Structural characterization of oligosaccharide from Spirulina platensis and its effect on the faecal microbiota in vitro

Bingn Ci, Xingxi Yi, Qin Hn, Jinyu Pn, Hu Chen, Huili Sun, Peng Wn,*

a Key Laboratory of Tropical Marine Bio-Resources and Ecology/Guangdong Key Laboratory of Marine Materia Medica, South China Sea Institute of Oceanology,Chinese Academy of Sciences, Guangzhou, 510301 Guangdong, China

b Southern Marine Science and Engineering Guangdong Laboratory (Guangzhou), Guangzhou, 511458 Guangdong, China

c School of Pharmacy, Guangxi University of Chinese Medicine, Nanning, 530200 Guangxi, China

d Innovation Academy of South China Sea Ecology and Environmental Engineering (ISEE), Chinese Academy of Sciences, Guangzhou, 510000 Guangdong, China

Keywords:

Oligosaccharide

Spirulina platensis

Gut microbiota

In vitro fermentationd

A B S T R A C T

In the present study, an oligosaccharide SPO-1 from Spirulina platensis was prepared by glycosidase from a marine bacterium.The prebiotic activity of SPO-1 on the growth of Lactobacillus paracasei and Bifidobacterium animalis, and its effect on human gut microbiota were examined in vitro.The molecular weight of the tetrasaccharide SPO-1 was 650.2 Da, and it was mainly composed of glucose with α-type glycosidic linkages.The prebiotic activity score of SPO-1 was the highest for the growth of probiotic strains L.paracasei and B.animalis.Furthermore, as fermentation proceeded, SPO-1 was gradually degraded and utilized by intestinal bacteria.The results showed that after treatment with SPO-1, carbohydrate consumption and short-chain fatty acids levels were increased, especially those of i-butyric and i-valeric acids.Moreover,SPO-1 significantly promoted the abundance, diversity and composition of gut microbiota, especially stimulating the growth of Bacteroides, Escherichia-Shigella and Megamonas.In addition, the change in intestinal microbiota function predicted by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt) after treatment with SPO-1 is mainly related to the terms “carbohydrate metabolism” and “amino acid metabolism”.These results suggest that SPO-1 is a potential oligosaccharide in regulation of intestinal microbiota.

1.Introduction

Accumulating evidence has revealed close associations between gut microbiota and a wide range of diseases, including colorectal cancer, metabolic syndrome, cardiovascular disease, inflammatory bowel disease, obesity and type 1 and type 2 diabetes [1].Dietary prebiotics are beneficial for host health, and are used as a selectively fermented ingredients and stimulate specific changes in the composition and activity of the gut microbiota [2].Polysaccharides or oligosaccharides are widely used as prebiotics, because they are not hydrolyzed by the enzymes of the gastrointestinal tract [3].Seaweeds and marine microalgae are one of the most important sources of polysaccharides or oligosaccharides for the discovery of potential prebiotic candidates [4-6].Oligosaccharides with a low degree of polymerization and a specific sequence of monosaccharide appear to be easier to ferment by gut microbiota [7].Research showed that agaro-oligosaccharides produced fromGracilaria lemaneiformisincreased the diversity of bacterial communities, and promoted the relative abundance of Bacteroidetes and Actinobacteria [8].Alginate oligosaccharides increased the abundance of beneficial gut microbiota, especially stimulated bothLactobacilliandBifidobacteria,and reduced the abundance of Enterobacteriaceae and Enterococci [9].The prebiotic effects of alginate oligosaccharides prepared by enzymatic method are more diverse than those produced by physical and chemical methods [10].In addition, marine microalgae oligosaccharides can be produced by enzymatic hydrolysis of polysaccharides, especially by new glycosidase from marine bacteria,which should be beneficial for changing polysaccharide structures and producing novel prebiotics [11,12].

