In Vitro Digestion and Fermentation of Three Polysaccharide Fractions

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Functional Structure/Activity Relationships

In vitro digestion and fermentation of three polysaccharide fractions from Laminaria japonica and their impact on lipid metabolism-associated human gut microbiota Jie Gao, Lianzhu Lin, Zijie Chen, Yongjian Cai, Chuqiao Xiao, Feibai Zhou, Baoguo Sun, and Mouming Zhao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.9b00970 • Publication Date (Web): 24 May 2019 Downloaded from http://pubs.acs.org on May 29, 2019

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Journal of Agricultural and Food Chemistry

Title and authorship In vitro digestion and fermentation of three polysaccharide fractions from Laminaria japonica and their impact on lipid metabolism-associated human gut microbiota Jie Gao a, c, Lianzhu Lin a, c, Zijie Chen a, c, Yongjian Cai a, c, Chuqiao Xiao a, c, Feibai Zhou a, c, Baoguo Sun b, d, Mouming Zhao a, b, c, d, * a

School of Food Science and Engineering, South China University of Technology Guangzhou 510640, China b Beijing

Advanced Innovation Center for Food Nutrition and Human Health，

Beijing Technology & Business University, Beijing 100048, China c Guangdong

Food Green Processing and Nutrition Regulation Technologies Research Center, Guangzhou 510640, China d

Beijing Advanced Innovation Center for Food Nutrition and Human Health, Beijing Technology & Business University, Beijing 100048 *Corresponding author Mouming Zhao (Tel./Fax: +86 20 87113914; E-mail: [email protected]). No.381, Wushan Road, Tianhe District, Guangzhou City, Guangdong Province, China

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ACS Paragon Plus Environment


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Abstract

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Our previous study has proved that the three polysaccharide fractions from L.

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japonica (LP-A4, LP-A6, and LP-A8) had significantly different structure

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characterization. Herein, we conducted in vitro simulated digestion and fermentation to

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study the digestive mechanism of LP-As. The results of gastrointestinal digestion

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indicated that LP-A6 and LP-A8 would be easier to trap the enzyme molecules for their

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denser interconnected macromolecule network compared with LP-A4. Fermentation of

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LP-As by human gut microbiota, especially for LP-A8, generated a large amount of

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short-chain fatty acids (SCFAs), which could upregulate the abundance of Firmicutes

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(Lachnoclostridium and Eubacterium). The high content of sulfate and highly branched

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sugar residue of LP-A8 might help it be easily used by Firmicutes in gut microbiota of

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hyperlipidemic patients. Functional analysis revealed that the increased metabolic

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activities of glycerophospholipid metabolism, ether lipid metabolism and fatty acid

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metabolism induced by LP-A8 treatment were closely associated with metabolic

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syndromes and hyperlipidemia.

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Keywords: Laminaria japonica polysaccharide fractions; Gastrointestinal digestion;

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Gut microbiota; Fermentation; Lipid metabolism

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INTRODUCTION

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There is a growing interest in understanding how soluble dietary fiber

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(polysaccharide) could be digested in the human gastrointestinal tract and affect the

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intestinal microbiota.1-4 Simulated gastrointestinal digestion is usually used to study the

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digestive mechanism of functional food, because human experiments are expensive,

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resource intensive and ethically disputable.5 Reproducibility, controlled conditions, less

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sample consumption and easy sampling at the site of interest make in vitro models very

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suitable for digestive and metabolic mechanism studies of purified polysaccharide

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fractions. Simulated digestion methods typically include the oral, gastric and small

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intestinal phases, and occasionally colon fermentation, taking into account the

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concentrations of digestive enzymes, pH, digestion time and salt concentrations.6-9

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Evidence is now accumulating to demonstrate that the gut microbiota plays a

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critical role in the development of diseases associated with lipid metabolism.10-17

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Animal models are commonly used to investigate the possible correlation between gut

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microbiota and lipid metabolism, which are much more convenient and affordable than

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conducting human trials.9-12 However, human gut microbiota may differ markedly from

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the animal models and it is not feasible to perform in vivo studies for the purified

