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Fiber-rich foods affected gut bacterial community and short-chain fatty acids production in pig model
Journal of Functional Foods 57 (2019) 266–274
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Journal of Functional Foods journal homepage: www.elsevier.com/locate/jff
Fiber-rich foods affected gut bacterial community and short-chain fatty acids production in pig model Jinbiao Zhao, Yu Bai, Shiyu Tao, Gang Zhang, Junjun Wang, Ling Liu, Shuai Zhang
T
⁎
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacterial community Fiber-rich foods Gut health Pig model Short chain fatty acids
This study aimed to explore effects of different fiber-rich foods on bacterial community and their metabolites in pig model. Thirty-six pigs were allotted to 6 dietary treatments including corn bran (CB), wheat bran (WB), oat bran (OB), soybean hulls (SH), sugar beet pulp (SBP) and rice bran (RB). The results demonstrated that lactate was mainly produced in the foregut, whereas propionate and butyrate were generated in the hindgut of pig. The ileal acetate and fecal butyrate production were positively correlated with the dietary acid detergent fiber and cellulose content, respectively. Different dietary fiber supplementation altered Filifactor and Intestinibacter abundances in ileal digesta and Ruminococcus_1 and Lachnoclostridium populations in feces of pigs. The differences in fiber fermentation and SCFAs production in ileal and fecal samples were associated with microbial compositions. Overall, this study would help to better understand how to promote the gut health through different fiber-rich foods intake in humans.
1. Introduction Some recent studies have reported that dietary fiber played an important role in improving gut health of human and animals, since it not only improved the epithelial barrier function of intestine (Chen et al., 2013; Liu, Ivarsson, Dicksved, Lundh, & Lindberg, 2012), but also maintained the homeostasis of intestinal microenvironment in host by modulating gut microbiota (Liu et al., 2012; Ma et al., 2018). Fiberdeprived microbiota was reported to impair the barrier function of colonic mucosa and increased the susceptibility of pathogens (Desai et al., 2016). Dietary fiber could be degraded by gut microbiota to generate short-chain fatty acids (SCFAs), which could induce the expression of porcine host defense peptides to improve the immune function and gut health (Koh, De Vadder, Kovatcheva-Datchary, & Backhed, 2016; Zeng et al., 2013). Therefore, it is necessary to include moderate level of fiber into diets to regulate the gut health of the host. However, different dietary fibers may play different roles in modualting bacterial community of the host considering their diverse physicochemical properties and compositions. Previous studies have reported that dietary fibers was mainly fermented in the hindgut of human and other monogastric animals (Jaworski & Stein, 2015; Williams, Verstegen, & Tamminga, 2001).
However, recent studies showed that substantial fiber fractions were also degraded in the foregut of monogastric animals (Jha & Leterme, 2012; Jha, Rossnagel, Pieper, Van Kessel, & Leterme, 2010). Moreover, little information is known on how dietary fibers affect the bacterial community and SCFAs production in the foregut. Therefore, the objective of this study was to test the hypothesis that different dietary fibers could alter the microbial community and SCFAs production in pig model, and to determine the effects of dietary fiber on microbial community in foregut and hindgut of pigs. 2. Materials and methods 2.1. Ethics The animal handling and all procedures of this study received approval from the Animal Care and Use Ethics Committee of the China Agricultural University (Beijing). 2.2. Animals, diets and experimental design Thirty-six Duorc × (Landrace × Yorkshire) growing barrows were surgically fitted with T-cannula in the distal ileum. Pigs were allotted to
Abbreviations: ADF, acid detergent fiber; ADL, acid detergent lignin; CB, corn bran; IDF, insoluble dietary fiber; NDF, neutral detergent fiber; OB, oat bran; OTUs, operational taxonomic units; RB, rice bran; SBP, sugar beet pulp; SCFAs, short-chain fatty acids; SDF, soluble dietary fiber; SH, soybean hulls; TDF, total dietary fiber; WB, wheat bran ⁎ Corresponding author. E-mail address: [email protected] (S. Zhang). https://doi.org/10.1016/j.jff.2019.04.009 Received 22 February 2019; Received in revised form 2 April 2019; Accepted 4 April 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 The chemical compositions of the fibrous ingredients used in this study (%, as fed basis). Items
Corn bran
Wheat bran
Soybean hulls
Rice bran
Sugar beet pulp
Oat bran
Neutral detergent fiber Acid detergent fiber Lignin Cellulose Hemicellulose Total dietary fiber Soluble dietary fiber Insoluble dietary fiber Starch
52.0 16.2 2.2 11.9 35.8 65.1 8.0 57.1 17.0
33.6 9.7 2.0 12.8 23.9 43.4 4.5 38.9 11.5
61.7 44.4 1.7 29.4 17.3 67.1 13.5 63.7 5.6
44.0 26.1 9.7 16.4 17.9 32.4 5.9 26.5 26.1
37.7 21.2 6.0 19.7 16.6 69.8 27.4 42.5 6.6
39.1 8.8 3.0 4.3 30.3 46.0 18.0 27.8 25.9
All data are the results of chemical analysis conducted in duplicate.
6 dietary treatments in a completely randomized design with 6 replicated pigs per treatment according to their body weight. The initial average body weight of the pigs was 48.5 ± 2.1 kg. The treatment diets consist of the same basal diet supplied with 6 different high-fiber food by-products, including corn bran (CB), wheat bran (WB), oat bran (OB), soybean hulls (SH), sugar beet pulp (SBP) and rice bran (RB), respectively. These high-fiber ingredients were the sole source of fiber components included in the experiment diets, and the level of total dietary fiber (TDF) were equal among all 6 treatment diets. Vitamins and minerals were supplemented to meet or exceed the nutrient requirements for growing pigs as recommended by the NRC (2012). The experimental period lasted for 21 days, including 15 days for dietary adaptation, 3 days for fecal collection and 3 days for digesta collection. The chemical components of the 6 fiber-rich ingredients were showed in Table 1. And the ingredients and chemical compositions of the 6 dietary treatments were showed in Supplementary Table 1. Before starting the animal trial, all pigs were fed pellet feed for 2 weeks, and then surgically fitted with T-cannula in the distal ileum referring to the procedures from Stein, Shipley, and Easter (1998). After the surgery, pigs were fed sucrose solution for 1 day and pellet feed for 2 weeks for recovery. During the trial, pigs were fed in individual crates (1.5 × 1.2 × 1.0 m) in an environmentally controlled room (20 °C ± 2 °C) with slatted floors, a self-feeder, and a nipple waterer. Pigs were provided ad libitum access to water, and fed a daily amount of treatment diet equivalent to 4% of their BW determined at the beginning of the animal trial. The diet fed everyday were divided into 2 equal meals and provided at 0900 and 1600 h each day.
The SCFA concentrations in the ileal digesta and fecal samples were analyzed according to the method described by Porter and Murray (2001). Briefly, about 1.0 g fecal samples were put into 10 mL centrifuge tube with 2.0 mL 0.10% hydrochloric acid addition, then incubated on ice for 25 min, mixed and centrifuged at 15,000 rpm. The supernate was obtained, and then filtered using a 0.20 mm Nylon Membrane Filter (Millipore, Bedford, OH, USA) and poured into a Gas Chromatograph System (Agilent HP 6890 Series, Santa Clara, CA, USA) for SCFA determination.
2.3. Samples collection
2.6. Statistical analysis
Ileal digesta and fresh fecal samples of pigs in each dietary treatment group were collected to determine the SCFA concentrations and bacterial community. Fresh fecal and ileal digesta samples from each pig were collected following the procedures described by CervantesPahm and Stein (2008). All samples were collected using 5 mL centrifuge tubes and placed into liquid nitrogen, and then stored at −80 °C.
Data for SCFA concentrations were checked for normality and outliers using the UNIVERIATE procedure of SAS (version 9.2, SAS Inst. Inc., Carry, NC, USA). Then data were analyzed using the procedure of PROC GLM. The treatment diet was the only fixed effect and the individual pig was treated as the experimental unit. The LSMEANS statement of SAS was used to separate treatment means, with Turkey’s test for adjustment. Correlation coefficients between dietary fiber components and SCFA concentrations were analyzed using the PROC CORR procedure of SAS. The diversity metrics of bacteria were represented from normalized Operational Taxonomic Units (OTU) reads. Bar plots on phylum and genus levels were generated to visualize the taxonomic distribution. Analyses for microbiota composition on relative abundant on the phyla and genera levels were conducted using the Kruskal-Wallis method. Principal coordinate analysis (PCoA) was used to compare the microbial community between ileal digesta and fecal samples using the unweighted UniFrac distances. Significant differences were considered when P < 0.05, and 0.05 ≤ P < 0.10 was considered as a tendency.