Spirulina platensisis an edible microalgae that has long been used as a health and functional food.Spirulinapossesses various biological activities such as antioxidant, antibacterial, antiviral, anticancer, antiinflammatory, and antidiabetic activities [13-15].In particular, the consumption ofSpirulinahas been shown to promote the growth of intestinal microflora [16].Chen et al.[17]reported that active substances ofSpirulinacould regulate gut microbes to improve lipid metabolism of the high-fat diet fed rats.Among the active substances ofSpirulina, polysaccharides have shown a significantly regulatory effect on intestinal microbiota, which can not only increase the proportion of beneficial bacteria such asLactobacillus,Akekermansia,Arthromitus,Butyricimonas,CandidatusandPrevotella, but also reduce the proportion of harmful bacteria such asDoreaandClostridium[18].However, scarcely any information was found on the structure and prebiotic activity of the oligosaccharides fromS.platensis.

Thus, the aim of this research was to obtain a prebiotic oligosaccharide with novel structural characterization fromS.platensis, which was produced by marine microbial enzymes.The prebiotic effect of oligosaccharide on the gut microbiotain vitrowas investigated.Furthermore, changes in intestinal microbiota function were predicted by phylogenetic investigation of communities by reconstruction of unobserved states (PICRUSt).

2.Materials and methods

2.1 Materials

S.platensiswas provided by Haiyikang Biotechnology Co., Ltd.(Guangzhou, China) in the form of a powder.The glycosidase from the marine bacteriumAquimarinasp.(SCSIO21287) with aβ-agarase activity of 500 U/mL and purity of 90% was a gift of Dr.Jian Yang(South China Sea Institute of Oceanology, Chinese Academy of Sciences, SCSIO, CAS).Lactobacillus paracasei(ATCCSD5275)andEscherichia coli(ATCC25922) were provided by Dr.Li Huang(Buffalo Research Institute, Chinese Academy of Agricultural Sciences and Guangxi Zhuang Nationality Autonomous Region).Bifidobacterium animalis(ATCC25527) was provided by Prof.Jianwen Teng (College of Light Industry and Food Engineering,Guangxi University).Monosaccharide standards including xylose(Xyl), galactose (Gal), glucose (Glc), mannose (Man), arabinose(Ara), galacturonic acid (GalA), glucuronic acid (GluA) and shortchain fatty acid (SCFA) standards including acetic, propionic,n-butyric,i-butyric,n-valeric, andi-valeric acids were purchased from Sigma Chemical Co., Ltd (St.Louis, MO, USA).MRS and LB broth were purchased from Guangdong Huankai Microbial Technology Co., Ltd.(Guangdong, China).All other chemical reagents used were analytical grade.

2.2 Preparation of polysaccharides

The polysaccharides fromS.platensis(SPP) were extracted as described previously by Kurb [19].S.platensispowder was stirred in a 30-fold volume of distilled water.Then, the suspension was freezethawed three times and subjected to ultrasound extraction for 40 min at 80 °C with an ultrasound power of 900 W.After the extraction with ultrasonic treatment, the supernatant was collected by centrifugation at 6 000 ×gfor 30 min and mixed with four volume of absolute ethanol at 4 °C overnight.The SPP was recovered by centrifugation at 1 800 ×gfor 15 min, washed with three volumes of acetone, and then deproteinized by the Sevage method (chloroform/butanol, 5/1,V/V)and freeze-dried.

2.3 Preparation of oligosaccharides

SPP (10 g) was dissolved in 1 L of deionized water and hydrolysed with glycosidase for 8 h under the following optimal hydrolysis conditions: ratio of substrate to enzyme 10:1, pH 8.0, and 40 °C.The extracting solution was adjusted to a neutral pH with 1 mol/L NaOH by centrifugation at 1 800 ×gfor 15 min.The resulting supernatant was mixed with 8 volumes of absolute ethanol at 4 °C overnight.The precipitates were collected, redissolved in distilled water, and then separated by 5 kDa ultrafiltration membrane.The filtrate was dialyzed and free-dried and then labelled as SPO.

2.4 Purification of SPO

SPO was further purified using a DEAE-Cellulose-52 column(2.6 × 40 cm) and eluted with deionized water and 0.1, 0.3, and 0.5 mol/L NaCl solution at a flow of 1.0 mL/min.The peak fractions were monitored by the phenol-sulfuric acid colorimetric method, and were collected, dialyzed and concentrated.A Sephadex G-15 column(2.6 × 90 cm) was used to further purify with distilled water at a rate of 0.5 mL/min.Three purified fractions SPO-1, SPO-2, and SPO-3 were lyophilized for further analysis.

