In vitro digestion and fermentation of released exopolysaccharides (r-EPS) from Lactobacillus delbrueckii ssp. bulgaricus SRFM-1

Journal Pre-proof In vitro digestion and fermentation of released exopolysaccharides (r-EPS) from Lactobacillus delbrueckii ssp. bulgaricus SRFM-1 Weizhi Tang (Conceptualization) (Methodology)Writing-Original draft preparation), Jianzhong Zhou (Data curation) (Software), Qian Xu (Data curation) (Software), Mingsheng DongWriting- Reviewing and Editing), Xia Fan (Software) (Validation), Xin Rui (Methodology) (Validation), Qiuqin Zhang (Methodology) (Investigation), Xiaohong ChenWriting- Reviewing and Editing), Mei JiangWriting- Reviewing), Junjun WuWriting- Reviewing), Wei Li (Conceptualization)Writing- Reviewing, Supervision)

PII:

S0144-8617(19)31261-5

DOI:

https://doi.org/10.1016/j.carbpol.2019.115593

Reference:

CARP 115593

To appear in:

Carbohydrate Polymers

Received Date:

5 August 2019

Revised Date:

8 November 2019

Accepted Date:

8 November 2019

Please cite this article as: Tang W, Zhou J, Xu Q, Dong M, Fan X, Rui X, Zhang Q, Chen X, Jiang M, Wu J, Li W, In vitro digestion and fermentation of released exopolysaccharides (r-EPS) from Lactobacillus delbrueckii ssp. bulgaricus SRFM-1, Carbohydrate Polymers (2019), doi: https://doi.org/10.1016/j.carbpol.2019.115593

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In vitro digestion and fermentation of released exopolysaccharides (r-EPS) from Lactobacillus delbrueckii ssp. bulgaricus SRFM-1

Weizhi Tanga, Jianzhong Zhoub, Qian Xuc, Mingsheng Donga, Xia Fana, Xin Ruia, Qiuqin Zhanga, Xiaohong Chena, Mei Jianga, Junjun Wua, Wei Lia,* [email protected]

College of Food Science and Technology, Nanjing Agricultural University, Nanjing, Jiangsu 210095,

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a

P.R. China. b

School of Food Science and Pharmacy, Xinjiang Agricultural University, Urumchi, Xinjiang

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College of Life Science, Tarim University, Alar, Xinjiang 843300, P.R. China.

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c

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830052, P.R. China.

Corresponding author: Dr Wei Li, College of Food Science and Technology, Nanjing Agricultural

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84399090

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University, 1 Weigang Road, Nanjing, Jiangsu, P.R. China , Tel: +86 25 84396989 , Fax: +86 25

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Graphical abstract

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Highlights

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In vitro fermentation study of three pure r-EPS fractions was carried out;

r-EPS1 and r-EPS2 exhibited high values of SI with strong bifidogenic effect;

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r-EPS1 produced the highest contents of lactic acid, acetic acid and total SCFA;

Abstract

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r-EPS could be good potential candidates for new functional food prebiotics.

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The aim of this study was to investigate the in vitro digestion and fermentation prebiotic properties of

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three released-exopolysaccharide fractions (r-EPS1, r-EPS2 and r-EPS3) from Lactobacillus delbrueckii ssp. bulgaricus SRFM-1. There were no free oligosaccharides and/or monosaccharides for r-EPS1 before and after simulated buccal, gastric and small intestinal (GSI) digestion in vitro. In contrast, r-EPS2 (13.4%) and r-EPS3 (10.6%) generated a few monosaccharides after digestion. Additionally, r-EPS1 and r-EPS2 seemed to present a strong bifidogenic effect comparing to inulin, as they exhibited high values of selectivity index (13.17 and 12.84, respectively). Furthermore, the 2

fermentation with r-EPS1 produced the highest contents of acetic acid and lactic acid (56.3 mM and 44.29 mM, respectively), which resulted in the highest amounts of total short chain fatty acid (145.51 mM) followed by r-EPS2 (135.57 mM) and inulin (99.28 mM). These results indicated that r-EPS from L. delbrueckii ssp. bulgaricus SRFM-1 could be a good potential candidate for new functional food prebiotic. Keywords: Lactobacillus delbrueckii ssp. bulgaricus SRFM-1; Released exopolysaccharide (r-EPS);

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In vitro digestion; In vitro fermentation; Selectivity index (SI); Short chain fatty acid (SCFA). 1. Introduction

A prebiotic has been defined as a ‘non-digestible food ingredients through the gastric and small

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intestine (GSI) to reach the large intestine in humans body in which they could be selectively utilized by some intestinal bacteria population, then benefit on host health by stimulating the growth and/or

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activity of intestinal flora’ (Gibson, Probert, Van Loo, Rastall & Roberfroid, 2004). This definition was

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later updated in 2017 and prebiotic is defined thereafter as ‘a substrate that is selectively utilized by host microorganisms conferring a health benefit’ (Gibson et al., 2017). They are generally

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galacto-oligosaccharides and fructo-oligosaccharides, although there exist with several other dietary carbohydrates and polyphenols and polyunsaturated fatty acids. The evidence available today supports

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the classification of inulin, -glucan and some other special polysaccharide candidate as prebiotics,

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since they are in accordance with three criteria of prebiotic (Gibson, Probert, Van Loo, Rastall & Roberfroid, 2004). Several studies have researched the influences of polysaccharides on changing intestinal flora composition and how this may benefit to the health of host (Dal Bello, Walter, Hertel & Hammes, 2001; He et al., 2016; Wang et al., 2015). However, there are few studies on the prebiotic properties of the exopolysaccharides (EPS) from lactic acid bacteria (LAB). As we all known, LAB have the ability to produce different kinds of EPS exhibiting a wide diversity 3

of physiological activities. EPS have also been identified as antioxidant, antiulcer, antitumoral agents and immunomodulator (Tang et al., 2017; Bao, Yuan, Wang, Liu & Lan, 2013； Zhou, Cui, & Qu, 2019). On account of LAB strains pass the GSI tract, symbiotic bacteria will be exposed to carbohydrate-based EPS synthesized by LAB which would be effective for fermentation (Khodaei, Fernandez, Fliss & Karboune, 2016; Van Bueren, Saraf, Martens & Dijkhuizen, 2015). For this reason, an extensive of EPS with potential of being selective fermented in the gastrointestinal tract is rising,

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which can be regarded as functional food ingredients candidate (Hongpattarakere, Cherntong,

Wichienchot, Kolida & Rastall, 2012; Turunen et al., 2011). In addition, it is also interesting to

correlate prebiotic effect with short chain fatty acids (SCFA) production such as formic, acetic,

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propionic, butyric acids and lactic acid secreted by several EPS-producing LAB (Lambo-Fodje &

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Nyman, 2006; Xu et al., 2019). Thus, further studies focusing on the structure-bioactivity relationship

2005; Snelders et al., 2014).

