Cross-feeding Activities Between Different Bifidobacterium Species With Inulin As The Sole Carbon Source

Bifidobacteria represent one of the first colonizers of the mammalian gut,where such colonization is facilitated by their saccharolytic capabilities.Bifidobacterial proliferation can be promoted by the intake of prebiotics.Clinical trials have indicated that inulin played an important role in promoting the proliferation of bifidobacteria in the human intestine when added to the diet.However,some bifidobacterial strains with probiotic functions cannot use inulin directly,so supplementation with inulin alone cannot promote their proliferation in the host intestine.Recent studies have reported that cross-feeding activities among Bifidobacterium and other intestinal bacteria,such as Lactobacillus rhamnosus and Streptococcus thermophilus,promoted the proliferation of bifidobacterial strains that cannot use inulin directly.Therefore,we hope to develop the strategy of the interspecies synergistic proliferation of bifidobacterial strains in inulin utilization and then provide a new method for bifidobacterial proliferation in the intestine,and the main conclusions are as follows:(1)By analyzing the carbohydrate-active enzyme genes of 60 Bifidobacterium strains,the number of genes that encode the GH32 family enzymes involved in inulin utilization differed significantly from bifidobacterial strains.For example,Bifidobacterium adolescentis R1 possessed two genes encoding GH32 family,while Bifidobacterium animalis FGZ9I1M1 possessed only one.By testing the capabilities of 25 Bifidobacterium strains in vitro inulin utilization,there were interspecies and inter-strain differences of Bifidobacterium strains in inulin utilization.This result of in vitro experiment was consistent with the results predicted at the gene level.Besides,different bifidobacterial strains have unique carbohydrate preference.(2)Giving the results of the above comparative genomics and in vitro experiments,we found that B.adolescentis R1 was capable of utilizing inulin,and animalis FGZ9I1M1 was unable to utilize inulin but can utilize oligofructose,glucose,and fructose.These two strains were selected for cross-feeding experiment.B.adolescentis R1 and B.animalis FGZ9I1M1 were in mono-culture and in co-culture on the medium with inulin as the sole carbon source.The results have shown that the growth rate of co-culture strains became faster than the mono-culture one.The bacteria number of co-culture strains was significantly increased compared with mono-culture one,while part of inulin was significantly degraded and acetic acid production increased in co-culture strains.Transcriptome analysis was performed to further elucidate the in vitro co-proliferation pattern of Bifidobacterium strains in inulin as the carbon source.The results showed that the expression of C8077_RS09170,a key gene of B.adolescentis R1 that encodes the enzymes involved in inulin utilization,increased from 29.63667 to 59.38333.The expression of C8077_RS04530,a key gene of B.animalis FGZ9I1M1 that encodes the enzymes involved in inulin utilization,increased from 155.5533 to 323.3967.The mentioned-above results suggested that B.adolescentis R1 degraded inulin and released sucrose,fructose,and glucose to promote the proliferation of B.animalis FGZ9I1M1,while the short-chain fatty acids produced by B.animalis FGZ9I1M1 also facilitated the proliferation of B.adolescentis R1.(3)The effects of gavage of mono-bifidobacterial strain and bi-bifidobacterial strains on the intestinal flora of C57BL/6J mice under the conditions of supplementation with different inulin doses were investigated.Compared with the experimental group supplemented with inulin only,the results showed that the experimental group supplemented with Bifidobacterium strains significantly increased the relative abundance of B.adolescentis and B.animalis in the mice intestine.Different gavage methods affected the proliferation ability of Bifidobacterium colonization,in which the experimental group with high doses of inulin and bi-bifidobacterial strains had the highest number of B.adolescentis and B.animalis colonies in the feces,reaching 1.357×109 CFU/g and 1.165×109 CFU/g,respectively.Compared with the control group,the experimental group with supplementation of Bifidobacterium strains were beneficial to increase the content of short-chain fatty acids in the feces.Besides,analysis of the fecal metabolome has revealed that the fecal metabolic network of mice in the bi-bifidobacterial group and mono-bifidobacterial group was significantly different(p<0.05),especially in metabolic pathways of linoleic acid.In summary,through carbohydrate gene analysis,in vitro inulin utilization experiments and transcriptome analysis,and evaluation of the colonization ability of bifidobacteria in the intestine of mice,a strategy to promote the synergistic proliferation of bifidobacteria was investigated in this project,which can be applied to more strains with health benefits but weak utilization of inulin and other oligosaccharides in the future to promote the intestinal proliferation of functional probiotics.