Effect of Whole Grain Qingke (Tibetan Hordeum vulgare L. Zangqing

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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

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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

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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]

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Short Title: Effect of Qingke on Serum Lipids and Intestinal Microbiota

18

1

ACS Paragon Plus Environment


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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

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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

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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

35

2


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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.

90

(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,

188

The Shannon diversity index, species richness estimator of Chao1, observed OTUs,

189

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

232

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.

243

The results, along with the calculated microbial community alpha diversity indexes,

244

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

252

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

264

other groups. Among all the samples, Firmicutes mainly consists of unclassified

265

Ruminococcaceae, Ruminococcus, Phascolarctobacterium, Anaerovibrio, Blautia,

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Streptococcus, Anaerostipes, unclassified Christensenellaceae, and unclassified

267

Lachnospiraceae

268

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

271

with increasing WGQ dosage. We selected 12 of the most abundant bacterial genera to

272

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

279

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

281

the BC and HD groups. These results indicate that HFD supplemented with WGQ,

282

particularly high-dose WGQ, can form bacterial communities distinct from those of

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HFD or normal diet.

284

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.

288

Extreme geographical conditions have led to the growth of crops with numerous

289

secondary metabolites.36 High β-glucan and dietary fiber content are reported in

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Qingke.8,37 Moreover,

291

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

297

(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,

299

rye, and millet.39

studies

showed

that

β-glucan

14


in

Qingke

exhibits

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The effect of WGQ on serum lipid was investigated in vivo through 8-week

301

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

308

hamsters. Conversely, Zhou et al.42 reported that whole grain oat decreases the weight

309

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

311

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

319

that consumption of high doses of WGQ significantly decreased (P < 0.05) the levels

320

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



322

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


Page 16 of 36

Page 17 of 36


344

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



366

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


Page 18 of 36

Page 19 of 36


388

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



410

atherogenic index; PCA, principal component analysis; OTUs, operational taxonomic

411

units

412

20


Page 20 of 36

Page 21 of 36


413

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414

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572

28


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573

Figure captions:

574

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.

29



Page 30 of 36

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.

30


Page 31 of 36


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.

31



Page 32 of 36

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).

32


Page 33 of 36


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

33



Figure 2

34


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Page 35 of 36


Figure 3

35



For Table of Contents Only:

Serum lipid WGQ

High-fat diet

Cecum

SCFA Volume

Prevotella Anaerovibrio

36


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Effect of Whole Grain Qingke (Tibetan Hordeum vulgare L. Zangqing

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