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of EPS will advance to the current acknowledge of their potential functionality (Sanz, Gibson & Rastall,

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In recent decades, the employ of in vitro batch-culture fermentation modelling inculcated with human faecal samples is diffusely accepted to pattern environmental conditions in the human intestine

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(Sarbini, Kolida, Gibson & Rastall, 2013). It provides a premier model to give an insight into the fermentation of carbohydrates by complex intestine bacterial and may offer prima facie evidence of the

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ability of the studied EPS to accelerate altering the human colonic microbiota. Using human faecal samples rather than mixed-, pure-or co-cultures as inoculum is a more significative method to assurance the multiplicity associated to colonic flora (Zhang et al., 2013; Snelders et al., 2014). Recently, three pure released-EPS (r-EPS) fractions were isolated from the Lactobacillus delbrueckii ssp. bulgaricus SRFM-1. The fine structural features and antioxidant activities of r-EPS fractions were

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characterized, and their structures were reported (Tang et al., 2017). Three r-EPS fractions had similar molecular weight (Mw) distributions, and they were composed of galactose and glucose with a molar ratio of 1.23: 1.00, 1.33: 1.00 and 1.00: 1.34, respectively. The r-EPS1 contained a backbone of →6- -D-Galp-(1→4)- -D-Glcp-(1→4)- -D-Galp-(1→4)- -D-Galp-(1→6)- -D-Galp-(1→4)- -D-Glc p-(1→4)- -D-Galp-(1→4)- -D-Galp-(1→4)- -D-Glcp-(1→, and had three branching points which existed in terminal with D-Glcp residues with / -D-(1→6) linkages. The r-EPS2 was composed of

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→6- -D-Galp-(1→4)- -D-Glcp-(1→6)- -D-Galp-(1→ as the backbone chain with a branching point which also existed in terminal D-Glcp residue with -(1→6) linkage. Therefore, in the present work,

our aim was to evaluate the digestion and influence of three purified r-EPS fractions on composition of

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human colonic microbiota. The changes of Mw and release of free oligosaccharides and/or

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monosaccharides during simulated buccal and GSI digestion in vitro were evaluated, and its

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fermentation behaviors with human fecal cultures in vitro were carried out by fluorescent in situ hybridization (FISH) and determining SCFA production.

2.1. Materials

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2. Materials and methods

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Salivary -amylase, pancreatic -amylase, amyloglucosidase, pancreatin, trypsin, SCFA standards including formic, acetic, propionic, butyric acids and lactic acid were obtained from Sigma-Aldrich Co.,

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Ltd. (St. Louis, Mo, USA). T-series dextrans (T-500, T-200, T-100, T-50 and T-10) were obtained from Pharmacia Co., Ltd. (Uppsala, Sweden). Inulin (source: chicory, Orafti HP, inulin content ≥ 94.5%,) from Beneo-Orafti NV (Tienen, Belgium) has a molecular weight (MW) of approximately 5,000 Da. All other reagents used were of analytical grade. 2.2. Isolation and purification of r-EPS

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r-EPS was isolated according to our previous method (Tang et al., 2017). In brief, cultivations of L. delbrueckii ssp. bulgaricus SRFM-1 for r-EPS production were performed as batch cultures with inoculum concentration of 4% (v/v) in milk at 40 °C for 28h in 5 L capacity fermenters. Culture medium was then kept at 4 °C for 12 h followed by centrifugation (4 °C, 12,000 rpm and 15 min) to remove cells and denatured proteins. The protein in supernatant was further removed by adding 4% (w/v) trichloroacetic acid and kept at 4 °C for 6-8 h followed by centrifugation. The r-EPS then was

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precipitated with 98% ethanol precipitation (1: 3 volumetric ratios) for overnight. After centrifugation, the precipitate was suspended in deionized water and dialyzed (Mw cut-off: 8,000-14,000 Da) against distilled water for 2 days. After centrifugation, the retentate was lyophilized to collect as crude r-EPS.

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The further purification of crude r-EPS (10 mL, 10 mg/mL) was subjected to a DEAE-52 anion

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exchange column (2.6 × 30 cm) and eluted with deionized water, 0.1 and 0.3 M NaCl at 1 mL/min flow rate. As a result, three r-EPS fractions were obtained, and the purity of these fractions was determined

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by high performance liquid chromatography (HPLC) and 1H nuclear magnetic resonance (NMR).

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2.3. Simulated buccal and GSI digestion of three r-EPS fractions in vitro In vitro buccal and GSI digestion of three r-EPS fractions was conducted using the method described

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by Li et al (2015b) with minor modifications. The subsequent digestion models were used to simulate human buccal, gastric and duodenal digestion. The pH of digestion solution was adjusted using a

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pH-meter model 744 (Metrohm, Herisau, Switzerland) with electrode Orion® 8220BNWP ROSS PerpHecT Micro Combination pH Electrode (Thermo Scientific, Waltham, MA, USA). In brief, 100 mg of r-EPS fraction was added with 100 U/mL salivary -amylase and amyloglucosidase, respectively, to 10 mL (1%, w/v) buffer solution (120 mM NaCl, 5 mM KCl, 6 mM CaCl2, pH 6.9) to simulate buccal digestion for 10 min. The pH was adjusted to 2.0 using 4 M HCl, then simulated gastric digestion

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was started by adding pepsin (Fluka, Buchs, Switzerland, >400 U/mg) to a final concentration of 3.0 mg/mL. The digestion was conducted at 37°C for 1 h in a shaking water bath at 55 rpm (SWB series, Biobase, Shandong, China). After that, pH of samples was adjusted to 6.8 using 4 M NaOH, pancreatin (0.4 mg/mL), trypsin (1.0 mg/mL) and pancreatic -amylase (100 U/mL) were added to simulate the small intestinal digestion and performed at 37°C for 8 h at 150 rpm. Then the samples were inactivated in boiling water for 5 min at once. The digestion products were filtrated through a 0.22 m membrane

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before analysis. Mw and digestion rate of three r-EPS fractions were determined by HPLC (Agilent

1100 series) equipped with a TSK GEL G4000 PW XL column (300 × 7.8 mm, Tosoh Corp., Tokyo,

Japan) on an Agilent 1100 system and an evaporative light-scattering detector (ELSD). Sample solution

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(40 L) was injected and eluted with distilled water at a flow rate of 0.6 mL/min with the temperature

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of 30°C. The linear curve was calibrated with standard T-series dextrans as mentioned above. Free

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oligosaccharides and/or monosaccharides were determined by Agilent 1100 series HPLC equipped a refraction index detector (RID) with a column of Shodex Sugar KS-801 (8.0 × 300 mm, Showa Denko

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Co., Tokyo, Japan). The column eluted with distilled water at a flow rate of 0.8 mL/min. Injection volume was 20 L. Sugar compounds in the sample were identified by comparing the retention times

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with those of standard mono- and di-saccharides. Quantification of each sugar was performed by an external calibration curve using its corresponding standard solutions (Li, et al., 2009).