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polysaccharide fractions. Recently, short chain fatty acids (SCFAs), produced by

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particular gut microbiota in colon, are being investigated in different ways to explain

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the association between gut microbiota and human body. These fatty acids not only

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serve as energy sources for the gut microbiota but also have impact on lipid and glucose

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metabolism.18-20

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L. japonica is the most widely cultivated and consumed commercial edible brown

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seaweed around the world. Numerous published papers have demonstrated that

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polysaccharides from L. japonica have beneficial bioactivities for human health, such

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as

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immunoregulation and hypolipidemic activities, which are particularly associated with

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their structural characteristics.21-27 Our previous study suggested that the three

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polysaccharide

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fucomannoglucan and fucogalactan, had notably distinct structure feature and bile acid-

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binding capacity.28 Compared with other two fractions, LP-A8 exhibited the highest

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bile acid-binding capacity, which might have correlation with their abundant sulfate,

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very tiny amounts of uronic acid and highly branched sugar residue such as (1→2, 3,

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4) linked β-D-ManpA. 28 However, limited information is focused on the connection

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between digestive mechanism and structural characteristics of purified polysaccharide

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fractions from L. japonica.

antioxidant,

antiallergic,

fractions

of

anticancer,

L.

japonica,

anti-thrombotic,

characterized

as

anticoagulant,

mannoglucan,

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Thus, the aim of this study was to evaluate how the LP-As digest in the

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gastrointestinal tract and alter the gut microbiota with SCFA production. Herein, we

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conducted in vitro simulated digestion and fermentation to study the digestive

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mechanism of three purified polysaccharide fractions (LP-A4, LP-A6 and LP-A8) from

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L. japonica. Fermentation of LP-A4, LP-A6 and LP-A8 by human gut microbiota

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generates large amount of short-chain fatty acids (SCFAs) and leads to the change of

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gut microbiota community composition, which could change the microbiota

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composition, uphold health and be a valuable food supplement.

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MATERIALS AND METHODS

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Materials

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L. japonica, harvested in July 2018 from Weihai (Shandong, China), was washed

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with tap water and distilled water, then dried at 60 °C in an oven. The dried samples

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were pulverized to get the powdered material using a 50-mesh screen. Pepsin from

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porcine gastric mucosa, α-amylase from human saliva, pancreatin, SCFAs standards

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including acetic, propionic, butyric, valeric, isobutyric and isovaleric acids were

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purchased from Sigma-Aldrich Corp. (St. Louis, USA). All the other reagents used in

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the present study were of analytical grade.

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Extraction and Purification of L. Japonica Polysaccharide

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The extraction and purification methods used in this study were as described in

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our previously published paper.24 Briefly, the dried L. japonica powder was extracted

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with 0.1 mol/L HCl solution. The L. japonica polysaccharide (LP-A) was got by freeze-

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drying of the final polysaccharide solution after precipitation and dialysis. The

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purification of LP-A was conducted with ion-exchange chromatography which was

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monitored with phenol-sulfuric acid reaction.2 The three purified LP-A fractions, LP-

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A4, LP-A6 and LP-A8, were obtained after dialysis, concentrating and lyophilization,

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which would be used in the subsequent digeston and fermentation.

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Simulated gastrointestinal digestion

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The simulated salivary, gastric and intestinal fluids were made according to the previously published procedures with some modifications. 1 The simulated salivary fluid (SSF) consisted of 15.1 mmol/L KCl, 13.6 mmol/L

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NaHCO3, 3.7 mmol/L KH2PO4, 0.15 mmol/L MgCl2(H2O)6 and 0.06 mmol/L

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(NH4)2CO3 and 1.1 mmol/L HCl 10 mg of LP-As were dissolved in 1 mL distilled water

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to make a solution for the simulated salivary digestion. The simulated salivary digestion

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(SSD) tube was the mixture of 5 mL of LP-As solution (10mg/mL), 3.5mL SSF, 0.5

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mL 1500 U/mL salivary alpha-amylase solution (alpha-amylase, 1000-3000 U/mg

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protein, human saliva Type IX-A, Sigma), 25 μL CaCl2(H2O)2 (0.3 mol/L) and 0.975

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mL distilled water. The simulated salivary digestion test was conducted at 37 °C for 5

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min and then heated in a boiling bath for 5 min.

