Abstract
Yeast mannan (YM) is an indigestible water-soluble polysaccharide of the yeast cell wall, with a notable prebiotic effect on the intestinal microbiota. We previously reported that YM increased Bacteroides thetaiotaomicron abundance in in vitro rat faeces fermentation, concluding that its effects on human colonic microbiota should be investigated. In this study, we show the effects of YM on human colonic microbiota and its metabolites using an in vitro human faeces fermentation system. Bacterial 16S rRNA gene sequence analysis showed that YM administration did not change the microbial diversity or composition. Quantitative real-time PCR analysis revealed that YM administration significantly increased the relative abundance of Bacteroides ovatus and B. thetaiotaomicron. Moreover, a positive correlation was observed between the relative ratio (with or without YM administration) of B. thetaiotaomicron and B. ovatus (r = 0.92), suggesting that these bacteria utilise YM in a coordinated manner. In addition, YM administration increased the production of acetate, propionate, and total short-chain fatty acids. These results demonstrate the potential of YM as a novel prebiotic that selectively increases B. thetaiotaomicron and B. ovatus and improves the intestinal environment. The findings also provide insights that might be useful for the development of novel functional foods.
Similar content being viewed by others
Introduction
Yeast has been widely consumed since ancient times in fermented foods and beverages such as bread, beer, and wine. Yeast mannan (YM) is an indigestible water-soluble polysaccharide of the yeast cell wall that has rarely been used as a food ingredient. YM is a densely branched α-linked mannose polymer with a molecular weight ranging from 20,000 to 200,000 Da1. It includes a linear α-1, 6-mannoside backbone branched with α-1, 2-mannoside and α-1, 3-mannoside bonds in the form of mono-, di-, tri-, and tetramers (Fig. 1)1,2. This structure is distinct from other plant cell wall mannans, such as konjac glucomannan and carob galactomannan, which include only β-linked mannose and not α-linked mannose3. YM has various effects in cells and mouse models, including immunomodulation, wound repair, and anti-inflammatory effects, and has potential health benefits in humans and animals4,5,6. YM appears to be utilised by specific intestinal bacteria due to its elaborate structure7, and the impacts of YM on the intestinal microbiota ecosystem have attracted research attention.
The human intestinal tract is colonised by trillions of microorganisms, which greatly contribute to host health by providing nutrients, energy, pathogen resistance, and immune response modulation8,9,10,11. Due to its importance in homeostasis, dysbiosis of the intestinal microbiota is associated with various multifactorial diseases, including metabolic, inflammatory bowel, cardiovascular, neoplastic, and neurological diseases11,12,13,14,15. Therefore, controlling the composition of the intestinal microbiota and maintaining a favourable intestinal environment with the diet plays a key role in maintaining the host’s health. Bacteroidetes, which is composed largely of members of the genus Bacteroides, is a dominant gut-associated bacterial phylum in healthy adult microbiota16 using a glycan-acquisition strategy; Bacteroidetes members employ multiple cell envelope-associated protein complexes called the starch utilisation system (Sus)17. Sus is the most studied glycan degradation system encoded by a polysaccharide utilisation locus (PUL)18. The proteins in the Sus are located in the outer membrane and periplasm of the cell. On the cell surface, the proteins bind glycan and degrade it into oligosaccharides and transport these oligosaccharides into the periplasm, where they are broken down into smaller saccharides and imported into the cell7,18,19,20. By utilising Sus-like systems, Bacteroidetes can degrade various indigestible carbohydrates from the human diet21,22,23. This process leads to the production of short-chain fatty acids (SCFAs)24, which act as both nutrients and energy sources for the host25.
Bacteroides thetaiotaomicron, a prominent human intestinal symbiont in the phylum Bacteroidetes, exhibits various distinctive functions, including anti-rotavirus activity26, induction of matrilysin27, and attenuation of colon inflammation28. Furthermore, clinical trials have been conducted using B. thetaiotaomicron as a live biotherapeutic candidate for the treatment of Crohn’s disease29. B. thetaiotaomicron is believed to confer various benefits on host health; therefore, it is desirable to increase its abundance. B. thetaiotaomicron has various PULs, including genomic sus genes, and thereby a broad ability to digest dietary fibre polysaccharides and host glycans30. YM is a polysaccharide that is digested and utilised by B. thetaiotaomicron, which metabolises it through a ‘selfish mechanism’ due its unique property of having three mannan-specific PULs; most other bacteria do not have such PULs and therefore cannot utilise it7. Unlike traditional prebiotics such as inulin, fructo-oligosaccharides, and galacto-oligosaccharides which increase Lactobacillus and Bifidobacterium31, we consider YM to be a valuable ingredient as a novel prebiotic candidate that increases the abundances of B. thetaiotaomicron and other Bacteroides spp. YM utilisation by B. thetaiotaomicron has been investigated in monoculture, co-culture, and gnotobiotic mice7, and our previous study showed that YM increases the abundance of B. thetaiotaomicron in in vitro rat faeces fermentation32. However, the effects of YM on human colonic microbiota are still unknown. Here, we investigated the effects of YM on human colonic microbiota using an in vitro human faeces fermentation system (referred to as the Kobe University Human Intestinal Microbiota Model, or KUHIMM), which maintains the diversity and richness of bacterial species in the original human faeces33. The KUHIMM reproduces the effects of prebiotics, in line with the results from human clinical trials34. The use of the KUHIMM prior to human clinical trials allows us to evaluate the effects of YM on human colonic microbiota and the relevant doses, without the influence of dietary intake. In addition, we can also evaluate the safety of YM for unexpected microbiota changes, such as an increase in harmful bacteria, and the effects on the metabolic profile of each individual. Thus, the KUHIMM serves as a tool for evaluating the effect of functional food components, such as prebiotics, on human colonic microbiota.
