 时对两种菌生长均有抑制作用 用高效液相色谱法测定几种有增菌作用的复合成分中嘌呤物质的含量 结果表明 啤酒 麦芽汁 酵母提取物中均含有一定浓度的腺嘌呤 而蛋白胨 牛肉粉在实验 条件下没有检测到 补充腺嘌呤后活菌数进一步提高 说明这些复合物除了能提供 碳源 氮源等物质外 其中含有的腺嘌呤可以更好的促进菌体细胞生长 本研究还对乳酸菌冻干过程中的损伤进行了初步研究 明确了 10 海藻糖与 1 甘氨酸组合作为冻干保护剂可以明显提高冷冻及冻干存活率 减少冷冻及干燥过 程中细胞膜上关键酶的损失 对维持细胞膜的完整性有一定作用 关键词 乳酸菌 中和剂 盐胁迫 增菌物质 冻干损伤保护 哈尔滨工业大学工学硕士学位论文 II Abstract In recent years there is a rapid development of China s dairy industry especially in the production of yoghurt starter cultures In order to improve the competitive of domestic yoghurt starter cultures we need to develop highly active high quality products to meet growing consumer s demands Streptococcus thermophilus and Lactobacillus bulgaricus are major strains to the production of yoghurt starter This paper study several key points including the selection of lactic acid neutralizer during culture process acclimate lactic acid bacteria tolerant sodium lactate the nutritional needs and freeze dried injury of lactic acid bacteria provides a theoretical basis for the production of the high activity yoghurt starter cultures In this study we compared the grows of Streptococcus thermophilus St sp1 1 and Lactobacillus bulgaricus Lb 34 5 in skim milk medium and synthetic medium respectively and determined 12 skim milk is the better condition for all the two strains the maximum viable count were 2 8 times and 2 6 times respectively compared with that in the synthesis medium Use skim milk as the based medium selected the best lactic acid neutralizer by fermentor the results showed that use 20 Na CO as 23 neutralizer the two bacteria can reach the highest viable cells and maximum specific growth rate Experiments also show that sodium lactate has a great negative impact on cell growth acclimated lactic acid bacteria to Sodium lactate tolerance can further enhanced cell density the viable count of St sp1 1 from 4 85 10 cfu mL increased to 5 83 10 cfu mL 9 9 Lb 34 5 from 1 28 10 cfu mL increased to 1 75 10 cfu mL 9 9 Choosed the stimulants for these two strains and determined adenine sodium formate L cysteine hydrochloride significantly increased the viable count 50 mol L adenine promoted growth significantly for both St sp1 1 and Lb 34 5 5 mmol L sodium formate obvious promoted growth of St sp1 1 10 mmol L sodium formate obvious promoted growth of Lb 34 5 50 mg L L cysteine hydrochloride promoted growth significantly for both St sp1 1 and Lb 34 5 when the concentration up to 250 mg L the growth of both strains were inhibited Use HPLC determined purine content of several materials which can promote growth of these two strains the results showed beer wort yeast extracts contain a certain concentration of adenine while the peptone beef powder were not detected in the experimental conditions and under the supplemented of adenine the viable count was further increased indicating that these compounds can not only 哈尔滨工业大学工学硕士学位论文 III provid carbon nitrogen and other substances but also the contain of adenine can better promote cell growth In this study we also preliminary study the damage of lactic acid bacteria during freeze dried cleared that 10 trehalose combination with 1 glycine as protectants can significantly improve the survival rate of frozen and freeze drying reduce the loss of key enzymes in cell membranes and have a role in maintaining the cell membrane integrity Key words lactic acid bacteria lactic acid neutralizer sodium lactate stress stimulants protection of freeze drying injury 哈尔滨工业大学工学硕士学位论文 I 目 录 摘 要 I Abstract II 第 1 章 绪论 1 1 1 立题背景 1 1 2 高活性乳酸菌发酵剂的概述 1 1 2 1 乳酸菌的种类及特性 1 1 2 2 乳酸菌发酵剂的分类 2 1 3 国内外研究现状 4 1 3 1 发酵剂产业的国内外现状 4 1 3 2 乳酸菌的高密度培养 5 1 3 3 乳酸菌的。