Chemical Compositions and Prebiotic Activity of Soy Hull Polysaccharides in Vitro

Abstract

Soy hull polysaccharides (SHPs) were extracted from soy hull using microwave-assisted extraction and their structures were investigated by Fourier transform infrared spectroscopy (FT-IR) and atomic force microscopy (AFM). Subsequently, the effects of SHPs on the proliferation of fecal microbiota were examined. The results indicated that microwave-assisted extraction had significantly higher efficiency in extracting SHPs. The yield of microwave-assisted oxalic acid extracted soy hull polysaccharide (MOS) and microwave-assisted ammonium oxalate extracted soy hull polysaccharide (MAS) increased by 33.4% and 17.4%, when compared with oxalic acid extracted soy hull polysaccharide (OS) and ammonium oxalate extracted soy hull polysaccharide (AS), respectively. High performance liquid chromatography showed that the four polysaccharides had significant differences in their monosaccharides composition. High performance gel filtration chromatography illustrated that the molecular weights of MAS and AS were the highest. FT-IR spectra indicated that the structures of MOS and OS were similar and mainly comprised furanose, whereas MAS and AS mostly consisted of pyranose. AFM showed that the molecular chain conformation of MOS and MAS was vermicular, OS was short-rod-like, and AS was rod-like. MOS significantly promoted proliferation and diversity of fecal bacteria. Interestingly, OS enhanced the growth of Bifidobacterium spp. and Lactobacillus spp. In summary, SHPs showed more potent prebiotic activity with microwave-assisted extraction method and could have prospective applications in functional food industry.

Introduction

Soy hulls are the major byproducts of soybean processing industry, and the insoluble carbohydrates fraction contains abundant polysaccharides (Kim et al., 2016). There has been a growing interest in the use of these soy hull polysaccharides (SHPs) as bioactive molecules, such as those involved in antioxidant, anti-tumor, immune-modulation, and hypoglycemic activities as well as intestinal micro-ecological equilibrium (Kang et al., 2016; Kozarski et al., 2011; Kozarski et al., 2012; Lee and Kim, 2005; Liu et al., 2005). However, little attention has been paid to the optimal extraction and function of SHPs, thereby hindering their further exploitation and utilization.

Microwave-assisted extraction of SHPs is an important strategy for potential applications of these molecules. In previous studies, acid (or alkali/enzyme)-assisted hot extraction had been the most common method employed to extract water-soluble polysaccharides from soy hull (Guang et al., 2011; Yan et al., 2011). However, the major limitation of this method is its low efficiency (Li et al., 2007). Therefore, there is a need to adopt an economical and highly efficient extraction process, such as microwave-assisted extraction, which has attracted much attention.

In general, bioactivities of polysaccharides are closely correlated with their chemical structures and molecular size (Zeng et al., 2015). Therefore, comprehensive understanding of the correlation between the structure and bioactivity of SHPs could be beneficial for potential utilization of these molecules in functional food industry. In this study, the SHPs were extracted using microwave-assisted extracting agents (oxalic acid or ammonium oxalate) (Liu et al., 2013; Wang et al., 2019), and their structures were investigated by Fourier transform infrared spectroscopy (FT-IR) and atomic force microscopy (AFM). Subsequently, the effects of SHPs on the proliferation of gut microbiota were examined.

Materials and Methods

Materials    Soy hulls were obtained from Yu Wang Group of Shan Dong in China. Lactobacillus bulgaricus (Strain designations IDCC 3601) was purchased from ATCC.

