In vitro digestion and human gut microbiota fermentation of longan pulp polysaccharides as affected by Lactobacillus fermentum fermentation

In vitro digestion and human gut microbiota fermentation of longan pulp polysaccharides as affected by Lactobacillus fermentum fermentation

Journal Pre-proof In vitro digestion and human gut microbiota fermentation of longan pulp polysaccharides as affected by Lactobacillus fermentum ferme...

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Journal Pre-proof In vitro digestion and human gut microbiota fermentation of longan pulp polysaccharides as affected by Lactobacillus fermentum fermentation

Fei Huang, Ruiyue Hong, Yang Yi, Yajuan Bai, Lihong Dong, Xuchao Jia, Ruifen Zhang, Guangjin Wang, Mingwei Zhang PII:

S0141-8130(19)37179-X

DOI:

https://doi.org/10.1016/j.ijbiomac.2020.01.059

Reference:

BIOMAC 14363

To appear in:

International Journal of Biological Macromolecules

Received date:

5 September 2019

Revised date:

31 December 2019

Accepted date:

6 January 2020

Please cite this article as: F. Huang, R. Hong, Y. Yi, et al., In vitro digestion and human gut microbiota fermentation of longan pulp polysaccharides as affected by Lactobacillus fermentum fermentation, International Journal of Biological Macromolecules(2018), https://doi.org/10.1016/j.ijbiomac.2020.01.059

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© 2018 Published by Elsevier.

Journal Pre-proof In vitro digestion and human gut microbiota fermentation of longan pulp polysaccharides as affected by Lactobacillus fermentum fermentation Fei Huanga, Ruiyue Honga, Yang Yib, Yajuan Baia, Lihong Donga, Xuchao Jiaa, Ruifen Zhanga, Guangjin Wangc and Mingwei Zhanga* a

Sericultural & Agri-Food Research Institute Guangdong Academy of Agricultural Sciences/Key Laboratory of Functional Foods, Ministry of Agriculture and Rural Key

Laboratory

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Agricultural

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College of Food Science & Engineering, Wuhan Polytechnic University, Wuhan

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430023, China c

Processing,

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Guangzhou 510610, PR China b

Products

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Affairs/Guangdong

Xiaogan, 432000, P. R. China *

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College of Chemistry and Materials Science, Hubei Engineering University,

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Corresponding author: Mingwei Zhang

Address: Sericultural and Agri-Food Research Institute, Guangdong Academy of

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Agricultural Sciences, Guangzhou 510610, P. R. China Telephone: +86-20-8723 7865, Fax: +86-20-8723 6354 E-mail: [email protected]

Journal Pre-proof Abstract: This study investigated the effect of Lactobacillus fermentum fermentation treatment on the gastrointestinal digestion and fermentation in vitro of polysaccharides from longan pulp. Polysaccharide isolated from unfermented and fermented longan pulp named LP and LP-F, respectively. The molecular weight of LP-F declined from 109.62±10.66 kDa to 51.46±6.26 kDa while that of LP declined from 221.63±2.41 kDa to 69.68±2.36 kDa with gastrointestinal digestion. At same

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time, the reducing sugars content of LP and LP-F were both increased significantly. In

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addition, after 48 h gut microbiota fermentation, there were more total short chain

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fatty acids and acetic acid, as well as more Enterococcus, Bifidobacterium, and

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Clostridium in LP-F fermentation culture than those in LP fermentation culture. Moreover, LP-F fermentation culture showed lower pH value and less residual

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carbohydrate percentage than that of LP. These results indicated that LP-F is easier

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than LP to be fermented by human gut microbiota.

Keywords: Longan polysaccharide; Digestion; Fermentation

Journal Pre-proof 1. Introduction Longan (Dimocarpus longan Lour.) is a subtropical and tropical fruit and its pulp has been used as a traditional Chinese medicine to promote blood metabolism, relieve insomnia and prevent amnesia in folk remedies. Previous studies showed that the numerous health benefits of longan pulp might be related to the polysaccharides, which exhibited immunostimulatory, antioxidant and antiproliferative activities [1-3]. Longan is a seasonal fruit and has a short shelf life due to its perishable nature

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and susceptibility to postharvest pathogens [4]. Many processing methods were used

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to expand longan fruit shelf life, such as drying, fermentation and so on [5, 6]. Some

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studies showed that processing treatment would affect functional ingredients in fruits