2.5 Prebiotic activity

The prebiotic activity was assessed with a quantitative score reported by Huebner [20].L.paracaseiATCCSD5275,B.animalisATCC25527 andE.coliATCC25922 were used for this study and were firstly maintained at −80 °C.Under sterile conditions, 50 μL frozen stock cultures ofL.paracaseiATCCSD5275 andB.animalisATCC25527 were inoculated in sterilized MRS broth medium(5 mL), while LB broth medium was used forE.coliATCC25922.L.paracaseiATCCSD5275 andE.coliATCC25922 were incubated at 37 °C for 18 h under aerobic conditions, whileB.animalisATCC25527 was incubated at 37 °C for 18 h in an anaerobic incubator.Then 1% (V/V) activated culture containing either 2% (m/V)samples or 2% (m/V) glucose was added to separate mediums.The cultures were kept at 37 °C for 24 h in anaerobic conditions forB.animalisATCC25527 and at ambient atmosphere for all other strains.Inoculated samples were enumerated using the serial dilution method in triplicate at 0 and 24 h, and the results were counted as CFU/mL of culture.

The prebiotic activity score was calculated by the following equation:

2.6 Average molecular weight determined for SPO-1

Matrix-assisted laser desorption ionization time-of-flight mass spectrometry (MALDI-TOF MS) analysis was carried out using the mass spectrometer (UltrafleXtreme, Bruker Daltonik GmbH,Leipzing, Germany) and confirmed the molecular weight and accurate degree of polymerization of SPO.1 mL of sample with the same volume of 2,5-dihydroxybenzoic acid (2,5-DHB, 20 mg/mL in acetonitrile/water (1:1)) was placed on a ground steel target and dried at room temperature.The spectra was found in reflection and positive ion mode with the following conditions: a reflector voltage of 26 kV, an acceleration voltage of 25 kV, ion extraction of 40 ns, and acquisition range of 70−3 000m/z[21].

2.7 Monosaccharide composition analysis

SPO-1 (5 mg) was hydrolyzed with 4 mol/L trifluoroacetic acid (TFA, 1 mL) at 120 °C for 2 h.Next, the hydrolysate was derivatized with 3-methyl-1-phenyl-2-pyrazolin-5-one (PMP), and then was analyzed by a previously reported HPLC method with some modifications [22].The samples or monosaccharide standards were mixed with an equal volume of 0.3 mol/L NaOH aqueous solution(100 μL) and 0.5 mol/L PMP methanol solution.The derivatization proceeded at 70 °C for 1.5 h after vortex blending.Then, the reaction mixture was neutralized with 50 μL of a HCl solution (0.3 mol/L),and the excess PMP was removed by chloroform for three times.Then the filtrate of aqueous layer was evaluated by an Agilent-1260 HPLC instrument with a YMC-Pack ODS-AQ column (250 × 4.6 mm,I.D.S-5 μm, 12 nm).Ammonium acetate solution (50 mmol/L) and acetonitrile in a ratio of 83:17 (V/V) were used as eluent and the flow rate was maintained at 1.0 mL/min.

2.8 Fourier transform infrared (FT-IR) and NMR analysis

The FT-IR spectra was obtained by a Shimadzu Spectrum 400 FT-IR(Japan) spectrometer in the frequency range of 4 000−400 cm-1.The freeze-dried SPO-1 (10 mg) was dissolved in 300 μL of D2O.The1H and13C NMR spectra of SPO-1 were detected with a Bruker AVANCE HD 700 MHz NMR spectrometer (Bruker Daltonik GmbH,Leipzing, Germany).

