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Effect of Whole Grain Qingke (Tibetan Hordeum vulgare L. Zangqing 320) on the Serum Lipid Levels and Intestinal Microbiota of Rats under High-fat Diet Xuejuan Xia, Guannan Li, Yongbo Ding, Tingyuan Ren, Jiong Zheng, and Jianquan Kan J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b05641 • Publication Date (Web): 16 Mar 2017 Downloaded from http://pubs.acs.org on March 18, 2017
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Journal of Agricultural and Food Chemistry is published by the American Chemical Society. 1155 Sixteenth Street N.W., Washington, DC 20036 Published by American Chemical Society. Copyright © American Chemical Society. However, no copyright claim is made to original U.S. Government works, or works produced by employees of any Commonwealth realm Crown government in the course of their duties.
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Journal of Agricultural and Food Chemistry
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Effect of Whole Grain Qingke (Tibetan Hordeum vulgare L. Zangqing 320) on
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the Serum Lipid Levels and Intestinal Microbiota of Rats under High-fat Diet
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Xuejuan Xia1, Guannan Li2, Yongbo Ding1, Tingyuan Ren1, Jiong Zheng1, Jianquan
4
Kan1*
5 6
1
College of Food Science, Southwest University, Chongqing 400715, China
7
2
College of Biotechnology, Southwest University, Chongqing 400715, China
8 9 10
*Corresponding author: Jianquan Kan
11
College of Food Science, Southwest University
12
Tiansheng Road 1, Beibei District, Chongqing, 400715, PR China
13
Tel.: +86 23 68 25 03 75
14
Fax: +86 68 25 19 47
15
E-mail:
[email protected] 16 17
Short Title: Effect of Qingke on Serum Lipids and Intestinal Microbiota
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ABSTRACT: This study investigated the hypolipidemic effect of whole grain Qingke
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(WGQ) and its influence on intestinal microbiota. Changes in the serum lipid,
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intestinal environment, and microbiota of Sprague−Dawley rats fed high-fat diets
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supplemented with different doses of WGQ were determined. Results showed that
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high doses of WGQ significantly decreased (P < 0.05) the Lee’s index, serum total
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cholesterol, low-density lipoprotein cholesterol, and non-high-density lipoprotein
25
cholesterol levels whereas increased the body weight of the rats. Cecal weight and
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short-chain fatty acid (SCFA) concentration increased with increasing WGQ dose. An
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Illumina-based sequencing approach showed that the relative abundance of putative
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SCFA-producing bacteria Prevotella and Anaerovibrio increased in the rats fed the
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WGQ diet. Principal component analysis revealed a significant difference in intestinal
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microbiota composition after the administration of the WGQ diet. These findings
31
provide insights into the contribution of the intestinal microbiota to the hypolipidemic
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effect of WGQ.
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KEYWORDS: Qingke, serum lipid, short-chain fatty acid, Illumina MiSeq
34
sequencing, Prevotella
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INTRODUCTION
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Whole grains are important sources of many bioactive compounds and health
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promoters.1 Barley (Hordeum vulgare L.) is the fourth most produced cereal
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worldwide and contains large amounts of β-glucans,2,3 which can decrease the
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concentrations of plasma lipids and reduce the risk of developing cardiovascular
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diseases.4,5 Whole grain barley also exerts a cholesterol-lowering potential.6
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Compared with regular hulled barley, hull-less barley provides more advantages to
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processing and food applications and has attracted attention as a food grain.7 Qingke
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is a hull-less barley cultivar that grows under highland conditions; this cultivar is the
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main staple food crop in Qinghai-Tibet Plateau, China and is also used as a brewing
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material and a feed source.8 However, little information is available about the
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cholesterol-lowering capacity of whole grain Qingke (WGQ). Hence, investigations
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on the hypolipidemic function of WGQ are crucial in determining its future
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development.
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The gastrointestinal tract (GIT) is the first organ susceptible to diet.9 The normal
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microflora within the GIT comprises diverse populations of bacteria, most of which
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are obligate anaerobes. These cecal bacteria primarily rely on dietary components that
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are undigested by enzymes in the upper GIT for energy and growth. These dietary
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components, which include resistant starch, non-starch polysaccharides, and
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oligosaccharides, are often loosely defined as dietary fiber.10 The complex intestinal
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microbiota ferments the dietary fiber and plays a key role in gut health.11 Studies
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suggested that both the composition and metabolism of the intestinal microbiota are 3
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strongly related to diet.10,12 Whole grains are rich in indigestible substrates,11 and a
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few of reports have examined the effects of whole grains on the intestinal
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microbiota.13,14 The microbiome enables complex interactions between the intestinal
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microbiota and its host during fat storage and maturation.14 Furthermore, the intestinal
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microbiota participates in the regulation of lipid metabolism.9,11,15,16 However, the
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effect of WGQ on the intestinal microbiota and the regulatory function of the
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intestinal microbiota in the lipid synthesis of WGQ remain insufficiently elucidated.
