Antiobesity Effect of Exopolysaccharides Isolated from Kefir Grains

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Anti-obesity Effect of Exopolysaccharides Isolated from Kefir Grains Juha Lim, Madhuvanti Kale, Dong-Hyeon Kim, Hong Seok Kim, Jung-Whan Chon, Kun-Ho Seo, Hyeon Gyu Lee, Wallace Yokoyama, and Hyunsook Kim J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b03764 • Publication Date (Web): 30 Oct 2017 Downloaded from http://pubs.acs.org on November 1, 2017

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Journal of Agricultural and Food Chemistry

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Anti-obesity Effect of Exopolysaccharides Isolated from Kefir

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Grains

3 4

Juha Lim1, Madhuvanti Kale2, Dong-Hyeon Kim3, Hong-Seok Kim3, Jung-Whan

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Chon3, Kun-Ho Seo3, Hyeon Gyu Lee1, Wallace Yokoyama2, Hyunsook Kim1,*

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1

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Korea, 2 Western Research Center, USDA, Albany, CA, USA, 3Center for One

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Health, College of Veterinary Medicine, Konkuk University, Seoul, Republic of

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Department of Food and Nutrition, Hanyang University, Seoul, Republic of

Korea

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CORRESPONDENCE:

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Hyunsook Kim, Ph. D.

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Department of Food and Nutrition, Hanyang University, 222 Wangsimni-ro,

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Seongdong-gu, Seoul 04763, Republic of Korea

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E-mail: [email protected]

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Phone: +82 2 2220 1208, Fax: +82 2 2220 1856

ACS Paragon Plus Environment


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Abstract

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Physiological properties of water-soluble exopolysaccharides (EPS) and

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residues after EPS removal (Res) from the probiotic kefir were determined in

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high-fat (HF) diet-fed C57BL/6J mice. EPS solutions showed rheological

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properties and lower viscosity compared to β-glucan (BG). EPS significantly

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suppressed the adipogenesis of 3T3-L1 preadipocytes in a dose-dependent manner.

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Mice were fed HF diets containing 5% EPS, 5% BG, 8% Res, or 5%

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microcrystalline cellulose (control) for 4 weeks. Compared with the control, EPS

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supplementation significantly reduced HF diet-induced body weight gain, adipose

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tissue weight, and plasma very low-density lipoprotein cholesterol concentration

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(P < 0.05). Res and BG significantly reduced body weight gain; however,

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reduction in adipose tissue weight was not statistically significant, suggesting that

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the anti-obesity effect of EPS occurs due to viscosity and an additional factor. EPS

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supplementation significantly enhanced abundance of Akkermansia spp. in feces.

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These data indicate that EPS shows significant anti-obesity effects possibly via

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intestinal microbiota alterations.

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Key words: Exopolysaccharides, β-glucan, obesity, Akkermansia, intestinal

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microbiota


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INTRODUCTION

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Obesity, a lipid metabolic disorder often associated with metabolic

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dysfunction, has become a global public health problem.1 The causes of obesity

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range from endocrine, genetic, and environmental factors to neural and infectious

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factors.1 Recently, an imbalance in gut microbiota has been suggested as an

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etiology for obesity and related diseases.2, 3 Several probiotics, which modulate

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gut microbiota, are reported to have beneficial effects against obesity, insulin

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resistance, type 2 diabetes, and nonalcoholic liver disease.4-6,7

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Kefir, a probiotic dairy beverage that originated in the Caucasus Mountains,

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has high acidity and viscosity.8, 9 Kefir grains, the fermented starters for kefir

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production, form a gelatinous symbiotic mass containing lactic acid bacteria

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(Lactobacillus, Leuconostoc, Lactococcus, and Streptococcus spp.), acetic acid

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bacteria (Acetobacter spp.), and yeasts (Saccharomyces, Kluyveromyces, and

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Torula spp.).8 Upon incubation, the kefir microorganisms form a slimy

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exopolysaccharide (EPS),10 which is composed of homo-polysaccharides,

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containing only one kind of monosaccharide (e.g., dextran, levan, and inulin) and

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hetero-polysaccharides, containing different kinds of mono-and disaccharides