2.5. Microbial detection The DNA Kit (Omega Bio-tek, Norcross, GA, USA) was used to extract the DNA of bacteria in the samples of digesta and feces. The PCR amplification technique was used to amplify the gene of bacterial 16S ribosomal RNA in V4-V5 region. And then amplicons were extracted, purified and quantified. Purified amplicons were pooled in equimolar and performed double-end sequencing using Illumina MiSeq platform. The sequences that its overlap is more than 10 bp were assembled. Operational taxonomic units (OTUs) were clustered with 97% similarity. Abnormal sequences were detected and removed using USEARCH soft (vsesion 7.0, http://drive5.com/uparse/). The RDP Classifier (http://rdp.cme.msu.edu/) against the silva (SSU115) 16S rRNA database was used to analyze the taxonomy of each 16S rRNA gene sequence using confidence threshold of 90%.
2.4. Chemical analysis and calculation According to the methods of AOAC (2007), soluble dietary fiber (SDF), insoluble dietary fiber (IDF) and TDF in all 6 ingredients and diets were analyzed using the Ankom Dietary Fiber Analyzer (Ankom Technology, Macedon, NY, USA). Neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) of ingredients and diets were detected using the filter bags (Model F57; Ankom Technology) and fiber analyzer equipment (ANKOM200 Fiber Analyzer, Ankom Technology) according to a modified procedure described by Van Soest, Robertson, and Lewis (1991). Based on the analyzed results, cellulose and hemicellulose contents were calculated as following:
Cellulose = ADF − ADL
Hemicellulose = NDF − ADF 267
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Table 2 Effects of different dietary fibers on the short-chain fatty acid concentrations in the intestine of pigs. Items
Diets Wheat bran
Corn bran
Sugar beet pulp
Oat bran
Soybean hulls
Rice bran
Ileal digesta Lactate Acetate Propionate Butyrate Total
4.62 ± 0.74ab 4.43 ± 0.98b 0.99 ± 0.31 0.09 ± 0.03 11.22 ± 1.31a
4.26 ± 0.82ab 5.20 ± 1.01ab 0.96 ± 0.25 0.19 ± 0.08 11.51 ± 1.42a
2.54 ± 0.63bc 6.17 ± 0.86a 0.98 ± 0.23 0.27 ± 0.11 10.97 ± 0.88a
6.33 ± 1.01a 3.64 ± 0.89b 0.87 ± 0.19 0.28 ± 0.09 12.25 ± 1.33a
3.60 ± 0.52b 6.39 ± 1.05a 1.34 ± 0.42 0.32 ± 0.15 12.21 ± 1.18a
1.59 4.61 1.14 0.23 8.32
Feces Lactate Acetate Propionate Butyrate Valerate Total
0.45 6.45 2.71 1.67 0.46 13.8
0.55 6.63 3.27 1.42 0.24 13.7
0.62 ± 0.16 8.81 ± 1.44b 3.49 ± 1.41b 2.57 ± 1.02b 0.75 ± 0.45b 18.09 ± 2.04b
0.44 6.58 2.15 1.74 0.67 13.5
0.70 ± 0.21 11.29 ± 2.01a 7.72 ± 1.94a 4.57 ± 1.22a 2.22 ± 0.63a 27.94 ± 2.62a
0.74 ± 0.25 5.89 ± 0.72c 1.66 ± 0.56c 0.86 ± 0.33b 0.24 ± 0.11b 10.87 ± 1.12d
± ± ± ± ± ±
0.11 1.24c 0.81bc 0.31b 0.28b 1.85c
± ± ± ± ± ±
0.04 1.57c 1.66b 1.35b 0.12b 1.97c
± ± ± ± ± ±
0.19 0.65c 0.68c 0.46b 0.49b 1.36c
± ± ± ± ±
P-value
0.45c 0.95b 0.38 0.09 0.79b
0.01 0.01 0.72 0.83 0.01 0.62 0.01 0.01 0.01 0.01 0.01
The results were presented as mean values ± SEM (n = 6 per treatment). Means in a row without a common superscript differ, P < 0.05.
NDF content (P < 0.10, r = −0.75), and the total SCFA concentration was negatively correlated with the hemicellulose content (P < 0.05, r = −0.81). However, the acetate concentration tended to positively correlate with the dietary NDF content (P < 0.10, r = 0.76), and was positively correlated with the dietary ADF content (P < 0.05, r = 0.80). In fecal samples, both acetate and butyrate concentrations tended to positively correlate with the dietary ADF content (P < 0.10, r = 0.71 and r = 0.75). In addition, dietary cellulose content tended to positively correlate with the acetate concentration (P < 0.10, r = 0.71), and was positively correlated with the butyrate concentration (P < 0.05, r = 0.80). Moreover, the propionate and total SCFA concentrations tended to positively correlate with the dietary IDF (P < 0.10, r = 0.75) and ADF (P < 0.10, r = 0.74) contents, respectively.
3. Results 3.1. The SCFA production The SCFA concentration in the ileal digesta and fecal samples were presented in Table 2. Lactate was mainly produced in the foregut, and propionate and butyrate were more produced in the hindgut of pigs. In ileal digesta samples, pigs fed the OB, SH, WB or CB diets had greater (P < 0.05) lactate content compared with pigs fed the RB diet. Pigs fed the SBP or SH diets had greater (P < 0.05) acetate content compared with pigs fed the WB, OB or RB diets. In addition, the total SCFA concentrations in ileal digesta of pigs fed the RB diet was lower (P < 0.05) than those fed the WB, CB, SBP, OB or SH diets. There were no significant differences in the propionate and butyrate contents in ileal digesta of pigs fed the 6 different dietary treatments. In fecal samples, pigs fed the SH diets showed greater (P < 0.05) acetate, propionate, butyrate and valerate contents compared with pigs fed the WB, CB, SBP, OB or RB diets. Moreover, the total SCFA concentrations in fecal samples of pigs fed the SH diet was the greatest (P < 0.05), while the total SCFA concentrations in fecal samples of pigs fed the RB diet was the lowest (P < 0.05) among the 6 dietary treatments.
3.3. Differences in microbial community among dietary treatments The ileal digesta samples of pigs fed diets containing WB, CB, RB, SH, SBP and OB had a total of 435, 332, 469, 412, 530 and 365 OTUs, respectively, which included 14, 7, 35, 16, 87 and 13 individual bacteria, respectively (Supplemental Fig. 1A). The fecal samples of pigs fed diets containing WB, CB, RB, SH, SBP and OB had a total of 896, 903, 888, 837, 925 and 899 OTUs, respectively, which contained 10, 27, 10, 13, 13 and 17 individual bacteria, respectively (Supplemental Fig. 1B). At the phylum level, 7 and 10 microbial communities in ileal digesta (Fig. 1A) and fecal samples (Fig. 1B) of pigs were detected, and the abundances of bacteria in phyla were not different among the 6 dietary treatments. At the genus level, the microbial composition and
3.2. Correlation between dietary fiber components and SCFA production The correlation coefficients between dietary fiber components and SCFA concentrations in ileal digesta samples and fecal samples of pigs were present in Tables 3 and 4, respectively. In ileal digesta samples, the lactate concentration tended to negatively correlate with the dietary
Table 3 Correlation coefficients between dietary fiber compoments and short-chain fatty acids (SCFAs) produced in ileal digesta after dietary fiber ingestion by pigs. Items
Lactate
Acetate
Propionate
Butyrate
Total SCFA
NDF
ADF
Lignin
Cellulose
Hemicellulose
TDF
SDF
IDF
Lactate Acetate Propionate Butyrate Total SCFA NDF ADF Lignin Cellulose Hemicellulose TDF SDF IDF
1 −0.53 −0.58 −0.14 0.61 −0.75* −0.24 −0.32 0.17 −0.65 0.01 −0.03 0.05
1 0.78* 0.42 0.33 0.76* 0.80** 0.27 0.55 −0.10 −0.20 −0.52 0.60
1 0.21 0.15 0.43 0.37 −0.25 0.24 0.06 0.08 0.42 0.69
1 0.21 0.24 0.66 0.03 0.41 −0.60 0.64 0.43 −0.16
1 −0.16 0.44 −0.19 0.03 −0.81** −0.14 −0.54 0.68
1 0.72 0.77* 0.70 0.31 −0.35 −0.26 0.13
1 0.52 0.41 −0.43 −0.05 −0.21 0.29
1 0.84** 0.28 −0.53 −0.11 −0.26
0.31 −0.62 −0.09 −0.33 0.49
1 −0.39 −0.05 −0.23
1 0.78* −0.35
1 −0.85**
1
ADF, acid detergent fiber; IDF, insoluble dietary fiber; NDF, neutral detergent fiber; SDF, soluble dietary fiber; TDF, total dietary fiber. ** P < 0.05. * P < 0.10. 268
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Table 4 Correlation coefficients between dietary fiber compoments and short-chain fatty acids (SCFAs) produced in feces after dietary fiber ingestion by pigs. Items
Lactate
Acetate
Propionate
Butyrate
Valerate
Total SCFA
NDF
ADF
Lignin
Cellulose
Hemicellulose
TDF
SDF
IDF
Lactate Acetate Propionate Butyrate Valerate Total SCFA NDF ADF Lignin Cellulose Hemicellulose TDF SDF IDF
1 0.43 0.38 −0.40 0.39 0.38 0.56 0.38 −0.01 −0.06 0.25 0.36 0.16 0.03
1 0.98** −0.90** 0.98** 0.99** 0.28 0.71* −0.21 0.71* −0.62 0.28 −0.28 0.66
1 −0.81** 0.97** 0.99** 0.17 0.60 −0.33 0.64 −0.61 0.23 −0.37 0.75*
1 0.96** 0.99** 0.29 0.75* −0.14 0.80** −0.67 0.22 −0.32 0.67
1 0.98** 0.12 0.60 −0.36 0.66 −0.66 0.43 −0.16 0.61
1 0.23 0.68 −0.25 0.74* −0.63 0.27 −0.3 0.69
1 0.72* 0.77* 0.41 0.31 0.35 −0.26 0.13
1 0.52 0.84** −0.43 −0.05 −0.21 0.29
1 0.32 0.28 −0.53 −0.1 −0.26
1 −0.62 −0.09 −0.33 0.49
1 −0.4 −0.05 −0.23
1 0.78* −0.35
1 −0.85**
1
ADF, acid detergent fiber; IDF, insoluble dietary fiber; NDF, neutral detergent fiber; SDF, soluble dietary fiber; TDF, total dietary fiber. ** P < 0.05. * P < 0.10.
