Study On Optimum Extraction Process And Antitumor Mechanisms Of Bifidobacterium Infantis Whole Peptidoglycan

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.