Accepted Manuscript In vitro fermentation of alginate and its derivatives by human gut microbiota Miaomiao Li, Guangsheng Li, Qingsen Shang, Xiuxia Chen, Wei Liu, Xiong’e Pi, Liying Zhu, Yeshi Yin, Guangli Yu, Xin Wang PII:
S1075-9964(16)30006-3
DOI:
10.1016/j.anaerobe.2016.02.003
Reference:
YANAE 1534
To appear in:
Anaerobe
Received Date: 29 November 2015 Revised Date:
9 February 2016
Accepted Date: 10 February 2016
Please cite this article as: Li M, Li G, Shang Q, Chen X, Liu W, Pi X’e, Zhu L, Yin Y, Yu G, Wang X, In vitro fermentation of alginate and its derivatives by human gut microbiota, Anaerobe (2016), doi: 10.1016/j.anaerobe.2016.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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In vitro fermentation of alginate and its derivatives by human gut microbiota
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Miaomiao Li1†, Guangsheng Li1†, Qingsen Shang1, Xiuxia Chen2, Wei Liu2, Xiong’e Pi2,
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Liying Zhu2, Yeshi Yin2, Guangli Yu1*, Xin Wang2*
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Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, Key
Laboratory of Marine Drugs of Ministry of Education, Ocean University of China, and
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Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine
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Science and Technology, Qingdao, 266003, China
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State Key Laboratory of Breeding Base for Zhejiang Sustainable Pest and Disease
Control, Institute of Plant Protection and Microbiology, Academy of Agricultural
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Sciences, Hangzhou, Zhejiang, China.
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Corresponding authors:
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E-mail: [email protected] or [email protected]
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ABSTRACT Alginate (Alg) has a long history as a food ingredient in East Asia. However, the
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human gut microbes responsible for the degradation of alginate and its derivatives have
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not been fully understood yet. Here, we report that alginate and the low molecular
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polymer derivatives of mannuronic acid oligosaccharides (MO) and guluronic acid
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oligosaccharides (GO) can be completely degraded and utilized at various rates by fecal
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microbiota obtained from six Chinese individuals. However, the derivative of propylene
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glycol alginate sodium sulfate (PSS) was not hydrolyzed. The bacteria having a
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pronounced ability to degrade Alg, MO and GO were isolated from human fecal
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samples and were identified as Bacteroides ovatus, Bacteroides xylanisolvens, and
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Bacteroides thetaiotaomicron. Alg, MO and GO can increase the production level of
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short chain fatty acids (SCFA), but GO generates the highest level of SCFA. Our data
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suggest that alginate and its derivatives could be degraded by specific bacteria in the
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human gut, providing the basis for the impacts of alginate and its derivates as special
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food additives on human health.
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Key words: human gut microbiota, alginate and its derivatives, B. ovatus,
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B. xylanisolvens, B. thetaiotaomicron, hydrolyze
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INTRODUCTION Alginates are naturally occurring anionic copolymers of a linear β (1→4)-linked
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glycuronan typically composed of residues of β-D-mannuronic acid (M) and its C-5
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epimer α-L-guluronic acid (G) [1]. They are extracted from brown seaweed and have
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been traditionally used as gelling agents in food, pharmaceuticals, cosmetics, feeds, and
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other industrial applications [2-4]. Under the natural growth condition, the following
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three types of polymers are present in the alginate according to the block chemical
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structures: poly β-D-mannuronic acid (MM), poly α-L-guluronic acid (GG) and
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heteropolymer (MG), the latter contains random arranged residues of M and G,
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Moreover, MM and GG demonstrate different gel forming properties compared to MG
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[5]. Alginate oligosaccharides were reported to have various activities such as
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antifungal activity [6], anti-inflammatory activity [7] and immunomodulatory activities
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[8]. Propylene glycol alginate sodium sulfate (PSS), a drug against cardiovascular
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disease, is a sulfated propylene glycol ester of low-molecular-weight alginate derivative.
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As other edible dietary fibers, alginate and its oligomer derivatives are resistant to
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the digestion by human endogenous enzymes, but can be utilized to a large extent by
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human gut microbiota [9]. Wang et al. found that the alginate oligosaccharides prepared
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through enzymatic hydrolysis of alginate enhanced the growth of intestinal
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bifidobacteria and lactobacilli of male Wistar rats after feeding for two weeks [10].
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Ramnani et al. [11] compared the fermentation patterns of three types of alginates with
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molecular weight of 212 kDa, 97 kDa, and 38 kDa and found that alginate of 212 kDa
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However, the human colonic bacteria that respond to the degradation of alginate and
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derivatives still remain unknown. It is desirable to investigate the fermentability of the
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different components of alginate and their derivatives by human gut microbiota and
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identify the degrading bacterial strains.
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In the current study, the fermentability of Alg, MO, GO and PSS by the gut
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microbes obtained from six Chinese individuals were compared in batch culture
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fermentation. The effects of the alginate and its derivatives on the bacterial composition
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were also evaluated by PCR-denaturing gradient gel electrophoresis (PCR-DGGE).
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Four alginate and alginate oligosaccharides hydrolyzing bacteria, Bacteroides
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xylanisolvens, Bacteroides thetaiotaomicron, and two strains of Bacteroides ovatus
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were also identified.
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MATERIALS AND METHODS
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Substrates. Alg with a molecular weight of 100 kD was purchased from Sangon
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Biotech Company (Shanghai, China,). MO (with a molecular weight of 2.5 kD) and GO
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(with a molecular weight of 4.0 kD) were obtained from Lantai Pharmaceutical
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Company (Qingdao, China) and prepared by using the following method with some
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modification [12]. In brief, alginate was hydrolyzed in 0.5 mol/L HCl at 100 °C for 8 h,
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the fraction insoluble in the hydrolysate was solubilized at neutral pH, followed by pH
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adjustment to 2.85. At this pH, the insoluble fraction G1 (G-rich fraction) was
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subsequently separated from the soluble fraction by centrifugation. M1 (M-rich fraction)
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and GO were obtained after M1 and G1 had been hydrolyzed in 0.1 mol/L HCl at 100°C
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for 3 h, then repeated fractionated precipitation at pH 2.85 and pH 1.50, respectively,
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was performed. The purity of GO and MO were at least 90% based on the
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monosaccharides
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1-phenyl-3-methyl-5-pyrazolone by HPLC [13, 14]. PSS, a derivative of alginate, with
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the molecular weight of 10-20 kD, was provided by Dalian Aosen Pharmaceutical
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corporation Ltd (Dalian, China). The structures of Alg, MO, GO and PSS were showed
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in Figure 1.
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Fecal sample preparation. Fecal samples were provided by six healthy human
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volunteers (aged 22 to 35 years). All volunteers provided informed, written consent, and
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the study was approved by the Ethics Committee of the Zhejiang Academy of
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Agricultural Sciences. The donors had not received antibiotics, probiotics, prebiotics, or
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synbiotics treatments for at least three months prior to sample collection. After
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collection, the fresh fecal samples were immediately homogenized in stomacher bags
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with 0.1 M anaerobic phosphate-buffered saline (PBS, pH 7.0) to create 20% (wt/vol)
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slurries. Large food residues were removed by passing the mixture through a 0.4 mm
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sieve, and the suspensions were used as inocula.