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Short-chain fatty acids (SCFAs), which mainly including acetate, propionate, and
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butyrate, are principal fermentation products ensuing from fiber breakdown.11
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Previous observations have collectively suggested that SCFAs can effectively reduce
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plasma cholesterol concentration.15 Studies even proposed that SCFAs participate in
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the mechanisms underlying the association between regular whole grain intake and
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reduced risk of cardiovascular diseases.11,17 Thus, the present study investigated the
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serum lipid and cecal SCFA concentrations of rats fed high-fat diets (HFDs)
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supplemented with or without different doses of WGQ. Changes in the intestinal
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microbiota were determined by high-throughput sequencing. Multiple factors (cecal
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weight, surface area, content weight, and cecal content moisture and pH) influencing
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the intestinal environment of rats were also investigated.
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MATERIALS AND METHODS
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Chemicals. β-glucan assay kit was obtained from Megazyme Int. Ireland Ltd.
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(Wicklow, Ireland). Corn starch was purchased from Unilever Ltd. (Shanghai, China).
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Soy bean oil, lard, and sucrose were purchased from a local market in Chongqing, 4
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China. Cholesterol, casein (99% protein), and cellulose were obtained from Henan
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Datian Industry Co., Ltd. (Henan, China). L-cystine, choline chloride, and minerals
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were procured from Kelong Chemical Reagent Factory (Chengdu, China). Vitamins
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were obtained from Henan Xingyuan Chemical Products Co., Ltd. (Henan, China).
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Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C),
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and high-density lipoprotein cholesterol (HDL-C) assay kits were purchased from
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Sichuan Maker Biotech Co., Ltd. (Chengdu, China). Acetate, propionate, and butyrate
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(> 99%) were obtained from TCI (Shanghai) Development Co. Ltd. (Shanghai, China).
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TIANamp Stool DNA Kit was obtained from Tiangen Biotech Co., Ltd. (Beijing,
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China). Qubit 2.0 DNA Assay Kits were obtained from Thermo Fisher Scientific Inc.
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(Shanghai, China). All other chemicals used were of analytical grade.
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Sample Preparation and Composition Analysis. WGQ (Tibetan Hordeum
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vulgare L. Zangqing 320) samples were provided by Jun Pro Food Co., Ltd.
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(Chongqing, China). After drying (55 °C) in an oven for 24 h, WGQ samples were
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ground and passed through an 80-mesh sieve (0.5 mm). The moisture, ash, and fat
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contents of WGQ were analyzed in accordance with Method 44-16, Method 08-01,
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and Method 30-10, respectively, of the Approved Methods of the AACC.18 Protein
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content was determined using a KjelFlex K-360 nitrogen determination system (Buchi
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Laboratory Equipment Trading, Ltd., Shanghai, China).19 The amounts of β-glucans
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in WGQ were analyzed using a β-glucan assay kit. The total dietary fiber (TDF)
100 101
contents of WGQ were determined in accordance with AOAC Method 991.43.20 Animals and Diets. A total of 36 male, specific pathogen-free Sprague−Dawley 5
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rats weighing 151±12 g (4 weeks old) were purchased from Chongqing Tengxin
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Biotech Co., Ltd. (permitted by SCXK 2012-0005 [Chongqing]). The rats were
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housed in stainless steel screen-bottomed cages. The room was illuminated with a 12
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h dark/light cycle (08:00 on–20:00 off) at a constant temperature of 23±2 °C and a
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relative humidity of 45%–65%. The rats were acclimated by feeding an AIN-93G
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diet21 for 1 week and given free access to food and water. After acclimation, the 36
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rats were randomly assigned to the following four dietary groups (n = 9 per group,
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three rats in the same group housed per cage): normal control (NC) group fed a
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normal AIN-93G diet, blank control (BC) group fed an HFD with additional 10% lard
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and 1% cholesterol),22 low-dose (LD) group fed an HFD containing low-dose (10%)
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WGQ, and high-dose (HD) group fed an HFD containing high-dose (49%) WGQ. The
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animals were fed the abovementioned experimental diets for 8 weeks; the
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composition of the experimental diets is shown in Table 1. Food intake was recorded.