Results
YM was utilised in the KUHIMM
YM was prepared from yeast cell wall slurry as described previously32, with a final mannan concentration of 50.5%. The KUHIMM was set up by adding a 0.4% YM preparation (0.2% mannan) (referred to as YM) under anaerobic conditions, and each of the eight human faecal samples (HS1, HS2, HS3, HS4, HS5, HS6, HS7, and HS8) (referred to as FEC) was used as the inoculum. A control culture without YM was also prepared (referred to as CUL). We investigated whether mannan was consumed by the human colonic microbiota in the KUHIMM after 30 h of fermentation. Mannan consumption was confirmed in all samples (Supplementary Fig. S1).
YM administration did not alter bacterial genus-level composition and selectively stimulated the growth of Bacteroides thetaiotaomicron and Bacteroides ovatus
The effects of YM on human colonic microbiota were investigated using next-generation sequencing (NGS) and quantitative real-time PCR (qPCR). DNA was extracted from KUHIMM samples with and without YM collected after 30 h of fermentation. The eubacterial copy numbers, evaluated by qPCR, reached 2.81–4.90 × 1011 copies/mL (Supplementary Table S1), which were comparable to the reported cell densities in the human colon (approximately 1011 cells/mL)35.
NGS was used for the V3–V4 region of bacterial 16S rRNA for gene sequence analysis of faecal samples and the corresponding cultures with and without YM using the Illumina MiSeq system. In total, 4,407,318 quality reads were obtained from the eight faecal samples and the corresponding KUHIMMs with and without YM (Table 1). The numbers of operational taxonomic units (OTUs) and the Chao1 values for species richness were lower in the CUL and YM groups than in the FEC group (Kruskal–Wallis test, p < 0.05); however, there was no significant difference between the CUL and YM groups (Kruskal–Wallis test, p > 0.05). The Shannon index for species diversity was lower in the CUL group than in the FEC group (Kruskal–Wallis test, p < 0.05); however, there was no significant difference between the CUL and YM groups (Kruskal–Wallis test, p > 0.05). The Simpson index for species diversity was not significantly different among the FEC, CUL, and YM groups (Kruskal–Wallis test, p > 0.05). Thus, the microbial diversity in the KUHIMMs did not change with the addition of the 0.4% YM preparation.
Principal coordinate analysis (PCoA) revealed that the microbiota in each KUHIMM was shifted in the same direction from the original faeces, and individual faecal samples and corresponding KUHIMMs with and without YM were assigned to the same cluster (Fig. 2). Microbiota β-diversity based on unweighted UniFrac distances was not significantly different between CUL and YM (permutational multivariate analysis of variance, PERMANOVA, p = 0.98). Bacterial genus-level compositional analyses of microbiota in the FEC, CUL, and YM are shown in Fig. 3. Almost all bacterial genera in the original faeces were also detected in the KUHIMMs. Comparing the relative abundance of 26 representative bacterial genera between CUL and YM, no significant differences were observed in any genus (Kruskal–Wallis test, p > 0.05). Thus, the microbial composition in the KUHIMMs did not change with the addition of the 0.4% YM preparation.
Principal coordinate analysis (PCoA) of 16S rRNA metagenomics data of bacterial species in eight human samples (HS1–HS8). Eight human faecal samples (FEC), the corresponding cultures without yeast mannan (CUL), and the corresponding cultures with a 0.4% yeast mannan preparation (YM) were sampled 30 h after the initiation of fermentation. The circles, triangles, and squares in the PCoA plot represent microbiota in the FEC, CUL, and YM groups, respectively.
Genus-level compositional views of bacteria in eight human samples (HS1–HS8). Eight human faecal samples (FEC), the corresponding cultures without yeast mannan (CUL), and the corresponding cultures with the 0.4% yeast mannan preparation (YM) were sampled 30 h after the initiation of fermentation. Genera with low abundance (< 1.0%) and low similarity (< 97%) were included in ‘Others’ and ‘Unclassified bacteria’, respectively.
We then evaluated the effect of YM administration on bacteria of the genus Bacteroides, which are the most predominant anaerobes in the gut36, using the KUHIMM. B. thetaiotaomicron, B. ovatus, B. caccae, B. uniformis, B. fragilis, and B. vulgatus of the genus Bacteroides are commonly found at high densities in human colonic microbiota37. After 30 h of fermentation, the numbers of six Bacteroides species were estimated by qPCR analysis (Fig. 4). As expected, the relative abundance of B. thetaiotaomicron was significantly increased in the YM group compared to that in the CUL group (Wilcoxon signed-rank test, p = 0.036). Remarkably, the relative abundance of B. ovatus was also significantly increased in the YM group compared to that in the CUL group (Wilcoxon signed-rank test, p = 0.036). Conversely, the relative abundance of the other Bacteroides species, B. caccae, B. uniformis, B. fragilis, and B. vulgatus, was not significantly different (Wilcoxon signed-rank test, p = 0.48, 0.61, 0.69, and 0.35, respectively) between CUL and YM. Thus, B. thetaiotaomicron and B. ovatus in the KUHIMMs were selectively increased by the addition of the 0.4% YM preparation.
