Nitrogen Source Utilization Mechanism Of Akkermansia Muciniphila And The Effect Of Bifidobacterium On Its Growth

With the gradual deepening of research,people found that probiotics play a vital role in the regulation of systemic diseases.Akkermansia muciniphila(Akk),a typical representative of the next generation of probiotics,and is also a marker of diseases such as obesity,inflammatory bowel disease,type 2 diabetes.Akk plays an integral role in the prevention and management of these diseases.Its safety had been proved in several clinical trials and animal studies.Pendulum company from the USA has been marketed as a viable Akk product for the treatment of type 2diabetes.However,the domestic industrial high-density cultivation of Akk is immature.The discovery of ovalbumin can promote its proliferation and provide the condition for the high-density culture of Akk.In addition,bacterial metabolites can act as the "messenger" of intestinal flora to regulate host health.It is very important for the research and development of Akk probiotics to know how the protein affects the Akk metabolite,how these metabolite will affect the body,and how to regulate the expression of Akk gene,but there are no reports.In this study,the Akk model strain DSM 22959 was used as the research object.Non-targeted metabolomics methods were used to investigate Akk metabolizing metabolites produced by ovalbumin and its influence on metabolic pathways,and the roles of key metabolites and metabolic pathways in disease regulation were analyzed.Transcriptomics was used to analyze the influence of proteins on Akk gene expression,as well as GO and KEGG enrichment analysis was conducted to explore its potential function.Then,based on our research group’s previous optimization of liquid thioglycolate medium,the influence of single amino acid on the growth of Akk was explored through in vitro culture,and whether amino acid could be used as the proliferation factor of Akk.In addition,taking Bifidobacterium as an example,this study investigated how the growth of Akk was affected by probiotics,their metabolites and inactivated bacteria,and the population effect between Akk and Bifidobacterium.The metabolomics results of this study showed that,compared with the thioglycolate liquid medium,Akk metabolized ovalbumin in positive and negative ion modes to produce 902 different metabolites,these metabolites were enriched in protein digestion and absorption,aminoacyl t RNA biosynthesis,bile secretion,ABC transporter,amino acid biosynthesis,and other metabolic pathways.Akk is involved in disease regulation through these pathways.332 differential genes were identified by transcriptomics,including 138 up-regulated genes and 194 down-regulated genes,which were related to their molecular functions,biological processes,and cell composition.These differential genes encoded different proteases and were enriched into different KEGG pathways.In vitro,culture results showed that neither single amino acid nor amino acid combination could meet the nutritional requirements of Akk,but a variety of amino acids could be used as proliferation factors.As the concentration was high,cysteine and proline promoted the growth of Akk(P<0.05),while tyrosine,phenylalanine,and glycine inhibited its proliferation(P<0.05),and the rest had no significant effect(P>0.05).When the concentration was lower,valine,threonine,and lysine promoted the growth of Akk(P < 0.05),and lysine had the best effect.Tyrosine,glycine,and isoleucine still inhibited the growth of Akk(P < 0.05),but the inhibition effect was relieved,and the rest had no significant effect(P > 0.05).All the live bacteria,inactivated bacteria,and metabolites of the six Bifidobacterium species inhibited the growth of Akk,and the inhibitory effect of metabolites increased with the increase in metabolite concentration.This study investigated the product and metabolic pathway of Akk metabolizing ovalbumin at the level of small molecule metabolites,and explained the gene regulation rule of Akk under the high-protein conditions from the perspective of genes,providing conclusive evidence for the function of Akk as probiotics and postbiotic agent in the regulation of many diseases in the future.It laid a theoretical foundation for the extensive application of optimized culture medium in industrial production.The study about the influence of Bifidobacterium on the growth of Akk provides a direction for exploring the relationship between Akk and probiotics and the quorum sensing of intestinal flora.