Microwave-assisted extraction    After smashing and screening, the soy hull was dissolved in 10-times volume of 1% (v/v) ethanol and stirred for 30 min at room temperature. Then, the mixture was filtered and the residue was dried at 65 °C. Subsequently, 50 g of the soy hull residues were extracted in 20-times volume of 0.6% (w/v) oxalic acid (analytical reagent) or ammonium oxalate (analytical reagent) with/without 85 °C microwave assistance for 20 min at 450 W. The microwave oven (WD800G) was purchased from Guangzhou Galanz Group Co., Ltd. After cooling and filtering, the extracts were centrifuged at 1,500 ×g for 15 min, and the supernatant was collected and its pH was adjusted to 4.0 with 1 M HCl. Subsequently, the supernatant was concentrated to one-third of its initial volume using a rotary evaporator at 60 °C and anhydrous ethanol was added twice and mixed continuously, and then the solution incubated at 4 °C for 40 min. The precipitated polysaccharides were collected through centrifugation (1,800 ×g for 30 min) and lyophilized, generating four SHPs samples (microwave-assisted oxalic acid extracted soy hull polysaccharide, MOS. oxalic acid extracted soy hull polysaccharide, OS. microwave-assisted ammonium oxalate extracted soy hull polysaccharide, MAS. ammonium oxalate extracted soy hull polysaccharide, AS).

Analysis of chemical properties of SHPs    The total carbohydrates content in SHPs was determined using phenol-sulfuric acid method with glucose as the standard. In brief, the SHPs solution was mixed with phenol and concentrated sulfuric acid to reach a color, and the light absorption was recorded at 490 nm (Dubois et al., 1956). The moisture and protein contents were determined by direct drying and bicinchoninic acid assay (BCA), respectively (Kong et al., 2015; Malgorzata et al., 2013).

The monosaccharide composition of SHPs was determined by high performance liquid chromatography (HPLC). Each SHPs sample (5 mg) was hydrolyzed using trifluoroacetic acid (TFA) (4 M, 1 mL) at 110 °C for 6 h in an oven (Zhang et al., 2009; Yang et al., 2012). Subsequently, the acid was completely removed by flash evaporation in a water bath at 50 °C, and the residues were co-distilled using methanol. The hydrolyzed samples and monosaccharide alditol acetate were prepared and injected (0.1 µL) into an Agilent 6890 N GC system (Agilent Technologies, Wilmington, USA) equipped with Carbopac PA20 column (150 mm×3 mm) and flame ionization detector. The column temperature was programmed to increase from 140 °C (maintained for 2 min) to 170 °C at a rate of 5 °C/min, and then to 250 °C at a rate of 2 °C/min. The injection temperature was 280 °C, detector temperature was 300 °C, and flow rate of the N₂ carrier gas was 0.5 mL/min.

Determination of molecular weight distribution    MOS, MAS, OS, and AS were respectively dissolved in 0.1 mol/L NaNO₃ ultrapure water solution, and their molecular weight distribution was determined by high performance gel filtration chromatography (HPGFC). In brief, 20 µL of the SHPs sample were injected into the HPGFC column and eluted with distilled water at a flow rate of 0.9 mL/min (45 °C, 1.6 MPa). The peaks were detected using a Shimadzu CLSS-VP 6 system equipped with ULtrahydrogel™ linear Ultrahydrogel™ Linear (300 mm×7.8 mm) and refractive index detector (RID-10A). The molecular weight and peak time of the SHPs sample was determined using a standard curve generated using Dextran T standards of known molecular weights (1.338×10⁵, 4.11×10⁴, 2.5×10³, and 1×10³). Number-average molecular weight (Mn) is the statistical average value of molecular weight according to the distribution function of molecular number. Weight-average molecular weight (Mw) is the statistical average value of molecular weight according to the distribution function of molecular mass. Molecular weight distribution index (Wi) denotes the ratio of weight-average molecular weight (Mw) to Mn.

FT-IR spectroscopy    FT-IR spectra were recorded using the KBr-disk method (Yu et al., 2017; Kong et al., 2015). The samples were ground with spectroscopy-grade KBr and pressed into a 1-mm pellet for FT-IR analysis at a frequency range of 4000–400 cm⁻¹ and resolution of 4 cm⁻¹.