2.9 SPO-1 fermentation with faecal microbiota in vitro

Fresh faecal inoculums were obtained from 3 healthy donors(2 female and 1 male, 20−30 years old, with normal diet, without digestive disease, without antibiotics, probiotics, prebiotics for at least 3 months).The final 20% (m/m) human faecal inoculum was obtained by diluting with 10% (V/V) Dulbecco’s PBS, and then homogenized at high speed.The homogenized medium was filtered with 4 layers of sterile gauze sponges.The growth medium was prepared and sterilized at 121 °C for 30 min and then put into the pre-sterilized anaerobic tubes [23].

A 100 mL volume of the final fermentation was composed of 45% faecal inoculums, 45% growth medium, and 10% SPO-1 solution or ultrapure water (the control), sealed in anaerobic fermentation tube and incubated under conditions of 10% CO2, 10% H2, and 80% N2in a LAI-3T anaerobic incubator (Drawell Artist of Science, Shanghai,China).The fermentations were collected at 0, 6, 12 and 24 h for analysis.The phenol-sulfuric acid method was used for analysis of the total carbohydrate content.

2.10 Determination of SCFA by GC-MS

The production of SCFAs during fermentation was determined by gas chromatography-mass spectrometry (GC-MS) as reported previously [24].1 mL of fermentation product and 2-ethylbutyric acid as internal standard (0.3 mg/mL) were added with H2SO4(0.5 mol/L, 25 μL) and diethyl ether (0.5 mL) and shaken energetically for 5 min.Then, the upper phase was collected after centrifugation at 4 800 ×gfor 20 min at 4 °C.After two extractions, the upper phases were combined and analyzed by GC-MS (GC-MS-QP2010 Ultra,SHIMADZU, Japan) equipped with a Rtx-Wax capillary column(30 metres, 0.25 mmID, 0.25 μm df, Restek, USA).The initial temperature of the column was kept at 100 °C for 1 min, subsequently increased to 180 °C at a rate of 5 °C/min and held for 5 min.Helium was chosen as the carrier gas, with a flow rate of 1.0 mL/min, and the injection volume was 1 μL, with a split ratio of 1:18.The ion source temperature of the MS parameters was 200 °C, and the ionization energy was 70 eV.The SCFA contents of the fermentation product was calculated according to standard calibration curves (Fig.1).

Fig.1 The standard calibration curves of short-chain fatty acids.

2.11 Gut microbiota analysis

The microbial DNA was extracted using the HiPure Soil DNA kits (Magen, Guanghzou, China) based on the manufacturer’s protocols.The V3−V4 regions of the ribosomal RNA gene were amplified by PCR using primers 341F (CCTACGGGNGGCWGCAG)and 806R (GGACTACHVGGGTATCTAAT) to analyse the microbial communities.The product were extracted from 2% agarose gels and purified with the AxyPrep DNA Gel Extraction Kit (Axygen Biosciences, Union City, CA, USA) based on the manufacturer’s instructions and quantified with an ABI Step One Plus Real-Time PCR System (Life Technologies, Foster City, USA).Sequencing was performed on an Illumina HiSeq 2500 platform (San Diego, CA,USA) by Gene Denovo Technology Co., Ltd (Guanghzou, China).

High quality and reliability sequences, which were clustered into operational taxonomic units (OTUs) of ≥ 97% similarity using the UPARSE pipeline, were obtained and used for downstream analyses.The representative OTU sequences were classified by a naive Bayesian model using the Ribosomal Database Project (RDP)classifier [25].OTUs and taxonomic ranks were used forα-diversity,β-diversity, and detection of different species analyses.Predictive functional analysis of the microbial communities was performed using phylogenetic investigation of communities by reconstruction(PICRUSt) [26].

2.12 Statistical analysis

The results are presented as the mean ± SD of three independent experiments.One-way ANOVA with the Bonferroni was used for comparing the data using IBM SPSS v.22.0.P-values less than 0.05 were considered as statistically significantly.

