Interaction Of Indigestible Glucans With Gut Microbiota And The Macrophage Immunomodulatory Activity Of In Vitro Fermentation Broth

Indigestible glucans(IGs)are representatives of dietary fiber with wide range of sources and complex structures,which has been known to have abundant physiologically active functions such as lowering postprandial blood glucose,lowering cholesterol,immunomodulation,anti-inflammation and anti-tumor.Currently,the relationship between beneficial effects of IGs and their capacities to regulate gut microbiota have been exploring.However,it is unclear how the structural and properties of IGs impact on the gut fermentation,the regulation of gut microbiota,and their role in the health of the gut.Therefore,six IGs with different physicochemical properties were used to analyze the fermentation characteristics of IGs with diverse structures based on an in vitro anaerobic fermentation model,and the changes in the structural composition of the gut microbiota were also studied,to investigate the interaction between IGs and gut microbiota and decode the key physico-chemical factors of IGs.Further investigation on gut immunomodulatory activity of gut fermentation broths of IGs was performed by cell culture technique,and the main active components were discussed.The main results of this study are summarized as follows:(1)The fermentation characteristics such as degrees of fermentation,monooligosaccharide release,p H change,gas production and SCFAs productions of IGs with various physicochemical properties were analyzed using an in vitro anaerobic fermentation model.The final degree of fermentation varied among the six IGs,L was almost completely fermented,while 14% of BG,23% of BY and PAC,and 35% of R and Lit remaining unfermented at the end of fermentation.The BG and L with high solubility or simple structure had higher initial fermentation rates,final fermentation degrees,SCFAs concentrations and lower p H than PAC,R and Lit with the insolubility or complex structures.Additionally,the release of mono-and oligosaccharides from the fermentation process also differed significantly,compared to other IGs,BG and L could be rapidly degraded by gut microbiota and release mono-and oligosaccharides more quickly during fermentation,with the highest levels of 3.1 ± 0.1 and 2.19 ± 0.2mmol/L,respectively,in contrast,despite of having similar glycosidic bond linkages to L,the high-viscosity BY and insoluble PAC produced much less monosaccharides,with the highest levels of 0.5 ± 0.1 and 0.11 ± 0 mmol/L,respectively.The SCFAs were produced by the fermentation of all IGs,more propionic acid was produced by L and more butyric acid was produced by PAC compared to the other IGs groups.The above results showed that the physicochemical properties such as solubility and glycosidic bonding mode have important effect on fermentation characteristics of IGs.Higher solubility and simpler glycosidic chain structure lead to higher fermentation rates and final fermentation degrees,and bigger changes of p H,gas and SCFAs productions,etc.(2)Effect of IGs with different physicochemical properties on gut microbiota were analyzed based on 16 S r RNA gene sequencing and real-time quantitative PCR.Except for PAC,the fermentation of IGs significantly reduced the species diversity of gut microbiota.Except for the L and Lit groups,significant differences in microbial composition structure were detected among all groups,except for the L and Lit groups,which were similar.While in terms of total gut bacteria,IGs enzymes significantly increased the abundance of total gut bacteria,except for Lit with complex glycosidic bond structure.and each IG significantly increased the abundance of at least one beneficial bacteria.Correlation analysis showed that the high production of carboxylic acids such as SCFAs and lactic acid was strongly correlated with gut microbiota interfered by IGs.These results suggest that the fermentation of IGs substantially enriches beneficial gut bacteria and that the differences in the type and content of carboxylic acids produced by the fermentation of different IGs correlate with the composition of the gut microbiota in which they are enriched.(3)Immunoactivity of IGs fermentation broths was analyzed by cell culture technique.IGs fermentation broths significantly enhance the proliferation rate of RAW264.7 macrophage,productions of NO and ROS,and cytokine TNF-α,IL-1β,IL-6release.In addition,treatment with polymyxin B showed that the cellular immunomodulatory effects of the fermentation broths were mainly derived from IGs fermentation products rather than from LPS lysed by gut bacteria.The results of the study showed that different IGs could effectively interacted with gut microbiota,and the physicochemical properties of IGs,such as solubility,types of glycosidic linkage and branched chain complexity,had important effects on its fermentation characteristics including enzymatic rate,enzymatic degree and metabolite production during the fermentation.IGs with different physicochemical properties influenced the structural changes of gut microbiota and differentially increased the abundance of beneficial gut bacteria,thus determining the types and content of carboxylic acid production by different groups of IGs.In addition,RAW 264.7 cell experiments showed that fermentation products of IGs by gut microbiota could significantly enhance cellular immunomodulatory activity.These results provide a scientific basis for revealing the differences in the fermentation characteristics and gut functional effects of IGs with different physicochemical properties under the action of gut microbiota,and provide a theoretical reference for the development of functional foods for IGs.