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The simulated gastric fluid (SGF) consisted of 47.2 mmol/L NaCl, 25 mmol/L

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NaHCO3, 8.5 mmol/L HCl, 6.9 mmol/L KCl, 0.9 mmol/L KH2PO4, 0.5 mmol/L

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(NH4)2CO3 and 0.1 mmol/L MgCl2(H2O)6. The simulated gastric digestion (SGD) tube

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was the mixture of 5 mL of salivary chyme from the simulated salivary digestion, 3.75

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mL SGF, 0.8 mL 25000 U/mL porcine pepsin solution (pepsin, 3200-4500 U/mg

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protein, porcine gastric mucosa, Sigma), 2.5 μL CaCl2(H2O)2 (0.3 mol/L), 0.1mL HCl

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(1.0 mol/L) and 0.348 mL distilled water. The simulated salivary digestion test was

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conducted at 37 °C for 2h and then heated in a boiling bath for 5 min.

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The simulated intestinal fluid (SIF) consisted of 85 mmol/L NaHCO3, 38.4

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mmol/L NaCl, 8.4 mmol/L HCl, 6.8 mmol/L KCl, 0.8 mmol/L KH2PO4 and 0.33

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mmol/L MgCl2(H2O)6. The simulated gastric digestion (SGD) tube was the mixture of

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5 mL of gastric chyme from the simulated gastric digestion, 2.75 mL SIF, 1.25 mL

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pancreatin solution (800 U/mL trypsin, porcine pancreas, Sigma), 0.625mL fresh bile

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(160 mM), 10 μL CaCl2(H2O)2 (0.3 mol/L), 38 μL NaOH (1.0 mol/L) and 0.328 mL

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distilled water. The simulated salivary digestion test was conducted at 37 °C for 2h and

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then heated in a boiling bath for 5 min.

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After simulated digestion, the molecular weight distribution of LP-As was

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determined by high performance size exclusion chromatography equipped with multi-

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angle laser light scattering and refractive index detector (HPSEC-MALLS-RID). Each

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sample was replicated three times.

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Microscopic observation and molecular weight distribution

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The simulated salivary, gastric and intestinal digestion solutions were put on slide

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glass substrates at room temperature and observed at 400× magnification by optical

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microscope. The molecular weight distribution of the LP-As after simulated salivary,

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gastric and intestinal digestion were measured and compared by using HPSEC-

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MALLS-RID according to the previous published method.29

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HPSEC-MALLS-RID measurements were conducted on a multiangle laser light

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scattering detector (MALLS, DAWN HELEOS-Ⅱ, Wyatt Technology Co., Santa

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Barbara, CA, USA) with a 2998 PDA detector in a Waters e2695 HPLC system coupled

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with TSK-GEL G5000PWXL (300 mm × 7.8 mm, i.d.) and TSK-GEL G3000PWXL

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(300 mm × 7.8 mm, i.d.) columns in series. Bovine serum albumin (BSA, 5 mg/mL)

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was used to normalize the diodes of the MALLS detector. A dn/dc value of 0.185 mL/g

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was determined for the BSA in our carrier liquid. NaCl aqueous solution (0.9%) was

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used as mobile phase at the flow rate of 0.6 mL/min. The simulated digestion solution

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was filtered by a 0.22 mm membrane and tested at an injection volume of 50 μL and a

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concentration of 2 mg/mL. The Astra software (Version 6.0.2, Wyatt Tech. Corp. Santa

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Barbara, CA, USA) was used for data analysis.

