| Since the concept of"probiotics"has been proposed,people have become more and more concerned about various probiotic functions such as"lowering blood sugar,lowering blood pressure,preventing diarrhea and obesity".When entering the human gastrointestinal tract,probiotics encounter a series of adverse conditions:such as the gastric acid environment in the stomach,the bile salt environment in the gastrointestinal tract and the duodenum.These adverse conditions may reduce the physiological activity of probiotics and even directly lead to the death of some probiotics,so that they can not play their role well in the human body.Lactobacillus plantarum NCU116 is an excellent probiotic strain isolated from Sichuan traditional sauerkraut,which has the function of lowering blood sugar,regulating intestinal microflora and alleviating constipation.In order to study the adaption mechanism of L.plantarum NCU116 to bile salt and provide a basis for its probiotic characteristics to some extent,changes of associated gene transcription level and key physiological indicators of NCU116 regulated by bile salt were analysed.The main research contents and conclusions are as follows.(1)Growth status of different lactic acid bacteria strains after bile salt stress.There are 4 strains of lactic acid bacteria isolated from the southwestern region of sauerkraut,including Lactobacillus plantarum NCU116,L.plantarum NCU001013,Lactobacillus brevis NCU002005 and Lemon Leuconostoc NCU027042,they were cultured for 2 h in MRS medium with bile salt mass concentrations of 0,0.3,0.5,0.7,1 g/100 mL,respectively.Among them,The number of viable bacteria of L.plantarum NCU116 was significantly higher than that of the other three strains after different concentrations of bile salt stress,and the viable cell count was maintained at about 10~7 CFU/mL when the concentration of bile salt was as high as 1 g/100 mL,which was significantly higher than the other three strains.It indicated that the bile salt resistance of L.plantarum NCU116 was significantly better than that of the other three strains.Therefore,L.plantarum NCU116 was selected for subsequent study.(2)Changes of the transcriptional level of L.plantarum NCU116 gene after bile salt stress.Fluorescence quantitative PCR was used to study,results showed that the transcription level of genes including molecular chaperone protein dnaK,groES,heat shock protein hsp,6-phosphofructose kinase pfk and ATP-dependent DNA helicase uvrD1 were significantly up-regulated by bile salt at the concentration of 0.3 g/100 mL.With the increase of the concentration of bile salts,the transcription levels of the seven genes continued to rise to varying degrees;and the transcription level of genes including pyruvate kinase pyk,Lactate dehydrogenase ldh,transcription elongation factor greA,and mitosis protein ftsZ etc.were significantly had varying degrees of decline by bile salt.(3)Changes in key physiological indicators of L.plantarum NCU116 after bile salt stress.Physiological analysis showed that the cellular surface hydrophobicity and self-aggregation characteristics of NCU116 decreased after bile salt stress.The cellular surface hydrophobicity decreased from 37%of the blank control to 14%after the gallstone stress of 1 g/100 mL of the mass concentration,the self-aggregation characteristics of the cells decreased from 75.5%of the blank control to 51.7%after the gallstone stress of 1 g/100mL of the mass concentration;The activity of glucose metabolism were found to be inhibited by bile salt stress,the glucose utilization rate was reduced,the intracellular glucose residue increased from 24.9 ug/mL in the blank control to 317 ug/mL in the 1 g/100mL bile salt stress.And the lactic acid content produced by this metabolic process was also decreased to 31 ug/mL.However,the impact of NCU116 weakened as the bile salt concentrations continued to rise to 0.7?1 g/100 mL.In addition,the content of nine amino acids in intracellular amino acids,serine,tyrosine,isoleucine and lysine etc.increased after bile salt stress,and the total intracellular amino acid content is increased by bile salt stress.But the amino acid changes were not obvious under different concentrations of bile salt stress.The determination of?-galactosidase activity and fluorescence microscopy showed that the cell membrane of NCU116 was damaged after bile salt stress,and the number of bacteria that died or died increased with the increase of bile salt mass concentration,however,there was still a certain distance from the complete lethal concentration.All of the above studies implied that L.plantarum NCU116 owns several mechanisms to resist bile salt and has the potential to exert probiotic functions in the gastrointestinal tract. |
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