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The experiment design was approved by the Animal Care and Use Committee of
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Southwest University (Permit SYXK2009-0002) and strictly conducted in accordance
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with the guidelines for animal care of the National Institute of Health.23
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Sample Collection. After treatment, the rats were weighed, fasted overnight
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(12−14 h), and lightly anesthetized with ethyl ether.4 The tail and body distance
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(anal-to-nasal length) were measured.9 After decapitation, blood was collected from
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the neck of each rat into a blood collection tube (Vacutainer, Liuyang City Medical
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Instrument Factory, Hunan, China) containing heparin as an anticoagulant. The
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plasma was centrifuged at 1400 ×g for 15 min at 4 °C (5810 centrifuge, Eppendorf 6
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China Ltd., Shanghai, China), and the obtained serum was stored at −80 °C until
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analysis.24 The cecum of each rat was removed and weighed together with the
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contents. Approximately 0.2–0.4 g of fresh cecal contents of each rat was placed in 10
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mL test tubes to determine the pH; 0.2–1.0 g of fresh cecal contents of each rat was
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placed in weighing bottles to determine the water content. Up to 0.2 g of cecal
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contents of each rat was placed in 2 mL micro-centrifuge tubes and then stored at
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−20 °C for SCFA determination. To determine the microbiota, the cecal contents of
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three rats per cage were pooled, and 0.2 g samples were stored at −80 °C for DNA
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extraction. Finally, the cecum of each rat was washed, dried, weighed, and then stored
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at 4 °C for surface area determination.
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Serum Lipid Analysis. Feed efficiency ratio was evaluated as follows: total weight
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gain (g)/total feed intake (g) ×100.25 Lee’s index, which reflects body fat percentage,
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was calculated from the following equation: body weight (g)1/3 × 1000/ naso-anal
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length (cm).26 The levels of TG, TC, LDL-C, and HDL-C in the serum were analyzed
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using assay kits, and measurements were performed using a 7020 Automatic Analyzer
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(Hitachi, Tokyo, Japan) in accordance with the manufacturer’s instructions.
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Non-high-density lipoprotein cholesterol (non-HDL-C) was evaluated as TC –
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(HDL-C),27
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(non-HDL-C)/(HDL-C).5
and
the
atherogenic
index
(AI)
was
calculated
as
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Analysis of Intestinal Environment Factors. The pH and water content of fresh
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cecal content were measured as described by Shen et al.12 The surface area of the
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cecum was determined as described by Loeschke et al.28 with some modifications. In 7
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brief, the caeca were spread and delineated on A3 papers. The profiles were copied to
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new papers and then dried to constant weights (Ws, accurate to 0.001 g).
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Simultaneously, the per unit area (1cm2) weights (Wu, accurate to 0.001 g) for each
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paper were determined to calculate the surface area as follows: surface area of cecum
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(cm2) = Ws/Wu. The SCFA concentrations of cecal contents were analyzed using a
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7890A gas chromatograph (GC, Agilent Technologies, California, USA) equipped
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with a DB-WAX capillary column (122-7032, 30 m × 0.25 µm × 0.25 mm, Agilent
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Technologies) as previously described.15 The initial oven temperature (90 °C) was
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maintained for 30 s, raised to 150 °C at 5 °C/min, and then held for 3.0 min.
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DNA Extraction and Barcoded Pyrosequencing. Approximately 0.2 g of pooled
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cecal contents of three rats per cage was subjected to DNA extraction by using a
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TIANamp Stool DNA Kit following the manufacturer’s instructions. The extracted
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DNA was dissolved in 50 µL of elution buffer. Concentration and quality were
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checked using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington,
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USA). Universal primers 341F (5′- CCT ACG GGN GGC WGC AG -3′) and 805R
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(5′- GAC TAC HVG GGT ATC TAA TCC -3′) were used to amplify the hypervariable
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V3–V4 regions of the 16S rRNA gene.29 The reverse primer contained a 6 bp
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error-correcting barcode unique to each sample.30 Qubit 2.0 DNA Assay Kits were
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used to measure the concentrations of amplification products. Pyrosequencing was
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performed on Illumina MiSeq platforms following the manufacturer’s manuals at
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Sangon Biotech Co., Ltd., Shanghai, China. Raw sequence data were deposited into
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the NCBI sequence read archive database (https://www.ncbi.nlm.nih.gov/sra) under 8
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accession no. SRP071820.
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Bioinformatics and Statistical Analysis. Raw pyrosequencing reads were
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assigned to each sample according to the unique barcode. Reads with low-quality
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scores and short lengths, along with reads that did not contain exact matches with the
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primer
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http://prinseq.sourceforge.net/).15 Pairs of reads from the original DNA fragments
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were merged by FLASH (Version 1.2.3, http://sourceforge.net/projects/flashpage/).30
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The quality filtering of reads was analyzed by using MOTHUR (Version 1.31,
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http://mothur.org/) and QIIME software (Version, 1.7.0, http://qiime.org/).31 The
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remaining high-quality 16S rRNA sequences were clustered into operational
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taxonomic units (OTUs) with 97% identity by UCLUST (Version 1.1.579,
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http://www.drive5.com/uclust/downloads1_1_579.html).32 Taxonomy was assigned
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using the RDP classifier (Version 2.2, http://rdp.cme.msu.edu/).33 The taxonomy of all
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high-quality sequences at the phylum and genus levels was selected to recalculate the
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proportion with the R software package (http://cran.r-project.org/).30 We created
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histograms at the phylum level and major genus composition of dominant phyla by
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using Microsoft Excel 2010 (Microsoft, Washington, USA). Subsequently, a heat map
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at the genus level was generated using custom R scripts. Alpha and beta diversities of
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intestinal microbiota on the basis of the microbial OTUs were analyzed using
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MOTHUR software.