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Batch culture fermentation of alginate and its derivatives with human fecal
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slurries. The effect of alginate and its derivatives on the composition of gut microbiota
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and fermentation properties were evaluated by using the batch culture fermentation 5
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anaerobic medium VI with the following ingredients (g/L): peptone, 3.0; tryptone, 3.0;
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yeast extract, 4.5; bile salts no. 3, 0.4; NaCl, 4.5; L-cysteine, 0.8; Hemin, 0.05; KCl, 2.5;
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KH2PO4, 0.4; MgCl2·6H2O, 4.5; CaCl2·6H2O, 0.2; resazurin, 0.25; Tween 80, 1 ml.
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Two milliliters of a solution of trace elements (g/L) containing MgSO4·7H2O, 3.0;
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MnCl2·4H2O, 0.32; FeSO4·7H2O, 0.1; CoSO4·7H2O, 0.18; CaCl2·2H2O, 0.1;
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ZnSO4·7H2O, 0.18; CuSO4·5H2O, 0.01 and NiCl2·6H2O, 0.092 was also added. To
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assess the degradation and utilization of alginate and its derivatives by human fecal
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microbiota, 5.0 g/L of Alg, 8 g/L of MO, GO or PSS were added as the sole carbon
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source and the medium without any carbon substances was used as a control. The final
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pH 6.5 was adjusted by adding 1 M HCl. After being autoclaved, the medium
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maintained its anaerobic condition by placing the hot bottle into the anaerobic cabinet
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(anaerobic workstation AW 500, Electrotek Ltd., UK). The human fecal slurry (7 ml)
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was inoculated into the serum bottle containing growth medium and the bottle incubated
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at 37 °C for 48 h in anaerobic chamber. Samples were removed at 0, 3, 6, 9, 12, 24, and
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48 h for further analysis. The pH value after 48 h fermentation was measured by pH
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probe (Eutech, Singapore).
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Analysis of carbohydrates degradation. To evaluate the degradation of alginate
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and its derivatives, thin layer chromatography (TLC) was used [16]. Samples (0.2 µl)
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were loaded on a pre-coated silica gel-60 TLC aluminum plates (Merck, Germany).
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After development with a solvent system consisting of n-butanol/formic acid/water 6
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(4:6:1 (v:v:v)), the plates were stained by dipping in the orcinol reagent (900 mg of
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orcinol, 25 ml of H2O, 375 ml of cool ethanol, 50 ml of concentrated sulfuric acid) and
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visualized by heating at 105 ℃ for 5 minutes. SCFA analysis. Analysis of SCFA was carried out by HPLC [17]. Fermentation
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samples obtained at 48 h was centrifuged at 14,000 g and the supernatant (20 µl) was
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subjected to Agilent 1260 infinity HPLC system (Agilent Scientific, USA). The SCFA
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was analyzed using HPLC on an ion-exclusion Aminex HPX-87H (7.8 × 300 mm;
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Bio-Rad, UK) with 0.005 mol/l H2SO4 at a flow rate of 0.6 ml/min at 50 °C. The
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detection wavelength was set to 215 nm.
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DNA extraction and PCR-DGGE analysis. Bacterial genomic DNA from
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fermentation samples obtained at 0 and 48 h were extracted respectively using a
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QIAamp DNA Stool Mini Kit according to the manufacturer’s instructions (Qiagen,
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Germany). The concentration of extracted DNA was determined using a NanoDrop
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ND-2000 (NanoDrop Technologies, USA), and its integrity and size were confirmed by
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agar gel electrophoresis (1.0%).
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For analysis of the microbial communities, the V3 region of the 16S rRNA gene
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(positions 341 to 534 of the Escherichia coli gene) of all fermentations at 0 and 48 h
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was analyzed using PCR-DGGE, as described previously [15, 18, 19]. DGGE was
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performed using a DCode universal mutation detection system (Bio-Rad, USA) in an
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8% (wt/vol) polyacrylamide gel containing a linear 35-65% denaturant gradient with a
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constant voltage of 200 V at 60 °C for 4 h [19]. The gels were then visualized by 7
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staining with SYBR green I nucleic acid (Sigma, USA) for 45 min and washing twice
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with deionized water. Blocks of polyacrylamide gels containing selected DGGE bands were cut with a
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sterile scalpel. The blocks were then transferred in 40 µl of sterile water, and the DNA
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of the bands was allowed to diffuse overnight at 4 °C. The water containing the eluted
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DNA was used for the reamplification. The PCR products were purified and cloned in
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PMD-18T vector (Takara, China). Three colonies were selected per DGGE band and
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sequenced by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Searches in the
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NCBI database were performed with the BLAST program to identify the closest known
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relatives of the partial 16S rRNA sequence obtained [20, 21].
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Statistical analysis. The DGGE gels were analyzed using Quantity One software
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(Bio-Rad, USA) with a match tolerance of 2% to compare the community structures of
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controls, Alg, MO and GO. After matching all bands in each lane, Richness (S) values
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were calculated as the number of DNA bands detected in the respective line of the
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DGGE profile, while the Shannon-Wiener index (H) and evenness (E) values were
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calculated according to the equations H= −∑Pi (ln Pi) and EH= H/Hmax= H/lnS,
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respectively, where Pi is the ratio between the specific band intensity and the total
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intensity of all bands and S is the total number of bands in each sample.
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The pH value and SCFAs of each fermentation sample were measured. Means and
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standard deviations (SD) were calculated. Differences were separately assessed using
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the unpaired two-tailed Student t test; P values of 0.05 were considered statistically 8
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significant. Isolation and identification of alginate and its derivatives degradation bacteria.
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Two grams of fresh fecal sample No. 1 and No. 4 were homogenized separately in
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stomacher bags with 10 ml of 0.1 M anaerobic PBS (pH 7.0) to produce 20% (wt/vol)
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slurries. 7 ml of slurry was inoculated into 63 ml of VI growth medium containing 5 g/L
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of Alg. After incubation in the medium at 37 °C for 24 h in an anaerobic chamber,
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fermentation samples were spread on an Alg agar plate (basic growth medium VI plus 5
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g/L Alg and 15 g/L agar) using a 10-fold dilution method. Thirty single colonies each
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for both No. 1 and No. 4 were randomly picked and re-inoculated into VI-Alg growth
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medium separately. Alg degradation was assessed using TLC analysis of the
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supernatant. Positive colonies were inoculated into growth medium separately
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containing Alg (5 g/L), GO and MO (8 g/L), and incubated at 37 °C for 192 h in
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anaerobic chamber. A total of 300 µl of sample were removed at 0, 12, 24, 48, 96, 144,
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and 192 h for analysis of carbohydrate degradation respectively.
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The isolates that demonstrated an ability to degrade Alg and its derivatives were
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identified by sequencing their 16S rRNA gene. Briefly, genomic DNA was extracted
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and
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(5′-CAGAGTTTGATCCTGGCT-3′) and 1492R (5′-AGGAGGTGATCCAGCCGCA-3′)
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[22]. DNA sequencing was conducted by Shanghai Sangon Biotech Co., Ltd. (Shanghai,
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China). Bacterial species were identified using aligned 16S rRNA sequences with the
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BLAST server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the National Center for
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Biotechnology Information (NCBI).