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(e.g., gellan, xanthan, and kefiran).10 The compositional, structural, and

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rheological characteristics of kefiran and the methods to improve kefiran

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production have been studied extensively.9, 11, 12 Kefiran is a water-soluble hetero-

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polysaccharide produced by Lactobacillus kefiranofaciens and is composed of



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almost equal amounts of glucose and galactose.10 Health benefits of kefiran, such

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as maintaining normal blood glucose, blood pressure, and plasma lipid levels;

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relieving constipation; and improving gut immune systems as well as its anti-

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pathogenic properties have been reported in animal models.13-18 Our previous

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study showed that kefir administration prevented high-fat (HF)-induced obesity,

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hepatic steatosis, and dysbiosis of gut microbiota in a mouse model.19, 20 In

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particular, Lactobacillus kefiri DH5, isolated from kefir, was resistant to gastric

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acidity and improved obesity-related parameters in HF diet-fed mice.21 To the best

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of our knowledge, EPS and its residue have not yet been reported to prevent

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obesity.

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The aim of the present study is to investigate the anti-obesity potential of

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EPS isolated from kefir grains in vitro and in a mouse model, as compared with β-

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glucan (BG), a viscous polysaccharide.

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MATERIALS AND METHODS

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Kefir grain preparation. Kefir grains were provided by the KU Center for

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Food Safety, College of Veterinary Medicine, Konkuk University. These grains

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were combined with ultra-high temperature-sterilized milk (1:10, w/w) (Seoul

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dairy cooperative, Seoul, Korea). The kefir culture was incubated at 30°C

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overnight. The milk was filtered off each day and fresh sterilized milk was added

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daily for 6weeks to obtain 1000 g of kefir grain. After fermentation, the grains

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were separated from the culture using a plastic filter (1 mm pore size), lyophilized,

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and stored at -20°C until use.

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Isolation and chemical analysis of EPS. EPS from the kefir grains was

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extracted as previously described8, 22 with slight modifications. Briefly, dried kefir

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grains were placed in hot water (1:10 w/w) and boiled for 1 h with continuous

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stirring (500 rpm). The mixture was centrifuged at 10,000 × g for 20 min at 20°C.

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Two volumes of cold food-grade ethanol (Daejung Chemical Co., Seoul, Korea)

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was added to the supernatant to precipitate the EPS, and the mixture was kept

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overnight at -20°C. The mixture was then centrifuged at 10,000 × g for 20 min,

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and the pellet was dissolved in boiling water (1:10) for 1 h under continuous

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stirring. The precipitation process was repeated twice. The centrifuged precipitate

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was dissolved in hot water (1:3) and lyophilized. The last EPS precipitate was

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dissolved in three volumes of hot water, analyzed for the absence of single sugars

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by the reducing sugar method.23 The purity of the EPS was assessed by Bradford’s

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method to confirm the absence of proteins.24 The total carbohydrate content of

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EPS was measured by the phenol-sulfuric method using a glucose standard curve,

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as described previously.8

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Rheological measurements of EPS and BG solutions. The apparent

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viscosity (ηa) and flow behavior of EPS solutions of various concentrations (3, 5,

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11, 13, 15%) were measured using a controlled-strain rheometer (RheoStress,

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Thermo HAAKE, Karlsruhe, Germany) equipped with parallel plate geometry (1

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mm gap and 35 mm diameter).25 The EPS solution was prepared by dissolving the

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dried EPS in 70°C water that was maintained at 25°C. The shear rate was

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increased from 0 to 500 s-1 at 25°C.25 Flow ( ) and consistency ( ) indexes were

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determined by fitting the experimental data to the power law model using

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RheoWin data manager (RheoWin pro v. 2.96, Thermo HAAKE), as follows:

109 is shear stress (Pa),

is the flow consistency index (Pa·sn),

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where

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shear rate (s-1), and

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viscosity (ηa) of each EPS solution was calculated at 300 s-1.

is the

is the flow behavior index (dimensionless). The apparent

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EPS and BG (Cargil, Minneapolis, MN) solutions (2% w/v) were

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prepared by suspending the polysaccharides in water and placing the containers in

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boiling water for about 10 min, until the polysaccharides were dissolved. The

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solutions were allowed to cool, and the apparent viscosity was measured. The

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flow behavior of EPS and BG was measured at 25°C using a rotational rheometer

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(AR 2000, TA Instruments, Newcastle, Delaware) equipped with concentric

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cylinder geometry. The shear rate was varied from 1 to 100 s-1.