sequence,
were
removed
using
PRINSEQ
(Version
0.20.4,
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The Shannon diversity index, species richness estimator of Chao1, observed OTUs,
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and rarefaction of OTUs were generated to compute the alpha diversities. Principal 9
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component analysis (PCA) was conducted on basis of weighted UniFrac distance
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matrices to compare the beta diversities.29 Data are presented as mean values with
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their standard errors. Statistical analysis was conducted through one-way ANOVA
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using SPSS 20.0 software (IBM, New York, USA). Significant differences between
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groups were determined through Duncan’s multiple range tests. Statistical
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significance was considered at P < 0.05.
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RESULTS
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Composition of WGQ and Its Effect on Body Weight. The moisture content of
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WGQ was 8.71%±0.03%. The ash, fat, protein, β-glucan, and TDF contents (on a dry
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weight basis) of WGQ were 1.95±0.08 g/100 g, 1.03±0.02 g/100 g, 17.00±0.26 g/100
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g, 5.77±0.28 g/100 g, and 19.01±0.54 g/100 g, respectively. On this basis, detailed
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characterizations of the fat, protein, dietary fiber, and β-glucan composition of diets
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are listed in Table 1.
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The effects of WGQ administration on the body weight, feed efficiency ratio, and
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Lee’s index of the rats are presented in Table 2. All HFD groups, including the BC,
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LD, and HD groups, gained higher body weight, feed efficiency ratios, and Lee’s
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index than the NC group. The body weight gain in both LD and HD groups was
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higher than that in the BC group, with that of the HD group being significantly higher
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(P < 0.05). Given that higher feed efficiency ratio corresponds to increased growth,25
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the ratios of the HD group were significantly higher (P < 0.05) than those of the other
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groups. The Lee’s indexes of the HD group were significantly lower (P < 0.05) than
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those of the BC and LD groups. 10
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Serum Lipid Concentration. Changes in serum lipid levels are shown in Table 2.
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The serum levels of TC, TG, HDL-C, LDL-C, and non-HDL-C were significantly
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higher (P < 0.05) in the BC, LD, and HD groups than in the NC group. Compared
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with the BC group, the HD group showed significantly lower (P < 0.05) TC, LDL-C,
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and non-HDL-C levels while significantly higher (P < 0.05) HDL-C levels. The AIs
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of the BC, LD, and HD groups were significantly higher (P < 0.05) than those of the
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NC group. Moreover, the AI levels were significantly lower (P < 0.05) in the LD and
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HD groups than in the BC group.
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SCFA Concentration. The generation of SCFAs in the cecal content was examined
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by measuring the concentrations of acetate, propionate, and butyrate. As shown in
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Table 3, the concentrations of the total SCFAs and each acid were significantly lower
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(P < 0.05) in the BC group than in the NC group. The propionate and butyrate
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concentrations were significantly higher (P < 0.05) in the LD group than in the BC
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group. The acetate, propionate, butyrate, and total SCFA concentrations were
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significantly higher (P < 0.05) in the HD group than in the BC and LD groups.
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Cecal Indexes. As indexes of indigestible residues and fermentative activity,34 the
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cecal weight, surface area, and content weight were measured (Table 3). No
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significant difference in these indexes was observed between NC and BC groups.
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These indexes increased in the LD group compared with the BC group, but the
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difference was not significant (P > 0.05). By contrast, the same indexes significantly
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increased (P < 0.05) in the HD group, and this increase was proportionally greater
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than those in the other groups. The water contents of each group showed no 11
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significant difference (Table 3). The pH values of the cecal contents were significantly
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higher (P < 0.05) in the BC and LD groups than in the NC and HD groups. In addition,
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the pH values of the HD group showed no significant difference compared with those
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of the NC group.
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Alpha Diversity of Microbial 16S rRNA Genes. Twelve samples from the four
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groups were evaluated. After the sequence optimization process, a total of 834,517
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reads were generated, corresponding to an average of 208,629 reads per group. After
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quality filtering, the average length of each read was more than 420 bp. Sequences
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were clustered into 2136–5965 OTUs per sample observed at a 97% similarity level.