The relative abundance of six Bacteroides species after 30 h of fermentation in the KUHIMM. Bacteroides thetaiotaomicron, Bacteroides ovatus, Bacteroides caccae, Bacteroides uniformis, Bacteroides fragilis, and Bacteroides vulgatus without yeast mannan (CUL) and with the 0.4% yeast mannan preparation (YM) are shown. Asterisks (*) represent significant differences (*p < 0.05) (n = 7 for B. uniformis, n = 5 for B. fragilis, and B. vulgatus, n = 8 for others) using the Wilcoxon signed-rank test.
YM administration reduced the pH and enhanced acetate and propionate production
The pH reflects the intestinal environmental condition, and low pH inhibits the growth of pathogenic bacteria, resulting in the reduction of putrefactive compounds38. Supplementary Figure S2 shows the results of continuous monitoring of pH during culture. After 30 h of fermentation, the pH was significantly reduced in the presence of YM compared to that of the CUL group (Wilcoxon signed-rank test, p = 0.025, Fig. 5a).
Changes in pH (a) and production of short-chain fatty acids (SCFAs) (b): acetate, propionate, butyrate, and total SCFAs after 30 h of fermentation in the KUHIMM without yeast mannan (CUL) and with the 0.4% yeast mannan preparation (YM). Asterisks (*) represent significant differences (*p < 0.05) (n = 8) using the Wilcoxon signed-rank test.
SCFAs are metabolic products of human gut microbiota, which act as signalling molecules and provide beneficial effects for host health39. Acetate, propionate, and butyrate are the most abundant (≥ 95%) SCFAs in the human colon40. The impact of YM administration on the production of SCFAs was examined after 30 h of fermentation (Fig. 5b). The concentrations of acetate, propionate, and total SCFAs were significantly higher in the YM group than in the CUL group (Wilcoxon signed-rank test, p = 0.036, 0.017, and 0.025, respectively). In contrast, the concentration of butyrate was not significantly different between YM and CUL (Wilcoxon signed-rank test, p = 0.67).
Discussion
The most recent definition of prebiotics is ‘a substrate that is selectively utilised by host microorganisms, conferring a health benefit’41, and numerous studies on prebiotics have found health benefits not only for the gut but also for the host in general31,42. Most traditional prebiotics increase the number of specific bacteria, such as Lactobacillus and Bifidobacterium31. In addition, they selectively increase the abundance at the bacterial genus level; few studies have reported on prebiotics that selectively increase abundance at the bacterial species level. It has been reported that bacteria of the genus Bacteroides have various beneficial effects36; among these bacteria, B. thetaiotaomicron and B. ovatus are expected to be utilised as potential novel probiotics43,44. Conversely, several species are pathogens and associated with harmful effects on host health, e.g. B. fragilis with the induction of abscess formation45 and B. vulgatus with the development of ulcerative colitis46. Therefore, a product that selectively increases beneficial bacteria of the genus Bacteroides could be a functional food ingredient as a novel prebiotic candidate.
In this study, we investigated the effect of YM on human colonic microbiota and metabolic end products using an in vitro human faeces fermentation system, the KUHIMM. Bacterial 16S rRNA gene sequence analysis showed that YM administration did not change microbial α-diversity, β-diversity, or the relative abundance of representative bacterial genera. Analysis of the growth profiles of six Bacteroides species in the KUHIMM revealed that YM administration stimulated the growth of only two species, B. thetaiotaomicron and B. ovatus, through the consumption of mannan. These results indicate that YM selectively increases the abundance of B. thetaiotaomicron and B. ovatus. To the best of our knowledge, there are few prebiotics that increase microbes in a species-specific manner, and YM is the first material that selectively increases B. thetaiotaomicron and B. ovatus in the complex of human colonic microbiota. B. ovatus is reported to exhibit immunogenic and immunomodulatory functions, such as expression of the tumour-specific Thomsen–Friedenreich antigen as a target for a cancer vaccine47 and alleviation of lipopolysaccharide-induced inflammation48. In addition to B. thetaiotaomicron, several strains of B. ovatus, B. vulgatus, and B. caccae metabolised mannan in monoculture7, although among these species, only B. ovatus was increased in human colonic microbiota.
Both B. thetaiotaomicron and B. ovatus can degrade various indigestible polysaccharides utilising Sus-like systems22. These bacteria break down polysaccharides extracellularly to liberate polysaccharide breakdown products (PBPs). Some of them produce PBPs exclusively for their own use, while others produce PBPs that they do not necessarily require but can be used for growth by other Bacteroides spp. having limited or no ability to use the polysaccharides23,49,50. In addition, there are potential effects outside the genus Bacteroides; B. ovatus liberates PBPs during growth on xylan, which can support the growth of Bifidobacterium adolescentis that are normally unable to utilise it51. One study showed that B. thetaiotaomicron uses YM exclusively through a selfish mechanism, in which B. thetaiotaomicron degrades YM into PBPs extracellularly and incorporates them into the cell in a manner that prevents other bacteria from using it, implying that B. thetaiotaomicron does not support the growth of other Bacteroides that can use the mannose- and mannan backbone7. In addition, B. thetaiotaomicron grew more efficiently than B. ovatus on YM in monoculture22. However, unlike what was previously thought, we found the interesting result that YM increased both B. thetaiotaomicron and B. ovatus to the same extent. Notably, the relative abundances of B. thetaiotaomicron and B. ovatus in the YM group relative to the control group had a strong positive correlation (r = 0.92, p = 0.0012, Fig. 6), although there were no significant correlations between the relative abundances of B. thetaiotaomicron and B. ovatus in the culture (r = − 0.05, p = 0.86). Therefore, it is considered that B. thetaiotaomicron and B. ovatus utilise YM in a coordinated manner, rather than in a competitive manner.
