Addition of 0.2% prebiotics does not alter microbial diversity

DNA was extracted from human faeces and from KUHIMM samples collected at 30h of fermentation. In all KUHIMM samples, eubacterial copy numbers, evaluated by quantitative PCR, reached 9.15×10¹⁰ copies/mL (Supplementary Table ^S1) and were comparable to the reported cell densities in human faeces (approximately 10¹¹/wet-g)²².

NGS was used for bacterial 16S rRNA gene sequence analysis of faecal samples and corresponding cultures with and without prebiotics. In total, 5,922,873 quality reads with an average of 148,072 reads were obtained (Table 2). The numbers of operational taxonomic units (OTUs) and α-diversity values (Chao1, Shannon index, and Simpson index) were not lower in cultures treated with or without prebiotics than in faecal samples (Kruskal-Wallis test). Thus, as described previously, bacterial numbers in human faeces were maintained in the corresponding cultures of our KUHIMM¹³. There were fewer OTUs in cultures treated with DEX and αCD than in control cultures (P<0.05, Kruskal-Wallis test); however, OTU numbers were higher in the presence of DRX than in the absence of prebiotics (P<0.05, Kruskal-Wallis test). Chao1 values, which indicate species richness, were lower in cultures with DEX and αCD than in controls (P<0.05, Kruskal-Wallis test), but were not significantly different between DRX-treated cultures and controls (P>0.05, Kruskal-Wallis test). Moreover, the Shannon and Simpson indexes were not significantly different between cultures containing prebiotics and controls (P>0.05, Kruskal-Wallis test). Therefore, species diversity in our KUHIMM did not change following addition of 0.2% prebiotics.

Table 2

Summary of 16S rRNA gene sequencing data and α-diversity values (Chao1 estimator, Shannon index, and Simpson index).

	KUHIMM (n=8)
	FEC	CUL	DEX	αCD	DXR
Read counts	123,622±29,146	166,869±35,152	151,911±30,120	79,656±12,693	218,300±42,461
Observed OTUs	1,168±291	1,525±204*	1,294±167#	1,083±151##	1,954±326#
Chao1	1,834±441	2,362±321*	2,024±253#	1,896±255#	2,751±510
Shannon index	5.39±0.26	5.24±0.25	5.14±0.32	5.10±0.27	5.31±0.36
Simpson index	0.94±0.01	0.94±0.02	0.94±0.02	0.93±0.01	0.94±0.02

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Eight human faeces samples (FEC), corresponding cultures (CUL), and corresponding cultures treated with 0.2% indigestible dextrin (DEX), α-cyclodextrin (αCD), or dextran (DXR) were sampled at 30h of fermentation.

The values show the mean±standard deviation.

Statistical differences between samples were evaluated with respect to observed operational taxonomic units (OTUs), Chao1 estimator, Shannon index, and Simpson index.

Asterisks (*) indicate significant differences between faeces from eight healthy subjects and corresponding cultures without or with prebiotics (*P<0.05 and **P<0.01, Kruskal-Wallis test).

Pounds (^#) indicate significant differences between cultures with and without prebiotics (^# P<0.05 and ^## P<0.01, Kruskal-Wallis test).

Addition of 0.2% prebiotics does not change microbial composition

Principal coordinate analysis (PCoA) of unweighted UniFrac distances revealed that microbiota in faecal samples shifted in the same direction as in the corresponding cultures without prebiotics (P=0.454, multivariate analysis of variance (MANOVA)) (Fig. 2). No significant changes were detected between the microbiota of cultures with and without prebiotics (DEX: P=0.571; αCD: P=0.986; DXR: P=0.996, MANOVA).

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Figure 2

Principal coordinate analysis (PCoA) of 16S metagenomics data of bacterial species in eight human volunteers’ faeces (designated as F26, F40, F62, M27, M37, M38, M39, and M60) and corresponding cultures with and without 0.2% prebiotics: indigestible dextrin (DEX), α-cyclodextrin (αCD), and dextran (DXR). Cultures were sampled at 30h after initiation of fermentation. (a) Human faeces (FEC); (b) corresponding culture without prebiotics (CUL); (c) corresponding culture with 0.2% DEX; (d) corresponding culture with αCD; (e) corresponding culture with DXR.

Bacterial composition was compared in cultures with and without prebiotics (Fig. 3). Approximately 99% of the total bacterial abundance was assigned to five phyla Actinobacteria, Bacteroidetes, Firmicutes, Fusobacteria, and Proteobacteria. Relative abundances of the genera Bifidobacterium, Collinsella, Bacteroides, Parabacteroides, Prevotella, Enterococcus, Lactococcus, Streptococcus, uncultured Clostridiaceae, Clostridium, uncultured Lachnospiraceae, Blautia, Coprococcus, Dorea, uncultured Peptostreptococcaceae, uncultured Ruminococcaceae, Faecalibacterium, Oscillospira, Ruminococcus, Acidaminococcus, Dialister, Megamonas, Megasphaera, Phascolarctobacterium, uncultured Erysipelotrichaceae, Fusobacterium, Sutterella, Succinivibrio, uncultured Enterobacteriaceae, and Citrobacter did not differ significantly between cultures with and without prebiotics (P<0.05, Kruskal-Wallis test). Thus, bacterial compositions in our KUHIMM did not change following addition of 0.2% prebiotics.