AFM    The SHPs (0.5 mg) were dissolved in distilled water (100 mL), and a drop of the sample was placed on the surface of a mica carrier and dried at 70 °C under ambient pressure. The AFM images were captured using an XE-70 atomic force microscopy (Park Systems Co., Korea) under tapping mode (Kong et al., 2015).

Bacterial culture    Lactobacillus bulgaricus were seeded in Φ100 culture dishes and thermostatic cultured at 37 °C. Those bacterial were photographed after 48 h. Fecal bacteria were obtained from two different donors. The donors had not accepted antibiotics and prebiotic treatment for at least 3 months prior to sample collection. Fresh fecal samples from these donors were collected immediately after defecation. A portion of each sample was stored at −80 °C for DNA extraction and sequencing analysis. Another portion of each sample was used for batch culture fermentation. Fecal bacteria were cultured in nutritional medium (Truchado et al., 2017). The nutritional medium consist of 2.25 g/L yeast extract, 0.75 g/L peptone, 12.5 g/L NaCl, 6.0 g/L bile salt, 0.9 g/L trypsin, 3 g/L mucin and 0.37 g/L L-cysteine, pH 6.8. 1% MOS, MAS, AS, OS and 1% inulin were added to the culture medium respectively as experimental group. The group with no polysaccharide served as control. The bacteria were incubated in an anaerobic environment (90% N₂, 5% H₂ and 5% CO₂) at 37 °C to maintain exponential growth. Subsequently, the proliferation of fecal bacteria was determined under 650 nm through spectrophotometer after treatment with 1% MOS, MAS, OS, AS, and inulin for 0, 12, 24, 36 and 48 h, respectively, and the growth curves were recorded.

Fecal bacterial DNA extraction, amplification, and 16S rDNA gene sequencing    The fecal bacterial DNA was extracted from 2 mL of stool homogenate using QIAamp Fast DNA Stool Mini Kit (QIAGEN, Duesseldorf, Germany), following manufacturer's instructions, and then sent to SANGO Biotechnology (Shanghai) Co., Ltd., Shanghai, China. The purity and quality of the genomic DNA were checked using 0.8% agarose gel, and each sequenced sample was prepared to amplify the V3 and V4 region (519F-806R) of the 16S rRNA gene. The barcoded fusion primers 341F (5′-CCT ACG GGN GGC WGC AG-3′) and 805R (5′-GAC TAC HVG GGT ATC TAA TCC-3′) were used for the amplification. The final purified product was quantified by quantitative PCR (qPCR) according to the qPCR Quantification Protocol Guide, and the quality was ascertained using Lab-Chip GX HT DNA High Sensitivity Kit (Perkin Elmer, Hopkinton, MA, USA). Subsequently, paired-end (2 × 300 bp) sequencing was performed using the MiSeq platform (SANGO Biotechnology Co., Ltd, China).

The 16S rDNA high-quality reads were separated using sample-specific barcode sequences and trimmed with Illumina Analysis Pipeline Version 1.8.0 for analysis using QIIME. The raw data were first screened and sequences shorter than 200 bp or with a low-quality score (≤ 20) were removed. The remaining sequences were clustered into operational taxonomic units (OTUs) at a similarity level of 97% to generate rarefaction curves as well as to calculate richness and diversity indices. Mothur (version V.1.30.1) was applied to perform the refraction and alpha-diversity analysis. The structure of different microbial communities was compared by principal component analysis (PCA), 3D-principal coordinate analysis (3D-PCoA), and Venn diagram based on the OTUs from each SHPs sample using R packages (version 3.1.0).

Statistical analysis    The data analysis utilized multiple comparisons performed using SPSS 19.0 software. All of the experiments were conducted in triplicate, and the data are expressed as mean ± SD. The groups were evaluated using one-way ANOVA. A value of p < 0.05 was considered to indicate statistically significant difference.