Three putative PULs (MAN-PUL1, MAN-PUL2, and MAN-PUL3) are important for B. thetaiotaomicron to utilise YM as the sole carbon source7. Bioinformatics studies found that B. ovatus possesses a putative PUL corresponding to MAN-PUL2 but no PULs corresponding to MAN-PUL1 or MAN-PUL3 in its genome (Fig. 7, Supplementary Table S2). A model has been proposed wherein YM is degraded extracellularly by at least two GH76s (endo-α-1, 6-mannanases BT2623 and BT3792) and a GH99 (endo-α-1, 2-mannosidase and endo-α-1, 2-mannanase BT3862) within these PULs to liberate PBPs, which are then transported into the periplasm, where they are depolymerised to mannose7. Of these three proteins, B. ovatus possesses only one GH76 (BO3915), with a relatively lower degree of homology with the other GHs in MAN-PUL2, suggesting that the extracellular degradation of YM is incomplete. Thus, in the in vitro human colonic microbiota model, B. ovatus appears to have utilised PBPs generated by B. thetaiotaomicron from the YM by incorporating them into the periplasm, revealing a novel cooperative relationship between Bacteroides species. This interesting phenomenon might have evolved cooperatively between B. thetaiotaomicron and B. ovatus in complex gut microbial ecosystems where various microbes compete for limited nutrients.
Bacteroides thetaiotaomicron VPI-5842 and Bacteroides ovatus ATCC 8483 yeast mannan polysaccharide utilisation loci (PULs). Each gene is depicted to reflect its orientation and scale on the genome. The number below each gene is its locus tag. Genes encoding known or predicted functionalities are colour-coded: glycoside hydrolase (GH; green), SusC-like proteins (SusC-like; purple), SusD-like proteins (SusD-like; orange), phosphatase (phosphatase; pink), regulatory proteins (regulator; blue), and other proteins (others; grey).
An increase in the production of acetate, propionate, and total SCFAs was observed in the culture with YM. The phylum Bacteroidetes is known to primarily produce acetate and propionate as metabolic end products40. Therefore, it was suggested that YM administration stimulated the growth of B. thetaiotaomicron and B. ovatus, increased the relative abundance of the phylum Bacteroidetes, and resulted in an increase in acetate and propionate. These SCFAs are thought to have reduced the pH. Acetate and propionate are the most potent activators of GPR43, a receptor on the cell surfaces of adipose tissue52. Because one SCFA is utilised by intestinal bacteria to produce another SCFA, and changes in the intestinal microbiota compositions are associated with the production of SCFAs39, an increase in one SCFA ideally should not reduce the levels of another beneficial SCFA. Therefore, YM might be a useful prebiotic because it increased the production of acetate, propionate, and total SCFAs and did not decrease that of butyrate.
Because there are various factors in the complex intestinal microbial ecosystem, including competition for prebiotics and cross-feeding interactions among microorganisms, even if a material is utilised by certain intestinal bacteria in monoculture, it may not necessarily increase the bacteria in the intestinal microbiota. Furthermore, the intake of prebiotics by humans may cause a considerable increase in the abundance of bifidobacteria, even if they do not affect the growth of bifidobacteria in in vitro monoculture53. Therefore, it was important to confirm that YM selectively increased B. thetaiotaomicron and B. ovatus abundance in the in vitro human colonic microbiota fermentation system, which reproduces the in vivo microbiota changes induced by prebiotics. The prebiotic effects of YM were confirmed at doses as low as 0.4% (0.2% mannan). Previous studies using this system have confirmed the bifidogenic effects of prebiotic oligosaccharides at a concentration of 0.5%34, while 0.2% did not change the colonic microbiota composition as reported in human and animal studies33. For this reason, compared to conventional prebiotics, YM may also exert prebiotic effects at lower doses in in vivo human clinical studies. However, when YM is ingested by humans, it may be affected by variation in diets; therefore, it is not clear whether YM exhibits the same prebiotic effect. To develop YM as a microbiota-directed food ingredient for human consumption that selectively increases the abundance of B. thetaiotaomicron and B. ovatus, clinical studies are required to verify its prebiotic effect, the resulting health benefits, and the doses at which these effects are produced.
Conclusion
YM selectively increased the relative abundance of B. thetaiotaomicron and B. ovatus in the human colonic microbiota model. In addition, YM increased the production of acetate, propionate, and total SCFAs. These results show the potential of YM as a novel prebiotic that selectively increases B. thetaiotaomicron and B. ovatus and improves the intestinal environment.
Methods
Preparation of YM
YM was produced from yeast cell wall slurry provided by Asahi Group Foods, Ltd. (Tokyo, Japan) as described previously32. The mannan concentration was measured by Japan Food Research Laboratories (Tokyo, Japan). The mannan concentration was 50.5%, calculated based on the mannose concentration after hydrolysis, which was quantified by high-performance liquid chromatography (HPLC).