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In vitro fermentation

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In vitro fermentation of LP-As was performed in triplicate according to the

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published methods with some modification.1-4, 30 Fecal samples were collected from

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two healthy donors (group NL, normal-lipid control, one female and one male, 45 - 60

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years old, TG 1.52 ± 0.1 mmol/L, TC 5.97 ± 0.49 mmol/L) and two hyperlipidemic

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patients (group HL, hyper-lipid control, one female and one male, 45 - 60 years old,

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TG 8.8 ± 0.28 mmol/L, TC 6.64 ± 1.40 mmol/L) who were eating their regular diets

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without taking antibiotics for more than 3 months. Fecal samples were tightly sealed in

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plastic tubes, kept on ice prior to rapidly being stored in -80 °C refrigerator, and used

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within 7 days of collection. The fecal samples were homogenized with sterilized

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phosphate-buffered saline, pH 7.4 (feces: PBS buffer= 1: 9 (w/v)) and then filtered and

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pooled to make the fecal slurries under anaerobic conditions maintained by Anaero

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Pack (Mitsubishi Gas Chemical Co., Tokyo, Japan). Then 100 μL of pooled fecal slurry

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and 400 μL of LP-As (17.5 mg LP-As dissolved in 400 μL PBS buffer, the final

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concentration for fermentation was 0.5%, w/v,) were inoculated into 3mL brain heart

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infusion (BHI) and incubated in a 37 °C incubator (120 rpm) for 120 h anaerobically

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(AnaeroPack‐Anaero; Mitsubishi Gas Chemical Co., Tokyo, Japan). The preparation

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of pooled fecal samples for fermentation experiments does not lead to a bacterial

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community with an aberrant profile and activity compared to that normally prepared

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from single donors.4, 9, 30 And according to the previous published studies, every steps

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of making fecal slurries should be kept in anaerobic conditions. At the end of the

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fermentation for each LP-As fraction, two aliquots were collected from each sample for

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DNA extraction (1 mL) and SCFA analysis (1 mL).

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SCFA analysis

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SCFA analyses were conducted as previously described.31 1 mL of each sample

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with 1% of formic acid were suspended and stored at -20 °C immediately after

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collection. Once thawed, the sample suspensions were homogenized and then

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centrifuged for 10 min at 17949 × g. One mL supernatant of each sample was extracted

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with 1 mL of ethyl acetate for 2 min and then separated by centrifugation for 10 min at

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17949 × g. Organic extracts were obtained and stored at -20 °C. Before analysis, 600

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μL organic extracts was transferred into a test tube with 500 μmol/L 4-methyl valeric

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acid as IS. The IS was employed for the correction of injection variability between

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samples and instrument stability.

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Analysis of the SCFAs was conducted by using Trace GC-MS system coupled

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with a Trisplus automated sampler, an Ultra GC and a quadrupole DSQ II MS (Thermo

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Finnigan, San Jose, CA). Separation was conducted with a TR-Wax column (30 m ×

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0.32 mm × 0.25 μm, Thermo Scientific, Waltham, MA, USA). SCFAs were analyzed

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under the following conditions: injector temperature at 230 °C, ion source and interface

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temperature at 250 °C; initial oven temperature at 90 °C, temperature increased to

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150 °C at 15 °C / min, to 170 °C at 5 °C/min and finally to 250 °C at 20 °C / min and

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maintain for 2 min (total time 14 min). Helium was used as carrier gas at 1 mL/min.

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The detector was operated in electron impact ionization mode (electron energy 70 eV),

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scanning the 30 - 350 m/z range. Identification of the SCFAs was conducted according

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to the retention time of standard compounds based on the NIST 08 and Wiley7N

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libraries. Three independent replicate extractions were performed per sample.

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DNA extraction

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One milliliter fermentation for each LP-As fraction was stored at -80 °C before

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DNA isolation. Total DNA was isolated by using the PowerMag Soil DNA isolation

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kit optimized for epMotion (Mo Bio Laboratories, Carlsbad, CA, USA) according to

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the manufacturer’s instructions. Samples stored at -80 °C were thawed and centrifuged

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at 13000 rpm for 10 mins. Precipitate was homogenized and transferred into the Lysing

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Matrix E tube for the DNA extraction.