Results and Discussion

Chemical properties of SHPs    The yields of MOS, MAS, OS, and AS were calculated from the quality of the obtained polysaccharides divided by the quality of the original soy hulls, which were 11.93%, 11.08%, 8.94%, and 9.44%, respectively. MOS showed 33.4% increase in relation to OS, while MAS presented 17.4% increase in relation to AS. Microwave-assisted extraction exhibited significantly enhanced efficiency in extracting SHPs. Table 1 lists the moisture, total carbohydrates, and proteins contents in the four SHPs samples. No significant difference in the moisture content was noted among the four SHPs samples (p > 0.05). However, the total carbohydrates content in MOS and MAS was significantly higher than that in OS and AS (p < 0.05). The proteins content in MOS was significantly higher than that in MAS, OS, and AS (p < 0.05). The results of HPLC analysis indicated that SHPs comprised arabinose, galactose, glucose, mannose, rhamnose, and galacturonic acid, and that microwave-assisted extraction produced higher arabinose content (MOS vs. OS=28.60 vs. 20.56, MAS vs. AS=17.16 vs. 10.88) and lower galactose (MOS vs. OS=15.27 vs. 17.76, MAS vs. AS=23.63 vs. 24.57), mannose (MOS vs. OS=52.50 vs. 58.51, MAS vs. AS=54.00 vs. 55.91), and glucuronic acid contents (MOS vs. OS=0.67 vs. 0.78, MAS vs. AS=1.17 vs. 2.21). It must be noted that microwave-assisted extraction with oxalic acid or ammonium oxalate could sharply increase the intracellular temperature of soy hull, which could induce exceedingly high pressure in the cell that the cell wall can endure, resulting in plasmatorrhexis. Consequently, the intracellular polysaccharides components diffused and dissolved in the extraction solvent, thus achieving high efficiency extraction (Zhang et al., 2014; Cheong et al., 2016). Oxalic acid and ammonium oxalate changed the content of arabinose and galactose, and had no obvious effect on the other monosaccharides of SHP. Mannose, arabinose and galactose are main glycosyl unit of hemicellulose, and galactose, arabinose, rhamnose and galacturonic acidis are the characteristic monosaccharide of pectin (Schädel et al., 2010; Harper, 1977). The distribution of hemicellulose is dominant in soy hull with more mannose, arabinose and galactose.

Table 1. Chemical composition and molecular weight distributionof MOS, MAS, OS and AS.

	Moisture (%)	Total carbohydrate (%)	Protein (%)	Monosaccharides composition (%)						Mn (g/mol)	Mw (g/mol)	Wi
				Ara	Gal	Glc	Man	Rha	GalA
MOS	10.46±0.03a	82.82±0.17a	3.06±0.05d	28.6	15.27	1.39	52.5	1.58	0.67	5.2×104	2.3×105	4.4
MAS	11.13±0.05a	69.37±0.08b	5.82±0.02a	17.16	23.63	2.2	54	1.84	1.17	5.1×104	2.6×105	5.1
OS	10.93±0.05a	63.15±0.03d	4.01±0.04c	20.56	17.76	1.28	58.51	1.12	0.78	4.2×104	2.4×105	5.7
AS	11.69±0.09a	68.49±0.05c	5.13±0.07b	10.88	24.57	2.96	55.91	3.47	2.21	4.2×104	2.6×105	6.2

Note: 1. Different letters represent significant differences between four groups, p < 0.05.

2. Ara, arabinose; Gal, galactose; Glc, glucose; Man, mannose; Rha, rhamnose; GlcA, glucuronic acid. Mn, number-average molecular weight; Mw, weight-average molecular weight; Wi, Molecular weight distribution index.

Mannose had prebiotic effect as the principal component of SHPs, and induced expression of pro- and anti-inflammatory cytokines to give evidence of immunostimulating properties of the monosaccharide (Korneeva et al., 2012). The partial physiological effects of mannose also arise from mannose-based polymers. Oligomers of mannose are able to enhance the growth of beneficial bacteria, decrease blood pressure, reduce fat absorption, or decrease attachment of pathogenic bacteria to the intestinal mucosa (Asano et al., 2007; Chauhan et al., 2014).