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Monosaccharide composition of three r-EPS fractions after digestion was determined by gas

chromatography (GC) as described by Li et al (2014). Briefly, 5 mg of r-EPS sample was hydrolyzed with 2 mL of 2 M trifluoroacetic acid (TFA) at 120 °C for 2 h. The hydrolysate was repeatedly co-concentrated with methanol to dryness and converted to its trimethylsilyl (TMS) derivative. The TMS derivative of hydrolyzate was prepared by adding 0.2 mL trimethylchlorosilane, 0.4 mL

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hexam-ethyldisilazane and 1 mL pyridine and heating at 80 °C for 30 min. Standard sugars and internal standard (inositol) were converted to their TMS derivatives in the same way. After cooling, samples were analyzed on Agilent HP 6890 GC (Palo Alto, CA, USA) equipped with flame ionization detector (FID) and a HP-5 fused silica capillary column (30 m × 0.32 mm × 0.25 mm, J&W Scientific Inc., Folsom, CA, USA). The following chromatographic conditions were used: N2 was used as the carrier gas at a flow rate of 1 mL/min; the temperature of injector and detector were set at 250 °C and 280 °C,

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respectively; initial column temperature was held at 100 °C for 5 min, then programmed at a rate of

5 °C/min to 150 °C and held at 150 °C for 5 min, subsequently programmed at 5 °C/min to 240 °C and held at 240 °C for 2 min. Calculation of the molar ratio of the monosaccharide was carried out on the

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basis of the peak area of the monosaccharide.

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2.4. Fecal slurry preparation

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The fresh fecal samples were obtained from three healthy volunteers (two women and a man, ages 21-25), who have not received antibiotic treatment for 6 months before participating in this study and

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no history of gastrointestinal diseases. The collected fecal samples were mixed and diluted in phosphate buffer solution (PBS, 1.0 mol/L, pH 7.0) in the ratio of 1: 5 (w/v) with sterile operation, and

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homogenized for 5 min to prepare the fecal slurry. 2.5. In vitro fermentation

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In vitro fermentation was carried out according to our previous method with minor modification

(2015c). The basal growth medium, comprised of 2.0 g of yeast extract, 2.0 g of peptone water, 0.5 g of L-cysteine HCl, 0.04 g of K2HPO4, 0.4 g of NaHCO3, 0.1 g of NaCl, 0.01 g of CaCl2, 2.0 g of NaHCO3, 0.01 g of MgSO47H2O, 0.5 g of bile salt, 2.0 mL of Tween 80, 10 L of Vk and 4.0 mL of resazurin solution (0.025%, w/v) per liter (pH 7.0) was used for static batch culture fermentation. In brief, the

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tested pure r-EPS fraction (1%, w/v) was filtrated through a 0.22 m membrane and then added into the each autoclaved medium (10mL) just before the addition of fecal slurry (10%, w/w). The incubation was carried out inside an anaerobic cabinet (Model Plas Labs 855-AC; PLAS LABS, INC., Lansing, MI, USA) at 37°C maintained at atmosphere of 85% N2, 10% H2 and 5% CO2. After 0, 6, 12, 24 and 48 h of fermentation, the liquid samples were taken for enumeration of bacterial populations and SCFA analysis, respectively. All experiments were repeated three times.

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2.6. Enumeration of bacterial populations

FISH technique was carried out as described by Li et al (2015c). Briefly, 100 L aliquots were

obtained from each culture vessels and were diluted in 4% (w/v) filtered paraformaldehyde (pH 7.2)

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and fixed for 10 h at 4°C. Fixed bacterial cells were then centrifuged for 5 min and washed twice with 1

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mL of sterile filtered PBS (0.1 M, pH 7.2). The cells were pelleted by centrifugation, resuspended in

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300 L of PBS and stored mixed with 300 L of ethanol at -20°C until further using. Hybridization was carried out using appropriate 16S rRNA-targeted oligonucleotide probes labelled

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with the cyanine-3 (Cy3) fluorescent dye (50 ng/ L stock solution) for the quantification of specific bacterial groups and the nucleic acid stain DAPI for total bacterial counts. The oligonucleotide probes

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used here are detailed in Table 1. The probes synthesized by Shanghai Sangon Biological Engineering Technology and Service Co., Ltd. (Shanghai, China), were Bif164 (specific for Bifidobacterium spp.),

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Bac303 (specific for Bacteroides-Prevotella group), His150 (specific for most species of the Clostridium histolyticum group, Clostridium clusters I and II), Lab158 (specific for Lactobacillus/Enterococcus spp.), Str493 (specific for Streptococcus/Lactococcus ssp.) and EC1531 (specific for Escherichia coli). An Axio Imager A1 epifluorescence microscope (Carl Zeiss, Göttingen, Germany) was used to count the bacterial cells. For each well, 10 to 20 random fields were counted,

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and the counts of the bacterial numbers were expressed as log10 cells per millilitre ± standard deviation (SD). Table 1. Specific 16s rRNA-targeted oligonucleotide probes used in this study. Hybridization

Probe

Target organism

Sequence from 5’ to 3’

Bif 164

Bifidobacterium

CATCCGGCATTACCACCC

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Bac 303

Bacteroides-Prevotella group

CCAATGTGGGGGACCTT

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His 150

Clostridium histolyticum group

TTATGCGGTATTAATA T(C/T)CCTT

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temperature (℃)

(Clostridium clusters Ⅰand Ⅱ） Lactobacillus/Enterococcus ssp.

GGTATTAGCATCTTCCA

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Str 493

Streptococcus/Lactococcus ssp.