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Figure 3

Genus-level compositional view of bacteria in cultures after 30h of fermentation. One each of eight human faecal samples (designated as F26, F40, F62, M27, M37, M38, M39, and M60) was used as the inoculum in each KUHIMM. Samples were obtained from fermentation cultures without prebiotics (CUL) and cultures with 0.2% indigestible dextrin (DEX), α-cyclodextrin (αCD), or dextran (DRX) at 30h after initiation of fermentation. Genera of lower abundance (<1.0%) and lower similarity (<97%) were included in Others and Uncultured Bacteria, respectively.

Addition of 0.2% prebiotics increases production of SCFAs

SCFAs are the end-products of microbial fermentation in the colon and act as signalling molecules between gut microbiota and the host, with important implications for host health^3,23. The effect of adding 0.2% prebiotics on SCFA production in the KUHIMM was evaluated (Fig. 4). At 30h after initiating fermentation and in the absence of prebiotics, the production of acetate, propionate, and butyrate was 101.0±23.2mM, 35.9±9.69mM, and 26.9±11.5mM, respectively. The concentration of lactate was lower than 0.02mM. Interestingly, the concentrations of acetate and propionate were significantly higher following addition of prebiotics (P<0.05, Dunnett test), whereas that of butyrate remained unchanged.

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Figure 4

Changes in production of (a) acetate, (b) propionate, and (c) butyrate at 30h after initiation of fermentation in the KUHIMM supplemented with 0.2% prebiotics: indigestible dextrin (DEX), α-cyclodextrin (αCD), and dextran (DXR). Changes are presented as the ratio of the concentrations in the KUHIMM with prebiotics normalised to those without prebiotics. Asterisks (*) indicate significantly different values (*P<0.05 and **P<0.01) (n=8) using Dunnett test. Error bars show the standard deviation of the mean.

Operation of the KUHIMM with and without prebiotics

The KUHIMM was operated using a multi-channel fermenter (Bio Jr.8; ABLE, Tokyo, Japan), as described previously^13,21 with some modifications. The KUHIMM consisted of eight parallel and independent vessels. Each vessel contained 100mL of Gifu Anaerobic Medium (GAM; Nissui Pharmaceutical Co., Ltd., Tokyo, Japan). The medium was autoclaved at 115°C for 15min and the initial pH was adjusted to 6.5. Anaerobic conditions in the vessel were achieved by purging with a mixture of N₂ and CO₂ (80:20; 15mL/min) that was filter-sterilised through a 0.2-µm PTFE membrane (Pall Corporation, Port Washington, NY, USA) at 37°C for 1h prior to cultivation. To prepare the inoculum, the faecal sample in the swab was suspended in 2.0mL of 0.1M phosphate buffer (pH 6.5, consisting of 0.2M NaH₂PO₄ and 0.1M Na₂HPO₄) supplemented with 1.0% L-ascorbic acid (Wako Pure Chemical Industries, Osaka, Japan).

Cultivations were initiated by inoculating one faecal suspension (100µL) into each vessel. During fermentation at 37°C, the culture broth was stirred at 300rpm with a magnetic stirrer and continuously purged with a filter-sterilised mixture of gas to maintain anaerobic conditions. Aliquots (1mL) of culture broth were sampled from the vessel at 30h after the initiation of cultivation. Faeces and culture broth samples were stored at −20°C until use.

To evaluate the effect of prebiotics (Table 1), one type of prebiotics was added into one of the vessels at a final concentration of 2.0g/L (0.2% per 100-mL vessel) prior to fermentation. The following prebiotics were used: DEX (Fibersol-2; Matsutani Chemical Industry Co., Ltd., Hyogo, Japan), αCD (Cyclochem Co., Ltd., Hyogo, Japan), and DXR (Dextran 40,000; Wako Pure Chemical Industries). A control vessel without prebiotics was prepared.