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and vegetables [7, 8]. In our previous study, the physicochemical and biological properties of longan pulp polysaccharides were modified by Lactobacillus fermentum More

in

details,

compared

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fermentation.

with

unfermented

longan

pulp

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polysaccharides (LP), fermented longan pulp polysaccharides (LP-F) had lower molecular weight, and contained less neutral sugar, uronic acid and glucose, but more

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arabinose, galactose, rhamnose and mannose. Moreover, LP-F exhibited stronger immunomodulatory and prebiotic activities [9]. As we known, the bioactive effect of functional ingredient is closely related to its digestion and absorption in the gastrointestinal digestive system. For bioactive polysaccharides, some reports showed that they could pass through the gastrointestinal tract to reach distal intestine, where they can be fermented and degraded by symbiotic bacteria [10]. Bioactive polysaccharides intake would change the metabolites and composition of intestinal flora, especially for enhancement of short-chain fatty acids (SCFAs) and health-promoting gut microbiota [11, 12]. Compared with LP, LP-F exhibited different physicochemical properties and

Journal Pre-proof enhanced bioactivities, but little information was available about their gastrointestinal digestive traits, and what is the difference between LP and LP-F during the digestion process. Therefore, the aim of this study was to investigate the digestion and fermentation behaviour of LP and LP-F. The digestion and fermentation behaviours of LP and LP-F, including carbohydrate degradation, Mw and pH change, free monosaccharide and SCFAs production in gastrointestinal digestion models and gut microbiota fermentation models were investigated.

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2. Materials and methods

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2.1 Materials and chemicals

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Standard dextrans (including T-4: molecular mass, 4×103 Da, T-10: 1×104 Da,

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T-40: 4×104 Da, T-70: 7×104 Da, T-500: 5×105 Da, and T-2000: 2×106 Da), rhamnose, arabinose, glucose, xylose, galactose and mannose were purchased from Sigma

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Chemical Co. (St. Louis, MO, USA). Man-Rogosa-Sharpe (MRS) was purchased

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from Guangdong Huankai Microbial Technology Co., Ltd. (Guangzhou, China). All other chemicals used in this work were analytical grade a purchased from Guangzhou

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Qiyun Biological Co., Ltd. (Guangzhou, China). Lactobacillus fermentum GIM 1.191 was purchased from Guangdong Provincial Microbial Culture Collection Center. 2.2 Preparation of polysaccharide The fermented or unfermented longan polysaccharides were prepared according to our previous published methods [9]. In a short, dried longan (cv. Chu-liang) were homogenised with water (1:7 w/v) to obtained longan juice. Some of longan juice was fermented with Lactobacillus fermentum for 24 h at 37 °C while the leftover was inoculated with distilled water as the unfermented group. Both fermented and unfermented longan juice were used hot water to extract polysaccharide. Fermented or unfermented longan polysaccharides, named as LP-F and LP, respectively.

Journal Pre-proof 2.3 Digestion of longan polysaccharides in vitro 2.3.1 Determination of simulated gastric digestion The simulated gastric digestion was evaluated according to the published method with some modifications [12, 13]. The gastric electrolyte solution was prepared by dissolving 6.2 g NaCl, 2.2 g KCl, 0.3 g GaCl2•2H2O and 1.2 g NaHCO3 in 2 L distilled water and adjusted the pH to 3.0 by 0.1 M HCl solution. Then, the mixtures

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of 1500 g of gastric electrolyte solution with 350 mg of pepsin, 350 mg of gastric lipase with 3 mL CH3COONa (1 M, pH 5) were prepared and the final pH was

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adjusted to 3.0 using HCl (0.1 M). For simulated gastric digestion, 10 mL LP or LP-F

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solution (5 mg/mL) with 10 mL simulated gastric fluid, 10 mL LP or LP-F solution

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with 10 mL distilled water and 10 mL simulated gastric fluid with 10 mL distilled

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water were prepared and incubated in a water bath of 37 °C. During the digestion, the pH of the reaction solution was maintained at pH of 3.0. At 0, 2 and 4 h of digestion,

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2.0 mL digested sample was taken out and immersed immediately into a boiling water bath for 5 min to inactivate enzymes.

