Excavation Of Bifidobacterium As The Signature Microbe During In Vitro Fermentation Of Jackfruit Polysaccharide Based On Multi-omics Technology And Development Of Jackfruit Fermented Beverage

Jackfruit(Artocarpus heterophyllus Lam.)is extremely abundant in the tropical and subtropical regions in southern China.Its pulp is rich in bioactive components like polysaccharides,flavonoids,phenolic compounds,etc.In recent years,plant-derived polysaccharides have become hot research topics in related fields due to their excellent biological activity.It has been discovered in previous studies that the polysaccharide extracted from jackfruit pulp has significant antioxidant and immunoregulatory activities.The results of animal experiments and in vitro fermentation experiments also indicated that jackfruit polysaccharide utilized by the gut microbiota has a certain regulatory effect on the intestinal immune barrier and gut microbiota.However,the mechanism of the interaction between jackfruit polysaccharide and human gut microbiota as well as the existence of related intestinal bacteria that can respond quickly during fermentation are still unclear.Therefore,in this study,in vitro fermentation model was used to simulate the fermentation process of jackfruit polysaccharide in the intestinal tract,and signature gut bacteria with rapid response capacity based on this result was dug out.Subsequently,whole genome and transcriptome technology were used to preliminarily explore the rapid response mechanism of signature microbe during the fermentation process.Finally,the studied bifidobacterial strains were utilized to prepare a jackfruit fermented beverage.The whole study was aimed at deepening the correlation between jackfruit polysaccharide and related gut bacteria with rapid response capacity,exploring the relationship between jackfruit polysaccharide and human gut microbiota,and providing a theoretical basis for the deep processing industry of jackfruit polysaccharide and jackfruit to serve the large health industry.The main contents and results of this study are as follows:1.Explore the in vitro fermentation characteristics of jackfruit,and the signature microbe with rapid response during the fermentation process by using high-throughput sequencing technology and QIIME2 bioinformation analysis technology.The results showed that the decreasing trend of sugar content and p H was consistent with the increasing trend of OD600.Within 4–8 h,a large amount of sugar was consumed,the p H value rapidly decreased,and microbes rapidly proliferated.During the fermentation process,the microbiota produced short-chain fatty acids and acetic acid is the main product.The composition of microbiota showed significant differences at different time points,and the relative abundance of Bifidobacterium was found to increase significantly during fermentation,of which the relative abundance was increased from the initial 0.39% to a final 35.18% and an initial rapid proliferation period was seen within 4-8 h(a fold change of 271%),suggesting it is a characteristic genus with rapid response in the fermentation process of jackfruit,The above results indicated that jackfruit polysaccharides could be fermented by gut microbiota,produce a large number of beneficial metabolites(short-chain fatty acids),and regulate the composition of gut microbiota through the rapid response of Bifidobacterium,and thus exerting a positive effect on intestinal health.2.The signature microbe in the in vitro fermentation of jackfruit polysaccharide were screened and identified by the whole genome sequencing,the changes in the transcriptome spectrum of the dominant bacteria in the rapid response process were explored,and the mechanism that how the strains utilizing polysaccharide fermentation broth was investigated.Based on the phenomenon that Bifidobacterium responded differently from other bacteria in 4 h,the dominant Bifidobacterium strains in this period were isolated by selective culture medium,and 54 strains were obtained.By evaluating the utilization ability of the fermentation broth of jackfruit polysaccharide at 4 h,two Bifidobacterium strains were finally obtained.By using ANI analysis comparison between two strains and the relevant Bifidobacterium strain published on NCBI database,the strains were identified as Bifidobacterium breve and the Bifidobacterium longum,and the two Bifidobacterium strains are predicted to contain coding genes of glycosidic hydrolase,glycosyltransferase and enzyme related to carbohydrate binding of carbohydrate active enzyme.Subsequently,combining the transcriptome with the whole genome,we found that there were significant differences in the gene expression of the two Bifidobacterium strains when using the jackfruit polysaccharide fermentation broth: the carbohydrate active enzyme family corresponding to the up-regulated genes of Bifidobacterium longum was GH family,and that corresponding to the up-regulated genes of Bifidobacterium breve was GT family.Further functional analysis showed that the functions corresponding to the upregulated genes of Bif.breve were focused on cell amide metabolism,peptide metabolism,gene expression,and metastasis,while the functions corresponding to the up-regulated genes of Bif.longum were focused on pentose metabolism,hexose metabolism,monosaccharide metabolism,carbohydrate metabolism,etc.The KEGG pathway analysis showed that Bifidobacterium breve metabolic jackfruit metabolism,etc.The KEGG pathway analysis showed that Bifidobacterium breve metabolic jackfruit polysaccharide fermentation broth was rich in four pathways including environmental information processing,genetic information processing,human diseases and metabolism,among which the metabolic pathways included glycolysis or gluconeogenesis pathway and carbon fixation,while Bifidobacterium longum was mainly rich in metabolic pathways,including galactose metabolism,fatty acid degradation,glycan degradation,glycolysis or gluconeogenesis,etc.3.Preparation and optimization of fermented jackfruit beverage by the selected two Bifidobacterium strains.First,the single factor experiment was conducted for different influencing factors(fermentation temperature,fermentation time,and bacterial inoculation amount)to obtain the factors’ optimal range,and then the response surface methodology was used to determine the optimal process conditions: inoculation amount 1.7%,fermentation time 15 h,and breve: longum = 1: 2 and fermentation temperature 36.5℃.Under these conditions,a pale yellow beverage with sensory score of 84.2,soluble solid content of 5.2±0.1% Brix,and p H of 4.5±0.3 was obtained.In summary,jackfruit polysaccharide can increase the short-chain fatty acid which is beneficial to the regulation of intestinal health.In addition,Bifidobacterium is able to rapidly respond during the fermentation,and it is important for the interaction of gut microbiota and jackfruit polysaccharide.The two obtained Bifidobacterium strains could utilize the jackfruit polysaccharide fermentation broth,but they were different in the utilization mechanisms,which were mainly evidenced by the up-regulation and down-regulation of genes related to carbohydrate active enzyme,genes related to GO functionality and genes related to KEGG metabolic pathway.Finally,in this study,the two screened Bifidobacterium(which belongs to the category of edible probiotics stipulated by the state)were used to prepare the jackfruit fermented beverage,and a beverage with strong jackfruit flavor was obtained.This study not only provides a theoretical basis for the potential probiotic effects of jackfruit polysaccharides at the theoretical level,but also provides a theoretical basis and research basis for the jackfruit finishing industry at the industrial level.