Effect Of Lactic Acid Bacteria With High Purine Scavenging Ability On Purine-induced Hyperuricemia Model And Preparation Of Microcapsules

Hyperuricemia(HUA)is a metabolic disease,and the purine intake and metabolism disorders in the human body are the main causes of hyperuricemia.At present,the treatment of hyperuricemia mainly includes promoting the excretion of uric acid or inhibiting the production of uric acid,but it is often accompanied by rash,fever and other side effects.Therefore,it is of great significance to find natural ingredients to intervene in HUA.In recent years,it has been reported that lactic acid bacteria can reduce uric acid by eliminating purine substances and regulating the metabolism of uric acid substances by intestinal flora.The purpose of this study is to screen probiotics with purine scavenging characteristics from 17 lactic acid bacteria strains in the laboratory,verify their uric acid lowering effect in vivo,and explore their interaction with intestinal flora.The specific methods and results are as follows:(1)The in vitro scavenging characteristics of lactic acid bacteria on four purines(hypoxanthine,xanthine,adenine,guanine)were investigated for 17 strains of lactic acid bacteria in the laboratory.The clearance rates of four purines in the fermentation supernatant were determined.It was found that the four L.brevis strains had strong adenine clearance ability,ranging from 87% to 100%.The guanine clearance rate of Lactobacillus habersiella was higher than 50%.Most of the strains could also clear hypoxanthine and xanthine to some extent.Streptococcus lactis NCUF214.1 had the highest clearance rate of hypoxanthine.For xanthine,the highest clearance rate of Lactobacillus plantarum NCUF201.1 was 30.55%.Based on the clearance results of four purines,9 lactic acid bacteria with purine-clearance specificity were selected for subsequent exploration of their probiotic performance.(2)Nine lactic acid bacteria with strong purine clearing ability were screened out and evaluated for gastrointestinal tolerance,adhesion property,and antioxidant properties.Lactic acid bacteria NCUF207.7 and NCUF201.1 showed the best tolerance at p H=2.5(69.8% and 67%,respectively).Lactobacillus NCUF214.1,NCUF213.1 and NCUF207.7 showed the best tolerance to 0.3% bile salt(92%,77.3% and 74.1%,respectively).The results of Caco-2 cell adhesion assay combined with the results of hydrophobic self-cohesion suggested that NCUF207.7,NCUF208.7,NCUF213.1 and NCUF214.1 might be the lactic acid bacteria strains with better adhesion properties.The results of in vitro antioxidant experiments showed that NCUF207.7,NCUF214.1,NCUF207.7,NCUF214.1 might be the lactic acid bacteria strains with better adhesion properties.NCUF214.1 and NCUF201.1 showed better antioxidant properties.According to the above results,7 strains of lactic acid bacteria were selected for in vivo verification in the HUA mouse model.(3)To establish a mouse model of hyperuricemia induced by adenine and potassium oxonate,and to explore the intervention effects of 7 strains of lactic acid bacteria on the model.The weight growth rate of mice in HUA group was lower than that in CON group,and increased after the intervention with 7 strains of lactic acid bacteria,while the growth rate of mice in allopurinol group was lower than that in lactic acid bacteria intervention group.Compared with healthy mice,the kidney index of HUA mice was increased,and HE staining showed that the kidney morphology was abnormal and inflammatory cells were exudated in the tissue.After the intervention of7 strains of lactic acid bacteria,the edema and lesions of the kidney were alleviated.The kidney index of the 7 strains of lactic acid bacteria gavage group was lower than that of the model group.The intervention of 7 strains of lactic acid bacteria can reduce the levels of serum uric acid,creatinine and urea nitrogen and the activities of XOD and ADA in the intestine and liver of HUA mice.Although the uric acid of allopurinol group decreased the most,some indicators suggested that allopurinol might have side effects on the body.At the same time,the results of CAT,SOD,MDA and so on showed that lactic acid bacteria treatment could also alleviate the liver oxidative damage of HUA mice.(4)Hyperuricemia can reduce the richness,diversity and evenness of intestinal flora in mice.Compared with the HUA group,the evenness of all lactic acid bacteria intervention groups was improved,and the intestinal flora tended to be stable.The results of β-diversity also showed that the structure of gut microbiota in HUA mice was different from that in normal mice,and the Lactobacillus intervention significantly changed the structure of gut microbiota in HUA mice,forming a homeostasis more similar to that in CON group.At the same time,the contents of short-chain fatty acids such as acetic acid,propionic acid,butyric acid and isobutyric acid in the intestine of hyperuricemic mice increased under the intervention of most lactic acid bacteria,which may have a protective effect on the intestine.According to the Lefse differential analysis,Firmicutes after the lactobacillus administration became the dominant group,The relative abundance of beneficial bacteria such as Oscillospira,Prevotellaceae＿UCG＿001,Prevotellaceae＿NK3B31＿group,and Lachnospiraceae NK4A136 group was recovered;According to the results of the correlation analysis,The genus Ruminococcus showed a significant negative correlation with uric acid content in mouse serum,The relative abundance of the Prevotellaceae was inversely correlated with the levels of creatinine and blood urea nitrogen in mouse serum.Therefore,the intervention of HUA on the above strains may be an important way for lactic acid bacteria to intervene in hyperuricemia.(5)A probiotic microcapsule of Lactobacillus plantarum NCUF201.1 was developed by optimizing the conditions.Through single factor experiment and response surface experiment,the concentration of chitosan,sodium alginate and calcium chloride in the preparation of Lactobacillus plantarum NCUF201.1microcapsules was optimized by taking the embedding rate as the evaluation index.The optimal embedding conditions were 2.28 g/100 m L,0.7 g/100 m L and 1.97 g/100 m L of sodium alginate,chitosan and calcium chloride,respectively.Under these conditions,the embedding rate of Lactobacillus plantarum sustained-release microcapsules was 76.7%.When the microcapsules were treated in intestinal fluid for8 h,the release rate reached 92%.It has good sustained release characteristics.The accelerated storage experiment at 60℃ showed that the survival rate of the microcapsules lyophilized after adding 2% inulin was improved,and the microcapsules had better storage performance.In conclusion,all the 7 lactic acid bacteria used in this study had a certain uric acid-lowering effect and protective effect on the kidney in vivo.It can regulate the metabolism of short-chain fatty acids by intestinal flora,enhance the antioxidant performance of the body,and reduce the damage caused by oxidative stress.Based on the results of this study,a lactic acid bacteria microcapsule with the potential to reduce uric acid was developed,which laid a theoretical foundation for further application.