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RESULTS Comparative utilization of alginate and its derivatives by six human fecal
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samples. The degradation of alginate and its derivatives by fecal microbiota obtained
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from six volunteers was determined by TLC and presented in Figure 2. In general, Alg,
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MO and GO could be completely degraded into oligosaccharides or monosaccharide
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after 24 h fermentation by human fecal microbiota, but the rates of degradation, which
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was evidenced as the time point for disappears of the original dot on TLC plates, varied
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between the tested substrates. GO was degraded slower than Alg and MO, as the
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original GO dots were disappeared after 12 hours incubation in 4 out of 6 tested fecal
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samples (Figure 2C), whiles the Alg and MO dots were completely disappeared after 6
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hours for most of the samples (Figure 2A and 2B). PSS, which is a sulfated propylene
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glycol ester of low-molecular-weight alginate, was resistant to the degradation by the
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human gut microbiota (Figure 2D). No dots were observed in the control group,
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indicating that no carbohydrate was presented in the blank culture medium.
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pH value and SCFA production in vitro fermentation. The pH value of the
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fermentation of different substances was varied significantly (Table 1). Compared to the
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control group, the pH values of fermentation with Alg, MO and GO were all decreased.
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GO produced the lowest pH with a mean value of 5.96. In contrast, the pH value of PSS
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was nearly unchanged, which confirmed the TLC results that it was not degraded.
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The concentration of SCFA produced during the fermentation after 48 h is shown in 10
ACCEPTED MANUSCRIPT Table 1. The SCFA mainly included acetic, propionic and butyric acid. Compared to the
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control group, the fermentation of all substances except PSS showed a significant
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increase in acetate, propionate, and butyrate production, indicating that these substances
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were produced by the human gut microbiota. Acetate was the predominant SCFA
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produced during the fermentation, with the concentration at 40-50 mM. For these
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different substances, GO produced the highest levels of acetate (52.4 mM), propionate
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(23.2 mM) and butyrate (20.8 mM) (P < 0.05), while MO was similar to alginate in the
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concentration of the SCFA. PSS produced almost the same level of SCFA as the control
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group, which was in accord with the result of pH value and indicated that PSS was not
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fermented by the gut microbiota. The molar ratios of acetate, propionate, and butyrate
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were consistent between the control group and PSS, but an increase was observed in the
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molar ratio of acetate after Alg, MO and GO fermentation, while the proportion of
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butyrate was slightly decreased (Table 1).
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The effects of alginate and its derivatives fermentation on the structures of
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bacterial communities analyzed by PCR-DGGE profiles. The PCR-DGGE profiles
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of bacterial 16s rRNA V3 region after the fermentation of Alg, MO, GO and PSS were
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showed in Figure 3. As previously reported [23, 24], the community structures of gut
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microbiota from individuals were varied remarkably, and supplement of carbohydrates
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into the growth medium could influence the compositions of the community structures
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of microbiota. Compared with the control group, both the Richness and
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Shannon-Wiener index (Table 3) of Alg, MO and GO groups were improved, indicated
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other substrates, the concentrations of DNA extracted from PSS fermentations by six
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human fecal slurries were very low. There were no bands after PCR amplification of the
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16s rDNA V3 region. We assume that the lack of PCR products is likely caused by PSS
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as either blocking the extraction of DNA or inhibiting the growth of the microbiota.
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The enriched bacterial species were identified by sequencing the bands on
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PCR-DGGE gel (Table 2). Bacteroides xylanisolvens (band a) was found in the samples
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of No. 1, No. 2, No. 3, No. 5, and No. 6 after fermentation with Alg, MO, and GO;
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Clostridium clostridioforme/ Clostridium symbiosum (band d) was found in all of the
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fermentations; Bacteroides finegoldii (band k) and Shigella flexneri/ Escherichia coli/
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Escherichia fergusonii (band j) were detected in half of the fermentation samples;
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Bacteroides ovatus (band n) was also found in two samples, which indicated these
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bacteria may be associated with the degradation and utilization of alginate and its
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derivatives.
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Isolation of the degradation bacteria for alginate and its derivatives. Fecal
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sample No. 1 and No. 4 were selected further to isolate the degradation bacteria, the
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fecal culture (150 µl), which had been grown in medium containing Alg for 24 h, was
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spread on Alg agar plates to isolate the Alg hydrolyzing bacterium. Total of 41 (No. 1)
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and 52 (No. 4) colonies were obtained from 7-fold dilution plates respectively. Thirty
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colonies each for both No. 1 and No. 4 were picked up from the Alg agar plates and
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then re-inoculated into growth medium containing Alg. TLC analysis was conducted
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substrate had been obviously degraded from the supernatant of two isolates G19 and
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G25 for No.1 and two isolates A9 and A12 for No.4, suggesting that they may be the
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Alg degrading bacteria (Figure 4). Further incubation and TLC pattern analysis
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suggested that MO and GO could also be degraded by G19 and G25 (Figure 4). After
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sequencing 16S rRNA from the four strains, G19 and G25 were identified as
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Bacteroides ovatus G19 and Bacteroides xylanisolvens G25, A9 and A12 were
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identified as Bacteroides ovatus A9 and B. thetaiotaomicron A12 respectively (all
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showing ≥ 99% similarity under BLAST at the NCBI database). The sequences were
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submitted to NCBI under the accession number KM396275, KP202688, KT452899, and
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KT452900.
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DISCUSSION
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The human gut is inhabited by 1014 microbes, including 1,000 to 1,150 phylotypes
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[25]. Significant variations in the composition of colonic microbiota exist among
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individuals [23]. In this way, the ability to degrade and utilize complex polysaccharides
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may vary among the individuals due to the different compositions of gut microbiota.
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Previous results showed that the degradation of agarose and agaro-oligosaccharides by
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human gut microbiota was significantly different between individuals [24]. The present
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data confirmed that Alg, MO and GO could be hydrolyzed by all of the six human fecal
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samples tested, but at different degradation rates. For example, No. 6 fecal sample
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showed the slowest degradation rates on all of the substrates. Moreover, the chemical
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2, degradation of GO is more difficult than MO and Alg by human gut microbiota. That
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may be ascribed to the different chemical structures of fermented substrates. MM block
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is formed from equatorial groups at C-1 and C-4; therefore, it is a relatively straight
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polymer looking like a flat ribbon. In contrast, the GG block is formed from axial
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groups at both C-1 and C-4. Therefore, the chain of GG is buckled (Figure 1). The
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difference in the configuration of chemical structures leads to the formation of different
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gel properties. For example, GG block forms more rigid gels [26], whereas the MM
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block tends to form softer gels. The configuration difference may explain the reason
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why the degradation of GO is more difficult than MO and Alg. Among the tested
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carbohydrates, PSS cannot be degraded completely. PSS is the hydrolyzed fragment of
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alginate that is further esterified by propylene epoxide and chlorosulfonic acid;
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therefore, the change in chemical structure, particularly the introduction of sulfate may
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block the degradation by human gut microbiota.