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2.3.2 Determination of simulated small intestinal digestion The simulated small intestinal digestion was carried out as described previously with slight modifications [12]. The intestinal electrolyte solution was prepared by dissolving 5.4 g NaCl, 0.65 g KCl, 0.33 g GaCl2•2H2O in 1 L distilled water and adjusted the pH to 7.0 by 0.1 M NaOH solution. Then, the intestinal medium contained 500 g of intestinal electrolyte solution, 500 g of pancreatin solution (7%, w/w) and 1000 g of bile salt solution (4 %,w/w) and 0.13 g of trypsin, and the final pH was adjusted to 7.0 using NaOH (0.1 M). For simulated small intestinal digestion, the mixtures of 3 mL of simulated small intestinal fluid with 10 mL of digested simulated gastric solution, 3 mL of distilled water with 10 mL of digested simulated

Journal Pre-proof gastric solution, and 3 mL of simulated small intestinal fluid with 10 mL distilled water were prepared. All mixture was incubated in a water bath of 37 °C. During the digestion, the pH of the reaction solution was maintained at pH of 7.0. At 0, 2 and 4 h of digestion, 2.0 mL digested sample was taken out and immersed immediately into a boiling water bath for 5 min to inactivate enzymes. 2.3.3 Determination of molecular weight The digestion products were filtrated through a 0.45 μm membrane before

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analysis. The molecular weight (Mw) was determined by ACQUITY APC (Waters

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Corp., USA) Advanced Polymer Chromatography system equipped with refractive

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index detector, and the Waters ACQUITY APCTM AQ 450, 200 and 125 columns were

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applied for detection. Samples were eluted with sodium sulphate solution (0.05 M) at

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a flow rate of 0.9 mL/min, and the injection volume was 20 μL. The column temperature was kept at 35 ± 0.2 °C. Dextran standards (4.4 kDa、9.9 kDa、21.4 kDa、

curve.

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43.5 kDa、124 kDa、196 kDa、277 kDa、401 kDa) were used to establish a standard

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2.3.4 Determination of reducing sugar content (CR) The CR of digestion products were analyzed by dinitrosalicylic acid (DNS) method using glucose as the standard [14]. 2.3.5 Determination of free monosaccharide The digestion products were filtrated through a 0.45 μm membrane before analysis. The free monosaccharide standards and samples were detected by an Agilent 1260 series HPLC equipped with an evaporative light scattering detector and an Asahipak NH2P-50-4E column. The mobile phase was acetonitrile-water (70:30, v/v) with a flow rate of 0.8 mL/min. The injection volume was 10 μL. The temperature of column and detector were 35 ± 0.2 °C and 70 ± 0.2 °C, respectively.

Journal Pre-proof 2.4 Fermentation of longan polysaccharides in vitro 2.4.1 Preparation of fecal inoculums The fecal inoculums were prepared according to the published method [15]. The fresh faecal samples were collected from three healthy donors who never had bowel disease before, and those donors consumed normal diets with no history of antibiotics for at least 3 months. Collected fecal samples were mixed with an equal amount of fecal from each donor. The mixed fecal samples were immediately stored in an

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anaerobic jar before use.

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Mixed fecal samples (12 g) were precultured in 100 mL preculture medium (1 g

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tryptone, 0.5 g yeast, 1 g NaCl, 0.5 g glucose and 0.6 g maltose). After cultured 12 h,

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the preculture was filtered through sterile gauze sponges to remove large particles. All

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operations were carried out in an anaerobic condition (10% H2, 10% CO2 and 80% N2) with YQX-Ⅱanaerobic incubator (Yuejin medical instrument co., LTD, Shanghai,

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China). 2.4.2 In vitro Fermentation

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The fermentation medium was prepared according to Hu’s method [16]. LP and LP-F were added to carbohydrate-free growth medium (2%, w/v). After that, the medium was sterilized for 15 min at 121 °C and subjected to different sterile anaerobic vessels. Then, human fecal inoculums (1%) were inoculated into the fermentation medium and incubated at 37 °C for 48 h. The culture medium without LP or LP-F was set as the blank control. Samples were taken for analysis after 0, 6, 12, 24 and 48 h fermentation. Each sample was detected three times independently. 2.4.3 Determination of pH, the total carbohydrate and proportion of residual carbohydrate The pH value was measured by PB-10 pH meter (Dolly scientific instrument co.,

Journal Pre-proof LTD, Guangzhou, China). The contents of total carbohydrate was measured by phenol-sulphuric acid method with glucose as standard [17]. The proportion of residual carbohydrate (%) was based on the contents of carbohydrate before and after fermentation. 2.4.4 Microbial population enumeration by quantitative real-time PCR (QPCR) Microbial population enumeration by QPCR was carried out as previous study with some modifications [18]. After 48 h of fermentation, 2 mL aliquots of the