Molecular weight distribution of SHPs    The chemical composition, structure, and molecular weight of SHPs are important for understanding their bioactivities (Ma et al., 2015). Accordingly, the molecular weight distributions of MOS, OS, MAS, and AS were examined (Tab. 1). The molecular weights of MAS and AS were the highest (2.6×10⁵, respectively), similar to Mn of MOS and MAS (5.2×10⁴ and 2.6×10⁵, respectively). Wi denotes the ratio of Mw to Mn, indicating the distribution of molecular weight. Thus, although the four SHPs samples exhibited polydispersity, the molecular weight distribution of MOS was consistent.

FT-IR spectra of SHPs    To further characterize the chemical structure of MOS, OS, MAS, and AS, their respective FT-IR spectra were examined (Fig. 1). The O-H (hydroxyl) stretching vibration appeared at 3500–3300 cm⁻¹ and the C-H (alkyl) stretching vibration of CH₂ or CH₃ appeared at 2940 cm⁻¹. The absorption peaks at 1700 and 1430 cm⁻¹ represented asymmetric and symmetric stretching vibration of C=O (GalA carboxyl) and C-O, respectively. The absorption peaks at 1650–1500 cm⁻¹ indicated N-H scissoring bond vibration, and that at 1147 cm⁻¹ corresponded to C-O-C deformation vibration. The feature absorption at 770 cm⁻¹ and three feature absorptions at 1100–930 cm⁻¹ reflected furan ring and pyran ring symmetry stretching vibration, respectively. The structures of MOS and OS were similar and mainly comprised furanose, while MAS and AS were mostly composed of pyranose. The FT-IR results indicated that microwave-assisted extraction had little effect on the structure of SHPs (Kong et al., 2015; Chokboribal et al., 2015; Shang et al., 2012).

Fig. 1.

Fourier transform infrared (FT-IR) spectra of MOS, MAS, OS and AS in regions from 4000 to 500 cm⁻¹

AFM of SHPs    The AFM images of MOS, MAS, OS, and AS are shown in Fig. 2. The MOS molecular chain exhibited vermicular structure with a length of 20.4–433.5 nm, width of 21.5–54.8 nm, and height of 0.4–2.3 nm. Similarly, MAS also presented vermicular structure with a length of 27.2–550.3 nm, width of 14.8–27.6 nm, and height of 0.3–2.9 nm. In contrast, OS displayed short-rod-like structure with a diameter of 10.4– 174.5 nm, and AS presented rod-like structure with a diameter of 21.4–255.2 nm. The morphology of SHPs revealed that the molecular aggregation of MOS with low Wi was stronger, when compared with that of MAS. Furthermore, hydrogen bonds were assumed to have an important role in the aggregation of polysaccharide molecules, which can be ascribed to the hydroxyl groups on the chains of the polysaccharides, providing strong intermolecular and intramolecular interaction with each other or water molecule (Zhang et al., 2007). Thus, microwave-assisted extraction techniques had a remarkable effect on the morphology of SHPs.

Fig. 2.

AFM images of MOS, MAS, OS and AS. The image acquisition was carried out in carrier medium mica, scanning range 1×1 µm, scanning frequency 1.001 Hz and constant force mode.

SHPs promoted proliferation of bacteria    Fig. 3 shows the growth of gut bacteria treated with MOS, MAS, OS, AS, and inulin for 48 h in vitro, respectively. The fecal bacterial population significantly increased after 1% MOS treatment, when compared with that after other treatments. Inulin is considered as a nondigestible food component, which selectively stimulates the growth of probiotics in the colon (Kolida and Gibson, 2007; Campos et al., 2012; Franck, 2002). However, the results of the present study revealed that the ability of MOS to stimulate the growth of fecal bacteria was more obvious than that of inulin.

Fig. 3.

