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
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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。
The industrial extracting procedure of whole peptidoglycan in Bifidobacterium Infantis was studied in this study. Moreover, the quality of the whole peptidoglycan extracted and its antitumor mechanism were also been discussed, which may provide necessary theoretical basis on its application.
Applying fermentation engineering approach, we have determined the optimum conditions for culturing the selected colony of bifidobacterium infantis, including temperature, media, and length of incubation. The centrifugation setting and lyophilization protectant were also optimized. By utilizing the modified TPY medium, we have achieved viable count at 5×109cfu/mL of culture medium, and more than 2.8×1011～3.0×1011cfu/mL viable count per gram of lyophilized product. The effect of enriching is higher than that of high cell density fermentation.
According to the structural property of the Gramm-positive bacteria, that the inserted teichoic acid in between the peptidoglycan layers plays an important role in cell wall permeability, we hypothesized that depletion of teichoic acid will increase cellular permeability to facilitate entrance of applied extracellular contents and expelling of intracellular wastes. This will be followed by stripping off the attached fatty matter in the cell wall and digestion of intracellular proteins with polypeptidases. With repeated experiments, a finalized extraction process for the whole peptidoglycan has been determined: Treatment with 10% TCA at 70℃with stirring for 1 hr to depleting teichoic acid; Treatment with a mixture of acetic acid、chloroform、methanol for 24 hrs to strip off fat; Treatment with2000U trypsin and alkaline protease for 12 hrs to digest intracellular protein; Centrifugation to get rid of debris. The whole peptidoglycan extracted by Sekine method and improved method have been done a comprehensive analysis of the structure and composition.
Systemic structure and composition study of extracted whole peptidoglycan. We have determined that the bifidobacterium infantis whole peptidoglycan product contains 20.04 mg/g hexosamine, 500.01 mg/g total sugar, 36.84 mg/g total fatty acid, 339.6 mg/g protein, and 0.3 mg/g phosphate. Experiments showed that compared with Sekine method and improved method, the quality of whole peptidoglucan obtained was not degradation. Moreover, it also shortten the extracted time and saved the expense.
Antitumor studies employing a mice H22 liver carcinoma transplant model. We have analyzed the spleen index, ConA-induced spleen lymphocyte proliferation, phagocytation of Neutral Red by spleen macrophages, serum IFN-γ, IL-12 and TNF-αconcentration, inhibition rate of tumor growth, as well as expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in the cancer cells. We concluded that bifidobacterium infantis whole peptidoglycan has an antitumor effect on H22 liver carcinoma in the experimental mice. The mechanisms may involve increased cellular immunity against the cancer implant, inhibition of tumor cell proliferation, and induction of tumor cell apoptosis.
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。
The industrial extracting procedure of whole peptidoglycan in Bifidobacterium Infantis was studied in this study. Moreover, the quality of the whole peptidoglycan extracted and its antitumor mechanism were also been discussed, which may provide necessary theoretical basis on its application.
Applying fermentation engineering approach, we have determined the optimum conditions for culturing the selected colony of bifidobacterium infantis, including temperature, media, and length of incubation. The centrifugation setting and lyophilization protectant were also optimized. By utilizing the modified TPY medium, we have achieved viable count at 5×109cfu/mL of culture medium, and more than 2.8×1011～3.0×1011cfu/mL viable count per gram of lyophilized product. The effect of enriching is higher than that of high cell density fermentation.
According to the structural property of the Gramm-positive bacteria, that the inserted teichoic acid in between the peptidoglycan layers plays an important role in cell wall permeability, we hypothesized that depletion of teichoic acid will increase cellular permeability to facilitate entrance of applied extracellular contents and expelling of intracellular wastes. This will be followed by stripping off the attached fatty matter in the cell wall and digestion of intracellular proteins with polypeptidases. With repeated experiments, a finalized extraction process for the whole peptidoglycan has been determined: Treatment with 10% TCA at 70℃with stirring for 1 hr to depleting teichoic acid; Treatment with a mixture of acetic acid、chloroform、methanol for 24 hrs to strip off fat; Treatment with2000U trypsin and alkaline protease for 12 hrs to digest intracellular protein; Centrifugation to get rid of debris. The whole peptidoglycan extracted by Sekine method and improved method have been done a comprehensive analysis of the structure and composition.
Systemic structure and composition study of extracted whole peptidoglycan. We have determined that the bifidobacterium infantis whole peptidoglycan product contains 20.04 mg/g hexosamine, 500.01 mg/g total sugar, 36.84 mg/g total fatty acid, 339.6 mg/g protein, and 0.3 mg/g phosphate. Experiments showed that compared with Sekine method and improved method, the quality of whole peptidoglucan obtained was not degradation. Moreover, it also shortten the extracted time and saved the expense.
Antitumor studies employing a mice H22 liver carcinoma transplant model. We have analyzed the spleen index, ConA-induced spleen lymphocyte proliferation, phagocytation of Neutral Red by spleen macrophages, serum IFN-γ, IL-12 and TNF-αconcentration, inhibition rate of tumor growth, as well as expression of anti-apoptotic protein Bcl-2 and pro-apoptotic protein Bax in the cancer cells. We concluded that bifidobacterium infantis whole peptidoglycan has an antitumor effect on H22 liver carcinoma in the experimental mice. The mechanisms may involve increased cellular immunity against the cancer implant, inhibition of tumor cell proliferation, and induction of tumor cell apoptosis.
引文
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