Human faecal sample collection from volunteers
Faecal samples were obtained from eight healthy subjects in their thirties to forties who had not taken antibiotics for at least 2 months prior to sampling, as described previously33. Written informed consent was obtained from all participants. The study was performed in accordance with the principles of the Declaration of Helsinki and the guidelines of Kobe University and was approved by the Intestinal Ethics Review Board at Kobe University (research code 1902, approval date 10 May 2016). All methods used in this study were in accordance with the guidelines of the Medical Ethics Committee at Kobe University. Approximately 200 mg faecal samples were collected using anaerobic culture swabs (212559 BD BBL CultureSwab; BD Biosciences, Franklin Lakes, NJ, USA) and stored at room temperature according to the manufacturer’s protocol until inoculation.
Operation of the KUHIMM with and without YM
The model culture system was operated using a multi-channel fermenter (Bio Jr. 8; ABLE, Tokyo, Japan) to construct the KUHIMM as described previously33,34. Briefly, each vessel in the system contained autoclaved Gifu anaerobic medium broth (100 mL; Nissui Pharmaceutical Co.), with the initial pH adjusted to 6.5. Anoxic conditions were established by purging vessels with a filter-sterilised mixture of N2 and CO2 (80:20) gas (15 mL/min) at 37 °C prior to and during fermentation. To prepare the inoculum, each faecal sample was suspended in 2 mL of 0.1 M phosphate buffer (pH 6.5, 0.1 M NaH2PO4:0.1 M Na2HPO4 = 2:1), supplemented with 1% l-ascorbic acid (Wako Pure Chemical Industries, Osaka, Japan). Fermentation was initiated by inoculation of each medium-containing vessel with 100 μL of the abovementioned faecal suspension. To evaluate the effects of YM administration, YM was added into one of the vessels at a final concentration of 4 g/L (0.4% per 100 mL of medium) prior to fermentation. A control vessel without YM was also prepared. Faecal samples and aliquots of fermentation cultures were stored at − 20 °C until use.
Extraction of microbial genomic DNA
Microbial DNA was extracted from faecal samples and fermentation cultures of the KUHIMM using 0.1 mm glass beads, TE (10 mM Tris–HCl and 1 mM ethylenediaminetetraacetic acid [EDTA])-saturated phenol, and sodium dodecyl sulphate, as described previously34. A OneStep PCR Inhibitor Removal Kit (Zymo Research, Irvine, CA, USA) was used for further purification according to the manufacturer’s instructions. Purified DNA was stored at − 20 °C until use.
Next-generation sequencing and data processing
NGS analysis was performed by Macrogen Japan Corp. (Kyoto, Japan). Samples for sequencing were prepared according to the Illumina 16S Metagenomic Sequencing Library Preparation protocols to amplify the V3 and V4 regions of the 16S rRNA genes. Bacterial 16S rRNA genes (V3–V4 region) were amplified using genomic DNA as the template. The following primers were used: S-D-Bact-0341-b-S-17 (5′-CCTACGGGNGGCWGCAG-3′) and S-D-Bact-0785-a-A-21 (5′-GACTACHVGGGTATCTAATCC-3′)54. PCR was performed according to the manufacturer’s instructions. Amplicons were purified using AMPure XP beads (Beckman Coulter, Inc., CA, USA). Paired-end sequencing was performed on the Illumina MiSeq platform. Overlapping reads were merged using fast length adjustment of short reads55. Pre-processing and clustering of sequences to identify OTUs was performed using the CD-HIT-OTU software56. After short reads were filtered out and extra-long tails were trimmed, chimeric reads were identified and discarded. The remaining representative reads were clustered into OTUs based on a ≥ 97% similarity threshold. Taxonomic composition for each sample from phylum to species levels was generated using QIIME-UCLUST57 against the RDP-16S rRNA gene database58. The various α-diversity values (Chao1, Shannon index, and Simpson index) and PCoA of unweighted UniFrac distances59 were calculated using QIIME software60.
Quantitative real-time PCR
Quantitative real-time PCR (qPCR) analyses were performed using specific primers for all eubacteria and six Bacteroides species (B. thetaiotaomicron, B. ovatus, B. caccae, B. uniformis, B. fragilis, and B. vulgatus) as described previously37,61. qPCR analyses were conducted in duplicate using an Applied Biosystems QuantStudio 3 Real-Time PCR system (Thermo Fisher Scientific Inc., Waltham, MA, USA). The qPCR amplification program is described in Supplementary Table S3. We prepared a synthesised DNA fragment (191–265 bp) identical to the 16S rRNA gene sequence as a reference for absolute quantification for each method. Standard curves were prepared by diluting reference fragments (101–108 copies). To confirm the specificity of the amplification using SYBR Green, a melting-point-determination analysis was performed.
Measurement of mannan concentration in the model culture system
The remaining mannan in the culture medium was analysed at 0 h and 30 h after the initiation of fermentation and was calculated based on the mannose concentration after hydrolysis, which allows the measurement of mannan utilisation. Samples were prepared according to the method described by Goubet et al.62 with minor modification. Briefly, each culture broth was centrifuged at 10,000 × g for 5 min, and 100 μL of the supernatant was recovered, which was then hydrolysed using 1 mL of 2 M trifluoroacetic acid for 4.5 h at 100 °C. The samples were combined with 1 mL of 99.5% ethanol and dried using a centrifugal evaporator (Genevac Ltd., Ipswich, UK). The dry residue was resuspended in water, and the low-molecular-weight (< 10 kDa) fraction was recovered using Vivaspin 500 MWCO 10,000 PES (Sartorius Stedim Biotech, Göttingen, Germany).