Fig. 1. Microbial diversity on the phylum and genus levels in pigs fed 6 fibrous dietary treatments. (A) Barplot for microbial community on the phylum level with the abundance greater than 0.01% in the ileal digesta samples of pigs. (B) Barplot for microbial community on the phylum level with the abundance greater than 0.01% in the fecal samples of pigs. (C) Barplot for microbial community on the genus level with the abundance greater than 1% in the ileal digesta sample of pigs. (D) Barplot for microbial community on the genus level with the abundance greater than 1% in the fecal samples of pigs. All data were analyzed by Kruskal-Wallis test and presented as mean percentage of different bacteria (n = 4 per treatment). The RB_D, WB_D, CB_D, SH_D, SBP_D and OB_D respresented the ileal digesta samples from pigs fed diets containing rice bran, wheat bran, corn bran, soybean hulls, sugar beet pulp and oat bran, respectively. The RB_F, WB_F, CB_F, SH_F, SBP_F and OB_F respresented the fecal samples from pigs fed diets containing rice bran, wheat bran, corn bran, soybean hulls, sugar beet pulp and oat bran, respectively. 269
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Fig. 2. Effects of dietary fibers ingestion on bacterial community of pigs on the genus level. (A) (B) The abundance of bacteria in the ileal digesta. (C) (D) The abundance of bacteria in the feces. All data were analyzed by Kruskal-Wallis test and presented as mean percentage of different bacteria (n = 4 per treatment), and different letters means P < 0.05. The RB, WB, CB, SH, SBP and OB respresented pigs fed diets containing rice bran, wheat bran, corn bran, soybean hulls, sugar beet pulp and oat bran, respectively.
compared, and the results indicated that Firmicutes and Proteobacteria were the dominant bacteria in ileal digesta of pigs, and Firmicutes was the dominant bacteria in feces of pigs. At the phylum level, the abundance of Firmicutes (Fig. 4A) and Bacteroidetes (Fig. 4C) in fecal samples were greater (P < 0.05) than those in ileal digesta samples, and the population of Proteobacteria (Fig. 4B) was lower (P < 0.05) in fecal samples compared with that in ileal digesta samples. At the genus level, Escherichia, Acinetobacter and Enterobacteriaceae were the top three genera in population in small intestine, while the dominant genus in fecal sample was Clostridiales. The abundance of Lactobacillus (Fig. 4D) and Escherichia-Shigella (Fig. 4F) in fecal samples were lower (P < 0.05) than that in ileal digesta sample, and the population of Bifidobacterium (Fig. 4E) in fecal samples was greater (P < 0.05) compared to that in ileal digesta samples.
community in leal digesta (Fig. 1C) and fecal samples (Fig. 1D) of pigs were also presented. In ileal digesta samples, the abandance of Filifactor in WB group was greater (P < 0.05) than that in OB and RB groups (Fig. 2A), and the abundance of Intestinibacter in SBP and SH groups were greater (P < 0.05) than that in WB, CB and OB groups (Fig. 2B). In fecal samples, the population of Ruminococcus_1 in SH group was greater (P < 0.05) than that in WB, CB and OB groups (Fig. 2C), and the population of Lachnoclostridium was greater (P < 0.05) in SH group than that in the other treatment groups (Fig. 2D). 3.4. Differences in microbial community between ileal digeata and fecal samples The richness and biodiversity of microbial community in ileal digesta and fecal samples were presented as the α-diversity indices of Chao and Shannon. The result showed that the Shannon (Fig. 3A) and Chao (Fig. 3B) indexes of microbial community in the ileal digesta samples were lower (P < 0.05) than those in the fecal samples. The βdiversity of microbial community between ileal digesta and fecal samples were compared using PCoA, and the result showed different clustering of microbial communities (Fig. 3C). Furthermore, the microbial community between ileal digesta and fecal samples at the phylum (Supplemental Fig. 2A) and genus (Supplemental Fig. 2B) levels were
4. Discussion 4.1. The SCFA concentrations produced by dietary fibers fermentation Dietary fiber fermentation results in the production of SCFAs, including lactate, acetate, propionate and butyrate, and some gases such as hydrogen, carbon dioxide and methane (Macfarlane & Macfarlane, 1993; Williams et al., 2001). Among all the SCFAs produced in the 270
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Fig. 3. Bacterial α-diversity and β-diversity in ileal digesta and fecal samples of pigs. (A) The Shannon index of bacterial community. (B) Chao index of bacterial community. The results were analyzed by wilcoxon rank-sum test and different letters means P < 0.05 (n = 4). (C) The β-diversity of bacterial community.
in the small intestine, and could promote the development of Lactobacilli, a family of health-promoting bacteria. In addition, pigs fed the SBP and SH diets had greater acetate content in the ileal digesta compared with those fed the WB, OB, and RB diets. Those results could be attributed to the greater NDF and ADF concentrations in SBP and SH diets compared to WB, OB and RB diets, which also agreed with the positive correlation (or positive correlation tendency) between acetate concentration in ileal digesta and the dietary NDF and ADF contents. Therefore, the dietary fiber soucre could influence the amount and type of SCFAs produced in the gut. This was supported by the results of Carneiro, Lordelo, Cunha, and Freire (2008), who found higher acetate and lower butyrate production in the cecum of pigs when wheat bran was replaced by maize cobs in diets. In the current study, pigs fed the SH diet showed greater acetate, propionate, butyrate and valerate concentrations in fecal samples compared with pigs fed the other diets. This was consistent with the results of Freire, Guerreiro, Cunha, and Aumaitre (2000), who compared the effects of 20% dietary WB, SBP, SH or alfalfa meal inclusion on total SCFA concentrations in the cecum of pigs, and found that the
hindgut, acetate accounts for the largest proportation (about 60.0%), whereas propionate and butyrate account for smaller quantities (Lunn & Buttriss, 2007). This was consistent with our results. As the endproducts of dietary fiber fermentation, SCFAs could act as energy substrates for colonocytes, modulate satiety, and alleviate inflammation (Koh et al., 2016). Butyrate was reported to be beneficial to the host by promoting the proliferation of mucosal epithelial cells, increasing the differentiation of intestinal epithelial cells and enhancing the function of colonic barrier (Han et al., 2017; Morrison & Preston, 2016). Our results showed that lactate was mainly produced in the foregut, whereas butyrate was produced in the hindgut of pigs, which may be due to the specific microbial communities in different intestinal parts of pigs, such as Lactobacillus and Enterococcus in the foregut and Firmicutes in the hindgut. The lactate concentration in the ileal digesta of pigs fed the OB, WB and CB diets was greater than that fed the RB diet. This was consistent with the results of Bach Knudsen and Canibe (2000), who observed greater concentration of lactate in the ileum of cannulated pigs after feeding a diet supplemented with SDF from OB. As the major component of OB, the β-glycan could stimulate the lactate production 271
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Fig. 4. Analysis for differential bacteria between the ileal digesta and fecal samples of pigs. (A)–(C) Differential bacteria on the phylum level. (D)–(F) Differential bacteria on the genus level. The data were analyzed by wilcoxon rank-sum test and presented as mean percentage of different bacteria. Different letters means P < 0.05 (n = 24).
