Synergistic Effect Of Konjac Mannose Oligosaccharides And Bacillus Subtilis On Intestinal Epithelial Cell Injury

In animal production,a variety of intestinal diseases often accompany,which have brought huge economic losses to the animal husbandry.Intestinal tract is the largest bacterial library and endotoxin library of the body,and intestinal epithelial barrier plays an important role in maintaining the permeability of epithelial cells and the steady state of the environment in the body.As the first line of defense to fight against intestinal bacteria,the intestinal epithelial cells are exposure to bacteria or LPS.LPS or the pathogen invasion can mediate intestinal epithelial cell damage,which lead to the occurrence of a variety of diseases.Prebiotics and probiotics can improve the intestinal microstructure of the gastrointestinal tract and the body's immunity,inhibit the adhesion of pathogens.Probiotic will be one of the most promising alternatives of antibiotic in the breeding industry.However,there are many bottlenecks in the research and application of probiotics,especially how to make probiotics planting rapidly,growing and maintaining a rich concentration in the body.Once these problems are solved,probiotics will play a stable role.The konjac mannan oligosaccharide is a kind of functional oligosaccharides extracted from konjac,which has the function of regulating immune defense and prebiotics,may promote the growth and reproduction of probiotics,enhance the effect of probiotics.In view of the problems in the use of probiotics alone and the good prenatal characteristics and prospects of konjac mannan oligosaccharides,in this study,we used LPS to construct the model of intestinal epithelial cell injury,selected probiotics that could use konjac mannose oligosaccharides and investigated the effects of exogenous addition of konjac mannose oligosaccharides,probiotics and their oligosaccharides on the repair of intestinal epithelial cells.The main research contents are as follows:1.Culture and Identification of Probiotics: The four strains were cultured and purified,then identified by physiological and biochemical identification and Gram stain,spore staining.The results showed that all four strains were Gram-positive bacteria,three contained spores,and their physiological and biochemical characteristics were consistent with Bacillus licheniformis,Enterococcus faecium,Bacillus megaterium and Bacillus iv subtilis.The genomic DNA of each strain was extracted and amplificated by 16 sr RNA PCR.The results showed that the sequence of the tested strain was the highest with the above strain.Based on the above results,the four strains were Bacillus licheniformis,Enterococcus faecium,Bacillus megaterium and Bacillus subtilis.2.Filter the probiotics using konjac mannose oligosaccharides: The konjac mannose oligosaccharides of three different concentrations(1 g/L,1.5 g/L and 2 g/L)were mixed with each strain.The effect of konjac mannan oligosaccharides on the growth of probiotics was detected by ultraviolet spectrophotometer to screen probiotics strains that utilize oligosaccharides.The results showed that konjac mannose oligosaccharides had a significant effect on the growth of Bacillus subtilis compared with the control group,and the optimum concentration of konjac mannan oligosaccharides was 2 g/L,and the best time was 24 h.3.Construction of intestinal epithelial cell injury model: The expression of IL-1?,IL-6 and TNF-? was detected by q PCR in LPS-stimulated Caco-2 cells.The results showed that the expression of IL-1?,IL-6 and TNF-? was significantly up-regulated compared with the control group,when LPS concentration is 1?g / m L and the time is 6h,the increase is the most significant.Furthermore,the activity of Caco-2 cells was detected by MTT assay.The results showed that the activity of Caco-2 cells in 1 ?g/m L LPS group was significantly lower than that in the control group.The impedance of Caco-2 cells was detected by real-time cell analyzer.Compared with the control group,the impedance value of LPS treatment group was significantly decreased,indicating that LPS can increase permeability of Caco-2 cells.The expression of ZO-1 and Claudin-1 was detected by Western blot.The results showed that the expression of ZO-1 and Claudin-1 was down-regulated,Indicating that LPS also caused the epithelial cells tight connection injury.The results showed that 1 ?g/m L LPS stimulated Caco-2 cells for 6 h,can successfully build intestinal epithelial cell damage model.4.Repair and synergistic effect of konjac mannose oligosaccharides and probiotics on intestinal epithelial cell injury: Add different concentrations of konjac manna oligosaccharides,probiotics and their complexes to injured intestinal epithelial cells,MTT assay was used to detect cell viability,the expression of IL-6,TNF-?,IL-1?,MUC-2,Claudin-1,ZO-1 m RNA was detected by q PCR,the expression of ZO-1 protein was detected by Western blot.The results showed that the activity of Caco-2 cells was significantly up-regulated compared with LPS injury group,oligosaccharide addition group,probiotics addition group,oligosaccharide combined with bacteria group,the expression of ZO-1,Claudin-1 and MUC-2 m RNA was significantly up-regulated,and the expression of ZO-1 protein was up-regulated.Compared with probiotics,the expression of Claudin-1,ZO-1 and MUC-2 m RNA was significantly up-regulated in oligosaccharide probiotics group;compared with the oligosaccharide addition group,the activity of Caco-2 cells was significantly up-regulated by oligosaccharide probiotics.Indicating that probiotics,oligosaccharides and their combined action can repair LPS-induced Caco-2 cell damage,and the synergistic effect of oligosaccharides and probiotics is more significant on certain indicators than the use of probiotics and oligosaccharides alone.