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Bacteroides and Clostridium were the predominant bacteria in human gut
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microbiota, which could help the host decompose polysaccharides and improve the
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efficiency of nutrition [27]. In the present study, Bacteroides xylanisolvens, Bacteroides
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ovatus, Clostridium clostridioforme, Bacteroides finegoldii and Shigella flexneri were
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identified on the PCR-DGGE profiles, suggesting that they may be related to the
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utilization of Alg, MO and GO. The following isolation experiment confirmed that B.
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xylanisolvens and B. ovatus were involved in the degradation of alginate and its
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thetaiotaomicron is also a common gut bacterium able to degrade polysaccharides. The
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genome sequence and proteome analysis showed B. thetaiotaomicron contains 261
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glycoside hydrolases and polysaccharide lyases[28]. Here, we showed that B.
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thetaiotaomicron is involved in the alginate degradation.
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SCFA produced from fermentation of carbohydrates by human gut microbiota
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have a range of physiological and immunological effects on colonic homeostasis and
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health [29-31]. In the current study, alginate and its derivates can be fermented by
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human gut bacteria to produce SCFA. It is interesting to note that although Alg, MO
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and GO can increase the SCFA production levels significantly, the slow fermented GO
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produces the highest level of SCFA, which was in accordance with the previous report
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that slow-fermenting substrates may provide more SCFA to the distal colon than
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fast-fermenting substrates[32, 33]. The current results suggest that compared to Alg and
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MO, GO may possess a better potential application as a prebiotic and to regulate the
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colonic health.
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B. ovatus has been associated with immunogenicity. Mice immunized with B.
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ovatus D-6 in the absence of adjuvants develop specific anti-TFa IgM and IgG
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antibodies [34]. Moreover, monocolonization with B. ovatus could also protect the
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immunodeficient SCID mice from mortality in chronic intestinal inflammation, caused
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by long-lasting dextran sodium sulfate treatment [35]. B. xylanisolvens is a
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xylan-degrading bacterium isolated from human feces [36], Ulsemer [37, 38] studied 15
ACCEPTED MANUSCRIPT the safety of a Bacteroides xylanisolvens strain (DSM 23964) recently and reported that
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it is safe and tolerated by healthy human individuals. Alg, MO and GO was mainly
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extracted from Laminaria japonica, which is commonly consumed as food worldwide
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for hundreds of years and has been established as healthy food materials that are rich in
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minerals and dietary fibers. Our data present new evidence suggesting that alginate and
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its derivates could be degraded by special bacteria in the human gut microbiota. Further
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study will carry out to evaluate the impacts of alginate and its derivates plus with their
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degrading bacteria on host health.
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ACKNOWLEDGEMENTS
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This work was supported in part by National Natural Science Foundation of China
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(31370156 and 31070724), NSFC-Shandong Joint Fund for Marine Science Research
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Centers (U1406402), National Science & Technology Support Program of China
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(2013BAB01B02), Taishan Scholars Project Special Funds, and the Special Fund for
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Marine Scientific Research in the Public Interest (201005024).
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Figure 1 Chemical structures of Alg, MO, GO and PSS
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Figure 2 Degradation of Alg (A), MO (B), GO (C), and PSS (D) by fecal slurries of six
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volunteers in batch fermentation. Results were analyzed by TLC.
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Figure 3 PCR-DGGE profiles of in vitro fermentation of Alg, MO and GO by gut microbiota
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from six volunteers. Lanes 1-5 are: original inoculum, control and fermentation of Alg, MO and GO
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by fecal slurry obtained from No. 1 volunteer after 48 h. Line 6-10, 11-15, 16-20, 21-25 and 26-30
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are the fermentation samples with the same order as described for Lanes 1-5, but inoculated with the
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fecal slurries obtained from Nos. 2, 3, 4, 5, and 6 volunteers, respectively. Lettered bands were
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sequenced and BLAST with the NCBI database.
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xylanisolvens G25 (B), Bacteroides ovatus A9 (C) and Bacteroides. thetaiotaomicron A12 (D).
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Figure 4 Degradation of Alg, MO and GO by Bacteroides ovatus G19 (A), Bacteroides
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Table 1 pH value and SCFA production after fermentation of Alg, GO, MO and PSS for 48 h of
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culture by six human fecal microbiota in batch culture Mean SCFA concentration (mM) pH value Acetate
Propionate
Butyrate
6.44±0.11
30.1±2.4
17.0±3.5
15.4±3.2
Alg
6.24±0.11*
41.3±5.8*
21.0±2.4*
16.4±3.1
MO
6.00±0.12*
41.8±3.6*
19.5±3.6
18.9±3.9
GO
5.96±0.13*
52.4±3.7*
23.2±4.5*
20.8±3.7*
PSS
6.47±0.10
30.2±4.8
18.3±1.1
15.3±2.2
Total
48:27:25
62.5±5.1
53:27:21
78.6±5.9*
52:24:24
80.2±7.4*
54:24:22
96.4±7.3*
47:29:24
63.8±4.8
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Control
Molar ratio
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Substrates#
Data are shown as means ± SD; #: Alg, Alginate; MO, oligo-β-D-mannuronic acid；GO,
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oligo-α-L-guluronic acid; PSS, Propylene glycol alginate sodium sulfate; Control, control group
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without adding carbohydrates in the medium. *P < 0.05 by two-tailed Student t test compared to the
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control group
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Table 2 The BLAST results of the lettered bands Bands
Similar species
identity
a
Bacteroides xylanisolvens
100%
b
Bacteroides sp. HGA0251 Ruminococcaceae bacterium,
100% 100%
Clostridiales bacterium, Firmicutes bacterium
Clostridium clostridioforme, Clostridium symbiosum
100%
e
Clostridium propionicum
100%
f
Sutterella sp.
g
Lachnospiraceae bacterium, Faecalibacterium prausnitzii
100%
h
Parasutterella excrementihominis
100%
i
Barnesiella intestinihominis, Bacteroidetes bacterium
100%
j
Shigella flexneri, Escherichia coli, Escherichia fergusonii
100%
k
Bacteroides finegoldii
100%
l
Bacteroides uniformis, Bacteroides sp. S461
100%
m
Bacteroides finegoldii
99%
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n
Bacteroides ovatus
o
Parabacteroides distasonis
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100% 100%
Table 3 Diversity indices calculated from the DGGE bands profiles generated from V3 region Group
Richness (S)
Alg MO
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GO
12.67±2.88
Shannon-Wiener index (H)
Evenness (E)
3.49±0.39
0.96±0.03
15.83±2.32*
3.77±0.38
0.95±0.05
16.67±1.03*
3.89±0.16*
0.96±0.03
15.00±1.55
3.66±0.24
0.94±0.04
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100%
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343 344 345 346
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*significant increase compared to the control group, P < 0.05
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Human gut microbiota degrade alginate and its oligosaccharides Bacteria identified for degrading alginate and its oligosaccharides are Bacteroides
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Alginate and its oligosaccharides increase the production level of
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S1075-9964(16)30006-3
DOI:
10.1016/j.anaerobe.2016.02.003
Reference:
YANAE 1534
To appear in:
Anaerobe
Received Date: 29 November 2015 Revised Date:
9 February 2016
Accepted Date: 10 February 2016
Please cite this article as: Li M, Li G, Shang Q, Chen X, Liu W, Pi X’e, Zhu L, Yin Y, Yu G, Wang X, In vitro fermentation of alginate and its derivatives by human gut microbiota, Anaerobe (2016), doi: 10.1016/j.anaerobe.2016.02.003. This is a PDF file of an unedited manuscript that has been accepted for publication. As a service to our customers we are providing this early version of the manuscript. The manuscript will undergo copyediting, typesetting, and review of the resulting proof before it is published in its final form. Please note that during the production process errors may be discovered which could affect the content, and all legal disclaimers that apply to the journal pertain.