The mannose concentration was determined by high-performance anion-exchange chromatography with a pulsed amperometric detector (HPAEC-PAD). HPAEC-PAD analysis was performed using a Dionex ICS-5000 (Thermo Fisher Scientific, CA, USA). The system was equipped with a CarboPac PA1 column (2 × 250 mm) in combination with a CarboPac PA1 guard column (2 × 50 mm) (Thermo Fisher Scientific). The mobile phases consisted of 10 mM NaOH (A) and 500 mM NaOH (B). Samples (10 μL) were applied to the column and eluted at a flow rate of 0.25 mL/min using the following linear gradient: 0 min–0% B; 20 min–0% B; 20.01 min–60% B; 35 min–60% B; 35.01 min–0% B; 50 min–0% B.
Measurement of SCFA concentrations
The concentrations of lactate, succinate, acetate, propionate, and butyrate were measured using an HPLC system (Shimadzu, Kyoto, Japan) equipped with an Aminex HPX-87H column (Bio-Rad Laboratories, Hercules, CA, USA) and an RID-10A refractive index detector (Shimadzu), as described previously33.
Bioinformatics and statistical analyses
PULs similar to MAN-PUL1, MAN-PUL2, and MAN-PUL3 were searched using the PUL prediction tool described in PULDB63. The Kruskal–Wallis test and Wilcoxon signed-rank test were performed using SPSS software ver. 23 (IBM Japan, Ltd., Tokyo, Japan). PERMANOVA was performed using the R ver. 3.6.0 Vegan package. A p-value < 0.05 was considered statistically significant.
Change history
08 February 2021
A Correction to this paper has been published: https://doi.org/10.1038/s41598-021-83010-9
References
Liu, H. Z., Liu, L., Hui, H. & Wang, Q. Structural characterization and antineoplastic activity of Saccharomyces cerevisiae mannoprotein. Int. J. Food Prop. 18, 359–371 (2015).
Kocourek, J. & Ballou, C. E. Method for fingerprinting yeast cell wall mannans. J. Bacteriol. 100, 1175–1181 (1969).
Scheller, H. V. & Ulvskov, P. Hemicelluloses. Annu. Rev. Plant Biol. 61, 263–289 (2010).
Jin, X., Zhang, M., Cao, G. F. & Yang, Y. F. Saccharomyces cerevisiae mannan induces sheep beta-defensin-1 expression via Dectin-2-Syk-p38 pathways in ovine ruminal epithelial cells. Vet. Res. (Faisalabad) 50, 8 (2019).
Michael, C. F. et al. Airway epithelial repair by a prebiotic mannan derived from Saccharomyces cerevisiae. J. Immunol. Res. 2017, 8903982 (2017).
Lew, D. B. et al. Beneficial effects of prebiotic Saccharomyces cerevisiae mannan on allergic asthma mouse models. J. Immunol. Res. 2017, 3432701 (2017).
Cuskin, F. et al. Human gut Bacteroidetes can utilize yeast mannan through a selfish mechanism. Nature 517, 165–169 (2015).
Flint, H. J., Scott, K. P., Louis, P. & Duncan, S. H. The role of the gut microbiota in nutrition and health. Nat. Rev. Gastroenterol. Hepatol. 9, 577–589 (2012).
Cani, P. D. et al. Microbial regulation of organismal energy homeostasis. Nat. Metab. 1, 34–46 (2019).
Hooper, L. V. & Macpherson, A. J. Immune adaptations that maintain homeostasis with the intestinal microbiota. Nat. Rev. Immunol. 10, 159–169 (2010).
Pickard, J. M., Zeng, M. Y., Caruso, R. & Núñez, G. Gut microbiota: Role in pathogen colonization, immune responses, and inflammatory disease. Immunol. Rev. 279, 70–89 (2017).
Arora, T. & Bäckhed, F. The gut microbiota and metabolic disease: Current understanding and future perspectives. J. Intern. Med. 280, 339–349 (2016).
Wang, Z. et al. Gut flora metabolism of phosphatidylcholine promotes cardiovascular disease. Nature 472, 57–63 (2011).
Wong, S. H. & Yu, J. Gut microbiota in colorectal cancer: Mechanisms of action and clinical applications. Nat. Rev. Gastroenterol. Hepatol. 16, 690–704 (2019).
Vogt, N. M. et al. Gut microbiome alterations in Alzheimer’s disease. Sci. Rep. 7, 13537 (2017).
The Human Microbiome Project Consortium. Structure, function, and diversity of the healthy human microbiome. Nature 486, 207–214 (2012).
Bolam, D. N. & Koropatkin, N. M. Glycan recognition by the Bacteroidetes Sus-like systems. Curr. Opin. Struct. Biol. 22, 563–569 (2012).
Foley, M. H., Cockburn, D. W. & Koropatkin, N. M. The Sus operon: A model system for starch uptake by the human gut Bacteroidetes. Cell. Mol. Life. Sci. 73, 2603–2617 (2016).
Bågenholm, V. et al. Galactomannan catabolism conferred by a polysaccharide utilization locus of Bacteroides ovatus. J. Biol. Chem. 292, 229–243 (2017).
Martens, E. C., Koropatkin, N. M., Smith, T. J. & Gordon, J. I. Complex glycan catabolism by the human gut microbiota: The Bacteroidetes Sus-like paradigm. J. Biol. Chem. 284, 24673–24677 (2009).