the different constituents in diets (Awati et al., 2005; Jha & Leterme, 2012; Jha et al., 2010). The source of dietary fiber could affect the digestion site and gut environment, thereby influencing the proliferation of microbiota in the gut (Hogberg & Lindberg, 2004). In the present study, Firmicutes was the dominant bacteria whose abundance exceeded 60% in fecal samples. Many previous studies showed that Firmicutes and Bacteroidetes were the two dominant phyla that account for about 90% of the gut microbiota in pigs (Liu, Wang, et al., 2018; Liu, Zhao, et al., 2018; Bian et al., 2016). Clostridial clusters XIVa and IV within the phylum of Firmicutes could digest fiber components to produce SCFAs, such as butyrate. The phylum of Bacteroidetes also contained bacteria that are capable of degrading fibers (Flint, Bayer, Rincon, Lamed, & White, 2008). Therefore, the abundance of Firmicutes and Bacteroidetes provide a reference for the potential fibre-degrading and butyrateproducing bacteria in the gut microbiota. Our results showed that the 6 dietary fibers had no influence on Firmicutes and Bacteroidetes abundances in the ileal digesta and feces of pigs. However, Mu et al. (2014) reported that the alfalfa meal diet incresed the abundances of Firmicutes and Bacteroidetes compared to the WB diet in piglets. Maier et al. (2017) also showed that the high-resistant starch diet, as a dietary fiber source, caused an increase in the Firmicutes to Bacteroidetes ratio and the abundances of some specific members of Firmicutes in the gut of human. Nevertheless, Ferrario et al. (2017) reported an increase in Bacteroidetes abundance and a concurrent reduction in Firmicutes abundance with the addition of inulin in diets as a dietary fiber source. These different effects of dietary fiber on the diversity of bacterial community could be ascribed to the different types of dietary fiber available for fermentation and the various gut environment of the host. In addition, we observed increased abundance of Ruminococcus_1 when pigs were fed the SH diet. Ruminococcus_1 was reported to be able to produce SCFAs through fermenting carbohydrates (Pryde, Duncan, Hold, Stewart, & Flint, 2002), which may explain the greatest SCFA production in pigs fed SH in our study. The abundances of Filifactor and Intestinibacter were altered by different fiber ingestion in our study, and these two bacteria
dietary SH inclusion increased the total SCFA concentration by 11.2%, 30.5% and 27.2% compared with the dietary WB, SBP and alfalfa meal inclusion, respectively. Our results mentioned above also suggested that SH is highly degraded in the cecum of pigs. This was in agreement with the high NDF and ADF digestibility of SH in cecum reported by the previous study (Freire et al., 2000). The variation in fermentability of the dietary fibers could be attributed to the differences in their chemical compositions and physicochemical properties such as bulk, viscosity, solubility, and waterholding capacity. In our study, lactate and total SCFA concentrations in ileal digesta tended to negatively correlate or negatively correlated with the dietary NDF and hemicellulose contents, respectively, which was due to that the major fermentation substrates are monosaccharides, and the formation of SCFAs from hemicellulose is negligible in pigs (Drochner, 1993). Moreover, in our study, there was a trend that both acetate and butyrate contents in feces were positively correlated with the dietary ADF and cellulose contents, and the propionate and total SCFA concentrations tended to positively correlate with the dietary IDF and cellulose contents, respectively. These may be because monosaccharides could be easily formed by cleaving homogeneous polysaccharides, which are the major components of ADF (Mu, Zhang, He, Smidt, & Zhu, 2014). This may also partly explain the greater SCFA production in pigs fed the SH diet, which contains greater ADF and cellulose components. 4.2. Microbial community induced by dietary fiber The gastrointestinal microbiota is a vast and dynamic ecosystem, which plays important roles in intestinal morphology, immunity development, digestion, and modulating host gene expression (Guo et al., 2008; Turnbaugh et al., 2006). The types of diet would influence the population and activity of bacteria in the gut (Bach Knudsen, Hedemann, & Laerke, 2012). According to the previous studies, dietary fiber was the major factor that could affect the gut environment among 272
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Conflict of interest
were associated with the periodontitis and type 2 diabetes diseases in human (Aruni, Chioma, & Fletcher, 2014; Wang & Jia, 2016). The population of Lachnoclostridium was also influenced by different dietary fiber supplementation in our study, and the Lachnoclostridium had a connection with the obesity of human (Amadou, Hosny, La Scola, & Cassir, 2016). From these reports, we may infer that dietary fibers may have potentials to modulate the metabolic diseases in human by shaping the composition of gut microbiota in the host. In the current study, Firmicutes was the dominant phylum in feces while Proteobacteria was the dominant phylum in ileal digesta, which was consistent with the previous report (Zhao et al., 2014). The feces of pigs contained larger proportion of Firmicutes than the ileal digesta, suggesting that the large intestine, instead of small intestine, may undertake more function on substrate fermentation (DiBaise et al., 2008; Flint et al., 2008). Escherichia-Shigella, Lactobacillus, Streptococcus and Enterococcus, which were dominant genera in the ileal digesta in our study (shown on Supplemental Fig. 2B), formed the major gap between fecal and intestinal microbiota. These results indicated that these 4 genus are correlated with the fermentation ability of the small intestine in pigs. The high Escherichia-Shigella and Streptococcus abundance in the ileal digesta in our study, which are always considered as pathogenic bacteria associated with infection and enteric disease of the host, also indicated that the pathogen invasion may enrich in the foregut. In the current study, the population of Firmicutes and Bacteroidetes in feces was greater than that in ileal digesta, indicating that dietary fiber was mainly degraded in the hindgut. The fermentation products by Lactobacillus and Enterococcus are mainly lactate, leading to more lactate production in the ileal digesta of pigs. As probiotics, both Lactobacillus and Enterococcus could improve the immune function and inhibit the adhesion of enteric pathogens to the host (Michail & Abernathy, 2002; Scharek et al., 2005; Varma, Dinesh, Menon, & Biswas, 2010). The indexes of Chao and Shannon in the ileal digesta samples were lower than that in the fecal samples, indicating more richness and biodiversity of microbial composition in feces. The β-diversity of microbial community showed different clustering of microbial communities between the ileal digesta and fecal samples, and the results were consistant with the previous study (Zhao et al., 2014), who reported that the PCA analysis clustered the microbial communities into three categories: communites of the small intestine, large intestine, and feces on the genus level.
There are no potential conflicts of interest in the research. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.04.009. References Amadou, T., Hosny, M., La Scola, M., & Cassir, N. (2016). “Lachnoclostridium bouchesdurhonense”, a new bacterial species isolated from human gut microbiota. New Microbe and New Infect, 13, 69–70. https://doi.org/10.1016/j.nmni.2016.06.015. AOAC (2007). Official methods of analysis of AOAC international. Gaithersburg, MD: AOAC International. Aruni, W., Chioma, O., & Fletcher, H. M. (2014). Filifactor alocis: The newly discovered kid on the block with special talents. Journal of Dental Research, 93, 725–732. https:// doi.org/10.1177/0022034514538283. Awati, A., Konstantinov, S. R., Williams, B. A., Akkermans, A. D. L., Bosch, M. W., Smidt, H., & Verstegen, M. W. (2005). 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Protein and Peptide Letters, 24, 388–396. https://doi.org/10.2174/ 0929866524666170220113312. Hogberg, A., & Lindberg, J. E. (2004). Influence of cereal non-starch polysaccharides on digestion site and gut environment in growing pigs. Livestock Production Science, 87, 121–130. https://doi.org/10.1016/j.livprodsci.2003.10.002. Jaworski, N. W., & Stein, H. H. (2015). Disappearance of nutrients and energy in the stomach and small intestine, cecum, and colon of pigs fed corn-soybean meal diets containing distillers dried grains with solubles, wheat middlings, or soybean hulls. Journal of Animal Science, 93, 1103–1113. https://doi.org/10.2527/jas2016.0752.
5. Conclusion Different dietary fiber sources altered the microbial community and the short-chain fatty acid production in pig model. The lactate was mainly produced in the foregut, whereas the propionate and butyrate were mainly generated in the hindgut of pig. The acetate concentration in the ileal digesta was positively correlated with the dietary ADF content, and the butyrate concentration in feces was positively correlated with the dietary cellulose content. Dietary fibers shaped the abundances of gut microbiota in the fecal samples of pigs, such as Filifactor, Intestinibacter, Ruminococcus_1 and Lachnoclostridium. This study would contribute to a better understanding of how to promote gut health by different fiber-rich foods intake in humans. 6. Ethics statement The animal handling and all procedures of this study received approval from the Animal Care and Use Ethics Committee of the China Agricultural University (Beijing). Acknowledgments This research was financially supported by the National Natural Science Foundation of China (Grant No. 31702121) and by the Chinese Universities Scientific Fund (Grant No. 2019TC120). 273
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Fiber-rich foods affected gut bacterial community and short-chain fatty acids production in pig model Jinbiao Zhao, Yu Bai, Shiyu Tao, Gang Zhang, Junjun Wang, Ling Liu, Shuai Zhang
T
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State Key Laboratory of Animal Nutrition, College of Animal Science and Technology, China Agricultural University, Beijing 100193, China
A R T I C LE I N FO
A B S T R A C T
Keywords: Bacterial community Fiber-rich foods Gut health Pig model Short chain fatty acids
This study aimed to explore effects of different fiber-rich foods on bacterial community and their metabolites in pig model. Thirty-six pigs were allotted to 6 dietary treatments including corn bran (CB), wheat bran (WB), oat bran (OB), soybean hulls (SH), sugar beet pulp (SBP) and rice bran (RB). The results demonstrated that lactate was mainly produced in the foregut, whereas propionate and butyrate were generated in the hindgut of pig. The ileal acetate and fecal butyrate production were positively correlated with the dietary acid detergent fiber and cellulose content, respectively. Different dietary fiber supplementation altered Filifactor and Intestinibacter abundances in ileal digesta and Ruminococcus_1 and Lachnoclostridium populations in feces of pigs. The differences in fiber fermentation and SCFAs production in ileal and fecal samples were associated with microbial compositions. Overall, this study would help to better understand how to promote the gut health through different fiber-rich foods intake in humans.