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fermentation samples were centrifuged. Then DNA extraction of the sample was

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carried out using QIAamp DNA Stool Mini Kit (QIAGEN, Shanghai, China), based

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on the instruction of manufacturer. The primers for QPCR assays were synthesized

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according to reported sequences. The primer sequences and annealing temperatures of the target genes are shown in Table S1. QPCR assays were carried out using a

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Stratagene MX3005 thermal cycler (Stratagene, La Jolla, CA). Each DNA sample (1

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μL) was added into a reaction solution of 25 μL. Cycling parameters were set as follows: 95 °C for 3 min; 35 cycles of 0.5 min at 95 °C, 0.5 min at 55 °C, and 0.5 min

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at 72 °C; and extension for 5 min at 72 °C. The amplified products were cloned using the TOPO TA cloning kit for Sequencing (Invitrogen) and transformed into Escherichia coli DH5α competent cells (Invitrogen). Sequences were submitted to the ribosomal RNA database to confirm the specificity of primers. For quantification of target DNA copy number, standard curves were generated using serial 10-fold dilutions of the extracted products by using at least 6 non-zero standard concentrations per assay [19]. The bacterial concentration in each sample was measured as log10 copy number by the interpolation of the Ct values obtained by the fermentation mixture and the standard calibration curves. Each plate included triplicate reactions per DNA sample and the appropriate set of standards.

Journal Pre-proof 2.4.5 Determination of SCFAs The fermentation mixture was centrifuged at 4800 g for 10 min. The supernatants filtrated though a 0.45 μm membrane were used for determination of SCFAs. The SCFAs standards (acetic acid, propionic acid, n-butyric acid, i-butyric acid, n-valeric acid and i-valeric acid) and samples were detected by GC-2010 plus GC system (Shimadzu Corp., Japan) with flame ionization detector. The GC column DB-FFAP (Shimadzu Corporation, Japan) was used. Nitrogen was supplied as the

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carrier gas at a flow rate of 30 mL/min with a split ratio of 1:9. The flow rates of

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hydrogen, air were 30 mL/min and 400 mL/min, respectively. The temperatures of

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detector and injection port were 240 °C. The initial column temperature was held

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70 °C for 1 min, next raised to 240 °C at a rate of 5 °C/min. The injection volume was 1 μL and the detection time was 42.47 min. The GC/MSD ChemStation software was

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used.

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2.5 Statistical analysis

All experiments were run in triplicate. The results were expressed as mean ±

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standard deviations (SD) using SPSS 19.0 software. Data were evaluated by univariate analysis of variance (ANOVA), a p-value of 0.05 was chosen as the threshold for significance.

3. Results and discussions

3.1 Digestion of longan polysaccharides in vitro The total carbohydrate content of LP and LP-F were 78.42 and 69.31%, respectively. They both were heteropolysaccharides and mainly composed of rhamnose, arabinose, xylose, mannose, glucose and galactose with different molar ratios. The molar ratio of LP was 8.79: 43.63: 1.53: 2.35: 23.39: 20.32 while that of LP-F was 9.29: 48.55: 1.83: 3.28: 14.33: 22.72. The molecular weight of LP and LP-F

Journal Pre-proof were 221.63 and 109.62 kDa, respectively [9]. Polysaccharides from food sources should pass through the digested system (gastric and intestinal tract) to be utilized by body, thereby exerting their bioactivities. Previous study reported that enzymes, salts and pH might affect the structural and chemical properties of polysaccharides during human digestion [20]. Therefore, the digestion of LP and LP-F by stimulated gastric and intestinal were carried out in this study. As shown in Table 1 and Fig. S1, as the digestion time increased, a steady

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decrease in Mw was observed in LP and LP-F. Specifically, the Mw of LP decreased

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from 221.63±2.41 kDa to 125.33±2.16 kDa during gastric digestion, and it

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continuously dropped to 69.68±2.36 kDa after intestinal digestion. As for LP-F, the

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Mw declined from 109.62±10.66 kDa to 51.46±6.26 kDa after gastric and intestinal digestion.