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Dynamic rheological measurements of EPS solutions and cryogels.

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Unfrozen EPS solution and cryogels were both prepared with 2% (w/v) EPS

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solutions.25 For preparing EPS cryogels, the 2% EPS solution was immediately

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incubated at -20°C for 48 h and then at 4°C for another 48 h. The unfrozen EPS

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solutions were prepared by incubating 2% EPS solution at 4°C for 96 h.

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Small-amplitude oscillatory shear flow of unfrozen EPS solution and EPS cryogel was measured using the AR 2000 rotational rheometer equipped with

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concentric cylinders and parallel plate geometry, respectively. The gelation

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behavior of the EPS solution was measured at 25°C using small-amplitude

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oscillatory shear with a strain of 1% and a frequency of 1–100 Hz. The linear

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viscoelastic range was determined using a stress sweep test at frequency 10 Hz.

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Storage (G’) and loss (G’’) moduli were evaluated as a function of frequency (1–

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100 Hz) within the linear range.

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Cell culture and differentiation. 3T3-L1 preadipocyte cells were purchased

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from the American Type Culture Collection (ATCC, Manassas, VA). Cells were

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incubated in 6-well plates (Corning Inc., NY) in Dulbecco’s modified Eagle’s

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medium (DMEM) with high glucose (Gibco, Grand Island, NY), 10% bovine

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serum (BS; Gibco), and 1% penicillin streptomycin (P/S; Gibco) until confluence.

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Two days after confluence (day 0), cells were cultured with MDI medium

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[DMEM supplemented with 10% fetal bovine serum (FBS; Gibco), 1% P/S, 0.5

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mM 3-isobutyl-1-methylxanthine, 1 µM dexamethasone, and 10 µg/mL insulin

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solution (Sigma-Aldrich, St. Louis, MO)] for 48 h to induce differentiation. The

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cell culture medium was then replaced with complete medium (DMEM containing

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10% FBS, 1% P/S, and 10 µg/mL insulin), and incubated for another 48 h. On day

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4, the cells were cultured with DMEM containing 10% FBS and 1% P/S for 24 h

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more until differentiation was complete. Cells were then incubated at 37°C and

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5% CO2 atmosphere in a CO2 incubator (Thermo Fisher Scientific, Seoul, Korea).

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Cell viability assay. EPS cytotoxicity in 3T3-L1 preadipocytes was

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determined by the 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide

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(MTT) assay, as previously described by Ho, et al.26 with slight modification. The

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cells were cultured in 96-well plates (Corning Inc., NY) at a density of 1 × 104

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cells/well in DMEM with 10% FBS and 1% P/S for 24 h, followed by treatment

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with 20 µL samples or phosphate buffered saline (PBS; Lonza, Walkersville, MD)

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as control. After 24 h, 20 µL of 0.5 mg/mL MTT (Sigma-Aldrich) in PBS was

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added, and the samples were incubated in the dark for 4 h. The well plates were

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then centrifuged at 2000 × g for 5 min and the supernatants were removed. The

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formazan crystal pelleted in the wells was dissolved in 150 µL of dimethyl

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sulfoxide (Daejung Chemical Co., Seoul) for 15 min, and the absorbance was

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measured at 540 nm in a microplate reader (Synergy HT Multi-microplate Reader,

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BioTek Instruments, Winooski, VT) to determine formazan content.