1. Introduction Some recent studies have reported that dietary fiber played an important role in improving gut health of human and animals, since it not only improved the epithelial barrier function of intestine (Chen et al., 2013; Liu, Ivarsson, Dicksved, Lundh, & Lindberg, 2012), but also maintained the homeostasis of intestinal microenvironment in host by modulating gut microbiota (Liu et al., 2012; Ma et al., 2018). Fiberdeprived microbiota was reported to impair the barrier function of colonic mucosa and increased the susceptibility of pathogens (Desai et al., 2016). Dietary fiber could be degraded by gut microbiota to generate short-chain fatty acids (SCFAs), which could induce the expression of porcine host defense peptides to improve the immune function and gut health (Koh, De Vadder, Kovatcheva-Datchary, & Backhed, 2016; Zeng et al., 2013). Therefore, it is necessary to include moderate level of fiber into diets to regulate the gut health of the host. However, different dietary fibers may play different roles in modualting bacterial community of the host considering their diverse physicochemical properties and compositions. Previous studies have reported that dietary fibers was mainly fermented in the hindgut of human and other monogastric animals (Jaworski & Stein, 2015; Williams, Verstegen, & Tamminga, 2001).
However, recent studies showed that substantial fiber fractions were also degraded in the foregut of monogastric animals (Jha & Leterme, 2012; Jha, Rossnagel, Pieper, Van Kessel, & Leterme, 2010). Moreover, little information is known on how dietary fibers affect the bacterial community and SCFAs production in the foregut. Therefore, the objective of this study was to test the hypothesis that different dietary fibers could alter the microbial community and SCFAs production in pig model, and to determine the effects of dietary fiber on microbial community in foregut and hindgut of pigs. 2. Materials and methods 2.1. Ethics The animal handling and all procedures of this study received approval from the Animal Care and Use Ethics Committee of the China Agricultural University (Beijing). 2.2. Animals, diets and experimental design Thirty-six Duorc × (Landrace × Yorkshire) growing barrows were surgically fitted with T-cannula in the distal ileum. Pigs were allotted to
Abbreviations: ADF, acid detergent fiber; ADL, acid detergent lignin; CB, corn bran; IDF, insoluble dietary fiber; NDF, neutral detergent fiber; OB, oat bran; OTUs, operational taxonomic units; RB, rice bran; SBP, sugar beet pulp; SCFAs, short-chain fatty acids; SDF, soluble dietary fiber; SH, soybean hulls; TDF, total dietary fiber; WB, wheat bran ⁎ Corresponding author. E-mail address: [email protected] (S. Zhang). https://doi.org/10.1016/j.jff.2019.04.009 Received 22 February 2019; Received in revised form 2 April 2019; Accepted 4 April 2019 1756-4646/ © 2019 Elsevier Ltd. All rights reserved.
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Table 1 The chemical compositions of the fibrous ingredients used in this study (%, as fed basis). Items
Corn bran
Wheat bran
Soybean hulls
Rice bran
Sugar beet pulp
Oat bran
Neutral detergent fiber Acid detergent fiber Lignin Cellulose Hemicellulose Total dietary fiber Soluble dietary fiber Insoluble dietary fiber Starch
52.0 16.2 2.2 11.9 35.8 65.1 8.0 57.1 17.0
33.6 9.7 2.0 12.8 23.9 43.4 4.5 38.9 11.5
61.7 44.4 1.7 29.4 17.3 67.1 13.5 63.7 5.6
44.0 26.1 9.7 16.4 17.9 32.4 5.9 26.5 26.1
37.7 21.2 6.0 19.7 16.6 69.8 27.4 42.5 6.6
39.1 8.8 3.0 4.3 30.3 46.0 18.0 27.8 25.9
All data are the results of chemical analysis conducted in duplicate.
6 dietary treatments in a completely randomized design with 6 replicated pigs per treatment according to their body weight. The initial average body weight of the pigs was 48.5 ± 2.1 kg. The treatment diets consist of the same basal diet supplied with 6 different high-fiber food by-products, including corn bran (CB), wheat bran (WB), oat bran (OB), soybean hulls (SH), sugar beet pulp (SBP) and rice bran (RB), respectively. These high-fiber ingredients were the sole source of fiber components included in the experiment diets, and the level of total dietary fiber (TDF) were equal among all 6 treatment diets. Vitamins and minerals were supplemented to meet or exceed the nutrient requirements for growing pigs as recommended by the NRC (2012). The experimental period lasted for 21 days, including 15 days for dietary adaptation, 3 days for fecal collection and 3 days for digesta collection. The chemical components of the 6 fiber-rich ingredients were showed in Table 1. And the ingredients and chemical compositions of the 6 dietary treatments were showed in Supplementary Table 1. Before starting the animal trial, all pigs were fed pellet feed for 2 weeks, and then surgically fitted with T-cannula in the distal ileum referring to the procedures from Stein, Shipley, and Easter (1998). After the surgery, pigs were fed sucrose solution for 1 day and pellet feed for 2 weeks for recovery. During the trial, pigs were fed in individual crates (1.5 × 1.2 × 1.0 m) in an environmentally controlled room (20 °C ± 2 °C) with slatted floors, a self-feeder, and a nipple waterer. Pigs were provided ad libitum access to water, and fed a daily amount of treatment diet equivalent to 4% of their BW determined at the beginning of the animal trial. The diet fed everyday were divided into 2 equal meals and provided at 0900 and 1600 h each day.
The SCFA concentrations in the ileal digesta and fecal samples were analyzed according to the method described by Porter and Murray (2001). Briefly, about 1.0 g fecal samples were put into 10 mL centrifuge tube with 2.0 mL 0.10% hydrochloric acid addition, then incubated on ice for 25 min, mixed and centrifuged at 15,000 rpm. The supernate was obtained, and then filtered using a 0.20 mm Nylon Membrane Filter (Millipore, Bedford, OH, USA) and poured into a Gas Chromatograph System (Agilent HP 6890 Series, Santa Clara, CA, USA) for SCFA determination.
2.3. Samples collection
2.6. Statistical analysis
Ileal digesta and fresh fecal samples of pigs in each dietary treatment group were collected to determine the SCFA concentrations and bacterial community. Fresh fecal and ileal digesta samples from each pig were collected following the procedures described by CervantesPahm and Stein (2008). All samples were collected using 5 mL centrifuge tubes and placed into liquid nitrogen, and then stored at −80 °C.
Data for SCFA concentrations were checked for normality and outliers using the UNIVERIATE procedure of SAS (version 9.2, SAS Inst. Inc., Carry, NC, USA). Then data were analyzed using the procedure of PROC GLM. The treatment diet was the only fixed effect and the individual pig was treated as the experimental unit. The LSMEANS statement of SAS was used to separate treatment means, with Turkey’s test for adjustment. Correlation coefficients between dietary fiber components and SCFA concentrations were analyzed using the PROC CORR procedure of SAS. The diversity metrics of bacteria were represented from normalized Operational Taxonomic Units (OTU) reads. Bar plots on phylum and genus levels were generated to visualize the taxonomic distribution. Analyses for microbiota composition on relative abundant on the phyla and genera levels were conducted using the Kruskal-Wallis method. Principal coordinate analysis (PCoA) was used to compare the microbial community between ileal digesta and fecal samples using the unweighted UniFrac distances. Significant differences were considered when P < 0.05, and 0.05 ≤ P < 0.10 was considered as a tendency.
2.5. Microbial detection The DNA Kit (Omega Bio-tek, Norcross, GA, USA) was used to extract the DNA of bacteria in the samples of digesta and feces. The PCR amplification technique was used to amplify the gene of bacterial 16S ribosomal RNA in V4-V5 region. And then amplicons were extracted, purified and quantified. Purified amplicons were pooled in equimolar and performed double-end sequencing using Illumina MiSeq platform. The sequences that its overlap is more than 10 bp were assembled. Operational taxonomic units (OTUs) were clustered with 97% similarity. Abnormal sequences were detected and removed using USEARCH soft (vsesion 7.0, http://drive5.com/uparse/). The RDP Classifier (http://rdp.cme.msu.edu/) against the silva (SSU115) 16S rRNA database was used to analyze the taxonomy of each 16S rRNA gene sequence using confidence threshold of 90%.