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The results, along with the calculated microbial community alpha diversity indexes,
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are shown in Table 3. The sequence number and OTUs, as well as the Shannon and
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Chao indexes, of the HD group were significantly lower (P < 0.05) than those of the
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other groups. However, the NC, BC, and LD groups showed no significant differences.
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These results indicate that HFD did not influence the alpha diversity within the
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microbial community, whereas high doses of WGQ reduced this diversity.
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Taxonomic Analyzes of Bacterial Communities. A total of 23 bacteria phyla were
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identified in all samples. Bacteroidetes and Firmicutes were the two most dominant
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phyla, accounting for > 92.08% of the reads, followed by Proteobacteria (accounting
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for < 3.74%, Figure 1A). In the NC group, the relative abundance of Bacteroidetes
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(44.52%) was lower than that of Firmicutes (50.22%). However, the average values of
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Bacteroidetes in the BC, LD, and HD groups were 49.43%, 50.50%, and 55.78%,
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respectively, which are higher than those of Firmicutes (46.67%, 45.47%, and 41.37%, 12
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correspondingly). At the genus level, all 204 detected genera were shared by all
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samples. Bacteroidetes in all samples mainly consists of Prevotella, unclassified
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Prevotellaceae, Alloprevotella, Bacteroides, unclassified Porphyromonadaceae, and
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Alistipes (Figure 1B).35 Prevotella is the most dominant genus in all groups, and its
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average relative abundance values in the NC, BC, LD, and HD groups were 19.04%,
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21.20%, 32.30%, and 39.38%, respectively. These results suggest that the relative
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abundance of Prevotella increased remarkably with increasing WGQ dosage. In
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addition, the HD group presented a lower relative abundance of Bacteroides than the
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other groups. Among all the samples, Firmicutes mainly consists of unclassified
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Ruminococcaceae, Ruminococcus, Phascolarctobacterium, Anaerovibrio, Blautia,
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Streptococcus, Anaerostipes, unclassified Christensenellaceae, and unclassified
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Lachnospiraceae
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Ruminococcaceae decreased in the HD group. The relative abundance of
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Anaerovibrio increased with increasing WGQ dosage. By contrast, Ruminococcus,
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Blautia, Streptococcus, Anaerostipes, and unclassified Christensenellaceae decreased
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with increasing WGQ dosage. We selected 12 of the most abundant bacterial genera to
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construct a heat map showing an intuitionistic relative abundance and the differences
273
in abundance (Figure 2). Except for incertae sedis bacteria, all these abundant genera
274
belong to Bacteroidetes and Firmicutes. At the species level, the uncultured bacteria
275
accounted for > 85.60% of the reads within all samples. Therefore, no further analysis
276
was conducted at the species level.
277
(Figure
1C).
The
relative
abundance
of
unclassified
To further compare the microbiota among the different samples, we performed PCA 13
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on the relative abundance of bacterial genera (Figure 3). Data are presented as a 2D
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plot to illustrate the relationship. The NC group plotted close to the BC group, and
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both of them were far from the HD group. In addition, the LD group plotted between
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the BC and HD groups. These results indicate that HFD supplemented with WGQ,
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particularly high-dose WGQ, can form bacterial communities distinct from those of
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HFD or normal diet.
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DISCUSSION
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Qingke accounts for more than 97.7% of the total varieties of Tibetan barley. The
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Tibetan Plateau has an average elevation exceeding 4000 m, with extreme
287
geographical conditions such as intense UV radiation, seasonal drought, and hypoxia.