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16S rRNA sequencing

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For 16S rRNA sequencing, the V3–V4 region of 16S rRNA gene was amplified

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by PCR using the universal bacterial primers: 341F(CCTACGGGNGGCWGCAG) and

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806R (GGACTACHVGGGTATCTAAT) and then sequenced by using 500 bp paired-

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end sequencing (Illumina MiSeq). For shotgun metagenomic sequencing, libraries were

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sequenced by using 50 bp single-read sequencing (Illumina HiSeq). 32-33

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Sequence processing and community analysis

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Sequences were processed with Mothur v.1.39.1 according to the MiSeq SOP

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using the described 454 standard operating procedure (SOP).34-35 All OTUs were

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clustered at a cutoff of 0.02 (98% similarity) and classified using the Ribosomal DNA

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Project (RDP) database (v9).36 Heat maps and bar plots were created in R using the

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packages vegan and ggplots.37-38

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For PICRUSt, we normalized the OTU results by subsampling to the lowest

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sequence count (3,500 OTUs/sample). This table was input into the QIIME pipeline,

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which was used to reference the Greengenes 16S rRNA database (v9), the required

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input for PICRUSt.39 Subsequently, OTUs were normalized by 16S rRNA copy number,

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and metagenomes were predicted based on the Kyoto Encyclopedia of Genes and

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Genomes (KEGG) catalogue.40 HUMAnN was used to predict downstream pathway

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coverage based on the KEGG Ortholog results.41

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Statistical Analysis

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The data was presented as mean ± SD (n = 3) and evaluated by one-way analysis

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of variance (ANOVA) followed by the Duncan’s test. All statistical analyses were

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carried out using R statistical package and SPSS for Windows, Version 17.0 (SPSS

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Inc., Chicago, IL, USA).

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RESULTS AND DISCUSSION

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Microscopic observation and molecular weight distribution

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The microscopic observation of the simulated salivary, gastric and intestinal

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digestion solutions of LP-As was showed in Fig. 1A. The remarkable aggregation of

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LP-A4 molecules has been observed after simulated gastric digestion for 2 hours,

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compared with LP-A6 and LP-A8. Furthermore, the precipitate formed in gastric

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digestion disappeared in the next intestinal digestion, which might due to the changing

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of pH value. According to our previous study, there is a large amount of uronic acids

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in the molecules of LP-A4 (89.64 ± 2.27%).24 Considering that the ionization of

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mannuronic acid and glucuronic acid in LP-A4 molecules could be inhibited by the

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sharply increased concentration of H+ in simulated gastric digestion solution, the

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intermolecular repulsive force of the macromolecular was weaker, thus stimulated the

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LP-A4 gathering of precipitation, decreasing the stability of the solution system.42

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As presented in Fig. 1B, HPSEC profiles with the static light scattering signals

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(SLS 90◦) indicate that there were no significant changes in molecular weight

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distribution of LP-As after simulated salivary (SSD) and gastric (SGD) digestion.

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However, the HPSEC profile of LP-A4 in gastric digestion couldn’t be recorded in this

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experiment, because LP-A4 molecules gathered and precipitated in gastric digestion

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solution revealed by the results of microscopic observation.

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During the simulated intestinal digestion (SIG), the average Mw of LP-A6

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decreased slightly from 1.959±0.06×106 g/mol to 5.244±1.92×105 g/mol after 2-hour

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digestion. The same downtrends could be found in the average Mw of LP-A8, which

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decreased from 1.332±0.32×106 g/mol to 1.109±0.60×106 g/mol after digestion, but

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there was no distinct variety in molecular weight distribution of LP-A4 after intestinal

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digestion. The different digestive characteristics of LP-As might indicate the possible

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connection between structure feature and digestibility. Compared with LP-A4, LP-A6

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and LP-A8 revealed a denser interconnected macromolecule network with higher

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content of sulfate and more branched sugar residue, which would be easier to trap the

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enzyme molecules in pancreatin and expose more cutting site for the enzyme digestion.

