| As an important component of diet,dietary fiber can’t be digested by digestive enzymes in the small intestine,and they are mainly used by intestinal microorganisms as fermentation substrate in the hindgut.They could stabilize intestinal environment and improve body health.Dietary fibers with different physical and chemical properties have different effects on the intestinal environment.Relevant studies showed that,as three different types of common dietary fibers,resistant starch,inulin and pectin can affect the composition and function of intestinal microbiota at different degrees.However,most of the current studies focus on the influence of dietary fiber on microbiota composition only,and few studies involving comparative analysis of both the composition and function of microbiota.Therefore,our study took the metatranscriptome high-throughput sequencing method to clarify the composition and function of pig colonic microbiota in response to different dietary fibers.The paper includes the three parts as follow:1.Effects of different dietary fibers on colonic microbiota composition in pigsThe purpose of this experiment was to compare the effects of different dietary fibers on colonic microbiota composition in growing pigs.Twenty-eight 35-day-old Duroc ×Landrace × Large White growing pigs with similar weight(8.79 ± 0.09 kg)were selected for this experiment,and they were randomly divided into 4 groups according to the principle of no difference in body weight: control group(CON),raw potato starch group(RPS),inulin group(INU)and pectin group(PEC),with 7 replicates in each group.Control group was fed corn-soybean meal basal diet,and experimental groups replaced 8% corn starch with raw potato starch,inulin and pectin,respectively.The experimental period is 40 days.after the period,all growing pigs were slaughtered and colonic chyme was collected and stored at-80°C for intestinal microbiota analysis.Extracted the RNA of the samples for metatranscriptome sequencing analysis.Compared the metatranscriptome sequencing results with 16 S r RNA gene sequencing results in our previous study,we found that metatranscriptome sequencing can observe more differences in the composition of the colonic microbiota in dietary fiber groups.The PCo A analysis of 16 S r RNA gene sequencing showed that microbiota composition of colonic chyme changed significantly in the pectin group only,while raw potato starch group and inulin group were not separated from the control group significantly.But the PCo A analysis of metatranscriptome sequencing showed that the inulin group and the pectin group were both different from the control group significantly.The metatranscriptome sequencing results showed that all three types of dietary fiber changed the composition of colonic microflora significantly(FDR <0.05).Compared with the control group,at the phylum level,the relative abundance of Verrucomicrobia reduced significantly(FDR < 0.05)in the raw potato starch group;and the relative abundance of Verrucomicrobia was also reduced significantly(FDR < 0.05)in the inulin group,the relative abundance of Fusobacteria and Actinobacteria increased significantly(FDR < 0.05)in the inulin group;and the relative abundance of Proteobacteria,and Spirochaetes increased significantly(FDR < 0.05)in the pectin group.While the 16 S r RNA gene sequencing results only showed that the addition of pectin changed the composition of colonic microbiota significantly(P < 0.05),and the addition of raw potato starch and inulin had no significant effect on the composition of colonic microbiota of growing pigs(P > 0.05).Furthermore,and changes observed in the 16 S r RNA gene sequencing results,that the relative abundance of Firmicutes increased significantly(P <0.05)and the abundance of Bacteroidetes decreased significantly(P < 0.05)in the pectin group,were not observed in the metatranscriptome sequencing results.At the genus level,the addition of raw potato starch significantly increased the relative abundance of Parabacteroides and Faecalibacterium(FDR < 0.05),while significantly reduced the relative abundance of 9 genera(FDR < 0.05),including Sutterella and Acinetobacter.The addition of inulin significantly increased the relative abundance of Fusobacterium and Rhodococcus(FDR < 0.05),and significantly reduced the relative abundance of 13 genera(FDR < 0.05),including Bacillus and Fusicatenibacter.The addition of pectin significantly increased the relative abundance of Streptococcus and Bacteroidetes＿norank(FDR < 0.05),and significantly reduced the relative abundance of 21 genera(FDR < 0.05),including Clostridium and Intestinibacter.In the metatranscriptome sequencing results,the number of changed microbiota in each fiber group was greater than that in the corresponding 16 S r RNA gene sequencing results.There was no overlap in the changes of microbiota in these two sequencing results,but they both showed that pectin had greater effect on colonic microbiota composition.The results showed that dietary fibers could affect the colonic microbiota composition in growing pigs,and compared with raw potato starch and inulin,pectin had a greater impact on colonic microbiota composition in growing pigs.2.Effects of different dietary fibers on the annotation of carbohydrate active enzymes of colonic microbiota in pigsThe purpose of this experiment was to compare and analyze the effects of three different types of dietary fibers on the gene expression of carbohydrate active enzymes of colonic microbiota