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Extreme geographical conditions have led to the growth of crops with numerous
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secondary metabolites.36 High β-glucan and dietary fiber content are reported in
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Qingke.8,37 Moreover,
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hypocholesterolemic effects.38 In the present study, the effect of WGQ on serum lipids
292
and intestinal microbiota was investigated. Nutrient composition analysis showed that
293
WGQ presents a relatively higher content of proteins and a lower content of fat
294
compared with other whole grains, such as winter wheat, rye, barley, millet, and
295
sorghum.19,39 The low fat and high protein contents of WGQ are consistent with those
296
of hull-less barley reported by Damiran and Yu.40 The β-glucan content of WGQ
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(5.77%) is higher than the mean of Chinese Tibet barleys (4.58%) reported by Zhang
298
et al.37 The TDF content of WGQ is higher than those of whole grain winter wheat,
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rye, and millet.39
studies
showed
that
β-glucan
14
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The effect of WGQ on serum lipid was investigated in vivo through 8-week
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administration of WGQ on HFD rats. Body weight and feed efficiency ratio tests
302
showed that compared with control HFD, high doses of WGQ increase the body
303
weight and feed efficiency ratios. Consistent with our results, Karl and Saltzman41
304
reviewed the evidence for the function of whole grains in body weight regulation and
305
reported that recent clinical trials have failed to support the role of whole grains in
306
promoting weight loss or maintenance. Moreover, Kim et al.6 reported that whole
307
grain barley does not significantly influence the body weight of HFD Syrian Golden
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hamsters. Conversely, Zhou et al.42 reported that whole grain oat decreases the weight
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gain of mice after 7-week administration. Lee’s index, which correlates with body
310
composition, was calculated in the current study to assess the obesity degree of
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rats.9,26 The Lee’s indexes of the HD group were significantly lower (P < 0.05) than
312
those of the BC and LD groups. These results suggest that WGQ decreased the
313
obesity degree of HFD rats. Consistent with our results, epidemiological studies
314
consistently demonstrate that high intakes of whole grains are associated with reduced
315
risk of obesity.41 However, the mechanism underlying the inverse association
316
observed between the increased body weight and reduced obesity degree of HFD rats
317
after WGQ intake should be further studied. The intake of whole grains, such as oat
318
and wheat, decreases serum lipid concentrations.43,44 Consistently, our study found
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that consumption of high doses of WGQ significantly decreased (P < 0.05) the levels
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of TC, LDL-C, and non-HDL-C. In the last 20 years, strong evidence from clinical
321
studies has demonstrated that the reduction of TC, LDL-C, and non-HDL-C is critical 15
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in decreasing the incidence of coronary events.27
323
In an anaerobic environment, bacteria rapidly ferment undigested carbohydrates to
324
SCFAs. Acetate serves as a substrate for liver cholesterol and fatty acid synthesis,
325
increases colonic blood flow and oxygen uptake, and enhances ileal motility by
326
affecting ileal contractions.45 Propionate is largely taken up by the liver and is a good
327
precursor for gluconeogenesis, liponeogenesis, and protein synthesis.11 Moreover,
328
propionate is thought to lower lipogenesis, serum cholesterol levels, and
329
carcinogenesis in other tissues.45 Butyrate has received much attention as an energy
330
source for colonocytes, and it has been described as an anticarcinogenic agent
331
preventing the growth and stimulating the differentiation of colon epithelial cells.45
332
The amounts and profiles of SCFAs can be influenced by the availability of dietary
333
fibers.2 Studies showed that barley brans increase fecal SCFA concentrations, with
334
particularly high amounts of butyrate.34 Cereal β-glucans stimulate butyric and
335
propionic acid formation in the cecum.12 Whole grain barley increases plasma butyric
336
acid concentrations in healthy subjects.2 Consistent with these reports, our results
337
suggest that WGQ increases the concentrations of acetate, propionate, butyrate, and
338
total SCFA. Corresponding to the changes in SCFAs, HFD increased the pH values of
339
the cecal contents, whereas addition of high doses of WGQ decreased the cecal pH to
340
normal levels. The cecal weight, surface area, and content weight were also measured.
341
Results showed that high doses of WGQ diet proportionally increased the cecal
342
weight, surface area, and content weight, suggesting that high doses of WGQ diet lead
343
to a large mass of indigestible residue and high fermentation activity.34 16
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The results of high-throughput sequencing suggest that lower alpha diversity
345
indexes were generated in the HD group than in the other groups. This finding may be
346
attributed to dominant bacterial communities restraining other populations.46
347
Consistent with our results, Zhong et al.2 investigated the effect of whole grain barley
348
on cecal microbiota in HFD rats and reported that the alpha diversity in the barley
349
group is lower than that in the control group. De Angelis et al.13 reported that diet
350
intervention with whole grain barley markedly decreases the total number of fecal
351
anaerobic cultivable bacteria. Taxonomic analyses of bacterial communities showed
352
that Bacteroidetes and Firmicutes were the most dominant phyla in all samples, and
353
this finding is consistent with previous reports.11,47 The current results further
354
demonstrated that all the HFD groups presented a higher relative abundance of
355
Bacteroidetes than the NC group. Consistent with our results, Wu et al.16 reported that
356
Bacteroidetes is positively associated with fats, whereas Firmicutes shows the
357
opposite association. It has been hypothesised that an increased ratio of Firmicutes to
358
Bacteroidetes may make a significant contribution to the pathophysiology of
359
obesity.11,12 However, a growing number of recent studies did not reproduce these
360
findings.15,47 Accordingly, more attention was set based on lower classification levels
361
of intestinal microbiota.47
362
The OTUs obtained in the present study were assigned to known genera by deeper
363
sequencing. The relative abundance of Prevotella and Anaerovibrio increased after
364
feeding with WGQ diet. The remarkable increase in Prevotella abundance may be
365
ascribed to the high dietary fiber content of WGQ because studies have suggested that 17
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high fiber intake is associated with increased levels of Prevotella.16,48 Moreover,
367
studies showed that Prevotella and Anaerovibrio can produce SCFAs.44,48 These
368
findings indicate that the high SCFA concentrations of cecal contents after WGQ diets
369
may be attributed to the increase in Prevotella and Anaerovibrio abundance. Several
370
studies need to be performed to elucidate the molecular mechanisms by which
371
Prevotella and Anaerovibrio participates in the hypolipidemic effect of WGQ. For
372
instance, Prevotella is a large genus with high species diversity; furthermore, species
373
can have high levels of genomic diversity between strains. To predict its function will
374
require a finer-grained understanding of these species’ genetic potential and
375
interactions with their host.49
376
After WGQ diets, many bacterial genera decreased. The decrease in Bacteroides
377
abundance and increase in Prevotella abundance were consistent with previous
378
reports, and these findings reinforce the implication that taxa from these two genera
379
compete for the same niche in the gut.48 PCA also revealed that a WGQ diet,
380
especially the high-dose diet, generated a significantly different composition of the
381
intestinal microbiota compared with those with a high fat or NC diet.