Effect of SHPs treatment on proliferation of bacteria. (a) The proliferation curve of fecal bacteria were drawed after 1% MOS, MAS, OS, AS and inulin treatment for different times. (b) The growth of Lactobacillus bulgaricus was photographed after 1% MOS, MAS, OS, AS and inulin treatment for 48 h. Lactobacillus bulgaricus were seeded in Φ100 culture dishes and thermostatic cultured at 37 °C. Control meant no addition of SHPs.

SHPs modulated the structure of gut microbiota    To further prove the ability of SHPs to modulate the structure of fecal microbiota, relative abundance of phylum was constructed, and 3D-PCoA (Fig. 4) were performed. The OTUs showed the modulatory effect of SHPs on fecal microbiota, with different SHPs samples exhibiting unique OTUs that were not shared with the negative control. MOS treatment presented the highest number of OTUs (858 OTUs). MAS, OS, AS and control group had 524, 660, 608 and 478 OTUs respectively. Given that SHPs could change the structure of fecal microbiota, the bacterial populations in each treatment group were compared at the phylum level. As indicated in Fig. 4a, the SHPs changed the growth of Proteobacteria, Firmicutes, Bacteroidetes, and Fusobacteria. However, the response of these bacteria to different SHPs samples varied. When compared with the control, MOS and OS treatment showed a decrease in the abundance of Bacteroidetes and an increase in the amount of Proteobacteria, while MAS and AS decreased the population of Bacteroidetes and increased the abundance of Fusobacteria and Proteobacteria. Zhang et al. found 10,000 Da Dioscorea opposita Thunb. polysaccride specifically promoted proliferation of B. thetaiotaomicron and B. ovatus execpte except 3,000 Da polysaccride (Zhang et al., 2019). Gao et al. found that the macromolecule network of L. japonica polysaccharide (LP-A8 fraction) could upregulate the abundance of Firmicutes (Lachnoclostridium and Eubacterium) (Gao et al., 2019). The main structural features of the pectin-mediated shifts in microbiota abundance (such as Faecalibacterium prausnitzii, Coprococcus, Ruminococcus, Dorea, Blautia, Oscillospira, Sutterella, Bifidobacterium, Christensenellaceae, Prevotella copri, and Bacteroides spp.) included composition of neutral sugars, distribution of homogalacturonan and rhamnogalacturonan fractions, and degree of branching (Larsen et al., 2019).

Fig. 4.

Response of the fecal microbiota to SHPs treatment in vitro. (a) The relative abundances of the fecal bacteria at the phylum levels. (b) 3D-PCoA of the fecal microbiota-based weighted UniFrac metric. MOS, MAS, OS, AS meant the fecal bacteria was determinated after 1% MOS, MAS, OS and AS treatment for 48 h. Control meant fecal bacteria without SHPs treatment.

3D-PCoA score plot demonstrated that the microbial composition of each group was quite different and the MOS-treated microbiota exhibited a more significant structural shift than the other treatment groups, both along the first and second principal components (Fig. 4b). This finding further proved that the fecal microbiota was more vulnerable to MOS intervention. MOS has the following characteristics: the molecular weights were the lowest, the structure was mainly consisted of furanose, and the molecular chain conformation was vermicular. Maybe, those characteristics were the contributing factor of the fecal bacteria response. Many previous feeding studies have reported distinct changes in the microbial community after polysaccharides treatment. For instance, Frederick et al. found that many OTUs of the gut microbiota, including Clostridium, Trepenoma, Oscillospira, Phascolarctobacterium, S24-7, and Ruminococcus, showed a shift following recalcitrant starch treatment in vitro (Warren et al., 2018). Shang et al. noted that Enteromorpha clathrata polysaccharide increased the abundances of Alistipes spp., Oscillibacter spp., Flavonifractor spp., Parabacteroides spp., and Akkermansia spp. (Shang et al., 2018). In the present study, some difference in the gut microbiota was observed following SHPs treatment, indicating the ability of SHPs to modulate gut microbiota.