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Polysaccharides are easy to form aggregates in solutions. It’s difficult to judge

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whether the decline in Mw of polymers was resulted from disruption of aggregates or chemical bond breakdown in the polymer chains. Therefore, the amounts of CR and

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free monosaccharide were further analysed. The CR of LP significantly increased from 24.74 ± 1.14 mg to 46.96 ± 0.87 mg with 4 h gastric digestion but retain stable during intestinal digestion. Similarly, the CR of LP-F increased from 29.44 ± 1.06 mg to 56.83 ± 1.30 mg with gastric digestion and then showed no change after intestinal digestion (Table 1). There was no free monosaccharide in both LP and LP-F after gastric and intestinal digestion conditions (Fig. S2). These results were similar with the polysaccharides from seeds of Plantago asiatica L [13] and this may be due to the acidic pH of gastric fluid, which breakdown covalent bonds of polysaccharides and formation of reducing ends. The results showed that the digestion behaviour of LP and LP-F showed

Journal Pre-proof similarity on the one hand, but there were some differences on the other hand. On the one hand, the trends of their Mws declined and CR increased with gastric digestion treatment, and their resistant to intestinal digestion were similar. On the other hand, the magnitude of this change in Mw and CR of LP and LP-F was different. The difference may be related with physicochemical properties of polysaccharide, such as monosaccharide composition and Mw. For example, pacific abalone polysaccharides [21] and Fuzhuan brick tea polysaccharides [12], both rich in acid polysaccharides,

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remained unchanged during the gastrointestinal digestion. But polysaccharides from

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mulberry fruit [22] and Inonotus obliquus [23] mainly contained neutral sugar such as

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rhamnose, arabinose, glucose, galactose and xylose, showing decline in Mw with

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gastrointestinal digestion. Moreover, the fraction A of bee collected pollen of Chinese wolfberry polysaccharide with larger Mw (1340 kDa) was not broken down during

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gastrointestinal digestion while the small fraction B (523 kDa) was degraded [24]. As

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for LP-F, it has lower uronic acid and smaller Mw than LP [9], may correspond to its different digestion behaviour.

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3.2 Fermentation of longan polysaccharides in vitro 3.2.1 pH change during fermentation It has been reported that the indigestible polysaccharides by gastric and small intestinal fluids could be utilized by gut microbiota. Thus, the fermentation of longan polysaccharides by microbiota was further studied. The change in pH value of fermentation culture reflect the fermentation process. As shown in Fig.1a, the pH value of LP, LP-F and blank groups showed a significant decline during fermentation from 0 to 48 h (p < 0.05). After fermentation for 48 h, the pH value of LP fermentation culture declined from 7.30 (0 h) to 4.39 (48 h), while that of LP-F decreased from 7.30 (0 h) to 3.94 (48 h), both were significantly lower than that of

Journal Pre-proof blank group at all-time points except for the initial time. At the same fermentation time point, the pH value of samples and blank groups was in the order as: LP-F < LP < Blank during fermentation process. The pH value reduction of LP and LP-F fermentation culture may be due to their fermentation products from gut microbiota [25]. The difference between the pH value of LP and LP-F fermentation culture might be related with the different utilization of two polysaccharides by gut microbiota. 3.2.2 Changes of total carbohydrate

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Previous studies indicated that gut microbiota could secret carbohydrate enzymes

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to breakdown the glycosidic bonds of polysaccharides and utilize carbohydrate [16,

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26], which result in the decrease of total carbohydrate. Therefore, the content change

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of total carbohydrate is often used as an indication of fermentation extent of carbohydrates. As shown in Fig.1b, the concentration of total carbohydrate in LP

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fermentation culture declined from 5.09 mg/mL to 4.04 mg/mL within 48 h

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fermentation, while those decreased from 4.98 mg/mL to 3.01 mg/mL in LP-F fermentation culture at the same time. The proportion of residual carbohydrate at

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different time points of fermentation in vitro was showed in Table 2. As the time increased, the percentage of residual carbohydrates was declined. The percentage of total carbohydrates in LP utilized by faecal microbiota was 20.58%, while that was 39.37% in LP-F. They were both lower than 55.03% of the polysaccharides from Cyclocarya paliurus leaves [26] and similar to 38.18% of Gracilaria rubra polysaccharides [11] and 37.32% of Camellia sinensis flowers polysaccharides [27]. These results showed different polysaccharides had difference in utilization by gut microbiota and this difference may be related with the extraction methods, Mw, structure of polysaccharides, as well as the composition of the intestinal microorganisms [28]. In addition, as different bacteria have different ability to utilize

Journal Pre-proof carbohydrates, the changes in bacterial community may affect the total carbohydrate content. To utilize carbohydrates, bacteria should have the ability to encode and expression carbohydrate-active enzymes. It is found that only some bacteria have related genes, such as Bacteroidetes, Firmicutes (Lactobacillus, Roseburia) and Actinobacteria (Bifidobacteria) and so on [29, 30]. Thus, the change of bacterial community in longan polysaccharides fermentation would affect the total content of carbohydrate.