3.Results and discussion

3.1 Isolation and prebiotic activity score of SPO

The crude oligosaccharide SPO was prepared fromS.platensisby a marine microbial enzyme and purified with DEAE-cellulose-52 and Sephadex G-15 chromatography, and 3 fractions were obtained, SPO-1,SPO-2 and SPO-3 (Figs.2 and 3).The prebiotic activity scores of SPP and SPO ofL.paracaseiATCCSD5275 andB.animalisATCC25527 was shown in Table 1.The prebiotic activity score of the oligosaccharide SPO was obviously higher than that for the polysaccharide SPP (P< 0.05).More and more clinical evidence have proved that oligosaccharides produced by enzymolysis have prebiotic activity [4].Oligosaccharides with a low degree of polymerization appear to be easier to ferment by microbiota.Furthermore, the purified fraction SPO-1 had the highest prebiotic activity score for the growth of both probiotic strains.This result may be related to the different structural features, including the molecular weight,sugar residue composition, linkage type between monosaccharides,and stereochemistry [4,7].SPO-1 was chosen for further structural investigation and its impact on the faecal microbiotain vitro.

Fig.2 DEAE-Cellulose-52 anion-exchange chromatograms of the SPO.

Fig.3 Sephadex G-15 chromatograms of the (a) SPO-1, (b) SPO-2 and (c) SPO-3.

Table 1Prebiotic activity scores of SPP and SPO for L.paracasei ATCCSD5275 and B.animalis ATCC25527.

3.2 Structural characterization of SPO-1

The monosaccharide compositions of SPO-1 were measured by HPLC.As shown in Fig.4, the monosaccharide profile of SPO-1 was glucose and galactose with the molar percentage of 94.58% and 5.42%, respectively.It indicated that SPO-1 was mainly consisted of glucose which proved to be a kind of glucosan.

Fig.4 PLC chromatograms of monosaccharide compositions of(a) monosaccharide standards and (b) SPO-1.

The molecular weight of oligosaccharide was detected using MALDI-TOF MS.The molecular weight distribution range of SPO-1 was 510.8−997.0 Da and the degree of polymerization was 3−5 (Fig.5a).In addition, the interval between peaks was 162 mass units, suggesting the pyranose repeating fragment unit in SPO-1.This finding showed that them/zof SPO-1 and Na+was 673.2 Da, with a degree of polymerization at 4.Thus, the average molecular weight of SPO-1 was 650.2 Da.

Fig.5 (a) MALDI-TOF MS spectrum, (b) FT-IR spectrum, (c) 1H NMR spectrum and (d) 13C NMR spectrum of SPO-1.

As shown in Fig.5b, the FT-IR spectra of SPO-1 displayed absorption peaks typical of saccharides.The peak at 3 286.70 cm-1was attributed to the O—H stretching vibration, and the absorption at 2 900 cm-1was regarded as the C—H stretching vibration.The absorption bands at 1 022.27, 1 078.21, and 1 145.72 cm-1revealed a pyranose configuration of SPO-1 [27].The band at 844.82 cm-1indicatedα-type glycosidic linkages [28].

As shown in the1H NMR spectrum (Fig.5c), the chemical shifts atδ5.34−5.37 with3J1,2coupling constants value below 4.0 Hz indicated that SPO-1 hadα-D-Glu [29,30].In the13C NMR spectrum(Fig.5d), a chemical shift was not found in the region betweenδ160.00 andδ180.00, which indicated that there was no carboxyl group in SPO-1.The peaks atδ99.68, 99.53, 95.72 and 91.85(δ< 101) revealed that the SPO-1 had three different types ofα-configuration glycosidic linkage [30], which was consistent with the analysis results of FT-IR.

3.3 Carbohydrate consumption

Bacteria contains many enzymes that hydrolyze carbohydrates,including glycosyltransferases, glycoside hydrolases, and carbohydrate esterases.The monosaccharide composition, category of glycosidic bonds, branching of carbohydrates and degree of polymerization of the oligosaccharide affect the utilization of bacteria [31].As shown in Fig.6, the carbohydrate consumption of the control groups fermented by faecal bacteria exhibited an obvious increasing trend from 0 to 12 h;thereafter, a slow increase was observed.However, the carbohydrate consumption percentage of the SPO-1 group was obviously higher(P< 0.05) than that of the control group after 12 h of fermentation.The utilization rate of carbohydrates in the SPO-1 group increased gradually and reached (59.49 ± 1.16)% at 24 h.Therefore, SPO-1 was used as a carbon and energy source, and could be utilized by the faecal microbiota.