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Oil red O staining and intracellular lipid quantification. Oil red O

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staining was performed to visualize intracellular lipids in differentiated 3T3-L1

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cells, as described previously27 with some modification. Cells were cultured in 6-

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well plates and induced differentiation as described above. 0.01, 0.1, and 1 mg/mL

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EPS solutions were added to each well during the differentiation, except the

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control wells. After differentiation, each well was washed twice with PBS and

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treated with 4% paraformaldehyde (Yakuri Pure Chemicals Co., Kyoto, Japan) in

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PBS for 1 h to fix the cells onto the well surfaces. Then, the cells were rinsed with

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distilled water and treated with 60% isopropanol (Daejung Chemical Co. Seoul)

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for 5 min. After removing the isopropanol, filtered Oil red O solution was added,

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and incubated at room temperature for 10 min. The wells were washed with water,

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and 100% isopropanol was added to each well. The extracted Oil red O was

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centrifuged at 10,000 × g for 2 min, and the supernatant was transferred into 96-

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well plates. The absorbance of Oil red O dye was measured at 480 nm using a

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microplate reader. The relative amount of intracellular lipids was calculated as

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(ODsample/ODcontrol) × 100.

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Animals and diets. Four-week-old male C57BL/6J mice were obtained from

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Jackson Laboratories (Sacramento, CA). The mice were individually housed in a

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room with a controlled environment (temperature 20–22°C, humidity 60%, 12-h

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light/dark cycles). Mice were provided with mouse chow diet (LabDiet 5015, PMI

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International, Redwood, CA) and tap water ad libitum for 1 week during the

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acclimation period. After this period, the mice were divided into 4 groups of 9

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mice each. Mice in the control group were fed high-fat (HF) diets containing 5%

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microcrystalline cellulose (MCC), and those in the experimental groups were fed

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HF diets containing 5% BG, 5% EPS, or 8% kefir-grain residue obtained after

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EPS (Res) (Table 1). The supernatants and solid residues remaining after EPS

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isolation were combined and lyophilized; EPS to residue yield was 5:8. The diets

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contained about 17%, 37%, and 46% calories from protein, carbohydrates, and fat,

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respectively. The body weights and food intake of the mice were measured weekly.

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The experiment was approved by the Animal Care and Use Committee, Western

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Regional Research Center, USDA, Albany, CA, USA.

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Plasma and tissue collection and plasma cholesterol analysis. Mice were

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feed-deprived for 12 h and anesthetized with 4% isoflurane (Phoenix

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Pharmaceutical, St. Joseph, MO, USA) and 1 L/min oxygen flow through a

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vaporizer (VetEquip Inc., Livermore, CA). Recumbent animals were maintained

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at 2–4 % isoflurane via nose cone. Blood was collected by cardiac puncture into

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an EDTA solution-rinsed syringe. The livers and epididymal tissues were

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collected and weighed. Plasma was obtained by centrifuging the blood at 2,000 ×

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g for 30 min at 4°C. Plasma concentrations of total cholesterol (TOTAL-C), high-

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density lipoprotein cholesterol (HDL-C), low-density lipoprotein cholesterol

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(LDL-C), and very low-density lipoprotein cholesterol (VLDL-C) were measured

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by size-exclusion chromatography, as previously reported.28

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Fecal microbiota analysis. Genomic DNA was extracted from 200 mg of

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lyophilized fecal samples using the QiaAmp DNA stool mini kit (Qiagen,

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Valencia, CA). Amplicons spanning the 16S rRNA V3–4 region were prepared by

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using the following barcoded primers: Bakt_341F (5ʹ-

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CCTACGGGNGGCWGCAG-3ʹ) and Bakt_805R (5ʹ-

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GACTACHVGGGTATCTAATCC-3ʹ) containing 50 Illumina overhang adapter

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sequences (5’TCGTCGGCAGCGTCAGATGTGTATAAGAGACAG3’ and

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5’GTCTCGTGGGCTCGGAGATGTGTATAAGAGACAG3’, respectively). The

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reactions were pooled and quantified using the Quant-iT PicoGreen double-

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stranded DNA assay (Invitrogen/Life Technologies, Inc., Burlington, ON, Canada)

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and subsequently sequenced on an Illumina MiSeq. The reads were then

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processed with QIIME (Quantitative Insights Into Microbial Ecology,

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http://www.qiime.org) analysis. The resulting fastq files containing paired-end

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reads were aligned with fastq-join and then quality-filtered and demultiplexed in

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QIIME. Then, operational taxonomy units (OTUs) were created using the

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Greengenes database as a reference, and taxonomic assignments were performed

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using the Ribosomal Database Project (RDP) classifier. OTUs representing

