婴儿双歧杆菌完整肽聚糖生产工艺及其抗肿瘤作用机理的研究

发布时间：2018-01-20 20:04

本文关键词： 双歧杆菌完整肽聚糖工业化提取工艺抑瘤机理　出处：《内蒙古农业大学》2009年博士论文　论文类型：学位论文

【摘要】： 本课题研究了婴儿双歧杆菌(Bifidobacterium infantis 6069)完整肽聚糖在工业化生产条件下的提取工艺,并对提取的完整肽聚糖进行了质量及其抗肿瘤作用机理的研究,为其推广应用提供了必要的理论依据。采用发酵工程研究方法,对婴儿双歧杆菌菌株的适宜培养温度、适宜培养基、培养终点、离心条件、冷冻干燥保护剂进行了研究,确定了改良的TPY培养基,婴儿双歧杆菌菌株经培养后发酵液活菌数可达5×109cfu/mL,冻干菌粉活菌数可达2.8×1011～3.0×1011cfu/g,其增菌效果高于高密度发酵法。根据革兰氏阳性菌细胞壁结构可知磷壁酸与细胞壁通透性有很大的关系,因此,首先抽提磷壁酸使细胞膜通透性增加,有利于外界物质更好的作用于细胞和细胞内容物质更容易排出,然后脱掉胞膜上的脂肪类物质,最后用复合酶酶解细胞内的蛋白质类物质。通过试验确定完整肽聚糖提取工艺路线为:10%TCA在70℃搅拌1hr;用乙酸钠、氯仿、甲醇混合液脱脂24hrs;用浓度为2000U的胰酶和碱性蛋白酶共同处理12hrs,最后通过离心清洗去除杂质。针对改良法和Sekine法所提取的完整肽聚糖做了全面的结构和成分分析,结果表明,采用改良法所提取的完整肽聚糖其超微结构完整,氨基己糖含量为20.04 mg/g;蛋白质含量为339.6 mg/g;中性糖含量为500.01 mg/g;总脂含量为36.84 mg/g;磷含量为0.3 mg/g。实验证明,与传统Sekine法相比,改良法所获得完整肽聚糖不仅品质没有劣化,还缩短了提取时间并节约了成本。本试验通过测定脾脏指数、观察ConA促进脾淋巴细胞增殖作用、脾脏巨噬细胞吞噬中性红的能力、检测血清中IFN-γ、IL-12、TNF-α的含量、计算肿瘤生长抑制率以及肿瘤细胞中癌基因Bcl-2和抑癌基因Bax蛋白的表达程度,从而发现,婴儿双歧杆菌完整肽聚糖对试验小鼠的细胞免疫可产生有利于机体免疫功能状态的调节作用;对体外移植性小鼠肝癌H22肿瘤具有明显的抑制生长作用,其抗肿瘤的机制可能是通过增强荷瘤小鼠的免疫功能、抑制肿瘤细胞增殖、诱导肿瘤细胞凋亡而发挥作用。
[Abstract]:The research of Bifidobacterium infantis (Bifidobacterium infantis 6069) whole peptidoglycan extraction process in the industrial production conditions, and the extraction of whole peptidoglycan of quality and its antitumor mechanism, provide necessary theoretical basis for its application.
Using the research methods of fermentation engineering, the suitable culture temperature on infant Bifidobacterium strains, suitable medium, culture conditions of centrifugal, end point, freeze drying protective agent was studied, the improved TPY medium, Bifidobacterium infantis strains cultured in fermentation broth of viable count was 5 * 109cfu/mL, live freeze-dried powder. The number of bacteria is 2.8 * 1011 ~ 3 * 1011cfu/g, the enrichment effect is higher than that of high density fermentation.
According to the structure of the cell wall of gram positive bacteria that teichoic acid and cell wall permeability have a great relationship, therefore, the first extraction of teichoic acid makes the cell membrane permeability increased, is conducive to a better role in the outside cell and content more easily discharged, and take off the fat cell membrane proteins, the final solution materials in the cell by enzymatic. Through the test to determine the whole peptidoglycan extraction process route is: 10%TCA at 70 DEG C, stirring 1hr; using sodium acetate, chloroform, methanol mixture extract 24hrs; concentration of 2000U trypsin and alkaline protease treatment 12hrs, finally through the centrifugal cleaning to remove impurities. Whole peptidoglycan extracted for the improved method and Sekine method to do the analysis of the structure and composition of the comprehensive results show that the improved method of whole peptidoglycan extracted the ultrastructure of hexosamine content of 20.04 mg /g; protein content is 339.6 mg/g; neutral sugar content is 500.01 mg/g; total lipid content is 36.84 mg/g; phosphorus content is 0.3 mg/g.. Experiments show that compared with traditional Sekine method, the improved PPG not only has no deterioration in quality, but also shortens the extraction time and saves costs.
This experiment by measuring the spleen index, observe the promoting effect of ConA lymphocyte proliferation, phagocytosis of macrophages in the spleen, serum IFN- gamma, IL-12, content of TNF- alpha, calculate the inhibition rate of tumor growth and tumor cells in cancer gene Bcl-2 and tumor suppressor gene Bax protein expression level, and found the baby cell immunity whole peptidoglycan of Bifidobacterium in mice can produce beneficial immune function regulation; has an obvious effect on inhibiting the growth of in vitro transplanted hepatoma H22 tumor, the anti-tumor mechanism may be through enhancing the immune function of tumor bearing mice, inhibit tumor cell proliferation and induce the apoptosis of tumor cells and play a role.

【学位授予单位】：内蒙古农业大学
【学位级别】：博士
【学位授予年份】：2009
【分类号】：R371;TQ464

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