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3.2.3 SCFA production during fermentation

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The types and concentrations of SCFAs in LP, LP-F and blank groups are shown

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in Table 3. After 48 h incubation, the total SCFAs content of blank group showed no

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significant change (p > 0.05) while that of LP and LP-F showed marked increase (p < 0.05). The total SCFAs content in LP group at 48 h was 3.65-fold of that at 0 h, while

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that is 6.32-fold in LP-F group. Acetic acid was the main SCFAs in three groups

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(Table 3). Acetic acid content showed an increase trend in both LP and LP-F groups while that was stable in blank group. Acetic acid increased from 2.86 ± 1.13 mM (0 h)

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to 10.93 ± 0.27 mM (48 h) in LP group, and that of LP-F group increased from 2.48 ± 1.17 mM (0 h) to 17.37 ± 0.50 mM (48 h). As for propionic acid, there is no change in blank group while there was slight increase in LP and LP-F groups. No i-butyric acid was detected in the blank group while it is very small (less than 0.3 mM) in LP and LP-F. 3.2.4 Changes of bacterial groups during fermentation The changes of bacterial groups during fermentation were presented in Table 4. The population size of all tested bacterial groups showed increase in different extent after LP or LP-F fermentation (p < 0.05). After 48 h fermentation, the population size of Enterobacteriaceae and Bacteroidaceae in LP and LP-F fermentation cultures

Journal Pre-proof showed no significant differences, but they were larger than that in blank group (p < 0.05). Moreover, the population size of Eubacteria in fermentation culture of LP was similar to that of LP-F except at 24 h fermentation. As for Lactobacillus, its population size in LP-F group were larger than that of LP within 12 h fermentation, but turned to similar in the late stage of fermentation (24 h and 48 h). LP-F fermentation could improve the abundance of Enterococcus, Bifidobacterium, and Clostridium, compared with LP group (p < 0.05). Bifidobacterium and Lactobacillus

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are commonly regarded as markers of probiotics, which produce lactic and acetic

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acids as their major fermentation products [31]. Many polysaccharides could induce

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Bifidobacterium growth with human faecal slurry cultures fermentation, such as Rosa

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roxburghii Tratt fruit polysaccharide [31] and hemicellu-lose-derived soluble arabinoxylooligosaccharides from wheat bran [32]. Clostridium group is one of the

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main bacteria groups in microbiota and it plays an important role in the balance of the

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gut microbiota [33]. Enterococcus also is one of probiotic market members, which has some benefits for human health [34]. The effects of LP and LP-F on bacterial groups

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during fermentation were similar to some polysaccharides. Plantago asiatica L seed polysaccharide could improve the population size of Clostridium with human faecal microbiota fermentation [33], and the increase of Enterococcus was also found in the extracts of brown seaweed Ecklonia radiata fermentation by human gut bacteria [18]. The lower pH value and residual carbohydrate percentage, the more acetic acid and total SCFAs content, as well as the lager population size of Enterococcus, Bifidobacterium, and Clostridium in LP-F fermentation culture, indicated that LP-F is easier to be fermented by faecal microbiota than LP. Usually, chemical properties of polysaccharides could affect their utilization by microbiota, such as monosaccharides compositions, glycosidic linkage, molecular weight, branch chain, particle size,

Journal Pre-proof solubility and viscosity [35, 36]. Smaller polysaccharide particle size may result in its bigger surface area available for bacteria. Polysaccharide with higher viscosity would also have a lower ferment ability, which makes the bacteria more difficult to utilize the polysaccharide [37]. The lower molecular weight polysaccharide is more easily fermented by bacteria. For example, Plantago asiatica L. polysaccharides with smaller particle size and Mw, showed enhanced SCFA production and microbial growth with in vitro fermentation [33, 38]. As for LP-F, it had better solubility, lower

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apparent viscosity, particle size and molecular weight, as well as different

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monosaccharides compositions, which may correspond to its better utilization by

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microbiota.