SHPs increased the diversity of gut microbiota    To further examine the effect of SHPs on fecal microbiota, the noticeable changes of 12 bacterial communities were analyzed at the genus level. As shown in Tab.2 the fecal microbiota after MOS treatment was dominated by Klebsiella, Gemmiger, Clostridium XIVb, Ruminococcus 2 and Butyrivibrio. The OS-treated group presented higher abundances of Escherichia/Shigella, Bifidobacterium, and Lactobacillus. The AS-treated group showed increased abundances of Flavonifractor, Methanosphaera, and Sulfurovum. In contrast, only Fusobacterium was enriched by MAS treatment. These findings indicated that the diversity of fecal microbiota was enhanced by MOS treatment, and that Bifidobacterium spp. and Lactobacillus spp. showed significant response to OS treatment.

Table 2. Ratio of microbiota at the genus level

Name	Control	MOS	MAS	OS	AS
Klebsiella	0.09	45.89	0.12	5.28	1.91
Escherichia/Shigella	0.19	33.73	8.89	60.11	41.43
Fusobacterium	4.38	2.9	71.7	5.22	24.47
Gemmiger	0.25	1.08	0.06	0.22	0.05
Clostridium XIVb	0.14	0.45	0.02	0.01	0.04
Butyrivibrio	0.05	0.25	0.05	0.1	0.05
Flavonifractor	0.1	0.18	0.08	0.14	0.23
Ruminococcus 2	0.1	0.37	0.08	0.12	0.24
Methanosphaera	0	0	0	0	0.1
Sulfurovum	0	0	0	0	0.05
Bifidobacterium	0.08	0.04	0.03	0.11	0.06
Lactobacillus	0.01	0.01	0	0.05	0.02

Lactobacillus spp. and Bifidobacterium spp. are common probiotics that have been widely used in food and pharmaceutical industries (Gholizadeh et al., 2018; Kumar et al., 2015; Gallego et al., 2016). Shang et al. found that keratin sulfate increased the abundance of Lactobacillus spp., while E. clathrata polysaccharide increased the population of Bifidobacterium spp., Akkermansia muciniphila, and Lactobacillus spp. Similarly, as SHPs significantly modulated the diversity of microbiota and promoted the growth of probiotics, further studies are warranted to elucidate the use of these polysaccharides as a new dietary prebiotic to alleviate obesity, diabetes, diarrhea, and many other gut dysbiosis diseases.

Conclusions

Microwave-assisted extraction increased the yields of MOS and MAS to 11.93% and 11.08%, respectively. MOS contained high amount of total sugar and less protein, and was mainly composed of furanose. The molecular weight of MOS was 2.3×10⁵, with a narrow polydispersity index of 4.4. Based on the theory of polymer solution, the chain conformation of MOS was noted to exhibit vermicular structure, which significantly promoted proliferation and diversity of fecal bacteria. Interestingly, OS treatment enhanced the abundance of Bifidobacterium spp. and Lactobacillus spp. Microwave-assisted oxalic acid extraction had a remarkable effect on the conformation, molecular weight distribution, and even biological activity of the extracted SHPs. In conclusion, the fundamental structural information about SHPs could be helpful for better understanding of their bioactivities and for their potential applications in functional food and health industries. In our future studies, the molecular mechanism of prebiotic function of SHPs will be explored.

Acknowledgements    The present study was funded by the National Natural Science Foundation of China (grant no. 31701618 and 31601510). We thank International Science Editing for editing this manuscript.

Abbreviations

SHPs

soy hull polysaccharides

MOS

microwave-assisted oxalic acid extracted soy hull polysaccharide

OS

oxalic acid extracted soy hull polysaccharide

MAS

microwave-assisted ammonium oxalate extracted soy hull polysaccharide

AS

ammonium oxalate extracted soy hull polysaccharide

HPLC

high-performance liquid chromatography

HPGFC

high performance gel filtration chromatography

AFM

atomic force microscopy

FT-IR

fourier transform infrared region

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