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In vitro fermentation of alginate and its derivatives by human gut microbiota
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Miaomiao Li1†, Guangsheng Li1†, Qingsen Shang1, Xiuxia Chen2, Wei Liu2, Xiong’e Pi2,
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Liying Zhu2, Yeshi Yin2, Guangli Yu1*, Xin Wang2*
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Shandong Provincial Key Laboratory of Glycoscience and Glycotechnology, Key
Laboratory of Marine Drugs of Ministry of Education, Ocean University of China, and
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Laboratory for Marine Drugs and Bioproducts, Qingdao National Laboratory for Marine
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Science and Technology, Qingdao, 266003, China
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State Key Laboratory of Breeding Base for Zhejiang Sustainable Pest and Disease
Control, Institute of Plant Protection and Microbiology, Academy of Agricultural
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Sciences, Hangzhou, Zhejiang, China.
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Corresponding authors:
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E-mail: [email protected] or [email protected]
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ABSTRACT Alginate (Alg) has a long history as a food ingredient in East Asia. However, the
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human gut microbes responsible for the degradation of alginate and its derivatives have
18
not been fully understood yet. Here, we report that alginate and the low molecular
19
polymer derivatives of mannuronic acid oligosaccharides (MO) and guluronic acid
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oligosaccharides (GO) can be completely degraded and utilized at various rates by fecal
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microbiota obtained from six Chinese individuals. However, the derivative of propylene
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glycol alginate sodium sulfate (PSS) was not hydrolyzed. The bacteria having a
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pronounced ability to degrade Alg, MO and GO were isolated from human fecal
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samples and were identified as Bacteroides ovatus, Bacteroides xylanisolvens, and
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Bacteroides thetaiotaomicron. Alg, MO and GO can increase the production level of
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short chain fatty acids (SCFA), but GO generates the highest level of SCFA. Our data
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suggest that alginate and its derivatives could be degraded by specific bacteria in the
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human gut, providing the basis for the impacts of alginate and its derivates as special
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food additives on human health.
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Key words: human gut microbiota, alginate and its derivatives, B. ovatus,
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B. xylanisolvens, B. thetaiotaomicron, hydrolyze
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INTRODUCTION Alginates are naturally occurring anionic copolymers of a linear β (1→4)-linked
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glycuronan typically composed of residues of β-D-mannuronic acid (M) and its C-5
36
epimer α-L-guluronic acid (G) [1]. They are extracted from brown seaweed and have
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been traditionally used as gelling agents in food, pharmaceuticals, cosmetics, feeds, and
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other industrial applications [2-4]. Under the natural growth condition, the following
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three types of polymers are present in the alginate according to the block chemical
40
structures: poly β-D-mannuronic acid (MM), poly α-L-guluronic acid (GG) and
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heteropolymer (MG), the latter contains random arranged residues of M and G,
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Moreover, MM and GG demonstrate different gel forming properties compared to MG
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[5]. Alginate oligosaccharides were reported to have various activities such as
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antifungal activity [6], anti-inflammatory activity [7] and immunomodulatory activities
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[8]. Propylene glycol alginate sodium sulfate (PSS), a drug against cardiovascular
46
disease, is a sulfated propylene glycol ester of low-molecular-weight alginate derivative.
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As other edible dietary fibers, alginate and its oligomer derivatives are resistant to
48
the digestion by human endogenous enzymes, but can be utilized to a large extent by
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human gut microbiota [9]. Wang et al. found that the alginate oligosaccharides prepared
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through enzymatic hydrolysis of alginate enhanced the growth of intestinal
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bifidobacteria and lactobacilli of male Wistar rats after feeding for two weeks [10].
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Ramnani et al. [11] compared the fermentation patterns of three types of alginates with
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molecular weight of 212 kDa, 97 kDa, and 38 kDa and found that alginate of 212 kDa
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However, the human colonic bacteria that respond to the degradation of alginate and
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derivatives still remain unknown. It is desirable to investigate the fermentability of the
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different components of alginate and their derivatives by human gut microbiota and
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identify the degrading bacterial strains.
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In the current study, the fermentability of Alg, MO, GO and PSS by the gut
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microbes obtained from six Chinese individuals were compared in batch culture
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fermentation. The effects of the alginate and its derivatives on the bacterial composition
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were also evaluated by PCR-denaturing gradient gel electrophoresis (PCR-DGGE).
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Four alginate and alginate oligosaccharides hydrolyzing bacteria, Bacteroides
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xylanisolvens, Bacteroides thetaiotaomicron, and two strains of Bacteroides ovatus
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were also identified.
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MATERIALS AND METHODS
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Substrates. Alg with a molecular weight of 100 kD was purchased from Sangon
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Biotech Company (Shanghai, China,). MO (with a molecular weight of 2.5 kD) and GO
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(with a molecular weight of 4.0 kD) were obtained from Lantai Pharmaceutical
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Company (Qingdao, China) and prepared by using the following method with some
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modification [12]. In brief, alginate was hydrolyzed in 0.5 mol/L HCl at 100 °C for 8 h,
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the fraction insoluble in the hydrolysate was solubilized at neutral pH, followed by pH
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adjustment to 2.85. At this pH, the insoluble fraction G1 (G-rich fraction) was
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subsequently separated from the soluble fraction by centrifugation. M1 (M-rich fraction)
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and GO were obtained after M1 and G1 had been hydrolyzed in 0.1 mol/L HCl at 100°C
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for 3 h, then repeated fractionated precipitation at pH 2.85 and pH 1.50, respectively,
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was performed. The purity of GO and MO were at least 90% based on the
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monosaccharides
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1-phenyl-3-methyl-5-pyrazolone by HPLC [13, 14]. PSS, a derivative of alginate, with
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the molecular weight of 10-20 kD, was provided by Dalian Aosen Pharmaceutical
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corporation Ltd (Dalian, China). The structures of Alg, MO, GO and PSS were showed
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in Figure 1.
using
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Fecal sample preparation. Fecal samples were provided by six healthy human
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volunteers (aged 22 to 35 years). All volunteers provided informed, written consent, and
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the study was approved by the Ethics Committee of the Zhejiang Academy of
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Agricultural Sciences. The donors had not received antibiotics, probiotics, prebiotics, or
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synbiotics treatments for at least three months prior to sample collection. After
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collection, the fresh fecal samples were immediately homogenized in stomacher bags
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with 0.1 M anaerobic phosphate-buffered saline (PBS, pH 7.0) to create 20% (wt/vol)
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slurries. Large food residues were removed by passing the mixture through a 0.4 mm
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sieve, and the suspensions were used as inocula.
