Characterization Of The Non-covalent Complex Of Xylitol And Milk Proteins And Its Intervention Mechanism On Type 2 Diabetes Mellitus

Milk proteins often coexist with other food components in the same system,which inevitably leads to non-covalent interaction.The interactions can improve the functionalities and biological activities of food components to a certain extent,which is of great significance for the development of functional milk-base food and the expansion of application of food components in the food industry.Studies have shown that sugar alcohols have positive effects on the structural and functionalities of proteins.However,there is no research on its mechanism from the perspective of non-covalent interaction.Therefore,xylitol(XY)was selected as the typical sugar alcohol compound,whey protein isolate(WPI)and casein(casein)were used as typical milk proteins,and the non-covalent interaction mechanism between XY and WPI and CN was studied based on multispectral technology and molecular docking technology in this thesis.The secondary structure and functional properties of XY–WPI complexes(XW)and XY–CN complexes(XC)were also characterized.Then,the intervention mechanisms of the two complexes on type 2 diabetes(T2DM)were analyzed respectively from the perspectives of glucose metabolism,lipid metabolism and intestinal microbiota changes,and the related theoretical basis was provided for the application of XW and XC as functional food suitable for T2DM population.The main results were as follows:Study on the non-covalent interaction mechanism of XY with WPI,CN and their fractions.The results showed that XY had a strong quenching effect on the fluorescence intensity of WPI,CN and their fractions,and the quenching mechanism belonged to the static quenching,indicating that there was a non-covalent interaction between them and the formation of complex.The binding affinity of XY to WPI was slightly stronger than that of CN,with K_A values of 16.95×10~4 and 6.69×10~4L/mol,respectively.The order of binding affinity of XY to WPI fractions and CN fractions was:α-Lactalbumin(α-La)>bovine serum albumin(BSA)>β-Lactoglobulin(β-Lg),β-Casein(β-CN)>κ-Casein(κ-CN)>α-Casein(α-CN).According to the results of thermodynamic parameters,the non-covalent interaction forces of XY with two milk proteins and their fractions were mainly hydrogen bondings and van der Waals forces.Molecular docking results showed that XY had different binding sites with two milk proteins and their fractions,which theoretically confirmed that the main types of non-covalent interaction forces.This study showed that XY could strongly interact with two milk proteins and their fractions,especially WPI,and form relatively stable complexes.Characterization of secondary structure and functional properties of XY–milk protein complexes.The binding rates of the two complexes was determined by HPLC.The results showed that the binding rates of the two complexes were more than 85%and showed a certain dose-dependent.The secondary structure and microstructure results showed that compared with WPI and CN,the contents ofβ-sheet and random coils were increased,α-helix andβ-turn were decreased,and the microstructure became relatively layered from the original spherical(WPI)and massive(CN).Due to the introduction of hydrophilic hydroxyl group in XY and the change of protein structure after non-covalent interaction,the solubility,emulsifying activity,emulsifying stability,foaming activity and foaming stability of the two complexes increased significantly(XW:10.75%,45.18%,89.49%,8.80%and 26.10%;XC:14.97%,15.41%,58.71%,20.40%and 35.60%),while the surface hydrophobicity,solution surface charge characteristics and stability decreased significantly(XW:6.47%and 16.26%;XC:9.06%and 16.52%).The thermal stability of XW increased by 9.68%,while the thermal stability of XC decreased by 19.41%.Therefore,after the complexes formation of non-covalent interaction between XY and two milk proteins,the backbone of the protein polypeptide chain stretched,a new microstructure and shape formed,and some functional properties were also improved.Evaluate the effects of XW and XC on glucose and lipid metabolism in T2DM mice induced by high-fat diet and intraperitoneal injection of streptozotocin(HFD+STZ).The results showed that XW and XC could improve the body weight of T2DM mice.The two complexes could effectively down-regulate FBG,GHb and blood glucose levels after gavage of glucose,and helped relieve the symptoms of hyperglycemia and restore glucose tolerance.The two complexes could significantly reduce the insulin level and insulin sensitivity of T2DM mice.Compared with T2DM mice,the insulin level of XW and XC groups decreased by 16.41%and 23.87%respectively,the index of Homeostasis model assessment-insulin resistance was decreased by 28.53%and 31.94%respectively,and Quantitative insulin sensitivity check index was increased by 4.33%and 5.35%respectively.The results of serum related parameter levels(triglyceride,total cholesterol,free fatty acid,aspartate aminotransferase,Alanine aminotransferase,lactate dehydrogenase,creatine kinase and creatinine)were significantly decreased in mice after the intervention of the two complexes.This study shows that the two complexes help to alleviate the disorder of glycolipid metabolism,as well as organ,tissue and cell damage in T2DM mice.Based on the above results,this study also analyzed the regulatory effects of the two complexes on intestinal microbiota of T2DM mice by 16S r RNA high-throughput sequencing technology,and discussed their intervention mechanism on T2DM from this perspective.The results showed that XW and XC regulated the diversity,abundance and structure of intestinal microbiota in T2DM mice by affectingαandβdiversity indices.Analysis of key intestinal microbiota at different classification levels showed that the relative abundances of Firmicutes,Lactobacillus,Bifidobacterium and Turicibacter were significantly increased in the two complexes groups(p<0.05),while the relative abundances of Bacteroidetes,Actinobacteria,Faecalibaculum and Helicobacter were significantly decreased(p<0.05).PICRUSt analysis showed that the two complexes could significantly promote the changes of gene abundance in Carbohydrate metabolism and Amino acid metabolism pathways,and XC played a more significant role.Spearman’s analysis showed that the relative abundance of genus level bacterias were significantly correlated with T2DM related indexes(body weight,fasting blood glucose,insulin level and serum parameters)(p<0.01).Therefore,the two complexes improved the associated symptoms in T2DM mice mainly by affecting the overall diversity,composition of the intestinal microbiota and gene abundance in related biological functions.