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SCFA Production during the in vitro Fermentation

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Short-chain fatty acids (SCFA) are the major products of bacterial metabolism in

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the large intestine and colon, which mainly come from the breakdown of polysaccharide

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and are beneficial to human health. Acetic acid, propionic acid, butyric acid and

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isobutyric acid were the main fermentation products in fecal (Fig. 2).

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The fermentation of LP-A8 in group HL (hyper-lipid control) produced

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significantly higher concentrations of acetic acid (34.46 ± 1.37 mmol/L), butyric acid

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(22.24 ± 1.07 mmol/L), isobutyric acid (12.73 ± 0.39 mmol/L), valeric acid (1.56 ± 0.26

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mmol/L) and total SCFA (78.35 ± 2.44 mmol/L) compared with the blank (p < 0.05).

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Furthermore, the concentration of acetic acid (26.60 ± 0.64 mmol/L), propionic acid

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(14.32 ± 0.87 mmol/L) and total SCFA (58.96 ± 3.47 mmol/L) were notably increased

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in group NL (normal-lipid control) by the intervention of LP-A8 compared with the

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blank (p < 0.05). At the same time, the fermentation of LP-A8 in both groups generated

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more acetic acid, propionic acid and total SCFA than LP-A4 and LP-A6. Propionic acid

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in the fermentation of group HL treated with LP-A8 was significantly decreased from

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7.64 ± 0.54 mmol/L (blank) to 5.81 ± 0.40 mmol/L (p < .05). Significant decreasing

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was also observed between the blank and LP-A4 or LP-A6 treatment of group NL in

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the amount of isobutyric acid. In particular, treatment of LP-A4 and LP-A6 led to

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increases in the concentration of butyric acid and isovaleric acid in both group HL and

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group NL, and also the total SCFAs in group HL, compared with the blank.

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Recently, many host metabolic pathways have been found to be linked to dietary

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fiber and gut microbiota. Numerous published papers have proved that SCFAs, the final

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fermentation products of the anaerobic intestinal microbiota, are beneficial to the host

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energy metabolism by upregulating expression of host SCFA receptors and target

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molecules in metabolic tissues. GPR109A is expressed in adipose tissues and activated

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adipose tissue macrophages where it regulates lipid homeostasis, which was first

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identified as a receptor for niacin and is mainly activated by β-hydroxybutryate and

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butyrate.43-44 Furthermore, SCFAs inhibited isoproterenol-induced lipolysis in a

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concentration-dependent manner in mouse 3T3-L1-derived adipocytes.45 GPR43, also

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named as FFAR2 has been identified as a receptor of SCFA and is activated by acetate

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and propionate followed by butyrate.46-47 FFAR2 is expressed in intestinal endocrine L-

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cells, which could be activated by SCFAs and result in the inhibition of fat

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accumulation.48 In this study, acetate and butyrate were the most abundant SCFAs in

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group NL, and their generation was upregulated sharply by LP-A8, which might reveal

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that LP-A8 have some beneficial effects on the host lipid metabolism. These data also

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indicated that fermentation of different kinds of LP-As by human gut microbiota in HL

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and NL groups promoted the generation of different SCFAs, which might then change

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the microbiota composition in different rules.

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Taxonomic composition of gut microbiota

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We plotted average relative abundance of microbiota composition in the

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fermentation products of LP-As in group NL and HL at order, family, genus and species

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level (Fig. 3).

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Proportions of four frequently detected orders including Gram-negative

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Bacteroidales (Bacteroidetes phylum) and Enterobacteriales (Proteobacteria phylum),

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Gram-positive Clostridiales and Lactobacillales (all from Firmicutes phylum), totaled

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up to ~90% of the entire community (Fig. 3A). Moreover, there were some differences

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between group NL and HL gut microbiota contents. Gram-positive Clostridiales and

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Lactobacillales were mainly observed in the gut microbiota of hyperlipidemic patients

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(group HL), while Gram-negative Bacteroidales and Enterobacteriales were the

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principal orders in the gut microbiota of healthy donors (group NL). The relative

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abundance of Clostridiales was significantly increased by the treatment of LP-As in

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group HL(P

In Vitro Digestion and Fermentation of Three Polysaccharide Fractions

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