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4. Conclusion

This study showed the influence of fermentation with Lactobacillus fermentum in

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gastrointestinal digestion and fermentation of longan polysaccharide in vitro. The Mw

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of LP and LP-F were both declined with gastric and intestinal digestion while their reducing sugars content were increased significantly. Compared with LP, LP-F

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fermentation culture showed more total SCFAs content and acetic acid, and more Enterococcus, Bifidobacterium, and Clostridium, but lower pH value and residual carbohydrate percentage. These results suggested that LP-F is easier to be fermented by gut microbiota than LP. This effect may be attributed to its chemical properties and structure, including good solubility, low apparent viscosity, particle size and molecular weight, as well as different monosaccharides compositions, which could be easily utilized by microbiota. Acknowledgements This work was supported by National Natural Science Foundation of China (31771979),

Guangdong

Provincial

Science

and

Technology

Project

Journal Pre-proof (2017A030313163, 2018A050506050), and Scientific Research Program of Guangzhou (201704020039, 201803010079).

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Author Statement

All authors have read and approved the final version of the manuscript. This manuscript has not been published and is not under consideration for publication elsewhere. We have no conflicts of interest to disclose. The contributions of authors are list as follows:

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Fei Huang: Writing- Original draft preparation and Design of the experiment

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Ruiyue Hong: Conducting a research and investigation process

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Yang Yi: Analysis data and Funding acquisition

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Yajuan Bai: Analysis data

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Lihong Dong: Conducting a research

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Xuchao Jia: Investigation process

Ruifen Zhang: Writing- Reviewing and Editing

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Guangjin Wang: Reviewing and Editing Mingwei Zhang: Ideas, Project administration and Funding acquisition

Journal Pre-proof Table 1 Molecular weight distribution and reducing sugars of LP and LP-F at different time points in stomach and intestinal digestion. LP Stomach 0h 2h 4h Intestine 0h 2h 4h

LP-F

Mw(kDa)

CR(mg)

Mw(kDa)

CR(mg)

221.63±2.41e 149.17±6.50d 125.33±2.16c

24.74±1.14a 42.23±1.2b 46.96±0.87c

109.62±10.66c 102.48±0.97c 92.92±1.35b

29.44±1.06a 50.17±1.52b 57.51±2.95c

100.29±8.05b 73.44±2.86a 69.68±2.36a

47.68±0.06c 47.40±1.01c 46.68±1.35c

91.85±2.11b 83.62±3.13b 51.46±6.26a

56.54±1.31c 57.56±1.04c 56.83±1.30c

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Different lowercase letters represent significant differences between samples (p < 0.05).

Journal Pre-proof Table 2 The proportion of residual carbohydrate (%) during fermentation of LP and LP-F at different time points. Fermentation time (h)

LP

LP-F

0 6 12 24 48

100±2.05c 91.17±1.78b 83.48±2.23a 82.51±1.04a 79.42±2.19a

100±2.05d 82.79±2.43c 71.12±1.04b 70.11±1.49b 60.62±2.33a

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Different lowercase letters represent significant differences between samples (p < 0.05).

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Table 3 Change of SCFAs during fermentation of LP and LP-F at different time points as compared to blank group. Groups

Blank

LP

Time (h)