Fig.6 The proportion of carbohydrate consumption of the SPO-1 group at different time points of fermentation.Different letters indicate significant differences between the SPO-1 and the control group (P < 0.05).

3.4 The effects of SPO-1 on gut microbiota

The enzymatic production of oligosaccharides is beneficial for gut systems by modulating the gut microbiota and is characterized by functional foods [4,32].Thus, the effect of oligosaccharide SPO-1 on intestinal flora afterin vitrofermentation was analyzed by high-throughput sequencing analysis.The stable Shannon indices(Fig.7) suggested that the sequencing data could accurately reflect the microbial community of the samples.

Fig.7 α-Diversity analysis of samples (a) Chao index and (b) Shannon index.C0, the faecal fermentation at 0 h.C24 and S24, the control group and SPO-1 fermentation at 24 h, respectively.

The microbial community was compared in the Venn analysis(Fig.8a), the overlap of OTUs indicated that 427 OTUs coexisted in the three groups and that 655 OTUs coexisted in the C24 and S24 groups.The Chao and ACE indices, indicating the community abundance of the sample, were obviously higher in the C24 and S24 groups than those in the C0 group (Table 2).Furthermore,the Shannon and Simpson indices of the S24 group, representing community diversity, were significantly higher than those of the C0 and C24 groups.These results suggested that SPO-1 could improve the abundance and diversity of gut microbiota.As shown in principal component analysis (PCA, Fig.8b) and principal coordinate analysis(PCoA, Fig.8c), the first two principal coordinates interpreted 99.53% and 97.16% of the total inter sample variance and respectively,indicating significant differences in the microbiota community of C24 and S24 to C0 groups after fermentation.This result was also verified by the system clustering tree (Fig.8d) and the weighted UniFracβ-diversity heatmap analysis (Fig.8e), and the C24 group was similar to the C0 group, followed by S24.

Fig.8 Differences in microbial community based on (a) Venn analysis,(b) PCA, (c) PCoA, (d) Weighted UniFrac cluster analysis and (e) β-diversity heatmap analysis.

Table 2Alpha diversity of different treatment groups.

The phylum level of the composition distribution of the samples was shown in Fig.9a.The microbiota in the C0 group was mainly composed of Bacteriodetes, Firmicutes and Proteobacteria.After 24 h of fermentation, the relative abundance of Firmicutes, Proteobacteria and Actinobacteria was accelerated but resulted in a reduction in Bacteroidetes abundance in C24 and S24 at the same time.The different microbial communities between the C24 and S24 groups were shown in a heat map (Fig.9b).At the genus level, S24 obviously accelerated the relative abundance ofDialister,Bacteroides,Escherichia-Shigella,Megamonas,Megasphaera,Blautia,Holdemanella,Collinsella,Phascolarctobacterium,Bifidobacterium,Dorea, and so on, but notably reduced the proportion ofPrevotella_9andFaecalibacterium.An increasing number of studies have indicated thatBacteroides,Escherichia-ShigellaandMegamonasare beneficial gut microbiota [33,34].Bacteroidesproducing SCFA plays an important role in the immune system, specifically affecting the tolerance of intestinal commensal bacteria [35].In addition,BifidobacteriumandLactobacillus, were viewed to be advantageous to human health [36], which had a significantly higher proportion in the SPO-1 group (Fig.9c).These results indicated that SPO-1 could modulate the microbial community after faecal batch fermentationin vitroand was beneficial for the proliferation of specific bacteria.

Fig.9 Bacterial taxonomic analysis at the phylum level of each group (a), the heat map of differences in bacterial distribution at the genus level (b), and changes in the abundance of distinct genera (c).

