The Oxygen Domestication And High Cell Density Cultivation Of Bifidobacterium Isolated From Guizhou Xiang Pigs

Bfidobacteria have been recognized as one of dominant probiotic microbiota because of they have many physiologic functions such as assimilation of cholesterol,improvement of the intestinal microbial balance of the host,and immunomodulatory effects.In our study,a Bifidobacterium was isolated from the intestinal soluble content of Guizhou Xiang pigs which was identified and named as Bifidobacterium animalis subsp. lactis BZ11. Most of its physiological features include the oxygen-, acid- and bile salts-resistance, the rate of degrade cholesterol, the ability to antibiotic pathogenic bacterium and adhesion to Caco-2 cells were evaluated.Three ways were taken to oxygen domesticate the strain isolated.Several different kind of batch fermentation and fed-batch fermentation methods were used and compared,and finally determine an efficient way for high cell density cultivation.There are the following main conclusions:(1) The maximum cell concentration of B.animalis subsp. lactis BZ11 can reach3.80×108 cfu/mL after cultivating 24 h in aerobic conditions,the results showed the stains itself has a good ability to resistant oxygen. The survival rate of the strain was(98.65±0.11)% after cultivating 2 h under the condition of pH3,and the survival rate can also over 80% when cultivating it in the medium with 0.3% bile salts for 24 h,it was significant higher than B.animalis subsp.lactis BZ25 and Golden Bifidobacterium,which showed B.animalis subsp.lactis BZ11 has a good resistance to acid and bile salts.The adhesion rate to Caco-2 cells was(6.66±0.32) cfu/cell.The cholesterol assimilation rate of the strain in vitro was(27.10±0.92)%.B.animalis subsp.lactis BZ11 was sensitive to most kinds of antibiotic.The fermentation broth of the strains can inhabit pathogenic stains including Escherichacoli,Staphyloccus,Salmonella,Proteusmirabilis,Enterobacter sakazakii,Bacillus cereus Frankland in varying degrees.These excellent features,making strain B.animalis subsp.lactis BZ11 has the potential to be used as probiotics.(2) To improve the aerotolerant capacity of B.animalis subsp.lactis BZ11 in aerobic environment,three ways including gradual increase of the partial pressure of oxygen in medium, alternate incubation between aerobic and anaerobic conditions and chemical mutagenesis were taken.After analyzing the growth characteristics of the strain pre- and post- domestication, the first method obtained a better effect and the cell concentration of the domesticated strain can reach 6.30×108 cfu/mL after cultivating 20 h in aerobic conditions, which was 2.24 times the concentration of the original strain.Moreover,the domesticated B.animalis subsp.lactis BZ11 kept the similar characteristics of physiological and morphological to the original strain by identification. So it can be used as a potential probiotics for further in depth development and utilization.(3) The optimum medium contain carrot juice and cabbage juice combine with different kinds of batch and fed-batch fermentation ways were taken to cultivate the strain B.animalis subsp.lactis BZ11 after oxygen domestication in 5 L fermentor.The maximum cell concentration of living bacteria was obtained in the cross-filtration combine with fed-batch systems after cultivating 72 h, which was 2.29×1010 cfu/mL, the cell concentration of the strain can meet people's requirements when add it to foods or drugs.