Acetic Acid

Propionic Acid

i-butyric acid

Total SCFA

0

2.34±0.05a,A

0.21±0.01a,A

ND

2.35±0.05a,A

6

2.42±0.05a,A

0.18±0.02a,A

ND

2.40±0.07a,A

12

2.33±0.07a,A

0.21±0.02a,A

ND

2.24±0.09a,A

24

2.32±0.02a,A

0.18±0.02a,A

ND

2.20±0.04a,A

48

2.23±0.06a,A

0.17±0.05a,A

ND

2.30±0.05a,A

0

2.86±1.13a,A

0.26±0.01a,A

ND

3.17±1.15a,B

0.12±0.00a,A

6.34±0.14b,B

6

5.89±0.13b,BC

0.36±0.00bc,B

12

8.86±0.37c,B

0.41±0.03c,C

0.18±0.01b,A

9.50±0.40c,B

0.32±0.00b,B

0.18±0.00b,A

8.79±0.44c,B

0.37±0.04bc,B

0.24±0.01c,A

11.58±0.32d,B

0.31±0.04ab,B

ND

2.85±1.22a,A

0.29±0.00a,A

0.15±0.01a,A

5.73±0.84b,B

0.29±0.02a,B

0.18±0.01b,A

9.11±0.05c,B

0.35±0.01bc,B

0.26±0.01c,B

15.98±0.19d,C

0.33±0.02abc,B

0.27±0.01c,A

18.01±0.52e,C

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LP-F

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8.24±0.43c,B

48

10.93±0.27d,B

0

2.48±1.17a,A

rn

6

5.18±0.84b,B

12

8.60±0.06c,B

24 48

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15.33±0.19d,C

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17.37±0.50e,C

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ND means not detected. Different lowercase letters mean significant differences among the same sample of different times (p < 0.05) while different capital letters mean significant differences among the same time of different samples (p < 0.05).

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Table 4 Change of main bacterial groups during fermentation of LP and LP-F at different time points as compared to blank group. Groups

Time(h)

Bifidobacterium

Enterococcus

Enterobacteriaceae

Lactobacillus

Blank

0 6 12 24 48

5.15±0.03a,A 5.20±0.04a,A 5.16±0.06a,A 5.13±0.04a,A 5.17±0.02a,A

6.25±0.03a,A 6.23±0.13a,A 6.30±0.10a,A 6.28±0.01a,A 6.26±0.13a,A

10.28±0.03a,A 10.30±0.10a,A 10.30±0.13a,A 10.26±0.09a,A 10.29±0.01a,A

LP

0 6 12 24 48

5.16±0.06a,A 6.98±0.22b,B 7.08±0.19b,B 7.28±0.15bc,B 7.58±0.14c,B

6.22±0.13a,A 6.79±0.08b,B 7.17±0.20c,B 7.82±0.08d,B 7.90±0.10d,B

10.29±0.13a,A 12.84±0.03c,B 12.71±0.08bc,B 12.61±0.09bc,B 12.56±0.27b,B

LP-F

0 6 12 24 48

5.18±0.04a,A 7.28±0.09b,C 7.40±0.28b,BC 7.52±0.07bc,BC 7.89±0.18c,C

6.24±0.17a,A 7.13±0.15b,C 7.46±0.14c,C 8.31±0.13d,C 8.49±0.04d,C

10.29±0.05a,A 12.59±0.12b,B 12.62±0.14b,B 12.65±0.08b,B 12.49±0.13b,B

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Bacteroidaceae

Eubacteria

5.37±0.01a,A 5.40±0.04a,A 5.34±0.05a,A 5.37±0.11a,A 5.40±0.07a,A

3.18±0.19ab,A 3.36±0.03b,A 3.21±0.04ab,A 3.21±0.18ab,A 3.02±0.02a,A

7.03±0.01a,A 7.03±0.02a,A 7.01±0.01a,A 7.02±0.01a,A 7.02±0.01a,A

8.21±0.01a,A 8.21±0.01a,A 8.20±0.02a,A 8.20±0.00a,A 8.20±0.01a,A

5.43±0.00a, A 6.25±0.04b,B 6.27±0.01b,B 6.59±0.07d,B 6.46±0.02c,B

3.24±0.23a,A 5.10±0.07b,B 5.16±0.19b,B 5.40±0.24b,B 5.15±0.09b,B

7.04±0.02a,A 7.25±0.13ab,A 7.23±0.22ab,A 7.35±0.09b,AB 7.32±0.08b,B

8.20±0.01a,A 9.92±0.02b,B 9.94±0.03b,B 9.96±0.13b,B 9.92±0.21b,B

5.37±0.06a,A 6.34±0.12b,C 6.44±0.11bc,C 6.56±0.13c,B 6.49±0.11bc,B

3.29±0.05a,A 5.54±0.07b,C 5.75±0.28bc,BC 5.84±0.00c,C 5.50±0.10b,BC

7.05±0.01a,A 7.25±0.17ab,A 7.21±0.21ab,A 7.48±0.24b,AB 7.45±0.20b,B

8.20±0.01a,A 9.78±0.13b,B 9.84±0.09b,B 10.23±0.23c,C 9.94±0.03bc,B

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Clostridium

Different lowercase letters mean significant differences among the same sample of different times (p < 0.05) while different capital letters mean significant differences among the same time of different samples (p < 0.05).

Journal Pre-proof Figure caption: Fig.1. Changes of the pH value (a) and total carbohydrate (b) during fermentation of LP and LP-F at different time points as compared to blank group.

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Fig.1.

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Figure 1