382
Our results confirmed the hypolipidemic effects of WGQ and showed that high
383
doses of WGQ can change the intestinal microbiota by short-term (8 weeks) dietary
384
supplementation. Further research will be conducted to assess the contribution of
385
Prevotella to the hypolipidemic effect of WGQ.
386 387 18
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AUTHOR INFORMATION
389
Corresponding author
390
*Jianquan Kan. E-mail:
[email protected]. Mail: College of Food Science,
391
Southwest University, Tiansheng Road 1, Beibei District, Chongqing, 400715, PR
392
China. Phone: +86-23-68250375. Fax: +86-68251947.
393
Author contributions
394
X.X. and J.K. designed the study; X.X., Y.D. and T.R. performed the experiments;
395
X.X. and G.L. analyzed the data; J.Z. contributed to the discussion for interpreting the
396
data; X.X. and G.L. wrote and revised the manuscript. All authors reviewed the
397
manuscript.
398
Funding
399
This work was financially supported by the Science and Technology Support
400
Demonstration Project of Chongqing (CSTC2014JCSF-JCSSX004).
401
Notes
402
The authors declare no competing financial interest.
403 404
ABBREVIATIONS USED
405
WGQ, whole grain Qingke; HFD, high-fat diet; SCFA, short-chain fatty acid; GIT,
406
gastrointestinal tract; TDF, total dietary fiber; NC, normal control group; BC, blank
407
control group; LD, low-dose group; HD, high-dose group; TG, triglyceride; TC, total
408
cholesterol; LDL-C, low-density lipoprotein cholesterol; HDL-C, high-density
409
lipoprotein cholesterol; non-HDL-C, non-high-density lipoprotein cholesterol; AI, 19
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atherogenic index; PCA, principal component analysis; OTUs, operational taxonomic
411
units
412
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572
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Figure captions:
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Figure 1. Relative read abundance of major microbial phyla (A) and genus
575
composition of the two most dominant phyla, Bacteroidetes (B) and Firmicutes (C).
576
Data are presented as the average values of three samples in each group.
577
Figure 2. Heat map of the intestinal microbiota in rats at the genus level. N-1, -2, and
578
-3 indicate three pooled samples in the N group. The heat map shows normalized
579
relative abundance using the equation Z = (value in each spot – average of values in
580
each row)/(standard deviation of values in each row). The sequence number of each
581
OUT was transformed into Z-score.
582
Figure 3. Principal component analysis (PCA) at the genus level based on weighted
583
UniFrac distance matrices. Principal components (PCs) 1 and 2 explained 33.5% and
584
19.3% of the variance, respectively.
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Tables: Table 1 Composition of experimental diets. NC
BC
LD
HD
530
490
390
-
Ingredient (g/kg) Corn starch WGQ Soy bean oil Lard Cholesterol
-
-
100
490
70.0
-
-
-
-
100
100
100
-
10.0
10.0
10.0
Casein (99% protein)
200
200
200
200
Sucrose
100
100
100
100
Cellulose
50.0
50.0
50.0
50.0
L-cystine
3.00
3.00
3.00
3.00
Choline chloride
2.50
2.50
2.50
2.50
Mineral mixture
35.0
35.0
35.0
35.0
Vitamin mixture
10.0
10.0
10.0
10.0
Content (g/100g) Fat
7.00
11.0
11.1
11.5
Protein
20.1
20.1
21.7
27.7
Dietary fiber
5.00
5.00
6.74
13.5
β-glucan
0.00
0.00
0.53
2.58
Mineral and vitamin mixtures were prepared in accordance with the AIN-93G-MX and AIN-93G-VX, respectively.21 “-”: not added. Abbreviations: NC, normal control group; BC, blank control group; LD, low-dose group; HD, high-dose group; and WGQ, whole grain Qingke.
