耐氧益生菌高密度培养的研究

论文总字数：25996字

摘 要

双歧杆菌属作为益生菌越来越多的出现在公共的视野中，大量的研究指出双歧杆菌具有调节人体肠道微生物种群、预防癌症、调节人体免疫力等多种益生作用，现代食品工业和医疗行业对双歧杆菌的需求度也逐渐增加。因而，如何大量的获取活性双歧杆菌成为了微生物产业的研究热点之一。本文以长双歧杆菌菌株为菌种，研究了长双歧杆菌在锥形瓶和发酵罐中分批培养情况，在此基础上摸索了长双歧杆菌在锥形瓶和发酵罐中补料高密度培养的补料条件和菌体生长情况。本实验主要内容和结论有：

从双歧杆菌乳杆菌三联活菌片活化分离得到长双歧杆菌菌株，并经过专业机构鉴定为纯菌株。获得纯菌株后进行锥形瓶分批培养预实验，确定5%为最佳接种量，进行后续的分批培养和补料高密度发酵。

以长双歧杆菌为菌种，接种到500ml锥形瓶和5L发酵罐中分批培养，隔2h取样测活菌数量和发酵液pH值以及OD值，并绘制成生长曲线和发酵过程曲线，得出长双歧杆菌在分批培养条件下的生长情况。总的来说，两者的发酵初期也就是延滞期大约为4h，对数生长期都在4h到12h之间，发酵罐略有提前，大概提前2h。到18h时，菌体生长处于稳定期，之后开始有衰减迹象。分批培养条件下，发酵罐中菌体生长情况比锥形瓶要稍好些，最大的活菌浓度也更高一点。

在分批培养的基础上，确定了pH值下降和葡萄糖消耗是限制菌体生长的主要因素。在前面的分批培养的研究中，确定pH值在6.0-6.5时菌体生长最快，补料方案是以1mol·L-1浓氨水为碱料，及时补充到发酵液，使得整个发酵过程的pH值维持在6.0-6.5之间。同时在发酵进行到对数生长末期时补充10%的与原培养基相同浓度的葡萄糖溶液。总体来说，补料高密度培养相较于分批培养，延长了发酵过程中的对数生长期和平稳期，获得了更高的最大活菌浓度。

关键词：长双歧杆菌；分离纯化；分批培养；补料高密度培养

RESEARCH ON HIGH-DENSITY CULTIVATION OF BIFIDOBACTERIUM LONGUM

41111103 Jiqing Huang

Instructor: Xudong Xu

Abstract

Bifidobacteria probiotic appearing in the public field of vision, a lot of researches indicate that Bifidobacteria can prevent human intestinal microbial populations, cancer, regulating the human immune system and so on. The demands of the modern food industry and the medical industry for Bifidobacteria gradually are increasing. Therefore, how to obtain a large number of active Bifidobacteria become one of the hot research points in microbes industry. In this paper, Bifidobacterium longum is studied in flasks and fermentors case for batch culture. On the basis of Bifidobacterium longum batch culture, I still study the fed high-density culture conditions and cell growth. In this study, the main contents and conclusions as follows:

Bifidobacterium longum strain in this paper is isolated from triple viable Bifidobacterium Lactobacillus sheet activation and identified by professional organizations. Using pure strains do the pre-batch culture experiments to determine the optimum5% inoculum , for subsequent fed-batch culture and high density fermentation.

Bifidobacterium longum Strain is inoculated into 500ml flasks and 5L fermenters for batch cultures, every 2h sampling and measuring the number of viable broth pH value and OD values and plotted growth curve and fermentation process curve, for growth conditions of Bifidobacterium longum in batch culture. Overall, the both initial fermentation lag are about 4h, and the logarithmic growth phase are between 4h to 12h, fermentation tank slightly ahead, probably ahead of 2h. When 18h, cell growth is in a stable phase. Under batch culture conditions, cell growth fermenter is slightly better than the case of flasks, and maximum viable cell concentration is higher.

The decrease of pH value and glucose consumption is limiting the growth of bacteria. In previous studies, cell growth is fastest in pH value 6.0-6.5, and the feeding program is based on 1mol·L-1 concentration of ammonia, so that the pH value is maintained between 6.0-6.5 in the fermentation process. At the same time, in the late logarithmic growth of fermentation, 10% glucose solution is supplemented into the medium. Overall, comparing with fed-batch culture, high-density culture fermentation process extend the logarithmic growth phase and stationary phase, and obtain a higher maximum concentration of viable cells.

KEY WORDS: Bifidobacterium longum; separation and purification; batch culture; fed high density culture

目 录

摘要 I

目录 III

第一章 绪论 1

1.1 双歧杆菌属概述 1

1.1.1 双歧杆菌属生物学特性 1

1.1.2 不同生态环境中的种群 2

1.1.3 双歧杆菌对人体的作用 2

1.1.4 双歧杆菌在工业上应用........................................................................................2

1.2 双歧杆菌高密度培养的研究进展 3

1.2.1 双歧杆菌的培养特性 3

1.2.2 高密度培养方法介绍 4

1.3 本课题研究目的、意义及内容 4

第二章 实验材料与实验方法 5

2.1 实验材料 5

2.1.1 长双歧杆菌菌种 5

2.1.2 主要试剂 5

2.1.3 培养基 5

2.1.4 主要仪器 6

2.2 实验方法 6

2.2.1 菌种准备 7

2.2.2 培养方法 8

2.2.3 测定方法 8

第三章 实验结果与讨论 9

3.1 菌种的分离与鉴定 9

3.1.1 菌种的分离 9

3.1.2 菌种的鉴定 9

3.2 确定分批培养接种量 10

3.3 测定分批培养生长曲线和发酵过程曲线 11

3.3.1 分批培养生长曲线 11

3.3.2 分批培养PH值曲线 11

3.3.3 锥形瓶分批培养发酵过程曲线 12

3.3.4 发酵罐分批培养发酵过程曲线 13

3.4 测定补料高密度培养生长曲线和发酵过程曲线 13

3.4.1 确定补料培养方案 13

3.4.2 补料高密度培养生长曲线 15

3.4.3 补料高密度培养PH值曲线 16

3.4.4 锥形瓶补料高密度培养发酵过程曲线 17

3.4.5 发酵罐补料高密度培养发酵过程曲线 18

第四章 总结与展望 19

参考文献（References） 20

致谢 22

第一章 绪 论

1.1 双歧杆菌属概述

1.1.1 双歧杆菌属生物学特性

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