| Bifidobacterium pseudocatenulatum(B.pseudocatenulatum)is one of the common probiotics in the intestines of Chinese people,which can improve the host glucolipid metabolism,inflammation and nervous system.Recent studies have confirmed that specific oligosaccharides can improve the intestinal survival rate or colonization persistence of probiotics,but there are strains specificities in the effect of oligosaccharides on probiotics.Furthermore,the internal cause and specific mechanism of strains specificities are not clear,which affects the development of dietary strategies used for regulating proliferation or colonization of specific strains.Therefore,this study focused on typical functional oligosaccharides-galactose(GOS).B.pseudocatenulatum strains with different ability of using GOS were screened in vivo.Combined with the growth characteristics in vitro and genome analysis of strains,the related genes leading to the differences in utilization ability of GOS were identified and verified.Finally,the effects of GOS on the colonization ability of specific B.pseudocatenulatum strains were evaluated by animal experiments.Overall,the aim of this study is to analyze the growth and genetic characteristics of B.pseudocatenulatum strains which can be stimulated by GOS,thereby,the selection methods of oligosaccharides and probiotics targeted at promoting intestinal growth and colonization of specific probiotics strain can be optimized.First of all,the responses of different Bifidobacterium species to GOS were analyzed through the human test-meal experiment of GOS.Ten healthy adult volunteers were recruited to participate in the GOS intervention trial(give GOS with 10 g/d for 4 weeks),eight volunteers have completed the trial.Faeces were collected at the end of the baseline period,2 weeks and 4 weeks of GOS intervention.Intestinal microflora diversity and Bifidobacterium species level were analyzed based on the Illumina Mi Seq sequencing technique to explore the effect of GOS on human intestinal flora.The results showed that GOS significantly increased the abundance of Acfinobacteria phylum and Bifidobacterium genus in the intestines of healthy adults,but the responses of B.pseudocatenulatum in the intestines of different individuals were different to GOS.Therefore,four B.pseudocatenulatum strains were isolated from the feces of volunteers for follow-up experiments,three strains came from three responders with increased abundance of B.pseudocatenulatum,and one strain came from one non-responder with decreased abundance of B.pseudocatenulatum.In order to analyze the reasons for the different responses to GOS in vivo of four strains as stated above,the growth characteristics and genomic characteristics of the strains those responded to GOS were explored via single bacteria culture experiments in vitro and genomes comparative analysis.The growth characteristics of four strains in the culture medium with GOS as carbon source indicated that the strains responding in vivo could more adapt to the nutritional environment contained GOS,their utilization ability of GOS of was stronger.The growth was larger in the stable period of growth curve(up to 32.84%),and the generation time was shorter(up to 27.58%).The genomic information of four strains was obtained by whole genomes sequencing.After genomes comparative analysis,it was exhibited that the potential pathway of B.pseudocatenulatum using GOS was as follows:Extracellular endo-?-galactosidase(Gal A)preliminarily hydrolyzed GOS,then permease(Lac S)transported GOS into the cell,and finally intracellular?-galactosidase(Lac Z)further degraded GOS.Subsequently,compared the carbohydrate active enzyme genes of four strains,it was revealed that the number of carbohydrate esterase(CE1 and CE10)genes of the strains that responding in vivo was more,carbohydrate esterase may assist extracellular or intracellular?-galactosidase to degrade GOS,thus increasing the overall utilization ability of GOS.Then,the correlation between carbohydrate esterase(CE1 and CE10 families)and the utilization ability of GOS was verified.The strains with different numbers of genes encoding CE1 and CE10 were selected to investigate the ability to utilize GOS in single cell system,mixed bacteria system and fecal system.In addition,analyzed the differences of GOS structural were consumed and related genes transcription levels of strains when using GOS.B.pseudocatenulatum FGZ28MM6(CE1:9 and CE10:8)and B.pseudocatenulatum FSDWF3M4(CE1:3 and CE10:4)were selected from our own strains bank for experiments.The results showed that strain FGZ28MM6 had stronger utilization ability of GOS,higher growth in stable phase of growth curve(2.11 times as much as FSDWF3M4),more GOS compounds with high degree of polymerization consumed within the same time.Compared with the use of glucose,the relative expression of gene 3872(encoding CE1)and gene 3878(encoding CE10)increased significantly(3.94 times and 4.86 times,respectively).In addition,the absolute number of strains FGZ28MM6 was bigger in the process of fermentation of GOS in mixed bacteria system and fecal system.Finally,based on the genomic characteristics and the experimental results in vitro,the strains with differences in the number of related genes and the ability of GOS utilization(B.pseudocatenulatum 1M25 and FSDWF3M4)were selected for animal experiments to evaluate the effect of GOS on intestinal colonization and metabolic activity of the strains.The results showed that GOS could increase the colonization intensity and prolong the colonization time of strain 1M25(the number of bacteria cells increased by 10~2 CFU/g feces,the colonization time was more than 14 days).GOS could also improve the metabolic activity of strain 1M25(the contents of acetic acid,propionic acid and total short chain fatty acids in mouse feces were increased).However,for strain FSDWF3M4,GOS neither increased its colonization intensity nor prolonged its colonization time(about 1 day of colonization).What's more,GOS had no effect on the metabolic activity of strain FSDWF3M4.Overall,this study showed that there were differences in the stimulating effect of GOS on the growth of B.pseudocatenulatum in different hosts intestines.It was found that the ability of the strains isolated from different hosts to utilize GOS was different,which may be related to the genes encoding carbohydrate esterase(CE1 and CE10).GOS could improve the colonization intensity,colonization persistence and metabolic activity of specific B.pseudocatenulatum strains. |
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