In Vitro Interaction Study Of Litchi Pulp Polyphenols In Promoting The Proliferation Of Intestinal Bacteria Co-cultur

Lychee pulp is rich in phenolic compounds,mainly in flavonoids.Previous studies have confirmed that lychee pulp phenolics(LPP)elevated the abundance of Bacteroides uniformis and Akkermansia muciniphila,alleviated dextran sodium sulfate induced colitis in mice.However,the regulatory role and metabolic characteristics of LPP based on the specific gut microbiota that enriched by itself are still unclear.This study used lychee(cv.Guiwei)pulp as raw material to prepare high-quality LPP and clarify its interaction with specific intestinal bacteria by magnetic nanometer combined with microwave thawing and anaerobic fermentation experiment in vitro.On this basis,quercetin-3-O-rutinose-7-O-α-L-rhamnoside(QRR),the main flavonoid monomer of LPP was purified to further elucidated the regulatory effects and metabolic of lychee pulp phenolics based on in vitro co-culture of specific gut microbiota.(1)Study on the new thawing method of frozen fruits: The magnetic nanometer combined with microwave thawing(MN-MT)was applied to thaw frozen lychee pulp for the first time.The thawing efficiency of MN-MT increased by 80.67%,25.86% and 18.83%compared to water immersion thawing(WT),microwave thawing(MT),and water immersion combined microwave thawing(WI-MT),respectively.The results of high performance liquid chromatography(HPLC)showed that compared with WT,MN-MT was the only method that significantly increased the content of QRR.UPLC-MS analysis showed that among the three microwave-based heat treatments,thermosensitive phenolic proanthocyanidins B2,phenylpropanoic acid,and protocatechuic acid were not degraded only when MN-MT was used.In addition,MN-MT significantly improved the retention rate of ascorbic acid in lychee pulp and better maintained the physicochemical properties of lychee pulp compared with other thawing treatments.The above results indicate that MN-MT is a new rapid thawing method that can improve the quality of LPP.(2)Study on the interaction between LPP and specific intestinal bacteria: the frozen lychee pulp treated by MN-MT was used to extract high quality LPP and the latter was subjected to in vitro anaerobic fermentation experiments with specific intestinal bacteria B.uniformis and A.muciniphila.The results of bacteria enumeration showed that LPP promoted the synergistic proliferation of specific intestinal bacterial species,the viable cell of B.uniformis and A.muciniphila increased by 1.61 and 3.57 times compared to those without supplementing with LPP,respectively.The results of HPLC and UPLC-MS confirmed that co-cultured strains participated in the metabolism of LPP,and B.uniformis improved the growth of itself and A.muciniphila by catalyzing the breaking of glycosidic bond on various flavonol glycosides(including QRR)to release corresponding aglycones,and also could crack the C-C bond on proanthocyanidin B2.This study discovered that taxifolin is a key intermediate metabolite for specific gut microbiota to degrade quercetin for the first time.The secondary metabolic pathway for specific gut microbiota to metabolize LPP and produce isoquercetin was only found in coculture systems.(3)Study of regulation and metabolism of QRR based on co-cultured gut bacteria: As the interaction between LPP and co-cultured bacteria have been confirmed,we further purified the main phenolic monomer substance QRR of LPP to co-culture with B.uniformis and A.muciniphila,in order to elucidated the regulation and metabolism of QRR based on cocultured gut bacteria.Qualitative analysis by UPLC-ESI-Triple-TOF-MS/MS showed that B.uniformis could break the glycosidic bond on the QRR to produce the key intermediate taxifolin.HPLC quantitative analysis found that the content of this metabolite was improved from limit of quantification to 13.06 ± 0.04 and 13.50 ± 0.28 ug/m L respectively after the anaerobic fermentation of QRR by single strain culture of B.uniformis or co-culture of B.uniformis and A.muciniphila.The release of glycosides from QRR promotes the proliferation of specific gut microbiota,as the number of cells counts increased in B.uniformis and A.muciniphila in the co-cultured system by 2.10 and 2.75 times,respectively,compared to the absence of QRR.The quantitative analysis of short chain fatty acid content by gas chromatography also suggests that QRR promotes the synergistic proliferation of B.uniformis and A.muciniphila in vitro.Analysis of QRR metabolism based on specific gut microbiota revealed that the key intermediate metabolite,taxifolin,was further decomposed into phenolic acids through direct cleavage of the flavonol C-ring.During the fermentation of QRR by coculture of specific gut microbiota,a secondary metabolic pathway for synthesizing kaempferol occurs.This study confirmed that magnetic nanometer combined with microwave is a new rapid thawing method that can improve the quality of LPP.It was clear that LPP can be metabolized and degraded by the co-culture of B.uniformis and A.muciniphila,and can promoted the proliferation of these two bacteria in vitro.It also clarified QRR is an effective substance in LPP that interacts with the specific gut bacteria.This study can provide practical basis for the probiotic value of polyphenols in lychee pulp,and also provide new insights into the interaction between active substances in fruits and gut microbiota.