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Batch culture fermentation of alginate and its derivatives with human fecal
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slurries. The effect of alginate and its derivatives on the composition of gut microbiota
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and fermentation properties were evaluated by using the batch culture fermentation 5
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anaerobic medium VI with the following ingredients (g/L): peptone, 3.0; tryptone, 3.0;
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yeast extract, 4.5; bile salts no. 3, 0.4; NaCl, 4.5; L-cysteine, 0.8; Hemin, 0.05; KCl, 2.5;
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KH2PO4, 0.4; MgCl2·6H2O, 4.5; CaCl2·6H2O, 0.2; resazurin, 0.25; Tween 80, 1 ml.
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Two milliliters of a solution of trace elements (g/L) containing MgSO4·7H2O, 3.0;
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MnCl2·4H2O, 0.32; FeSO4·7H2O, 0.1; CoSO4·7H2O, 0.18; CaCl2·2H2O, 0.1;
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ZnSO4·7H2O, 0.18; CuSO4·5H2O, 0.01 and NiCl2·6H2O, 0.092 was also added. To
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assess the degradation and utilization of alginate and its derivatives by human fecal
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microbiota, 5.0 g/L of Alg, 8 g/L of MO, GO or PSS were added as the sole carbon
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source and the medium without any carbon substances was used as a control. The final
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pH 6.5 was adjusted by adding 1 M HCl. After being autoclaved, the medium
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maintained its anaerobic condition by placing the hot bottle into the anaerobic cabinet
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(anaerobic workstation AW 500, Electrotek Ltd., UK). The human fecal slurry (7 ml)
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was inoculated into the serum bottle containing growth medium and the bottle incubated
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at 37 °C for 48 h in anaerobic chamber. Samples were removed at 0, 3, 6, 9, 12, 24, and
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48 h for further analysis. The pH value after 48 h fermentation was measured by pH
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probe (Eutech, Singapore).
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Analysis of carbohydrates degradation. To evaluate the degradation of alginate
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and its derivatives, thin layer chromatography (TLC) was used [16]. Samples (0.2 µl)
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were loaded on a pre-coated silica gel-60 TLC aluminum plates (Merck, Germany).
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After development with a solvent system consisting of n-butanol/formic acid/water 6
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(4:6:1 (v:v:v)), the plates were stained by dipping in the orcinol reagent (900 mg of
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orcinol, 25 ml of H2O, 375 ml of cool ethanol, 50 ml of concentrated sulfuric acid) and
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visualized by heating at 105 ℃ for 5 minutes. SCFA analysis. Analysis of SCFA was carried out by HPLC [17]. Fermentation
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samples obtained at 48 h was centrifuged at 14,000 g and the supernatant (20 µl) was
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subjected to Agilent 1260 infinity HPLC system (Agilent Scientific, USA). The SCFA
123
was analyzed using HPLC on an ion-exclusion Aminex HPX-87H (7.8 × 300 mm;
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Bio-Rad, UK) with 0.005 mol/l H2SO4 at a flow rate of 0.6 ml/min at 50 °C. The
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detection wavelength was set to 215 nm.
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DNA extraction and PCR-DGGE analysis. Bacterial genomic DNA from
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fermentation samples obtained at 0 and 48 h were extracted respectively using a
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QIAamp DNA Stool Mini Kit according to the manufacturer’s instructions (Qiagen,
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Germany). The concentration of extracted DNA was determined using a NanoDrop
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ND-2000 (NanoDrop Technologies, USA), and its integrity and size were confirmed by
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agar gel electrophoresis (1.0%).
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For analysis of the microbial communities, the V3 region of the 16S rRNA gene
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(positions 341 to 534 of the Escherichia coli gene) of all fermentations at 0 and 48 h
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was analyzed using PCR-DGGE, as described previously [15, 18, 19]. DGGE was
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performed using a DCode universal mutation detection system (Bio-Rad, USA) in an
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8% (wt/vol) polyacrylamide gel containing a linear 35-65% denaturant gradient with a
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constant voltage of 200 V at 60 °C for 4 h [19]. The gels were then visualized by 7
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staining with SYBR green I nucleic acid (Sigma, USA) for 45 min and washing twice
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with deionized water. Blocks of polyacrylamide gels containing selected DGGE bands were cut with a
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sterile scalpel. The blocks were then transferred in 40 µl of sterile water, and the DNA
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of the bands was allowed to diffuse overnight at 4 °C. The water containing the eluted
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DNA was used for the reamplification. The PCR products were purified and cloned in
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PMD-18T vector (Takara, China). Three colonies were selected per DGGE band and
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sequenced by Shanghai Sangon Biotech Co., Ltd. (Shanghai, China). Searches in the
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NCBI database were performed with the BLAST program to identify the closest known
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relatives of the partial 16S rRNA sequence obtained [20, 21].
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Statistical analysis. The DGGE gels were analyzed using Quantity One software
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(Bio-Rad, USA) with a match tolerance of 2% to compare the community structures of
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controls, Alg, MO and GO. After matching all bands in each lane, Richness (S) values
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were calculated as the number of DNA bands detected in the respective line of the
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DGGE profile, while the Shannon-Wiener index (H) and evenness (E) values were
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calculated according to the equations H= −∑Pi (ln Pi) and EH= H/Hmax= H/lnS,
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respectively, where Pi is the ratio between the specific band intensity and the total
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intensity of all bands and S is the total number of bands in each sample.
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The pH value and SCFAs of each fermentation sample were measured. Means and
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standard deviations (SD) were calculated. Differences were separately assessed using
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the unpaired two-tailed Student t test; P values of 0.05 were considered statistically 8
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significant. Isolation and identification of alginate and its derivatives degradation bacteria.
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Two grams of fresh fecal sample No. 1 and No. 4 were homogenized separately in
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stomacher bags with 10 ml of 0.1 M anaerobic PBS (pH 7.0) to produce 20% (wt/vol)
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slurries. 7 ml of slurry was inoculated into 63 ml of VI growth medium containing 5 g/L
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of Alg. After incubation in the medium at 37 °C for 24 h in an anaerobic chamber,
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fermentation samples were spread on an Alg agar plate (basic growth medium VI plus 5
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g/L Alg and 15 g/L agar) using a 10-fold dilution method. Thirty single colonies each
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for both No. 1 and No. 4 were randomly picked and re-inoculated into VI-Alg growth
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medium separately. Alg degradation was assessed using TLC analysis of the
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supernatant. Positive colonies were inoculated into growth medium separately
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containing Alg (5 g/L), GO and MO (8 g/L), and incubated at 37 °C for 192 h in
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anaerobic chamber. A total of 300 µl of sample were removed at 0, 12, 24, 48, 96, 144,
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and 192 h for analysis of carbohydrate degradation respectively.
