Effect of Whole Grain Qingke (Tibetan Hordeum vulgare

Effect of Whole Grain Qingke (Tibetan Hordeum vulgare...
0 downloads 0 Views 761KB Size
Subscriber access provided by University of Newcastle, Australia

Article

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

Just Accepted “Just Accepted” manuscripts have been peer-reviewed and accepted for publication. They are posted online prior to technical editing, formatting for publication and author proofing. The American Chemical Society provides “Just Accepted” as a free service to the research community to expedite the dissemination of scientific material as soon as possible after acceptance. “Just Accepted” manuscripts appear in full in PDF format accompanied by an HTML abstract. “Just Accepted” manuscripts have been fully peer reviewed, but should not be considered the official version of record. They are accessible to all readers and citable by the Digital Object Identifier (DOI®). “Just Accepted” is an optional service offered to authors. Therefore, the “Just Accepted” Web site may not include all articles that will be published in the journal. After a manuscript is technically edited and formatted, it will be removed from the “Just Accepted” Web site and published as an ASAP article. Note that technical editing may introduce minor changes to the manuscript text and/or graphics which could affect content, and all legal disclaimers and ethical guidelines that apply to the journal pertain. ACS cannot be held responsible for errors or consequences arising from the use of information contained in these “Just Accepted” manuscripts.

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.

Page 1 of 36

Journal of Agricultural and Food Chemistry

1

Effect of Whole Grain Qingke (Tibetan Hordeum vulgare L. Zangqing 320) on

2

the Serum Lipid Levels and Intestinal Microbiota of Rats under High-fat Diet

3

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

18

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

19

ABSTRACT: This study investigated the hypolipidemic effect of whole grain Qingke

20

(WGQ) and its influence on intestinal microbiota. Changes in the serum lipid,

21

intestinal environment, and microbiota of Sprague−Dawley rats fed high-fat diets

22

supplemented with different doses of WGQ were determined. Results showed that

23

high doses of WGQ significantly decreased (P < 0.05) the Lee’s index, serum total

24

cholesterol, low-density lipoprotein cholesterol, and non-high-density lipoprotein

25

cholesterol levels whereas increased the body weight of the rats. Cecal weight and

26

short-chain fatty acid (SCFA) concentration increased with increasing WGQ dose. An

27

Illumina-based sequencing approach showed that the relative abundance of putative

28

SCFA-producing bacteria Prevotella and Anaerovibrio increased in the rats fed the

29

WGQ diet. Principal component analysis revealed a significant difference in intestinal

30

microbiota composition after the administration of the WGQ diet. These findings

31

provide insights into the contribution of the intestinal microbiota to the hypolipidemic

32

effect of WGQ.

33

KEYWORDS: Qingke, serum lipid, short-chain fatty acid, Illumina MiSeq

34

sequencing, Prevotella

35

2

ACS Paragon Plus Environment

Page 2 of 36

Page 3 of 36

Journal of Agricultural and Food Chemistry

36

INTRODUCTION

37

Whole grains are important sources of many bioactive compounds and health

38

promoters.1 Barley (Hordeum vulgare L.) is the fourth most produced cereal

39

worldwide and contains large amounts of β-glucans,2,3 which can decrease the

40

concentrations of plasma lipids and reduce the risk of developing cardiovascular

41

diseases.4,5 Whole grain barley also exerts a cholesterol-lowering potential.6

42

Compared with regular hulled barley, hull-less barley provides more advantages to

43

processing and food applications and has attracted attention as a food grain.7 Qingke

44

is a hull-less barley cultivar that grows under highland conditions; this cultivar is the

45

main staple food crop in Qinghai-Tibet Plateau, China and is also used as a brewing

46

material and a feed source.8 However, little information is available about the

47

cholesterol-lowering capacity of whole grain Qingke (WGQ). Hence, investigations

48

on the hypolipidemic function of WGQ are crucial in determining its future

49

development.