GTTAGCCGTCCCTTTCTGG

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EC 1531

Escherichia coli

CACCGTAGTGCCTCGTCATCA

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Lab 158

2.7. Slectivity index (SI)

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The SI value represents a comparative relationship between the growth of ‘beneficial’ fecal bacteria

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and ‘undesirable’ ones, related to the changes of the total number of bacteria. The SI equation used was

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based on the described by Díez-Municio, Kolida, Herrero, Rastall & Moreno (2016) with slightly modified as follows:

( Bif t / Bif 0) ( Labt / Lab0) ( Strt / Str 0) ( Bact / Bac 0) ( Hist / His 0) ( ECt / EC 0) Totalcount t / Totalcount 0

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SI

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where Bift /Bif0 is Bifidobacterium spp numbers at sample time (6, 12, 24 and 48 h fermentation, respectively.) divided by numbers at inoculation, Labt /Lab0 is Lactobacillus/Enterococcus spp numbers

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at sample time divided by numbers at inoculation, Strt /Str0 is Streptococcus/Lactococcus spp numbers at sample time divided by numbers at inoculation, Bact /Bac0 is Bacteroides-Prevotella group numbers

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at sample time divided by numbers at inoculation, Hist /His0 is Clostridium histolyticum group numbers at sample time divided by numbers at inoculation, ECt /EC0 is Escherichia coli numbers at sample time divided by numbers at inoculation, and Totalcountt /Totalcount0 is total bacteria numbers at sample time divided by numbers at inoculation. 2.8. SCFA analysis

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SCFA concentrations during faecal fermentations were determined by HPLC system (Agilent 1100 series) with a diode-array detector (DAD) at 210 nm. Five hundreds microlitre samples taken from batch culture vessels at each fermentation time point were centrifuged (13,500 g for 5 min) and supernatants were then filtered through a 0.22 m filter membrane. Two hundred L of each prepared sample were completed on a Beckman Ultrasphere column (4.6 × 250 mm, 5 m, Beckman Instruments Inc., Berkeley, CA, USA). The column oven temperature was set at 30°C, and the mobile

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phase consisted of H3PO4 solution adjusted to pH 2.5 with 20 mM KH2PO4 (A) and methanol (B).

Elution was performed with the linear gradient as follows: 0-16 min, A 95%, B 5%; 16-30 min, A from

95 to 70%, B from 5 to 30%; 30-40 min, A 70%, B 30%. The flow rate was 0.8 mL/min. Quantification

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of formic, acetic, propionic, butyric acids and lactic acid were obtained through external standard

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calibration curves using concentrations ranging between 0.5 and 100 mM.

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2.9. Statistical analysis

The results obtained were analyzed by using SPSS version 16.0 (SPSS Inc., Chicago, IL, USA). Any

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significant difference was determined by one-way analysis of variance (ANOVA) followed by Tukey test for multiple comparisons considering difference statistically at P < 0.05. SPSS was also employed

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for the layout of dendrograms based on hierarchical cluster analysis using average linkage (between-groups), the Euclidean distance coefficient and Z scores to standardize the variables. Data

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was obtained from three independent experiments, and each sample was analyzed in triplicate. 3. Results and discussion 3.1. In vitro buccal and GSI digestion of three r-EPS fractions As shown in Fig. 1, there was no free oligosaccharides and/or monosaccharides in the simulated buccal and GSI medium (Fig. 1A), and the Mw of r-EPS1 was the same (3.97 × 105 Da) before and

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after digestion in vitro. In contrast, the minor peak a (Fig. 1B) and peak b (Fig. 1C) appeared after GSI digestion which indicated that r-EPS2 (13.4%) and r-EPS3 (10.6%) generated few oligosaccharides and/or monosaccharides in the GSI medium. Furthermore, free oligosaccharides and/or monosaccharides were characterized by a column of Shodex Sugar KS-801 that could effectively separate various types of oligosaccharides and monosaccharides. Fig. S1 (Supporting Information) showed that r-EPS2 and r-EPS3 generated a few glucose and glactose after simulated buccal and GSI

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system with molar ratios of 1.0: 1.3 and 1.3: 1.0, respectively. In addition, the sugar composition of

r-EPS2 and r-EPS3 after the digestion have been determined and compared with the native r-EPS2 and r-EPS3 (Supporting information Fig.S2). The monosaccharide composition of r-EPS2 and r-EPS3

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was analyzed by gas chromatography (GC) by comparing the retention time with the reference sugar

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standards. The results indicated that r-EPS2 and r-EPS3 were composed of glucose and galactose with

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ratios of 1.0: 1.3 and 1.3: 1.0, respectively, which was the same compared to native r-EPS2 and r-EPS3. Carnachan, Bootten, Mishra, Monro and Sims (2012) reported that water-soluble polysaccharides from

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kiwifruit reduced slightly in Mw with simulated GSI digestion in vitro, which was similar to our results. Some studies have also been carried out to determine changes of other EPSs after simulated GSI

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digestion. They have shown that EPS digestibility could be influenced by source, Mw and ratio of - to -linkages (Salazar, Gueimonde, de los Reyes-Gavilan, & Ruas-Madiedo, 2016; Zhou, Cui, & Qu,

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2019). Our results showed that the value of in vitro digestibility of r-EPS1 was lower than that of other two r-EPS fractions (r-EPS2 and r-EPS3), which might be related to its higher -linkages in r-EPS1 (Tang et al., 2017). The above results also suggested that the glycosidic bonds of r-EPS2 and r-EPS3 were partially broken and released a few monosaccharides (glucose and galactose), resulting in a slightly decrease in Mw, but no significant effect on the repetitive structure units and the main structure

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of polysaccharides after digestion in simulated buccal and GSI system. Therefore, three r-EPS fractions from L. delbrueckii ssp. bulgaricus SRFM-1 could resist digestion through the GSI system to reach the large intestine.

Response (mV)

(A)

After

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Response (mV)

Before

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Response (mV)

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Before

(C) b After Before

Retention time (min)

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Fig. 1. HPLC chromatograms of r-EPS1 (A), r-EPS2 (B) and r-EPS3 (C) after in vitro simulated buccal and GSI digestion compared with chromatograms before simulated buccal and GSI digestion. 3.2. Effects of three r-EPS fraction on bacterial populations Table 2 showed the bacterial populations after 0, 6, 12, 24 and 48 h of incubation of a faecal inoculum supplemented with different purified r-EPS fractions as substrates in vitro anaerobic culture. The results of anaerobic incubation with r-EPS1 at 12 h by FISH were shown in Fig. 2. On the whole,

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quantities of total bacteria were maintained in the process of fermentation for all r-EPS fractions tested. The ubiquitous bifidobacteria within the intestinal flora has been mainly connected with maintaining a healthy gastrointestinal tract, as well as other various health advantages including prevention of colorectal cancer and prevention of microbial pathogen infections (van den Broek, Hinz, Beldman, Vincken & Voragen, 2008). There was a significant increase in the bifidobacterial population (Bif164) in the anaerobic fermentation following the response to r-EPS1 at 24 h compared with 0 h, and this

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upward tendency was maintained at 48 h of fermentation (Table 2). Furthermore, the bifidobacterial

population of r-EPS2 at 48 h fermentation was similar with inulin. To be specific, values added up to

0.8-1.0 log10 cells/mL in bifidobacterial group were realized following fermentation of the r-EPS1 (7.85