2.4. Chemical analysis and calculation According to the methods of AOAC (2007), soluble dietary fiber (SDF), insoluble dietary fiber (IDF) and TDF in all 6 ingredients and diets were analyzed using the Ankom Dietary Fiber Analyzer (Ankom Technology, Macedon, NY, USA). Neutral detergent fiber (NDF), acid detergent fiber (ADF) and acid detergent lignin (ADL) of ingredients and diets were detected using the filter bags (Model F57; Ankom Technology) and fiber analyzer equipment (ANKOM200 Fiber Analyzer, Ankom Technology) according to a modified procedure described by Van Soest, Robertson, and Lewis (1991). Based on the analyzed results, cellulose and hemicellulose contents were calculated as following:
Cellulose = ADF − ADL
Hemicellulose = NDF − ADF 267
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Table 2 Effects of different dietary fibers on the short-chain fatty acid concentrations in the intestine of pigs. Items
Diets Wheat bran
Corn bran
Sugar beet pulp
Oat bran
Soybean hulls
Rice bran
Ileal digesta Lactate Acetate Propionate Butyrate Total
4.62 ± 0.74ab 4.43 ± 0.98b 0.99 ± 0.31 0.09 ± 0.03 11.22 ± 1.31a
4.26 ± 0.82ab 5.20 ± 1.01ab 0.96 ± 0.25 0.19 ± 0.08 11.51 ± 1.42a
2.54 ± 0.63bc 6.17 ± 0.86a 0.98 ± 0.23 0.27 ± 0.11 10.97 ± 0.88a
6.33 ± 1.01a 3.64 ± 0.89b 0.87 ± 0.19 0.28 ± 0.09 12.25 ± 1.33a
3.60 ± 0.52b 6.39 ± 1.05a 1.34 ± 0.42 0.32 ± 0.15 12.21 ± 1.18a
1.59 4.61 1.14 0.23 8.32
Feces Lactate Acetate Propionate Butyrate Valerate Total
0.45 6.45 2.71 1.67 0.46 13.8
0.55 6.63 3.27 1.42 0.24 13.7
0.62 ± 0.16 8.81 ± 1.44b 3.49 ± 1.41b 2.57 ± 1.02b 0.75 ± 0.45b 18.09 ± 2.04b
0.44 6.58 2.15 1.74 0.67 13.5
0.70 ± 0.21 11.29 ± 2.01a 7.72 ± 1.94a 4.57 ± 1.22a 2.22 ± 0.63a 27.94 ± 2.62a
0.74 ± 0.25 5.89 ± 0.72c 1.66 ± 0.56c 0.86 ± 0.33b 0.24 ± 0.11b 10.87 ± 1.12d
± ± ± ± ± ±
0.11 1.24c 0.81bc 0.31b 0.28b 1.85c
± ± ± ± ± ±
0.04 1.57c 1.66b 1.35b 0.12b 1.97c
± ± ± ± ± ±
0.19 0.65c 0.68c 0.46b 0.49b 1.36c
± ± ± ± ±
P-value
0.45c 0.95b 0.38 0.09 0.79b
0.01 0.01 0.72 0.83 0.01 0.62 0.01 0.01 0.01 0.01 0.01
The results were presented as mean values ± SEM (n = 6 per treatment). Means in a row without a common superscript differ, P < 0.05.
NDF content (P < 0.10, r = −0.75), and the total SCFA concentration was negatively correlated with the hemicellulose content (P < 0.05, r = −0.81). However, the acetate concentration tended to positively correlate with the dietary NDF content (P < 0.10, r = 0.76), and was positively correlated with the dietary ADF content (P < 0.05, r = 0.80). In fecal samples, both acetate and butyrate concentrations tended to positively correlate with the dietary ADF content (P < 0.10, r = 0.71 and r = 0.75). In addition, dietary cellulose content tended to positively correlate with the acetate concentration (P < 0.10, r = 0.71), and was positively correlated with the butyrate concentration (P < 0.05, r = 0.80). Moreover, the propionate and total SCFA concentrations tended to positively correlate with the dietary IDF (P < 0.10, r = 0.75) and ADF (P < 0.10, r = 0.74) contents, respectively.
3. Results 3.1. The SCFA production The SCFA concentration in the ileal digesta and fecal samples were presented in Table 2. Lactate was mainly produced in the foregut, and propionate and butyrate were more produced in the hindgut of pigs. In ileal digesta samples, pigs fed the OB, SH, WB or CB diets had greater (P < 0.05) lactate content compared with pigs fed the RB diet. Pigs fed the SBP or SH diets had greater (P < 0.05) acetate content compared with pigs fed the WB, OB or RB diets. In addition, the total SCFA concentrations in ileal digesta of pigs fed the RB diet was lower (P < 0.05) than those fed the WB, CB, SBP, OB or SH diets. There were no significant differences in the propionate and butyrate contents in ileal digesta of pigs fed the 6 different dietary treatments. In fecal samples, pigs fed the SH diets showed greater (P < 0.05) acetate, propionate, butyrate and valerate contents compared with pigs fed the WB, CB, SBP, OB or RB diets. Moreover, the total SCFA concentrations in fecal samples of pigs fed the SH diet was the greatest (P < 0.05), while the total SCFA concentrations in fecal samples of pigs fed the RB diet was the lowest (P < 0.05) among the 6 dietary treatments.
3.3. Differences in microbial community among dietary treatments The ileal digesta samples of pigs fed diets containing WB, CB, RB, SH, SBP and OB had a total of 435, 332, 469, 412, 530 and 365 OTUs, respectively, which included 14, 7, 35, 16, 87 and 13 individual bacteria, respectively (Supplemental Fig. 1A). The fecal samples of pigs fed diets containing WB, CB, RB, SH, SBP and OB had a total of 896, 903, 888, 837, 925 and 899 OTUs, respectively, which contained 10, 27, 10, 13, 13 and 17 individual bacteria, respectively (Supplemental Fig. 1B). At the phylum level, 7 and 10 microbial communities in ileal digesta (Fig. 1A) and fecal samples (Fig. 1B) of pigs were detected, and the abundances of bacteria in phyla were not different among the 6 dietary treatments. At the genus level, the microbial composition and
3.2. Correlation between dietary fiber components and SCFA production The correlation coefficients between dietary fiber components and SCFA concentrations in ileal digesta samples and fecal samples of pigs were present in Tables 3 and 4, respectively. In ileal digesta samples, the lactate concentration tended to negatively correlate with the dietary
Table 3 Correlation coefficients between dietary fiber compoments and short-chain fatty acids (SCFAs) produced in ileal digesta after dietary fiber ingestion by pigs. Items
Lactate
Acetate
Propionate
Butyrate
Total SCFA
NDF
ADF
Lignin
Cellulose
Hemicellulose
TDF
SDF
IDF
Lactate Acetate Propionate Butyrate Total SCFA NDF ADF Lignin Cellulose Hemicellulose TDF SDF IDF
1 −0.53 −0.58 −0.14 0.61 −0.75* −0.24 −0.32 0.17 −0.65 0.01 −0.03 0.05
1 0.78* 0.42 0.33 0.76* 0.80** 0.27 0.55 −0.10 −0.20 −0.52 0.60
1 0.21 0.15 0.43 0.37 −0.25 0.24 0.06 0.08 0.42 0.69
1 0.21 0.24 0.66 0.03 0.41 −0.60 0.64 0.43 −0.16
1 −0.16 0.44 −0.19 0.03 −0.81** −0.14 −0.54 0.68
1 0.72 0.77* 0.70 0.31 −0.35 −0.26 0.13
1 0.52 0.41 −0.43 −0.05 −0.21 0.29
1 0.84** 0.28 −0.53 −0.11 −0.26
0.31 −0.62 −0.09 −0.33 0.49
1 −0.39 −0.05 −0.23
1 0.78* −0.35
1 −0.85**
1
ADF, acid detergent fiber; IDF, insoluble dietary fiber; NDF, neutral detergent fiber; SDF, soluble dietary fiber; TDF, total dietary fiber. ** P < 0.05. * P < 0.10. 268
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Table 4 Correlation coefficients between dietary fiber compoments and short-chain fatty acids (SCFAs) produced in feces after dietary fiber ingestion by pigs. Items
Lactate
Acetate
Propionate
Butyrate
Valerate
Total SCFA
NDF
ADF
Lignin
Cellulose
Hemicellulose
TDF
SDF
IDF
Lactate Acetate Propionate Butyrate Valerate Total SCFA NDF ADF Lignin Cellulose Hemicellulose TDF SDF IDF
1 0.43 0.38 −0.40 0.39 0.38 0.56 0.38 −0.01 −0.06 0.25 0.36 0.16 0.03
1 0.98** −0.90** 0.98** 0.99** 0.28 0.71* −0.21 0.71* −0.62 0.28 −0.28 0.66
1 −0.81** 0.97** 0.99** 0.17 0.60 −0.33 0.64 −0.61 0.23 −0.37 0.75*
1 0.96** 0.99** 0.29 0.75* −0.14 0.80** −0.67 0.22 −0.32 0.67
1 0.98** 0.12 0.60 −0.36 0.66 −0.66 0.43 −0.16 0.61
1 0.23 0.68 −0.25 0.74* −0.63 0.27 −0.3 0.69
1 0.72* 0.77* 0.41 0.31 0.35 −0.26 0.13
1 0.52 0.84** −0.43 −0.05 −0.21 0.29
1 0.32 0.28 −0.53 −0.1 −0.26
1 −0.62 −0.09 −0.33 0.49
1 −0.4 −0.05 −0.23
1 0.78* −0.35
1 −0.85**
1
ADF, acid detergent fiber; IDF, insoluble dietary fiber; NDF, neutral detergent fiber; SDF, soluble dietary fiber; TDF, total dietary fiber. ** P < 0.05. * P < 0.10.