Oligosaccharides are selectively utilized by host microbiotaviamaintaining intestinal homeostasis to confer health benefits, including promoting beneficial microbes, defence against pathogens, protection gastrointestinal function and immunoregulation [37].The metabolites of oligosaccharides are produced by the gut microbiota and play a substantial part in the potential health benefits [38].PICRUSt was used to predict the functional changes in the gut microbiota among the three groups.As shown in Fig.10a, in KEGG level 1, the differential microbes were mainly related to “metabolism”, “genetic information processing”, “human diseases” and “organismal systems” after the addition of SPO-1.The largest differences between S24 and the other two groups were “metabolism”, “carbohydrate metabolism”and “amino acid metabolism”.Within “environmental information processing”, the proportion rate of signal transduction was obviously higher in the SPO-1 group than that in the other two groups(Fig.10b).

Fig.10 (a) The functional composition of the gut microbiota in the C0, C24 and S24 groups, (b) significant changes were obvious in the gut microbiota KEGG pathway among the three groups via the Kruskal-Wallis test in the level-2 term.Compared with the C0 group, *P < 0.05.Compared with the C24 group, #P < 0.05.

3.5 Effects of SPO-1 on the production of SCFAs

Numerous studies have also reported that oligosaccharides used as prebiotics are beneficial to human health by increasing SCFA concentrations [39].SCFAs, including acetic, propionic,n-butyric,i-butyric,n-valeric andi-valeric acids, play an important role in the health of colonic health and are beneficial for immune regulation [38].The total SCFA levels in all groups increased during the fermentation process in Table 3.Obviously, acetic, propionic andn-butyric acids were the primary fermentation products.The total SCFA levels in the control groups increased rapidly from 0 h to 6 h and slowly increased at 12 and 24 h.However, the total SCFA concentration in the SPO-1 group remained significant raising from 6 h to 24 h and reached(53.09 ± 0.65) mmol/L, indicating that SPO-1 could modulate the intestinal microenvironment.Notably, after 24 h fermentation with SPO-1, there were significant increases in the concentration of all SCFAs except propionic acid compared with control group.Among the SCFAs,i-butyric andi-valeric acids exhibited the most significant concentration increases in the SPO-1 group.The concentrations ofi-butyric andi-valeric acids in the SPO-1 group at 24 h were 12.67- and 24.31-fold higher than those in the control group,respectively.Notably, SPO-1 was beneficial for human health because butyrate is the most important gut metabolite to protect the colonic mucosal functions [4].

Table 3The concentrations of SCFAs in fermentation solution at different time points of fermentation in vitro.

4.Conclusion

In conclusion, SPO-1 was hydrolyzed using glycosidase from a marine bacterium, showing a significantly high prebiotic score forB.animalisATCC25527.SPO-1 is a tetrasaccharide composed of glucose withα-type glycosidic linkages.SPO-1 significantly improved the microbial community by increasing the community richness and diversity.At the genus level, SPO-1 modulated the microbiota composition by increasing the abundance of more beneficial species, includingBacteroides,Escherichia-Shigella,Megamonas,Megasphaera,Blautia,BifidobacteriumandLactobacillus.In addition, the functional analysis indicated that the intestinal microbiota was mainly related to the terms “carbohydrate metabolism” and “amino acid metabolism” after SPO-1 intervention.Therefore, SPO-1 has the potential to be a prebiotic oligosaccharide for human health and disease prevention by regulating intestinal microbiota.

Acknowledgments

The authors are indebted to Dr.Yongli Gao, Shikun Dai, and engineer Yu Zhang (the Equipment Public Service Center, SCSIO,CAS) for their assistance with the high-speed refrigerated centrifuge and mass spectrometric analysis.This research was funded by the National Key R&D Program of China (No.2018YFC0311202),the Natural Science Foundation of China (No.21662006), the National Natural Science Foundation of Guangdong, China(No.2018A030313903, 2018A030313088, 2018A0303130144 and 2018A030313626), the Program of Department of Natural Resources of Guangdong Province (No.GDNRC[2020]038 and GDNRC[2020]036), the Key Special Project for Introduced Talents Team of Southern Marine Science and Engineering Guangdong Laboratory of Guangzhou, China (No.GML2019ZD0406) and the Science and Technology Program of Guangzhou, China (No.201804010321 and 201804010364).

Conflicts of Interest

The authors declare no conflict of interest.