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Table 2 Effect of different-dose WGQ diet on the body weight, Lee’s index and serum lipid of rats. NC
BC
LD
HD
Initial weight (0 week, g)
166±15.0a
166.±14.8a
167±15.0a
168±15.8a
Final weight (8 weeks, g)
360±16.4a
382±21.5b
387±43.9b
405±21.6c
Body weight
Body weight gain (8 weeks, g)
194±14.4a
215±17.5b
220±28.8b
237±15.8c
Feed efficiency ratio (8 weeks)
14.7±0.61a
15.5±1.10b
15.5±1.85b
16.0±1.26c
Naso-anal length (cm)
22.4±0.57a
22.1±0.90a
22.3±1.14a
22.9±0.83b
Lee’s index
318±15.8a
328±13.6b
327±6.64b
323±6.05c
Lee’s index
Serum lipid TC (mmol/L)
2.36±0.20a
4.68±0.33b
4.40±0.21b
3.60±0.27c
TG (mmol/L)
0.72±0.16a
1.60±0.13b
1.70±0.08b
1.59±0.08b
HDL-C (mmol/L)
1.06±0.08a
1.14±0.08b
1.20±0.07b
1.40±0.04c
LDL-C (mmol/L)
1.07±0.14a
1.74±0.10b
1.75±0.22b
1.32±0.05c
Non-HDL-C (mmol/L)
1.30±0.12a
3.54±0.26b
3.20±0.27b
2.20±0.28c
AI
1.23±0.08a
3.11±0.10b
2.67±0.27c
1.57±0.20d
Values are presented as the mean ± SD (n = 9). Values in the same row with different letters are significantly different (P < 0.05). Abbreviations: TC, total cholesterol; TG, triglyceride; HDL-C, high-density lipoprotein cholesterol; LDL-C, low-density lipoprotein cholesterol; non-HDL-C, non-high-density lipoprotein cholesterol; and AI, atherogenic index.
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Table 3 Effect of different-dose WGQ diet on the intestinal environment factors and alpha diversities of intestinal microbiota. NC
BC
LD
HD
5.56±1.13a
5.07±0.30a
5.30±1.84a
8.19±1.39b
38.8±6.99a
31.9±2.23a
35.7±1.35a
54.1±0.81b
5.11±1.23a
4.77±0.28a
4.95±1.76a
7.52±1.32b
Cecum Total wet weight (g) 2
Surface area (cm ) Cecal contents Wet weight (g) Water content (%)
77.3±7.74a
74.0±0.37a
80.1±1.22a
76.7±7.16a
pH
6.91±0.83a
7.50±0.35b
7.34±0.30b
6.84±0.37a
SCFA (µmol/g) Acetic acid
62.2±8.12a
43.2±5.23b
50.0±2.38b
70.5±9.22a
Propionic acid
24.1±2.54a
17.7±5.51b
20.3±5.38a
28.2±5.32c
Butyric acid
19.4±3.75a
15.3±1.76b
18.0±1.87a
24.2±3.69c
Total SCFAs
106±14.3a
76.3±12.5b
88.3±9.63b
123±18.3c
Observed OTUs (×1000)
5.48a
5.23a
5.19a
2.43b
Shannon
5.93a
5.83a
5.72a
4.84b
Chao 1 (×10000)
1.23a
1.21a
1.20a
5.24b
Coverage (%)
0.93a
0.93a
0.94a
0.94a
Intestinal microbiota
Values of intestinal environment factors are presented as the mean ± SD (n = 9). Data of intestinal microbiota are presented as the average values of three pooled samples in each group. Values in the same row with different letters are significantly different (P < 0.05).
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Figures: Figure 1
A Bacteroidetes
HD
Firmicutes LD
Proteobacteria Others
BC NC 0%
20% 40% 60% 80% Relative abundance of major phyla
100%
B
Prevotella
HD
Unclassified Prevotellaceae Alloprevotella
LD
Bacteroides BC
Unclassified Porphyromonadaceae Alistipes
NC 0
20 40 Genus composition of Bacteroidetes
60 Unclassified Ruminococcaceae
C
Ruminococcus
HD
Phascolarctobacterium Anaerovibrio
LD
Blautia Streptococcus
BC
Anaerostipes Unclassified Christensenellaceae
NC
Unclassified Lachnospiraceae 0
10
20
30
Genus composition of Firmicutes
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Figure 2
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Journal of Agricultural and Food Chemistry
Figure 3
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For Table of Contents Only:
Serum lipid WGQ
High-fat diet
Cecum
SCFA Volume
Prevotella Anaerovibrio
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