Identification And Inhibition Of Dipeptidyl Peptidase-4 Inhibitors In Metabolites Of Lactic Acid Bacteria

Diabetes mellitus(DM)is a metabolic disorder caused by many factors.With the change of people's lifestyle and dietary habits,the incidence of diabetes is increasing at an alarming rate.Drugs for treating diabetes are also increasing.Among them,dipeptidyl peptidase-4(DPP-4)is a new target for blood sugar control in type 2 diabetes.It has been reported that some probiotics have the ability to inhibit alpha-glucosidase and dipeptidyl peptidase-4(DPP-4),which can slow down the absorption and decomposition of carbohydrates in the intestine,thus delaying and delaying the time and process of postprandial blood glucose rise in diabetic patients and playing a role in lowering blood sugar.In this study,a strain BLP12 with DPP-4 inhibitory activity was screened from laboratory-preserved strains,and its metabolites were isolated and purified.Finally,the substances inhibiting DPP-4 in metabolites were identified by high performance liquid chromatography-mass spectrometry(LC-MS).At the same time,a Caco-2 cell model was established to validate its efficacy in inhibiting DPP-4.Finally,a kind of assistant with milk and xylitol was developed.Functional yogurt with hypoglycemic effect.The cell contents(CFE)and cell metabolites(CFS)of Lactobacillus plantarum BLP12 were prepared and their inhibitory effects on DPP-4 were determined.The results showed that the components of the metabolites of Lactobacillus plantarum BLP12 that inhibited DPP-4 mainly existed in cell metabolites.Then,organic solvent extraction,ultrafiltration,filtration chromatography,gel chromatography and semi preparative liquid chromatography were used to separate and purify the metabolites of BLP12.The samples obtained from the separation and purification were collected to determine their inhibitory effects on DPP-4.Finally,the DPP-was identified by high performance liquid chromatography-mass spectrometry(LC-MS).Substances of 4.The results of mass spectrometry showed that 50 substances were identified and 10 of them were related to the treatment of type 2 diabetes mellitus.They were dodecyl sulfate,nicotinic acid,dodecyl diacid,pyridine carboxylic acid,octadecatriene acid,allopurinol,guanine,pyrogallol,kojic acid and glutamic acid.Dodecyl sulfate is a reported inhibitor of DPP-4.Nicotinic acid can treat abnormal blood lipids in the course of diabetes treatment,and nicotinic acid derivatives are DPP-4 inhibitors;dodecanedioic acid can improve the metabolic inflexibility of diabetic patients;pyridine carboxylic acid mixed with zinc(II),maltol,threonine and other substances can reduce blood sugar;allopurinol can alleviate the symptoms of proteinuria in type 2 diabetes mellitus patients;pyrogallol can inhibit alpha.-Glucosidase;these substances work together to alleviate the symptoms of diabetes.Caco-2 monolayer cell model was established to simulate the intestinal absorption of drugs.Cell morphology was observed by electron microscopy to determine whether a closely connected cell monolayer was formed;fluorescein sodium transport test was used to detect cell permeability;alkaline phosphatase content in the upper and lower chambers was used to determine whether polarity was formed,and these tests were used to evaluate the success of the model establishment.After 24 hours of co-culture,the cell viability was measured to determine whether the samples were toxic to the cells.After confirming the non-toxicity of the sample,the sample was co-cultured with the cell for 3 hours.The cell culture medium and lysate were collected to determine the DPP-4 activity in the culture medium and lysate.Compared with the control group without the sample,the sample group BLP12 had a significant inhibitory effect on DPP-4 in the cell.The inhibitory rate was 48% at 50 g/ml and 54% at 100 g/ml.The inhibitory effect on DPP-4 in cell culture medium was not observed.The inhibitory activity of sample 1.6970 was not found in both intracellular and extracellular cells.Using fresh milk,xylitol,Lactobacillus bulgaricus,Streptococcus thermophilus and Lactobacillus plantarum BLP12,a Functional Yoghurt with hypoglycemic effect was developed.The optimum conditions of each factor were determined by single factor test.Then,according to single factor test results,orthogonal design experiments were carried out on four factors of different strain ratio(A),xylitol addition(B),inoculation(C)and fermentation time(D).The sensory evaluation was used to determine the optimum production process of yoghurt: 4% fermentation agent,Lactobacillus bulgaricus,Streptococcus thermophilus and D.The ratio of Lactobacillus plantarum was 1:1:2,the addition of xylitol was 8%,and the fermentation time was 6 hours.The content of fat,protein and carbohydrate,the number of viable bacteria and acidity value,microbial index and DPP-4 inhibition activity in yoghurt products were determined.It was found that the yoghurt fermented separately by Lactobacillus bulgaricus and Streptococcus thermophilus,Lactobacillus plantarum 1.6970 and Lactobacillus plantarum BLP12 had an inhibitory effect on DPP-4,but the inhibitory effect was significantly lower than that fermented by Lactobacillus bulgaricus,Streptococcus thermophilus and Lactobacillus plantarum BLP12.