in growing pigs.The experimental design and sample collection as the same with the first experiment.After metatranscriptome sequencing,compared the obtained gene set data with the carbohydrate active enzyme database to get the corresponding annotation information of carbohydrates active enzyme,then compared and analyzed the annotation results.Evaluate the gene expression difference of carbohydrate activity enzyme between experimental groups and control group according to FC.FC > 2or < 0.5,FDR < 0.05 used as screening criteria.The results showed that,the gene expression of 21 carbohydrate active enzymes down-regulated significantly in the raw potato starch group compared with the control group,such as AA7,CBM26 and GH101,while 9 carbohydrate active enzymes up-regulated significantly,such as CBM21,GH128 and GT10.In the inulin group,the gene expression of 14 carbohydrate active enzymes down-regulated significantly,including AA4,CBM41 and GH14.And in the pectin group,the gene expression of 22 carbohydrate active enzymes down-regulated significantly,including AA1,CBM21 and GH112,and the gene expression of 13 carbohydrate active enzymes up-regulated significantly,including AA12,CBM9 and CE14.The changes of these enzymes were also correlated with the abundance of the microbiota.Using Spearman’s correlation coefficient > 0.5 or <-0.5,FDR<0.05 as the screening criteria,we found that the relative gene expression of CBM21 and GT91 was negatively correlated with the relative abundance of Sutterella,and Faecalibacterium was the main microorganism encoding CBM74 and GH38.Streptococcus was the main contributor to CBM61,and CBM61 can effectively promote the fermentation of pectin in the colon.The results showed that the addition of three different types of dietary fiber significantly affected the gene expression of microbial carbohydrate active enzymes in the colon of growing pigs,and the expression of carbohydrate active enzymes was also affected by changes of the microbiota composition.The effects of raw potato starch and pectin on the microbiota function were more diverse than those in the inulin group,involving the promotion of degradation of starch and pectin substrates.The gene expression of enzymes in the inulin group were all significantly down-regulated,which may have a negative effect on the degradation of carbohydrates.3.Effects of different dietary fibers on KEGG pathway of colonic microbiota in pigsThe purpose of this experiment was to compare and analyze the effects of three different types of dietary fiber on the KEGG pathway of the colonic microbial in growing pigs.Based on the metatranscriptome sequencing results,the predicted genes were compared with the KEGG database,obtained the gene expression of 374 different KEGG pathways in different diet fiber treatment groups and control groups.Compared the gene expression of KEGG pathways in the different fiber groups with the control group,and FC > 2 or < 0.5,FDR < 0.05 was used as the screening condition.The results showed that,compared with the control group,91 functional categories changed significantly in the raw potato starch group,of which only the Biosynthesis of siderophore group nonribosomal peptides pathway was up-regulated,and the rest of the pathways showed a downward-regulated trend,the number of down-regulated pathways was larger than up-regulated pathways,and the down-regulated pathways mainly concentrated in Proteoglycans in cancer,Vibrio cholerae infection,Secondary bile acid biosynthesis,etc.In the inulin group,88 metabolic pathways were significantly affected,of which 3 pathways were up-regulated and 85 pathways were down-regulated,and the number of down-regulation was also larger than up-regulation.The functions of down-regulation are mainly concentrated in Fat digestion and absorption,Carbohydrate digestion and absorption,Styrene degradation,etc.There were 81 functional categories affected significantly in the pectin group,of which 33 were significantly up-regulated and 48 were significantly down-regulated.There were also more down-regulated pathways than up-regulated pathways.The up-regulated pathway functions were mainly concentrated in Polycyclic aromatic hydrocarbon degradation and Dioxin degradation,etc.While the down-regulated pathways were mainly concentrated in Vitamin digestion and absorption,Carbohydrate digestion and absorption,etc.The results showed that the intake of dietary fiber could make the colonic microbiota have different digestive functions,and the addition of raw potato starch and inulin down-regulated the gene expression of many KEGG pathway,reduced the metabolic activity of the microbiota,while the addition of pectin could change metabolism of the microflora more diversified,which affected the degradation of harmful substances and the digestion and absorption of nutrients,then benefits and negative effects coexist.In summary,the research in this paper showed that the composition and function of the colonic microbiota of pigs were responsive to three different dietary fibers.Compared with raw potato starch and inulin,the addition of 8% pectin had a greater impact on the colonic microflora composition and function.In addition,the impact of pectin on the function of the microflora was more diverse,involving the degradation of harmful substances and the digestion and absorption of nutrients.The raw potato starch group and inulin group had simpler impacts compared with the pectin group. |
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