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The isolates that demonstrated an ability to degrade Alg and its derivatives were
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identified by sequencing their 16S rRNA gene. Briefly, genomic DNA was extracted
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and
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(5′-CAGAGTTTGATCCTGGCT-3′) and 1492R (5′-AGGAGGTGATCCAGCCGCA-3′)
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[22]. DNA sequencing was conducted by Shanghai Sangon Biotech Co., Ltd. (Shanghai,
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China). Bacterial species were identified using aligned 16S rRNA sequences with the
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BLAST server (http://blast.ncbi.nlm.nih.gov/Blast.cgi) of the National Center for
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the
16S
rRNA
gene
was
amplified
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by
PCR
with
primers
27F
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Biotechnology Information (NCBI).
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RESULTS Comparative utilization of alginate and its derivatives by six human fecal
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samples. The degradation of alginate and its derivatives by fecal microbiota obtained
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from six volunteers was determined by TLC and presented in Figure 2. In general, Alg,
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MO and GO could be completely degraded into oligosaccharides or monosaccharide
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after 24 h fermentation by human fecal microbiota, but the rates of degradation, which
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was evidenced as the time point for disappears of the original dot on TLC plates, varied
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between the tested substrates. GO was degraded slower than Alg and MO, as the
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original GO dots were disappeared after 12 hours incubation in 4 out of 6 tested fecal
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samples (Figure 2C), whiles the Alg and MO dots were completely disappeared after 6
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hours for most of the samples (Figure 2A and 2B). PSS, which is a sulfated propylene
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glycol ester of low-molecular-weight alginate, was resistant to the degradation by the
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human gut microbiota (Figure 2D). No dots were observed in the control group,
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indicating that no carbohydrate was presented in the blank culture medium.
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pH value and SCFA production in vitro fermentation. The pH value of the
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fermentation of different substances was varied significantly (Table 1). Compared to the
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control group, the pH values of fermentation with Alg, MO and GO were all decreased.
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GO produced the lowest pH with a mean value of 5.96. In contrast, the pH value of PSS
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was nearly unchanged, which confirmed the TLC results that it was not degraded.
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The concentration of SCFA produced during the fermentation after 48 h is shown in 10
ACCEPTED MANUSCRIPT Table 1. The SCFA mainly included acetic, propionic and butyric acid. Compared to the
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control group, the fermentation of all substances except PSS showed a significant
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increase in acetate, propionate, and butyrate production, indicating that these substances
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were produced by the human gut microbiota. Acetate was the predominant SCFA
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produced during the fermentation, with the concentration at 40-50 mM. For these
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different substances, GO produced the highest levels of acetate (52.4 mM), propionate
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(23.2 mM) and butyrate (20.8 mM) (P < 0.05), while MO was similar to alginate in the
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concentration of the SCFA. PSS produced almost the same level of SCFA as the control
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group, which was in accord with the result of pH value and indicated that PSS was not
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fermented by the gut microbiota. The molar ratios of acetate, propionate, and butyrate
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were consistent between the control group and PSS, but an increase was observed in the
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molar ratio of acetate after Alg, MO and GO fermentation, while the proportion of
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butyrate was slightly decreased (Table 1).
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The effects of alginate and its derivatives fermentation on the structures of
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bacterial communities analyzed by PCR-DGGE profiles. The PCR-DGGE profiles
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of bacterial 16s rRNA V3 region after the fermentation of Alg, MO, GO and PSS were
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showed in Figure 3. As previously reported [23, 24], the community structures of gut
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microbiota from individuals were varied remarkably, and supplement of carbohydrates
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into the growth medium could influence the compositions of the community structures
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of microbiota. Compared with the control group, both the Richness and
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Shannon-Wiener index (Table 3) of Alg, MO and GO groups were improved, indicated
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other substrates, the concentrations of DNA extracted from PSS fermentations by six
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human fecal slurries were very low. There were no bands after PCR amplification of the
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16s rDNA V3 region. We assume that the lack of PCR products is likely caused by PSS
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as either blocking the extraction of DNA or inhibiting the growth of the microbiota.
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The enriched bacterial species were identified by sequencing the bands on
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PCR-DGGE gel (Table 2). Bacteroides xylanisolvens (band a) was found in the samples
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of No. 1, No. 2, No. 3, No. 5, and No. 6 after fermentation with Alg, MO, and GO;
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Clostridium clostridioforme/ Clostridium symbiosum (band d) was found in all of the
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fermentations; Bacteroides finegoldii (band k) and Shigella flexneri/ Escherichia coli/
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Escherichia fergusonii (band j) were detected in half of the fermentation samples;
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Bacteroides ovatus (band n) was also found in two samples, which indicated these
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bacteria may be associated with the degradation and utilization of alginate and its
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derivatives.
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Isolation of the degradation bacteria for alginate and its derivatives. Fecal
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sample No. 1 and No. 4 were selected further to isolate the degradation bacteria, the
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fecal culture (150 µl), which had been grown in medium containing Alg for 24 h, was
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spread on Alg agar plates to isolate the Alg hydrolyzing bacterium. Total of 41 (No. 1)
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and 52 (No. 4) colonies were obtained from 7-fold dilution plates respectively. Thirty
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colonies each for both No. 1 and No. 4 were picked up from the Alg agar plates and
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then re-inoculated into growth medium containing Alg. TLC analysis was conducted
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substrate had been obviously degraded from the supernatant of two isolates G19 and
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G25 for No.1 and two isolates A9 and A12 for No.4, suggesting that they may be the
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Alg degrading bacteria (Figure 4). Further incubation and TLC pattern analysis
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suggested that MO and GO could also be degraded by G19 and G25 (Figure 4). After
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sequencing 16S rRNA from the four strains, G19 and G25 were identified as
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Bacteroides ovatus G19 and Bacteroides xylanisolvens G25, A9 and A12 were
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identified as Bacteroides ovatus A9 and B. thetaiotaomicron A12 respectively (all
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showing ≥ 99% similarity under BLAST at the NCBI database). The sequences were
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submitted to NCBI under the accession number KM396275, KP202688, KT452899, and
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KT452900.
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DISCUSSION
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The human gut is inhabited by 1014 microbes, including 1,000 to 1,150 phylotypes
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[25]. Significant variations in the composition of colonic microbiota exist among
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individuals [23]. In this way, the ability to degrade and utilize complex polysaccharides
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may vary among the individuals due to the different compositions of gut microbiota.
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Previous results showed that the degradation of agarose and agaro-oligosaccharides by
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human gut microbiota was significantly different between individuals [24]. The present
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data confirmed that Alg, MO and GO could be hydrolyzed by all of the six human fecal
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samples tested, but at different degradation rates. For example, No. 6 fecal sample
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showed the slowest degradation rates on all of the substrates. Moreover, the chemical
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2, degradation of GO is more difficult than MO and Alg by human gut microbiota. That
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may be ascribed to the different chemical structures of fermented substrates. MM block
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is formed from equatorial groups at C-1 and C-4; therefore, it is a relatively straight
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polymer looking like a flat ribbon. In contrast, the GG block is formed from axial
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groups at both C-1 and C-4. Therefore, the chain of GG is buckled (Figure 1). The
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difference in the configuration of chemical structures leads to the formation of different
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gel properties. For example, GG block forms more rigid gels [26], whereas the MM
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block tends to form softer gels. The configuration difference may explain the reason
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why the degradation of GO is more difficult than MO and Alg. Among the tested
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carbohydrates, PSS cannot be degraded completely. PSS is the hydrolyzed fragment of
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alginate that is further esterified by propylene epoxide and chlorosulfonic acid;
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therefore, the change in chemical structure, particularly the introduction of sulfate may
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block the degradation by human gut microbiota.