Fig. 1. Microbial diversity on the phylum and genus levels in pigs fed 6 fibrous dietary treatments. (A) Barplot for microbial community on the phylum level with the abundance greater than 0.01% in the ileal digesta samples of pigs. (B) Barplot for microbial community on the phylum level with the abundance greater than 0.01% in the fecal samples of pigs. (C) Barplot for microbial community on the genus level with the abundance greater than 1% in the ileal digesta sample of pigs. (D) Barplot for microbial community on the genus level with the abundance greater than 1% in the fecal samples of pigs. All data were analyzed by Kruskal-Wallis test and presented as mean percentage of different bacteria (n = 4 per treatment). The RB_D, WB_D, CB_D, SH_D, SBP_D and OB_D respresented the ileal digesta samples from pigs fed diets containing rice bran, wheat bran, corn bran, soybean hulls, sugar beet pulp and oat bran, respectively. The RB_F, WB_F, CB_F, SH_F, SBP_F and OB_F respresented the fecal samples from pigs fed diets containing rice bran, wheat bran, corn bran, soybean hulls, sugar beet pulp and oat bran, respectively. 269
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Fig. 2. Effects of dietary fibers ingestion on bacterial community of pigs on the genus level. (A) (B) The abundance of bacteria in the ileal digesta. (C) (D) The abundance of bacteria in the feces. All data were analyzed by Kruskal-Wallis test and presented as mean percentage of different bacteria (n = 4 per treatment), and different letters means P < 0.05. The RB, WB, CB, SH, SBP and OB respresented pigs fed diets containing rice bran, wheat bran, corn bran, soybean hulls, sugar beet pulp and oat bran, respectively.
compared, and the results indicated that Firmicutes and Proteobacteria were the dominant bacteria in ileal digesta of pigs, and Firmicutes was the dominant bacteria in feces of pigs. At the phylum level, the abundance of Firmicutes (Fig. 4A) and Bacteroidetes (Fig. 4C) in fecal samples were greater (P < 0.05) than those in ileal digesta samples, and the population of Proteobacteria (Fig. 4B) was lower (P < 0.05) in fecal samples compared with that in ileal digesta samples. At the genus level, Escherichia, Acinetobacter and Enterobacteriaceae were the top three genera in population in small intestine, while the dominant genus in fecal sample was Clostridiales. The abundance of Lactobacillus (Fig. 4D) and Escherichia-Shigella (Fig. 4F) in fecal samples were lower (P < 0.05) than that in ileal digesta sample, and the population of Bifidobacterium (Fig. 4E) in fecal samples was greater (P < 0.05) compared to that in ileal digesta samples.
community in leal digesta (Fig. 1C) and fecal samples (Fig. 1D) of pigs were also presented. In ileal digesta samples, the abandance of Filifactor in WB group was greater (P < 0.05) than that in OB and RB groups (Fig. 2A), and the abundance of Intestinibacter in SBP and SH groups were greater (P < 0.05) than that in WB, CB and OB groups (Fig. 2B). In fecal samples, the population of Ruminococcus_1 in SH group was greater (P < 0.05) than that in WB, CB and OB groups (Fig. 2C), and the population of Lachnoclostridium was greater (P < 0.05) in SH group than that in the other treatment groups (Fig. 2D). 3.4. Differences in microbial community between ileal digeata and fecal samples The richness and biodiversity of microbial community in ileal digesta and fecal samples were presented as the α-diversity indices of Chao and Shannon. The result showed that the Shannon (Fig. 3A) and Chao (Fig. 3B) indexes of microbial community in the ileal digesta samples were lower (P < 0.05) than those in the fecal samples. The βdiversity of microbial community between ileal digesta and fecal samples were compared using PCoA, and the result showed different clustering of microbial communities (Fig. 3C). Furthermore, the microbial community between ileal digesta and fecal samples at the phylum (Supplemental Fig. 2A) and genus (Supplemental Fig. 2B) levels were
4. Discussion 4.1. The SCFA concentrations produced by dietary fibers fermentation Dietary fiber fermentation results in the production of SCFAs, including lactate, acetate, propionate and butyrate, and some gases such as hydrogen, carbon dioxide and methane (Macfarlane & Macfarlane, 1993; Williams et al., 2001). Among all the SCFAs produced in the 270
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Fig. 3. Bacterial α-diversity and β-diversity in ileal digesta and fecal samples of pigs. (A) The Shannon index of bacterial community. (B) Chao index of bacterial community. The results were analyzed by wilcoxon rank-sum test and different letters means P < 0.05 (n = 4). (C) The β-diversity of bacterial community.
in the small intestine, and could promote the development of Lactobacilli, a family of health-promoting bacteria. In addition, pigs fed the SBP and SH diets had greater acetate content in the ileal digesta compared with those fed the WB, OB, and RB diets. Those results could be attributed to the greater NDF and ADF concentrations in SBP and SH diets compared to WB, OB and RB diets, which also agreed with the positive correlation (or positive correlation tendency) between acetate concentration in ileal digesta and the dietary NDF and ADF contents. Therefore, the dietary fiber soucre could influence the amount and type of SCFAs produced in the gut. This was supported by the results of Carneiro, Lordelo, Cunha, and Freire (2008), who found higher acetate and lower butyrate production in the cecum of pigs when wheat bran was replaced by maize cobs in diets. In the current study, pigs fed the SH diet showed greater acetate, propionate, butyrate and valerate concentrations in fecal samples compared with pigs fed the other diets. This was consistent with the results of Freire, Guerreiro, Cunha, and Aumaitre (2000), who compared the effects of 20% dietary WB, SBP, SH or alfalfa meal inclusion on total SCFA concentrations in the cecum of pigs, and found that the
hindgut, acetate accounts for the largest proportation (about 60.0%), whereas propionate and butyrate account for smaller quantities (Lunn & Buttriss, 2007). This was consistent with our results. As the endproducts of dietary fiber fermentation, SCFAs could act as energy substrates for colonocytes, modulate satiety, and alleviate inflammation (Koh et al., 2016). Butyrate was reported to be beneficial to the host by promoting the proliferation of mucosal epithelial cells, increasing the differentiation of intestinal epithelial cells and enhancing the function of colonic barrier (Han et al., 2017; Morrison & Preston, 2016). Our results showed that lactate was mainly produced in the foregut, whereas butyrate was produced in the hindgut of pigs, which may be due to the specific microbial communities in different intestinal parts of pigs, such as Lactobacillus and Enterococcus in the foregut and Firmicutes in the hindgut. The lactate concentration in the ileal digesta of pigs fed the OB, WB and CB diets was greater than that fed the RB diet. This was consistent with the results of Bach Knudsen and Canibe (2000), who observed greater concentration of lactate in the ileum of cannulated pigs after feeding a diet supplemented with SDF from OB. As the major component of OB, the β-glycan could stimulate the lactate production 271
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Fig. 4. Analysis for differential bacteria between the ileal digesta and fecal samples of pigs. (A)–(C) Differential bacteria on the phylum level. (D)–(F) Differential bacteria on the genus level. The data were analyzed by wilcoxon rank-sum test and presented as mean percentage of different bacteria. Different letters means P < 0.05 (n = 24).