Larsbrink, J. et al. A discrete genetic locus confers xyloglucan metabolism in select human gut Bacteroidetes. Nature 506, 498–502 (2014).
Martens, E. C. et al. Recognition and degradation of plant cell wall polysaccharides by two human gut symbionts. PLoS Biol. 9, e1001221 (2011).
Rakoff-Nahoum, S., Foster, K. R. & Comstock, L. E. The evolution of cooperation within the gut microbiota. Nature 533, 255–259 (2016).
Flint, H. J., Bayer, E. A., Rincon, M. T., Lamed, R. & White, B. A. Polysaccharide utilization by gut bacteria: Potential for new insights from genomic analysis. Nat. Rev. Microbiol. 6, 121–131 (2008).
Koropatkin, N. M., Cameron, E. A. & Martens, E. C. How glycan metabolism shapes the human gut microbiota. Nat. Rev. Microbiol. 10, 323–335 (2012).
Varyukhina, S. et al. Glycan-modifying bacteria-derived soluble factors from Bacteroides thetaiotaomicron and Lactobacillus casei inhibit rotavirus infection in human intestinal cells. Microbes Infect. 14, 273–278 (2012).
López-Boado, Y. S. et al. Bacterial exposure induces and activates matrilysin in mucosal epithelial cells. J. Cell Biol. 148, 1305–1315 (2000).
Delday, M., Mulder, I., Logan, E. T. & Grant, G. Bacteroides thetaiotaomicron ameliorates colon inflammation in preclinical models of Crohn’s disease. Inflamm. Bowel Dis. 25, 85–96 (2019).
Hansen, R. et al. A phase I randomized, double-blind, placebo-controlled study to assess the safety and tolerability of (Thetanix) Bacteroides thetaiotaomicron in adolescents with stable Crohn’s disease. https://www.4dpharmaplc.com/application/files/1815/5824/8886/Thetanix_DDW_poster_2019.pdf. Accessed 15 July 2020 (2019).
Salyers, A. A., Vercellotti, J. R., West, S. E. & Wilkins, T. D. Fermentation of mucin and plant polysaccharides by strains of Bacteroides from the human colon. Appl. Environ. Microbiol. 33, 319–322 (1977).
Rawi, M. H., Zaman, S. A., Pa’ee, K. F., Leong, S. S. & Sarbini, S. R. Prebiotics metabolism by gut-isolated probiotics. J. Food Sci. Technol. 57, 1–14 (2020).
Oba, S. et al. Yeast mannan increases Bacteroides thetaiotaomicron abundance and suppresses putrefactive compound production in in vitro fecal microbiota fermentation. Biosci. Biotechnol. Biochem. 84, 2174–2178 (2020).
Sasaki, D. et al. Low amounts of dietary fibre increase in vitro production of short-chain fatty acids without changing human colonic microbiota structure. Sci. Rep. 8, 435 (2018).
Takagi, R. et al. A single-batch fermentation system to simulate human colonic microbiota for high-throughput evaluation of prebiotics. PLoS ONE 11, e0160533 (2016).
Sender, R., Fuchs, S. & Milo, R. Revised estimates for the number of human and bacteria cells in the body. PLoS Biol. 14, e1002533 (2016).
Wexler, H. M. Bacteroides: The good, the bad, and the nitty-gritty. Clin. Microbiol. Rev. 20, 593–621 (2007).
Tong, J., Liu, C., Summanen, P., Xu, H. & Finegold, S. M. Application of quantitative real-time PCR for rapid identification of Bacteroides fragilis group and related organisms in human wound samples. Anaerobe 17, 64–68 (2011).
Slavin, J. Fiber and prebiotics: Mechanisms and health benefits. Nutrients 5, 1417–1435 (2013).
Koh, A., De Vadder, F., Kovatcheva-Datchary, P. & Bäckhed, F. From dietary fiber to host physiology: Short-chain fatty acids as key bacterial metabolites. Cell 165, 1332–1345 (2016).
den Besten, G. et al. The role of short-chain fatty acids in the interplay between diet, gut microbiota, and host energy metabolism. J. Lipid Res. 54, 2325–2340 (2013).
Gibson, G. R. et al. The International Scientific Association for Probiotics and Prebiotics (ISAPP) consensus statement on the definition and scope of prebiotics. Nat. Rev. Gastroenterol. Hepatol. 14, 491–502 (2017).
Holscher, H. D. Dietary fiber and prebiotics and the gastrointestinal microbiota. Gut Microbes 8, 172–184 (2017).
Chang, C. J. et al. Next generation probiotics in disease amelioration. J. Food Drug Anal. 27, 615–622 (2019).
Tan, H. et al. Pilot safety evaluation of a novel strain of Bacteroides ovatus. Front. Genet. 9, 539 (2018).
Tzianabos, A. O., Onderdonk, A. B., Rosner, B., Cisneros, R. L. & Kasper, D. L. Structural features of polysaccharides that induce intra-abdominal abscesses. Science 262, 416–419 (1993).
Bamba, T., Matsuda, H., Endo, M. & Fujiyama, Y. The pathogenic role of Bacteroides vulgatus in patients with ulcerative colitis. J Gastroenterol. 30(Suppl 8), 45–47 (1995).
Ulsemer, P. et al. Specific humoral immune response to the Thomsen-Friedenreich tumor antigen (CD176) in mice after vaccination with the commensal bacterium Bacteroides ovatus D-6. Cancer Immunol. Immunother. 62, 875–887 (2013).