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log10 cells/mL) and r-EPS2 (8.07 log10 cells/mL) from the 0 h populations (7.01 log10 cells/mL). Kolida

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and Gibson (2007) reported that enhancements of 0.5-1.0 log10 cells/mL in bifidobacterial population

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might be considered as a significant change in the enteric microorganism towards a potentially healthier composition. Therefore, the r-EPS2 could be deemed to bifidus factor under these studied

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conditions, with bacteria values even higher than those stimulated by inulin. The Lactobacillus/Enterococcus populations (Lab158) were also stimulated, with the highest levels (7.59

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log10 cells/mL) observed for r-EPS1 at 48 h of fermentation. Comparing all r-EPS fractions assayed to the blank control, it was noticed that Lactobacillus/Enterococcus populations (Lab158) were

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significantly higher at fermentation time 12 h and 24 h (P < 0.05) in the presence of r-EPS3. As for r-EPS2, Lactobacillus/Enterococcus populations were significantly higher at 6 h, 12 h and 24 h (P < 0.05). After 48 h of fermentation, significant differences from the inoculation (0 h) persisted in the inulin fermentation. Streptococcus/Lactococcus population (Str 493) showed a very slow growth with all substrates. However, significant growth could be considered for all substrates after 24 h of

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fermentation when compared to the sample taken at 0 h. In this study, although Streptococcus/Lactococcus population occupied the minority of the intestinal flora, it remained substantial increase throughout the whole process of fermentation. Generally, all the substrates initially increased the number of Clostridia (His150), but a decrease trend was found after 6 h of fermentation. Escherichia coli (EC 1531) populations showed a very similar trend in all case. It showed a small increase after 6 h of fermentation, especially with the r-EPS1. And then for further fermentation, it did

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not exhibit any statistically significant changes resulting from the fermentation of the r-EPS fractions tested in this study. Meanwhile, Bacteroides (Bac303) dominated the bacterial populations at

inoculation. Bacteroides levels significantly increased in 6 h of fermentation with all r-EPS fractions.

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Apart from blank control, three r-EPS fractions and inulin showed no significant differences after 6 h

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that compared with 0 h.

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From the point of view of r-EPS utilization, the responses (mv) of r-EPS1, r-EPS2 and r-EPS3 peaks in HPGPC chromatograms significantly decreased after 48 h fermentation (Supporting Information

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Fig. S3). Thereinto, the response of peaks at 11.9 min and 14.2 min (due to breakdown of r-EPS1), peaks at 13.5 min (due to breakdown of r-EPS2) and peak at 12.9 min and 14.2 min (due to breakdown

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of r-EPS3) were increased, suggesting that r-EPS1, r-EPS2 and r-EPS3 were hydrolyzed and utilized to fragments with smaller MWs by intestinal microbiota. Thus, the results showed that the three r-EPS

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fractions tested could be utilized by intestinal microbiota, which is consistent with the result mentioned above.

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Fig. 2. The results of anaerobic incubation with r-EPS1 as substrate at 12 h by fluorescence in situ hybridization (FISH) using epifluorescence microscope (EC1531 (A), Str493 (B), Bac303 (C), His150 (D), Lab158 (E), Bif164 (F) and DAPI (G、H)).

16

Table 2. Different bacterial populations (log10 cells per milliliter) in vitro fermentation at 0 (inoculum), 6, 12, 24 and 48 h using three purified r-EPS fractions and inulin as substrates, compared with a no treatment control (n=3) a Carbon

Fermentation time (h)a

sources

0

6

12

24

48

DAPI

CK

9.53(0.19)A

9.55(0.13)a,A

9.56(0.18)a,A

9.57(0.16)a,A

9.60(0.20)a,A

(Total bacteria)

r-EPS1

9.28(0.11)b,AB

9.32(0.15)a,A

9.40(0.09)a,A

9.47(0.13)a,A

r-EPS2

9.38(0.19)ab,A

9.39(0.10)a,A

9.47(0.14)a,A

9.54(0.21)a,A

r-EPS3

9.32(0.18)a,A

9.35(0.15)a,A

9.46(0.11)a,AB

9.52(0.14)a,A

Inulin

9.29(0.11)a,AB

9.31(0.09)a,AB

9.42(0.14)a,A

9.50(0.13)a,A

6.98(0.29)a,A

7.09(0.14)a,A

7.11(0.11)a,A

7.14(0.13)a,A

Probe

CK

7.01(0.32)A

(Bifidobacteria)

r-EPS1

7.32(0.16)a,AB

7.41(0.21)b,AB

7.69(0.19)b,BC

7.85(0.18)b,C

r-EPS2

7.33(0.22)a,AB

7.45(0.13)b,BC

7.74(0.16)b,CD

8.07(0.19)b,D

r-EPS3

7.02(0.15)a,A

6.89(0.13)a,A

7.05(0.20)a,A

7.08(0.18)a,A

Inulin

7.35(0.12)a,AB

7.51(0.16)b,B

7.73(0.19)b,BC

8.04(0.22)b,C

6.88(0.19)a,A

6.90(0.21)a,A

6.92(0.19)a,A

6.95(0.14)a,A

Lab158

CK

(Lactobacilli)

r-EPS1

7.09(0.12)a,A

7.35(0.19)b,B

7.49(0.17)b,B

7.59(0.12)b,B

r-EPS2

7.14(0.13)a,AB

7.29(0.17)b,B

7.33(0.16)b,B

7.40(0.15)b,B

r-EPS3

7.02(0.11)a,A

7.25(0.09)b,B

7.29(0.07)b,B

7.35(0.10)ab,B

Inulin

7.12(0.11)a,AB

7.33(0.16)b,BC

7.45(0.16)b,C

7.55(0.14)b,C

6.33(0.13)a,A

6.36(0.12)a,A

6.40(0.16)a,A

CK

(Streptococcus)

r-EPS1

6.43(0.11)a,AB

6.54(0.09)ab,AB

6.59(0.08)b,AB

6.63(0.07)ab,B

r-EPS2

6.39(0.12)a,A

6.55(0.11)b,AB

6.62(0.10)b,B

6.66(0.15)b,B

r-EPS3

6.41(0.14)a,A

6.50(0.09)ab,AB

6.56(0.08)b,B

6.61(0.14)ab,B

Inulin

6.49(0.11)a,AB

6.58(0.13)b,AB

6.62(0.09)b,B

6.70(0.19)b,B

8.19(0.11)a,A

8.32(0.12)a,B

8.30(0.14)b,B

8.37(0.15)b,B

8.24(0.21)a,AB

8.21(0.19)a,AB

7.98(0.14)ab,A

7.96(0.13)a,A

8.32(0.23)a,B

8.19(0.14)a,AB

7.94(0.21)a,A

7.91(0.12)a,A

8.31(0.21)a,B

8.25(0.17)a,AB

8.09(0.19)b,A

8.02(0.09)a,A

8.19(0.11)a,A

8.21(0.13)a,AB

8.05(0.17)ab,A

7.95(0.11)a,A

7.40(0.14)a,A

7.35(0.13)a,A

7.30(0.15)a,A

7.28(0.10)a,A

(Bacteroides)