Human intervention study

All participants were recruited according to the inclusion criteria, which comprised an age of 20 to 50 years, being Japanese, non-smoking status, good health and physical condition, and a fasting triglyceride level of 120mg/dL to 199mg/dL. The exclusion criteria included significant clinical deviation from normal as determined by investigators; history or suspicion of diabetes, liver disease, kidney disease, or having a food allergy; or taking supplemental dietary fibre or lipid-lowering medications. A human intervention study was conducted on four volunteers (F26, M37, M38, and M39) (Supplementary Fig. ^S2). At first, the volunteer drank water (200mL) twice a day for 14 days (days 1–14, washout-1), and faeces were sampled using the swab on day 14. Then, the volunteer ate 3.0g of αCD dissolved in water (200mL) twice a day (6.0g/day) for seven days (days 15–21), and faeces were sampled on day 21. Next, the volunteer drank water (200mL) twice a day for 14 days (days 22–35, washout-2) again, and faeces were sampled on day 35. Thereafter, the volunteer ate 3.0g of DEX dissolved in water (200mL) twice a day (6.0g/day) for seven days (days 36–42), and faeces were sampled on day 42. During the study, the four volunteers avoided drinking alcohol or consuming other dietary fibres.

The study was performed in accordance with the guidelines of Kobe University Hospital, and approved by the institutional ethics review board of Kobe University. All methods in this study were in accordance with the Declaration of Helsinki and the approved guidelines by the Medical Ethics Committee at Kobe University (research code; 290001, approved date; 21 Mar 2017). The authors have no financial or personal relationships that could inappropriately influence this research.

Extraction of microbial genomic DNA

Microbial genomic DNA was extracted from suspended faeces and culture broth from the KUHIMM at 30h, as described previously²¹. Purified DNA was eluted into TE buffer (10mM TrisHCl, 1.0mM EDTA) and stored at −20°C until use.

Illumina library generation

Bacterial 16S rRNA genes (V3-V4 region) were amplified using genomic DNA as template and primers S-D-Bact-0341-b-S-17 (5′-CCTACGGGNGGCWGCAG-3′) and S-D-Bact-0785-a-A-21 (5′-GACTACHVGGGTATCTAATCC-3′)³⁸, as described previously¹³. Index primers (Nextera XT Index Kit; Illumina Inc., San Diego, CA, USA) overhanging the amplified sequences were added to the gene-specific sequences. The PCR reaction was performed according to the manufacturer’s instructions. Amplicons were purified with AMPure XP DNA purification beads (Beckman Coulter, Brea, CA, USA) and eluted in 25µL of 10mM Tris (pH 8.5). Purified amplicons were quantified using an Agilent Bioanalyzer 2100 with DNA 1000 chips (Agilent Technology, Santa Clara, CA, USA) and Qubit 2.0 (Thermo Fisher Inc., Waltham, MA, USA), and pooled in equimolar concentration (5nM). The 16S rRNA genes along with an internal control (PhiX control v3; Illumina) were subjected to paired-end sequencing using a Miseq sequencer (Illumina) with a Miseq reagent kit v3 (600 cycles; Illumina). The PhiX sequences were removed, and paired-end reads with Q scores≥20 were joined using the software package MacQIIME version 1.9.1³⁹. The UCLUST algorithm⁴⁰ was used to cluster filtered sequences into OTUs based on a≥97% similarity threshold. Chimeric sequences were checked and removed from the library using ChimeraSlayer⁴¹. Representative sequences from each OTU were taxonomically classified via the GreenGenes taxonomic database using the Ribosomal Database Project Classifier⁴². OTUs were used for α-diversity estimation of the Chao1, Shannon diversity, and Simpson indexes. PCoA plots were calculated using OTUs from each sample based on unweighted UniFrac distances.

Real-time PCR

Real-time PCR to quantify total bacterial growth during cultivation was performed using a TP700 Thermal Cycler Dice Real Time System Lite (Takara Bio Inc., Ohtsu, Japan) with a primer set targeting all eubacteria²¹. The PCR reaction and amplification were performed as described previously²¹.

Measurement of SCFAs

Concentrations of SCFAs such as acetate, propionate, butyrate, lactate, and succinate were measured using a high-performance liquid chromatograph (HPLC; Shimadzu Corporation, Kyoto, Japan) equipped with an Aminex HPX-87H column (Bio-Rad Laboratories, Inc., Hercules, CA, USA) and a RID-10A refractive index detector (Shimadzu Corporation). The HPLC was operated at 65°C using 5mM H₂SO₄ as the mobile phase with a flow rate of 0.6mL/min.

Bioinformatics and statistical analyses

The various α-diversity values (Chao1, Shannon index, and Simpson index) were calculated using the MacQIIME software package³⁹ because they best fit the data distribution. PCoA was conducted using OTU information from each sample and calculated based on unweighted UniFrac distances⁴³ using MacQIIME. The nonparametric Kruskal-Wallis test, Dunnett test, and MANOVA were performed using JMP 13 software (SAS Institute Inc., Cary, NC, USA). P<0.05 was considered statistically significant.

This work was supported by Japan Innovative Bioproduction Kobe (http://www.org.kobe-u.ac.jp/bioproduction/) and Japan Society for the Promotion of Science (JSPS) KAKENHI Grant Number 17K12897. The funders had no role in study design, data collection and analysis, decision to publish, or preparation of the manuscript.