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Bacteroides and Clostridium were the predominant bacteria in human gut
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microbiota, which could help the host decompose polysaccharides and improve the
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efficiency of nutrition [27]. In the present study, Bacteroides xylanisolvens, Bacteroides
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ovatus, Clostridium clostridioforme, Bacteroides finegoldii and Shigella flexneri were
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identified on the PCR-DGGE profiles, suggesting that they may be related to the
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utilization of Alg, MO and GO. The following isolation experiment confirmed that B.
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xylanisolvens and B. ovatus were involved in the degradation of alginate and its
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thetaiotaomicron is also a common gut bacterium able to degrade polysaccharides. The
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genome sequence and proteome analysis showed B. thetaiotaomicron contains 261
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glycoside hydrolases and polysaccharide lyases[28]. Here, we showed that B.
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thetaiotaomicron is involved in the alginate degradation.
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SCFA produced from fermentation of carbohydrates by human gut microbiota
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have a range of physiological and immunological effects on colonic homeostasis and
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health [29-31]. In the current study, alginate and its derivates can be fermented by
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human gut bacteria to produce SCFA. It is interesting to note that although Alg, MO
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and GO can increase the SCFA production levels significantly, the slow fermented GO
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produces the highest level of SCFA, which was in accordance with the previous report
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that slow-fermenting substrates may provide more SCFA to the distal colon than
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fast-fermenting substrates[32, 33]. The current results suggest that compared to Alg and
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MO, GO may possess a better potential application as a prebiotic and to regulate the
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colonic health.
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B. ovatus has been associated with immunogenicity. Mice immunized with B.
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ovatus D-6 in the absence of adjuvants develop specific anti-TFa IgM and IgG
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antibodies [34]. Moreover, monocolonization with B. ovatus could also protect the
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immunodeficient SCID mice from mortality in chronic intestinal inflammation, caused
304
by long-lasting dextran sodium sulfate treatment [35]. B. xylanisolvens is a
305
xylan-degrading bacterium isolated from human feces [36], Ulsemer [37, 38] studied 15
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it is safe and tolerated by healthy human individuals. Alg, MO and GO was mainly
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extracted from Laminaria japonica, which is commonly consumed as food worldwide
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for hundreds of years and has been established as healthy food materials that are rich in
310
minerals and dietary fibers. Our data present new evidence suggesting that alginate and
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its derivates could be degraded by special bacteria in the human gut microbiota. Further
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study will carry out to evaluate the impacts of alginate and its derivates plus with their
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degrading bacteria on host health.
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ACKNOWLEDGEMENTS
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This work was supported in part by National Natural Science Foundation of China
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(31370156 and 31070724), NSFC-Shandong Joint Fund for Marine Science Research
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Centers (U1406402), National Science & Technology Support Program of China
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(2013BAB01B02), Taishan Scholars Project Special Funds, and the Special Fund for
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Marine Scientific Research in the Public Interest (201005024).
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Figure 1 Chemical structures of Alg, MO, GO and PSS
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Figure 2 Degradation of Alg (A), MO (B), GO (C), and PSS (D) by fecal slurries of six
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volunteers in batch fermentation. Results were analyzed by TLC.
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Figure 3 PCR-DGGE profiles of in vitro fermentation of Alg, MO and GO by gut microbiota
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from six volunteers. Lanes 1-5 are: original inoculum, control and fermentation of Alg, MO and GO
329
by fecal slurry obtained from No. 1 volunteer after 48 h. Line 6-10, 11-15, 16-20, 21-25 and 26-30
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are the fermentation samples with the same order as described for Lanes 1-5, but inoculated with the
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fecal slurries obtained from Nos. 2, 3, 4, 5, and 6 volunteers, respectively. Lettered bands were
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sequenced and BLAST with the NCBI database.
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xylanisolvens G25 (B), Bacteroides ovatus A9 (C) and Bacteroides. thetaiotaomicron A12 (D).
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Figure 4 Degradation of Alg, MO and GO by Bacteroides ovatus G19 (A), Bacteroides
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Table 1 pH value and SCFA production after fermentation of Alg, GO, MO and PSS for 48 h of
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culture by six human fecal microbiota in batch culture Mean SCFA concentration (mM) pH value Acetate
Propionate
Butyrate
6.44±0.11
30.1±2.4
17.0±3.5
15.4±3.2
Alg
6.24±0.11*
41.3±5.8*
21.0±2.4*
16.4±3.1
MO
6.00±0.12*
41.8±3.6*
19.5±3.6
18.9±3.9
GO
5.96±0.13*
52.4±3.7*
23.2±4.5*
20.8±3.7*
PSS
6.47±0.10
30.2±4.8
18.3±1.1
15.3±2.2
Total
48:27:25
62.5±5.1
53:27:21
78.6±5.9*
52:24:24
80.2±7.4*
54:24:22
96.4±7.3*
47:29:24
63.8±4.8
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Molar ratio
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Substrates#
Data are shown as means ± SD; #: Alg, Alginate; MO, oligo-β-D-mannuronic acid；GO,
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oligo-α-L-guluronic acid; PSS, Propylene glycol alginate sodium sulfate; Control, control group
340
without adding carbohydrates in the medium. *P < 0.05 by two-tailed Student t test compared to the
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control group
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Table 2 The BLAST results of the lettered bands Bands
Similar species
identity
a
Bacteroides xylanisolvens
100%
b
Bacteroides sp. HGA0251 Ruminococcaceae bacterium,
100% 100%
Clostridiales bacterium, Firmicutes bacterium
Clostridium clostridioforme, Clostridium symbiosum
100%
e
Clostridium propionicum
100%
f
Sutterella sp.
g
Lachnospiraceae bacterium, Faecalibacterium prausnitzii
100%
h
Parasutterella excrementihominis
100%
i
Barnesiella intestinihominis, Bacteroidetes bacterium
100%
j
Shigella flexneri, Escherichia coli, Escherichia fergusonii
100%
k
Bacteroides finegoldii
100%
l
Bacteroides uniformis, Bacteroides sp. S461
100%
m
Bacteroides finegoldii
99%
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n
Bacteroides ovatus
o
Parabacteroides distasonis
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Table 3 Diversity indices calculated from the DGGE bands profiles generated from V3 region Group
Richness (S)
Alg MO
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GO
12.67±2.88
Shannon-Wiener index (H)
Evenness (E)
3.49±0.39
0.96±0.03
15.83±2.32*
3.77±0.38
0.95±0.05
16.67±1.03*
3.89±0.16*
0.96±0.03
15.00±1.55
3.66±0.24
0.94±0.04
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100%
d
343 344 345 346
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c
*significant increase compared to the control group, P < 0.05
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Human gut microbiota degrade alginate and its oligosaccharides Bacteria identified for degrading alginate and its oligosaccharides are Bacteroides
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Alginate and its oligosaccharides increase the production level of
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