r-EPS1

lP

CK

8.04(0.12)A

r-EPS2

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r-EPS3 Inulin His150

CK

(Clostridia)

r-EPS1

6.29(0.12)a,A

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Str493

Bac303

6.34(0.14)A

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6.89(0.21)A

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Bif164

7.32(0.09)A

7.46(0.11)a,AB

7.27(0.09)a,AB

7.20(0.13)a,C

7.48(0.15)a,AB

7.31(0.13)a,A

7.24(0.16)a,C

r-EPS3

7.52(0.16)a,B

7.53(0.21)a,B

7.39(0.14)a,A

7.32(0.12)a,A

Inulin

7.49(0.11)a,AB

7.45(0.12)a,AB

7.32(0.15)a,A

7.23(0.11)a,A

7.40(0.11)a,A

7.44(0.16)a,A

7.43(0.13)b,A

7.56(0.12)a,B

EC1531

CK

7.36(0.10)a,A

(Escherichia

r-EPS1

7.39(0.09)a,A

7.54(0.14)a,AB

7.64(0.18)b,B

7.30(0.16)a,A

coli)

r-EPS2

7.45(0.13)a,A

7.50(0.11)a,AB

7.43(0.14)a,A

7.39(0.18)ab,A

r-EPS3

7.40(0.11)a,A

7.51(0.19)b,AB

7.46(0.15)ab,A

7.38(0.17)ab,A

Inulin

7.49(0.12)b,AB

7.45(0.15)b,AB

7.39(0.11)a,A

7.28(0.16)a,A

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7.54(0.10)a,B

r-EPS2

a

7.34(0.12)A

One-way ANVOA and Tukey tests were uesd to determine significant differences for each bacterial

population. Different lowercases letters (a and b) indicate significant differences for each bacterial among the different carbohydrate sources. Different capital letters (A, B, C and D) indicate significant differences for each bacterial genus among the different time points. Standard error is given in

17

parentheses. 3.2. Selectivity index (SI) As shown in Fig. 3A, SI values obtained for all assayed r-EPS fractions after 48 h of fermentation resulted in a positive relative selectivity towards organisms recognized as health-promoting. The highest SI values, i.e., 13.17 and 12.84, were obtained for r-EPS1 and r-EPS2 respectively, after 48 h of fermentation. Remarkably, the SI score reached for the r-EPS1 at 48 h of fermentation was higher than

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the corresponding value of the well-established prebiotic inulin. There were differences in the selectivity of r-EPS1 and r-EPS2 for different bacteria, in which the latter resulted in a higher SI at 48 h of fermentation due to the increased populations of bifidobacteria. Meanwhile, the r-EPS1 resulted in

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similar increases in bifidobacteria at 48 h but a greater increase in lactobacilli, resulting in a much

higher SI. Conversely, r-EPS3 obtained the lowest values at different points. In addition, the control

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without carbohydrate source scored SI values that are highly negative, ranging from -1.6 to -0.8, at the

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four fermentation time points, which is indicative of low populations of bifidobacteria and/or lactobacilli when compared to the rest of studied bacterial groups.

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With the aim of obtaining a broader assessment of the effect of the chemical structure of the test r-EPS fractions on the human intestinal flora composition and activity, one dendrogram based on the SI

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scores was constructed. As it may be expected, all r-EPS fractions clustered separately from the control

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without carbon source (Fig. 3B). Remarkably, r-EPS1, r-EPS2 and inulin formed an individual cluster comparing to r-EPS3 that based on the SI scores. According to the in vitro GSI digestion experiment and primary chemical structures of r-EPS from L. delbrueckii ssp. bulgaricus SRFM-1 (Tang et al., 2017), it could potentially be regarded as prebiotic ingredients based on three conceptions of prebiotic (Gibson et al., 2017). Firstly, determinate glycosidic linkages in the structures of these r-EPS fractions mean that they might not be more digested than polysaccharides with -linkages by intestinal enzymes. 18

Specifically, the high resistance of -(1-4,6) linkages to in vivo and in vitro gastrointestinal digestion has been previously studied (Nakada, Nishimoto, Chaen & Fukuda, 2003). On the other hand, compared to well-known prebiotics such as lactulose or galactooligosaccharide, as well as with inulin, the purified r-EPS fractions could possess the prebiotic properties that might be attributed to these monosaccharide compositions. Furthermore, the general target microorganisms for prebiotic activities are bifidobacteria and lactobacilli (Rastall & Gibson, 2015). It has been reported that the prebiotic

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effects of polysaccharide were influenced by their chemical composition and structure of the polymeric backbone (Salazar, Gueimonde, de los Reyes-Gavilan, & Ruas-Madiedo, 2016). According to our

previous study (Tang et al., 2017), the r-EPS1 and r-EPS2 were homogenous, whereas r-EPS3 had two

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peaks, indicating that r-EPS3 was hetrogenous. The chemical composition and structure of r-EPS1 and

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r-EPS2 was greatly different from that of r-EPS3, although the Mw of r-EPS1 and r-EPS2 was similar

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with that of r-EPS3. r-EPS1 (1.2: 1.0) and r-EPS2 (1.3: 1.0) exhibited much higher molar ratios between galctose and glucose than r-EPS3 (0.8: 1.0). Moreover, differed from r-EPS3, r-EPS1 and

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r-EPS2 possessed more side chains which probably facilitated the exposure of active groups and/or its spatial structure. The degrees of branching of the different r-EPS fractions were calculated using the

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following formula. The degree of branching = number of branching points/ unbranching sugar residues from the polysaccharide main chain (Li et al., 2015a). In our study, the number of branching points of

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r-EPS1, r-EPS2 and r-EPS3 were 3, 1 and 1, respectively. The number of unbranching sugar residues from the polysaccharide main chain of r-EPS1, r-EPS2 and r-EPS3 were 9, 3 and 8, respectively. Thus, the degree of branching was 33.3% for both of r-EPS1 and r-EPS2 which was higher than r-EPS3 (12.5%), according to the molar ratios of branched to linear sugar residues. In addition, r-EPS1 and r-EPS2 included higher ratio of - to -linkages comparing to r-EPS3. These special features might

19

have made it possible that bifidobacteria and lactobacilli -glucosidases and/or

-galactosidases work

on one or on both terminal linkages, thus allowing a better bacterial growth and activity with r-EPS1 and r-EPS2 as substrate than with r-EPS3 (Xu, et al., 2019). Therefore, structural characteristics such as the molar ratios of monosaccharide composition, homogeneity, configuration and high degree of branching might attribute r-EPS1 and r-EPS2 a high SI and prebiotic activity.