Tan, H., Zhao, J., Zhang, H., Zhai, Q. & Chen, W. Novel strains of Bacteroides fragilis and Bacteroides ovatus alleviate the LPS-induced inflammation in mice. Appl. Microbiol. Biotechnol. 103, 2353–2365 (2019).
Luis, A. S. et al. Dietary pectic glycans are degraded by coordinated enzyme pathways in human colonic Bacteroides. Nat. Microbiol. 3, 210–219 (2018).
Rakoff-Nahoum, S., Coyne, M. J. & Comstock, L. E. An ecological network of polysaccharide utilization among human intestinal symbionts. Curr. Biol. 24, 40–49 (2014).
Rogowski, A. et al. Glycan complexity dictates microbial resource allocation in the large intestine. Nat. Commun. 6, 7481 (2015).
Le Poul, E. et al. Functional characterization of human receptors for short chain fatty acids and their role in polymorphonuclear cell activation. J. Biol. Chem. 278, 25481–25489 (2003).
Okubo, T. et al. Effects of partially hydrolyzed guar gum intake on human intestinal microflora and its metabolism. Biosci. Biotechnol. Biochem. 58, 1364–1369 (1994).
Klindworth, A. et al. Evaluation of general 16S ribosomal RNA gene PCR primers for classical and next-generation sequencing-based diversity studies. Nucleic Acids Res. 41, e1 (2013).
Magoč, T. & Salzberg, S. L. FLASH: Fast length adjustment of short reads to improve genome assemblies. Bioinformatics 27, 2957–2963 (2011).
Li, W., Fu, L., Niu, B., Wu, S. & Wooley, J. Ultrafast clustering algorithms for metagenomic sequence analysis. Brief. Bioinform. 13, 656–668 (2012).
Edgar, R. C. Search and clustering orders of magnitude faster than BLAST. Bioinformatics 26, 2460–2461 (2010).
Maidak, B. L. et al. The RDP-II (ribosomal database project). Nucleic Acids Res. 29, 173–174 (2001).
Lozupone, C. & Knight, R. UniFrac: A new phylogenetic method for comparing microbial communities. Appl. Environ. Microbiol. 71, 8228–8235 (2005).
Caporaso, J. G. et al. QIIME allows analysis of high-throughput community sequencing data. Nat. Methods 7, 335–336 (2010).
Furet, J. P. et al. Comparative assessment of human and farm animal faecal microbiota using real-time quantitative PCR. FEMS Microbiol. Ecol. 68, 351–362 (2009).
Goubet, F., Jackson, P., Deery, M. J. & Dupree, P. Polysaccharide analysis using carbohydrate gel electrophoresis: A method to study plant cell wall polysaccharides and polysaccharide hydrolases. Anal. Biochem. 300, 53–68 (2002).
Terrapon, N. et al. PULDB: The expanded database of polysaccharide utilization loci. Nucleic Acids Res. 46, D677–D683 (2018).
Acknowledgements
We are grateful to Naoko Watanabe and Hiroto Morita for providing technical and analytical support.
Author information
Authors and Affiliations
Contributions
S.O., T.S., R.T., K.A., H.S., and T.O. conceived the study and designed the experiments. A.K., D.S., and K.S. operated the culture system. S.O., T.S., R.T., and K.A. performed sequence analyses and real-time PCR. S.O. and T.S. performed the bioinformatics analysis. S.O. and K.S. wrote the paper, and T.S., R.T., and H.S. revised the manuscript. S.H., T.O., Y.N., and A.K. supervised the study. All authors have read and approved the final manuscript.
Corresponding author
Ethics declarations
Competing interests
The authors declare no competing interests.
Additional information
Publisher's note
Springer Nature remains neutral with regard to jurisdictional claims in published maps and institutional affiliations.
Supplementary information
Rights and permissions
Open Access This article is licensed under a Creative Commons Attribution 4.0 International License, which permits use, sharing, adaptation, distribution and reproduction in any medium or format, as long as you give appropriate credit to the original author(s) and the source, provide a link to the Creative Commons licence, and indicate if changes were made. The images or other third party material in this article are included in the article's Creative Commons licence, unless indicated otherwise in a credit line to the material. If material is not included in the article's Creative Commons licence and your intended use is not permitted by statutory regulation or exceeds the permitted use, you will need to obtain permission directly from the copyright holder. To view a copy of this licence, visit http://creativecommons.org/licenses/by/4.0/.
About this article
Cite this article
Oba, S., Sunagawa, T., Tanihiro, R. et al. Prebiotic effects of yeast mannan, which selectively promotes Bacteroides thetaiotaomicron and Bacteroides ovatus in a human colonic microbiota model. Sci Rep 10, 17351 (2020). https://doi.org/10.1038/s41598-020-74379-0
Received:
Accepted:
Published:
DOI: https://doi.org/10.1038/s41598-020-74379-0
- Springer Nature Limited
This article is cited by
-
Colonic in vitro fermentation of mycoprotein promotes shifts in gut microbiota, with enrichment of Bacteroides species
Communications Biology (2024)
-
Beneficial metabolic transformations and prebiotic potential of hemp bran and its alcalase hydrolysate, after colonic fermentation in a gut model
Scientific Reports (2023)
-
The Kobe University Human Intestinal Microbiota Model for gut intervention studies
Applied Microbiology and Biotechnology (2021)