Studies On Extracellular Polysaccharide Of Lactobacillus Casei And Inhibition The Growth Of Gastric Cancer Cell

Lactobacillus is probiotic in the human body at present.The function of it are that it can adjust the normal micro flora of the gastrointestinal tract,keep the balance of microflora,improve the digestibility of food,control the production of endotoxins,decrease the level of serum cholesterol,produce the substance of nutrients,inhibit the growth and reproduction of spoilage bacteria in the gut and stimulate the growth of tissue as well as other features.So that it can act on the level of nutrition,the function of physiological,the infection of cells,the effects of drug,the response of immune,the reaction of toxicity and reduce probability of the risk of tumors.There was an isolated strain from kimchi in the laboratory.The isolated strain was identified by the methods with observation of morphological,comparison the characteristics with physiological and biochemical and analysis 16S rDNA sequence.Through the results of single factor experiments,it was determined the optimization of culture conditions and media component to produce extracellular polysaccharide of the strain.It was purified by the chromatography of cellulose gel,and the result of purified was verified by the method with polyacrylamide gelelectrophoresis.The method of the studies of extracellular polysaccharides on the effects of gastric cancer cells were through MTT assay,Giemsa staining,DNA fragmentation analysis and protein changed.The results were as follows:1)Isolation and identification of the strain.It was observed that the diameter of strain was 18 mm,the surface of shape was raised and smooth.The color of colony turned from ivory white to pale yellow.After the colony was cultured in 24 hours,it began to appear the bubble circle,and gradually became much bigger in the MRS selective culture.The results of glycolysis experiment,salt tolerance experiment,and hydrogen sulfide experiment matched with the characteristics of physiological and biochemical of Lactobacillus in Bergey's Manual of Systematic Bacteriology.The sequence of 16S rDNA of the strain was analyzed,the result showed that the similarity with L.casei W56 was 100%.Therefore,the strain belongs to L.casei,so it was named as L.casei 20130913.Meanwhile,the serial number of the gene sequence of the strain was No.KJ598784.2)Optimization of extracellular polysaccharides condition.The orthogonal experiments including the factors of pH,inoculation,culture temperature and time were studied so as to confirm the optimization condition.Results suggested that extracellular polysaccharides conditions were temperature 37 ~oC,time 14 h,pH 6.5,inoculation 3%,the highest yield of extracellular polysaccharide was 248?g/mL.The orthogonal experiments including the factors of carbon,nitrogen,ratio of carbon and nitrogen,content of phosphate were studied so as to confirm the optimization condition.The results suggested that extracellular polysaccharides conditions were glucose as carbon source and peptone as nitrogen source.Ratio of carbon and nitrogen was 2?1.The content of phosphate was 6%.The highest yield of extracellular polysaccharide was 556?g/mL.3)Isolation and purification of extracellular polysaccharides.The crude polysaccharides were obtained by the methods with cells removed,protein precipitated and inorganic salts dialyed.The polysaccharides were purified by the method with DEAE-D52 cellulose gel chromatography.The components collected were verified by the method with polyacrylamide gel electrophoresis.The result showed a single band.4)Evaluation of biological activities for polysaccharides.The result of polysaccharide antioxidant experiment was that when concentration of purified extracellular polysaccharide was 200?g/mL,the inhibition radio of DPPH·radical was 40.89%.The radical of·OH was 37.84%.The result of MTT assay showed that polysaccharides had the effect on the inhibition of gastric cancer cells(823)in vitro.The inhibition efficiency of gastric cancer cell was 45.67%,when concentration of polysaccharide was 200?g/mL.Throughout observation of cells,it was shrinkage and turned round,there was vacuole around the cells after 6 hours in dosing.The nuclear concentrated after 12 hours.The nuclear envelope nucleolus was broken and contents were spilled after 24 hours.The DNA fragment size of 200 bp appeared after 6 hours.At this time the protein of 17 KD was appeared.In summary,the isolated strain from kimchi in this laboratory was identified which belonged to L.casei,which was named as L.casi 20130913.The production of extracellular polysaccharide was improved through optimization the conditions.It was showed capacity of antioxidant and inhibition of gastric cancer cells.This will provide evidence for representative of new products as probiotics.