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(A)

(B)

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Rescaled Distance Cluster Combine

Fig. 3. Comparison of the selectivity index (SI) with different purified r-EPS fractions during anerobic fermentation (A); dendrograms derived from selectivity index (SI) scores calculated at 48 h for three r-EPS fractions, inulin and a control without carbon source (B). Different lowercase letters (a, b, c and d) above bars indicate statistically significant differences (P < 0.05) for different polysaccharides at the same time points.

20

3.3. SCFA production during fermentation As we all konw, SCFA plays an important role in gastrointestinal and hepatic metabolism by improving the host health. Acetic acid is oxidized by heart and peripheral tissues. Propionic acid weakens cholesterol synthesis in liver and improves the lipid metabolism. Butyric acid usually serves as an energy source for intestinal microorganisms and provides prevention against colorectal cancer (Gibson et al., 2017). The HPLC chromatograms of SCFA with three r-EPS fractions at different times

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were shown in Fig. 4A. Additionally, mean SCFA concentrations at different fermentation time points were shown in Table 3. It was noticed that total SCFA increased during in vitro fermentation in the

present of the test r-EPS fractions. The acetic, propionic, butyric acids and lactic acid were found as the

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predominant components in the total SCFA. The r-EPS1 contained higher concentration of total SCFA

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as compared with inulin with the fermentation time increase. Finally, the total SCFA concentration

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reached the maximum level of 145.51 mM after 48 h of fermentation. Acetic acid was clearly the most prevalent SCFA produced during the fermentation of all substrates followed by lactic acid and

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propionic acid. Acetic acid levels increased significantly after 6 h of fermentation with all substrates. The highest acetic acid concentrations (56.37mM) were detected in r-EPS1 after 48 h of fermentation.

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Moreover, lactic acid, formic acid, propionic acid and butyric acid concentrations also increased for all tested substrates with incubation time. In this study, the concentrations of propionic acid and butyric

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acid at 48 h were 20.58 and 7.83mM (inulin), 23.45 and 9.21 mM (r-EPS1), 21.98 and 8.61 mM (r-EPS2), and 18.39 and 5.92 mM (r-EPS3), respectively. Fermentation of r-EPS1 and r-EPS2 resulted in comparatively high propionic and butyric acids. These results were similar to other studies (Hu, Nie, Li, Fu, & Xie, 2013; Xu et al., 2019). Table 3. Different kinds of SCFA and total SCFA in vitro fermentation at 0, 6, 12, 24 and 48 h using three r-EPS fractions and inulin, compared with a no treatment control (n=3)a

21

Carbon

Fermentation time (h)a

lactic acid

sources

0

6

12

24

48

Formic acid

CK

1.14（0.35）A

0.95(0.32)a,AB

1.17(0.67)a,AB

1.29(0.58)a,B

2.02(0.45)a,B

r-EPS1

4.98(0.65)c,B

8.03(1.20)c,C

10.01(0.93)c,CD

12.19(0.84)c,D

r-EPS2

5.02(0.54)c,B

7.14(0.74)bc,C

8.54(1.02)bc,CD

10.25(0.95)b,D

r-EPS3

2.74(0.67)b,B

5.98(0.83)b,C

7.81(0.89)b,CD

8.93(0.92)b,D

Inulin

4.85(0.83)c,B

6.69(0.59)bc,C

8.57(0.47)bc,D

9.02(0.43)b,D

2.94(0.58)a,B

4.07(0.78)a,BC

4.89(0.81)a,BC

6.02(1.21)a,C

r-EPS1

18.68(1.48)b,B

32.89(3.48)b,C

46.43(4.03)b,D

56.37(5.89)b,E

r-EPS2

18.22(1.93)b,B

30.48(3.58)b,C

43.78(4.25)b,D

52.28(5.47)b,D

r-EPS3

17.02(2.42)b,B

29.74(4.01)b,C

41.92(3.68)b,D

48.83(5.03)b,D

19.45(2.10)b,B

32.61(3.93)b,C

43.08(3.97)b,CD

51.79(6.36)b,D

2.02(0.35)a,B

3.61(0.45)a,C

4.29(0.67)a,CD

5.73(0.84)a,D

r-EPS1

14.43(3.01)b,B

23.34(2.30)b,C

r-EPS2

13.93(2.68)b,B

20.87(3.23)b,B

r-EPS3

10.92(2.35)b,B

18.47(2.67)b,C

Inulin

14.23(2.34)b,B

21.45(2.89)b,B

0.79(0.21)a,AB

1.02(0.24)a,AB

Acetic acid

CK

2.65（1.09）A

Inulin Lactic acid

CK

1.20（0.20）A

0.00（0.00）A

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SCFA and

34.73(3.83)b,D

44.29(4.26)b,E

32.28(3.48)b,C

42.45(4.83)b,D

29.83(2.94)b,D

36.49(4.12)b,D

32.89(3.69)b,C

40.74(4.23)b,C

1.82(0.35)a,BC

2.74(0.83）a,C

18.83(2.58)b,C

23.45(3.61)b,C

CK

acid

r-EPS1

5.37(1.68)b,AB

9.73(2.10)b,B

r-EPS2

4.98(1.74)b,AB

9.25(2.03)b,B

17.92(3.21)b,C

21.98(3.38)b,C

r-EPS3

2.93(0.83)ab,AB

7.98(1.94)b,B

15.29(2.17)b,C

18.39(3.54)b,C

5.63(1.45)b,AB

10.32(2.35)b,B

17.59(2.48)b,C

20.58(3.29)b,C

0.00(0.00)a,A

0.37(0.23)a,AB

0.79(0.42)a,B

0.92(0.31)a,B

CK

0.00（0.00）A

2.99(0.61)b,AB

5.20(1.42)b,BC

8.01(1.92)b,CD

9.21(2.14)b,D

r-EPS2

2.57(0.78)b,AB

4.85(0.91)b,BC

7.69(1.64)b,CD

8.61(1.47)b,D

1.82(0.69)ab,AB

3.73(0.83)b,BC

4.88(1.11)b,CD

5.92(0.94)b,D

3.29(0.95)b,B

5.05(1.20)b,BC

6.93(1.29)b,C

7.83(1.43)b,C

6.70(1.89)a,AB

10.24(2.46)a,BC

13.08(3.85)a,BC

17.43(4.12)a,C

46.45(5.29)b,B

79.19(6.32)b,C

118.01(6.37)c,D

145.51(6.98)c,E

44.72(5.93)b,B

72.59(5.93)b,C

110.21(7.29)bc,D

135.57(7.02)bc,E

35.43(5.26)b,B

65.90(5.33)b,C

99.73(5.73)b,D

118.56(7.53)b,E

47.45(5.36)b,B

76.12(6.24)b,C

109.06(6.96)bc,D

129.96(7.23)bc,E

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r-EPS1

r-EPS3 Inulin CK r-EPS1 r-EPS2 r-EPS3

4.99（1.75）A

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Inulin a

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Total acids

re

Inulin Butyric acid

-p

Propionic

One-way ANVOA and Tukey tests were used to determined significant differences for each SCFA.