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0.01%) significantly modulated by 5% microcrystalline cellulose (Con), 5% β-glucan (5% BG), 5% exopolysaccharides (EPS), or 8% residue remaining after the isolation of EPS from kefir grains (8% Res). Name Con

Classification level Phylum Family_Genus Actinobacteria

5% BG

5% EPS

8% Res

Bifidobacteriaceae_Bifidobacterium

9.11±0.01

3.65±0.02*

2.09±0.01#

7.55±0.02

Coriobacteriaceae_Adlercreutzia

0.13±0.00

0.06±0.00

0.03±0.00*

0.06±0.00

Erysipelotrichaceae_Allobaculum

8.76±0.05

32.9±0.05#

7.55±0.01

19.9±0.04

2.21±0.01

0.84±0.00

14.0±0.06

12.7±0.03*

Lachnospiraceae_Coprococcus

1.82±0.00

0.31±0.00*

0.14±0.00*

0.72±0.00

Ruminococcaceae_Oscillospira

7.94±0.02

3.01±0.00*

1.55±0.00*

3.21±0.00*

Ruminococcaceae_Ruminococcus

0.98±0.00

0.59±0.00*

0.34±0.00#

0.99±0.00

8.79±0.05

14.4±0.03

35.3±0.03#

7.25±0.05

Bacteroidetes Bacteroidaceae_Bacteroides Firmicutes

Verrucomicrobia Verrucomicrobiaceae_Akkermansia Data are means ± SEM. *

P < 0.05 vs. Con; # P < 0.01 vs. Con

32


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Fig. 1 A

B 400

1.2

Shear stress (Pa)

Apparent viscosity (Pa·s)

1.6

0.8 0.4 0.0

300

16% 13% 11% 5% 3%

200

100

0 55

155

255 355 Shear rate (s-1)

455

0

100

C

Apparent viscosity (mPa·s)

100

2% BG 2% EPS

10

1 0.1

1

10 ACS Paragon 100 Plus Environment -1 Shear rate (s )

200

300

Shear rate

400

(s-1)

500


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Fig. 2

Cell viability (%)

120

80

40

0 Con

0.01

0.1

EPS solution (mg/mL)


1

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Fig. 3 A

Con

B

0.1 mg/mL

0.01 mg/mL

120

Relative Oil red O staining (% of control)

a b

90

c 60

d 30

0 Con

0.01

0.1

1

EPS solution (mg/mL) ACS Paragon Plus Environment

1 mg/mL

Fig. 4


B

12

a b b b b

a 18

a

a

Weight gains (g)

Daily energy intake (calories)

A 24

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a 12

6

9

a a ab b b bab b

6

Con 5% BG

3

5% EPS 8 Res 5% 0

0

Con

5% BG

5% EPS

8 Res 5%

0

1

2

3

4

Age (weeks)

C 1.2

Epididymal fat mass (g)

D 1.2

Liver weight (g)

0.9

0.6

0.3

*

0.9

0.6

0.3

0.0

0.0

Con

5% BG

5% EPS


5% Res 8

Con

5% BG

5% EPS

5% Res 8

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Fig. 5

A

B 0.5

150

0.4

Con

5% BG 120

LDL/HDL ratio

Lipoprotein cholesterol (mg/dL)

180

5% EPS 5% 8 Res

90

60

0.3

0.2

0.1

30

* 0.0

0

VLDL-C

LDL-C

HDL-C

TOTAL-C

Con


5% BG

5% EPS

5% 8 Res


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Fig. 6

Taxanomy-Phylum

Abundance - % of Total

120 100 80 60 40 20 0

Con

5% BG

5% EPS

Actinobacteria

Bacteroidetes

Firmicutes

Tenericutes

Verrucomicrobia

Other


8% Kres Proteobacteria

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TOC Graphic

EPS: exopolysaccharides

Kefir grainderived EPS

Reduction of high-fatinduced body weight gain and adipose tissue weight

Remodeling high-fat-induced intestinal dysbiosis through increased Bacteroidetes and Akkermansia spp.

Induction of anti-obesity effect ACS Paragon Plus Environment

Antiobesity Effect of Exopolysaccharides Isolated from Kefir Grains

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