the different constituents in diets (Awati et al., 2005; Jha & Leterme, 2012; Jha et al., 2010). The source of dietary fiber could affect the digestion site and gut environment, thereby influencing the proliferation of microbiota in the gut (Hogberg & Lindberg, 2004). In the present study, Firmicutes was the dominant bacteria whose abundance exceeded 60% in fecal samples. Many previous studies showed that Firmicutes and Bacteroidetes were the two dominant phyla that account for about 90% of the gut microbiota in pigs (Liu, Wang, et al., 2018; Liu, Zhao, et al., 2018; Bian et al., 2016). Clostridial clusters XIVa and IV within the phylum of Firmicutes could digest fiber components to produce SCFAs, such as butyrate. The phylum of Bacteroidetes also contained bacteria that are capable of degrading fibers (Flint, Bayer, Rincon, Lamed, & White, 2008). Therefore, the abundance of Firmicutes and Bacteroidetes provide a reference for the potential fibre-degrading and butyrateproducing bacteria in the gut microbiota. Our results showed that the 6 dietary fibers had no influence on Firmicutes and Bacteroidetes abundances in the ileal digesta and feces of pigs. However, Mu et al. (2014) reported that the alfalfa meal diet incresed the abundances of Firmicutes and Bacteroidetes compared to the WB diet in piglets. Maier et al. (2017) also showed that the high-resistant starch diet, as a dietary fiber source, caused an increase in the Firmicutes to Bacteroidetes ratio and the abundances of some specific members of Firmicutes in the gut of human. Nevertheless, Ferrario et al. (2017) reported an increase in Bacteroidetes abundance and a concurrent reduction in Firmicutes abundance with the addition of inulin in diets as a dietary fiber source. These different effects of dietary fiber on the diversity of bacterial community could be ascribed to the different types of dietary fiber available for fermentation and the various gut environment of the host. In addition, we observed increased abundance of Ruminococcus_1 when pigs were fed the SH diet. Ruminococcus_1 was reported to be able to produce SCFAs through fermenting carbohydrates (Pryde, Duncan, Hold, Stewart, & Flint, 2002), which may explain the greatest SCFA production in pigs fed SH in our study. The abundances of Filifactor and Intestinibacter were altered by different fiber ingestion in our study, and these two bacteria
dietary SH inclusion increased the total SCFA concentration by 11.2%, 30.5% and 27.2% compared with the dietary WB, SBP and alfalfa meal inclusion, respectively. Our results mentioned above also suggested that SH is highly degraded in the cecum of pigs. This was in agreement with the high NDF and ADF digestibility of SH in cecum reported by the previous study (Freire et al., 2000). The variation in fermentability of the dietary fibers could be attributed to the differences in their chemical compositions and physicochemical properties such as bulk, viscosity, solubility, and waterholding capacity. In our study, lactate and total SCFA concentrations in ileal digesta tended to negatively correlate or negatively correlated with the dietary NDF and hemicellulose contents, respectively, which was due to that the major fermentation substrates are monosaccharides, and the formation of SCFAs from hemicellulose is negligible in pigs (Drochner, 1993). Moreover, in our study, there was a trend that both acetate and butyrate contents in feces were positively correlated with the dietary ADF and cellulose contents, and the propionate and total SCFA concentrations tended to positively correlate with the dietary IDF and cellulose contents, respectively. These may be because monosaccharides could be easily formed by cleaving homogeneous polysaccharides, which are the major components of ADF (Mu, Zhang, He, Smidt, & Zhu, 2014). This may also partly explain the greater SCFA production in pigs fed the SH diet, which contains greater ADF and cellulose components. 4.2. Microbial community induced by dietary fiber The gastrointestinal microbiota is a vast and dynamic ecosystem, which plays important roles in intestinal morphology, immunity development, digestion, and modulating host gene expression (Guo et al., 2008; Turnbaugh et al., 2006). The types of diet would influence the population and activity of bacteria in the gut (Bach Knudsen, Hedemann, & Laerke, 2012). According to the previous studies, dietary fiber was the major factor that could affect the gut environment among 272
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Conflict of interest
were associated with the periodontitis and type 2 diabetes diseases in human (Aruni, Chioma, & Fletcher, 2014; Wang & Jia, 2016). The population of Lachnoclostridium was also influenced by different dietary fiber supplementation in our study, and the Lachnoclostridium had a connection with the obesity of human (Amadou, Hosny, La Scola, & Cassir, 2016). From these reports, we may infer that dietary fibers may have potentials to modulate the metabolic diseases in human by shaping the composition of gut microbiota in the host. In the current study, Firmicutes was the dominant phylum in feces while Proteobacteria was the dominant phylum in ileal digesta, which was consistent with the previous report (Zhao et al., 2014). The feces of pigs contained larger proportion of Firmicutes than the ileal digesta, suggesting that the large intestine, instead of small intestine, may undertake more function on substrate fermentation (DiBaise et al., 2008; Flint et al., 2008). Escherichia-Shigella, Lactobacillus, Streptococcus and Enterococcus, which were dominant genera in the ileal digesta in our study (shown on Supplemental Fig. 2B), formed the major gap between fecal and intestinal microbiota. These results indicated that these 4 genus are correlated with the fermentation ability of the small intestine in pigs. The high Escherichia-Shigella and Streptococcus abundance in the ileal digesta in our study, which are always considered as pathogenic bacteria associated with infection and enteric disease of the host, also indicated that the pathogen invasion may enrich in the foregut. In the current study, the population of Firmicutes and Bacteroidetes in feces was greater than that in ileal digesta, indicating that dietary fiber was mainly degraded in the hindgut. The fermentation products by Lactobacillus and Enterococcus are mainly lactate, leading to more lactate production in the ileal digesta of pigs. As probiotics, both Lactobacillus and Enterococcus could improve the immune function and inhibit the adhesion of enteric pathogens to the host (Michail & Abernathy, 2002; Scharek et al., 2005; Varma, Dinesh, Menon, & Biswas, 2010). The indexes of Chao and Shannon in the ileal digesta samples were lower than that in the fecal samples, indicating more richness and biodiversity of microbial composition in feces. The β-diversity of microbial community showed different clustering of microbial communities between the ileal digesta and fecal samples, and the results were consistant with the previous study (Zhao et al., 2014), who reported that the PCA analysis clustered the microbial communities into three categories: communites of the small intestine, large intestine, and feces on the genus level.
There are no potential conflicts of interest in the research. Appendix A. Supplementary material Supplementary data to this article can be found online at https:// doi.org/10.1016/j.jff.2019.04.009. References Amadou, T., Hosny, M., La Scola, M., & Cassir, N. (2016). “Lachnoclostridium bouchesdurhonense”, a new bacterial species isolated from human gut microbiota. New Microbe and New Infect, 13, 69–70. https://doi.org/10.1016/j.nmni.2016.06.015. AOAC (2007). Official methods of analysis of AOAC international. Gaithersburg, MD: AOAC International. Aruni, W., Chioma, O., & Fletcher, H. M. (2014). Filifactor alocis: The newly discovered kid on the block with special talents. Journal of Dental Research, 93, 725–732. https:// doi.org/10.1177/0022034514538283. Awati, A., Konstantinov, S. R., Williams, B. A., Akkermans, A. D. L., Bosch, M. W., Smidt, H., & Verstegen, M. W. (2005). Effect of substrate adaptation on the microbial fermentation and microbial composition of faecal microbiota of weaning piglets studied in vitro. Journal of the Science of Food and Agriculture, 85, 1765–1772. https://doi. org/10.1002/jsfa.2178. Bach Knudsen, K. E., & Canibe, N. (2000). Breakdown of plant carbohydrates in the digestive tract of pigs fed on wheat- or oat-based rolls. Journal of the Science of Food and Agriculture, 80, 1253–1261. Bach Knudsen, K. E., Hedemann, M. S., & Laerke, H. N. (2012). The role of carbohydrates in intestinal health of pigs. Animal Feed Science and Technology, 173, 41–53. https:// doi.org/10.1016/j.anifeedsci.2011.12.020. Bian, G. R., Ma, S. Q., Zhu, Z. G., Su, Y., Zoetendal, E. G., Mackie, R., ... Zhu, W. (2016). Age, introduction of solid feed and weaning are more important determinants of gut bacterial succession in piglets than breed and nursing mother as revealed by a reciprocal crossfostering model. 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5. Conclusion Different dietary fiber sources altered the microbial community and the short-chain fatty acid production in pig model. The lactate was mainly produced in the foregut, whereas the propionate and butyrate were mainly generated in the hindgut of pig. The acetate concentration in the ileal digesta was positively correlated with the dietary ADF content, and the butyrate concentration in feces was positively correlated with the dietary cellulose content. Dietary fibers shaped the abundances of gut microbiota in the fecal samples of pigs, such as Filifactor, Intestinibacter, Ruminococcus_1 and Lachnoclostridium. This study would contribute to a better understanding of how to promote gut health by different fiber-rich foods intake in humans. 6. Ethics statement The animal handling and all procedures of this study received approval from the Animal Care and Use Ethics Committee of the China Agricultural University (Beijing). Acknowledgments This research was financially supported by the National Natural Science Foundation of China (Grant No. 31702121) and by the Chinese Universities Scientific Fund (Grant No. 2019TC120). 273
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