Different lowercases letters (a, b and c) indicate significant differences for each bacterial genus among

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the different carbohydrate sources. Different captial letters (A, B, C, D and E) indicate significant differences for each SCFA among the different time points. Standard error is given in parenthess.

22

(A) Response (mAU)

(I)

Retention time (h)

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Response (mAU)

(II)

(III)

Retention time (h)

(IV)

Response (mAU)

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na

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Response (mAU)

re

Retention time (h)

Retention time (h) 23

(B)

Rescaled Distance Cluster Combine

(II)

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(I)

(IV)

(V)

(VI)

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lP

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(III)

Fig. 4. High performance liquid chromatography (HPLC) chromatograms of SCFA (A) with standard (Ⅰ) and r-EPS1 (ⅠI), r-EPS2 (ⅡI) and r-EPS3 (IV) at different fermention times (0, 6, 12, 24 and 48 h); dendrograms derived from formic (Ⅰ), acetic (Ⅱ), propionic (Ⅲ), butyric (Ⅳ), lactic (Ⅴ) acids and total acid (Ⅵ) levels produced during human faecal fermentation (6, 12, 24 and 48 h) for three r-EPS fractions, inulin and a control without carbon source (B).

24

Furthermore, a dendrogram based on different SCFA levels throughout fermentation was constructed. As shown in Fig. 4B, all r-EPS fractions and inulin clustered separately from the blank control without carbon source. To be specific, r-EPS1, r-EPS2 and inulin formed an individual cluster based on either the propionic acid or butyric acid. r-EPS2, r-EPS3 and inulin were also clearly clustered together based on the formic acid. However, a remarkable dissimilarity of r-EPS3 with respect to the rest of substrates was found when correlating r-EPS structures from the propionic and butyric acid values throughout the

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fermentation process (6, 12, 24 and 48 h). This behaviour reflects the fact that r-EPS3 exhibited the

slowest rate of fermentation as it could be inferred from low SCFA values and SI scores obtained after 48 h of fermentation (Fig. 3A). As a whole, r-EPS1 and r-EPS2 were highly related to inulin based on

-p

both the SI scores and mean SCFA production. Therefore, from the results shown by the cluster

re

analysis, insights on relationships between structure and microbial growth could be inferred.

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Recently, several authors have linked the pathways of relevant groups of bacteria with the production of the main SCFA resulting from saccharolytic fermentation (Shang, Jiang, Cai, Hao, Li, & Yu, 2018).

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For instance, acetic acid and lactic acid are the major SCFA of bifidobacteria fermentation, as well as for lactobacilli and lactococci, whereas butyric acid is a major product of clostridia and eubacteria.

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Moreover, Bacteroides-Prevotella is a dominant group of the gut microbial communities and is known as a producer of propionic acid (Flint, Scott, Duncan, Louis, & Forano, 2012). In addtion, the different

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SCFA compositions of different r-EPS fractions could be connected with some especial structural properties, including Mw, monosaccharide composition, branch chain and glycosidic linkage (Zhou, Cui, & Qu, 2019). Guillon and Champ (2000) reported the EPS with smaller Mw might be utilized more effectively by enteric microorganism. The total SCFA was also significantly increased. It was also reported that -glucan could be highly fermented by human inoculums, and promote the growth of

25

bifidobacteria and lactobacilli which produced SCFA, especially acetic acid (Chaikliang, Wichienchot, Youravoug, & Graidist, 2015). High levels of acetic and lactic acids accumulated in the presence of EPS1 and EPS2, which is in agreement with the high numbers of bifidobacteria and lactobacilli in these cultures. This may be related to r-EPS1 and r-EPS2 containing more galactose residue and higher degree of branching than r-EPS3 (Tang, et al., 2017). The present results agreed that the report of Khodaei, Fernandez, Fliss and Karboune (2016), who reported that acetic acid was the predominant

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acid produced with the galactose-rich polysaccharide and its oligosaccharides using a continuous

culture system inoculated with immobilized fecal microbiota. Therefore, the enhancement of SCFA may be due to specific structure in r-EPS from L. delbruecckii ssp. bulgaricus SRFM-1 after

-p

fermentation with human inoculums in vitro. Overall, key structural features such as monosaccharide

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composition and branching degree seem to be critical for determining the prebiotic property of the

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non-digestible r-EPS. 4. Conclusions

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In this study, we provided information about the GSI digestion and prebiotic potential effects of three r-EPS fractions from L. delbruecckii ssp. bulgaricus SRFM-1 in vitro. The results indicated there was

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no (r-EPS1) or a few free oligosaccharides or monosaccharides (r-EPS2 and r-EPS3) released throughout the GSI digestion period. r-EPS1 and r-EPS2 exerted stronger promoting activity for

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bifidobacteria and were high producers of SCFA following their fermentation on human faecal samples. Both of r-EPS1 and r-EPS2 showed high SI values with the increased populations of bifidobacteria, lactobacilli and lactococci, which were even higher than inulin. This finding might point out that r-EPS derived from L. delbruecckii ssp. bulgaricus SRFM-1 could be regarded as candidate prebiotic. Further in vivo studies using animal model and human volunteer trials are necessary in order to assess the

26

functionality of these r-EPS fractions.

Authors contribution Weizhi Tang: Conceptualization, Methodology, Writing-Original draft preparation; Jianzhong Zhou: Data curation, Software; Qian Xu: Data curation, Software; Mingsheng Dong: Writing-

Reviewing and Editing; Xia Fan: Software, Validation; Xin Rui: Methodology, Validation; Qiuqin

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Zhang: Methodology, Investigation; Xiaohong Chen: Writing- Reviewing and Editing; Mei Jiang:

Writing- Reviewing; Junjun Wu: Writing- Reviewing; Wei Li: Conceptualization, Writing-

re

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Reviewing, Supervision.

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Acknowledgements

This work was co-financed by National Natural Science Foundation of China (No. 31871771 and

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31571818), Fundamental Research Funds for the Central Universities (KYYJ201807 and

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KYLH201702) and Natural Science Foundation of Jiangsu Province (No. BK20161448).

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