50

The gastrointestinal tract (GIT) is the first organ susceptible to diet.9 The normal

51

microflora within the GIT comprises diverse populations of bacteria, most of which

52

are obligate anaerobes. These cecal bacteria primarily rely on dietary components that

53

are undigested by enzymes in the upper GIT for energy and growth. These dietary

54

components, which include resistant starch, non-starch polysaccharides, and

55

oligosaccharides, are often loosely defined as dietary fiber.10 The complex intestinal

56

microbiota ferments the dietary fiber and plays a key role in gut health.11 Studies

57

suggested that both the composition and metabolism of the intestinal microbiota are 3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

58

strongly related to diet.10,12 Whole grains are rich in indigestible substrates,11 and a

59

few of reports have examined the effects of whole grains on the intestinal

60

microbiota.13,14 The microbiome enables complex interactions between the intestinal

61

microbiota and its host during fat storage and maturation.14 Furthermore, the intestinal

62

microbiota participates in the regulation of lipid metabolism.9,11,15,16 However, the

63

effect of WGQ on the intestinal microbiota and the regulatory function of the

64

intestinal microbiota in the lipid synthesis of WGQ remain insufficiently elucidated.

65

Short-chain fatty acids (SCFAs), which mainly including acetate, propionate, and

66

butyrate, are principal fermentation products ensuing from fiber breakdown.11

67

Previous observations have collectively suggested that SCFAs can effectively reduce

68

plasma cholesterol concentration.15 Studies even proposed that SCFAs participate in

69

the mechanisms underlying the association between regular whole grain intake and

70

reduced risk of cardiovascular diseases.11,17 Thus, the present study investigated the

71

serum lipid and cecal SCFA concentrations of rats fed high-fat diets (HFDs)

72

supplemented with or without different doses of WGQ. Changes in the intestinal

73

microbiota were determined by high-throughput sequencing. Multiple factors (cecal

74

weight, surface area, content weight, and cecal content moisture and pH) influencing

75

the intestinal environment of rats were also investigated.

76

MATERIALS AND METHODS

77

Chemicals. β-glucan assay kit was obtained from Megazyme Int. Ireland Ltd.

78

(Wicklow, Ireland). Corn starch was purchased from Unilever Ltd. (Shanghai, China).

79

Soy bean oil, lard, and sucrose were purchased from a local market in Chongqing, 4

ACS Paragon Plus Environment

Page 4 of 36

Page 5 of 36

Journal of Agricultural and Food Chemistry

80

China. Cholesterol, casein (99% protein), and cellulose were obtained from Henan

81

Datian Industry Co., Ltd. (Henan, China). L-cystine, choline chloride, and minerals

82

were procured from Kelong Chemical Reagent Factory (Chengdu, China). Vitamins

83

were obtained from Henan Xingyuan Chemical Products Co., Ltd. (Henan, China).

84

Total cholesterol (TC), triglyceride (TG), low-density lipoprotein cholesterol (LDL-C),

85

and high-density lipoprotein cholesterol (HDL-C) assay kits were purchased from

86

Sichuan Maker Biotech Co., Ltd. (Chengdu, China). Acetate, propionate, and butyrate

87

(> 99%) were obtained from TCI (Shanghai) Development Co. Ltd. (Shanghai, China).

88

TIANamp Stool DNA Kit was obtained from Tiangen Biotech Co., Ltd. (Beijing,

89

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.

91

Sample Preparation and Composition Analysis. WGQ (Tibetan Hordeum

92

vulgare L. Zangqing 320) samples were provided by Jun Pro Food Co., Ltd.

93

(Chongqing, China). After drying (55 °C) in an oven for 24 h, WGQ samples were

94

ground and passed through an 80-mesh sieve (0.5 mm). The moisture, ash, and fat

95

contents of WGQ were analyzed in accordance with Method 44-16, Method 08-01,

96

and Method 30-10, respectively, of the Approved Methods of the AACC.18 Protein

97

content was determined using a KjelFlex K-360 nitrogen determination system (Buchi

98

Laboratory Equipment Trading, Ltd., Shanghai, China).19 The amounts of β-glucans

99

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

102

rats weighing 151±12 g (4 weeks old) were purchased from Chongqing Tengxin

103

Biotech Co., Ltd. (permitted by SCXK 2012-0005 [Chongqing]). The rats were

104

housed in stainless steel screen-bottomed cages. The room was illuminated with a 12

105

h dark/light cycle (08:00 on–20:00 off) at a constant temperature of 23±2 °C and a

106

relative humidity of 45%–65%. The rats were acclimated by feeding an AIN-93G

107

diet21 for 1 week and given free access to food and water. After acclimation, the 36

108

rats were randomly assigned to the following four dietary groups (n = 9 per group,

109

three rats in the same group housed per cage): normal control (NC) group fed a

110

normal AIN-93G diet, blank control (BC) group fed an HFD with additional 10% lard

111

and 1% cholesterol),22 low-dose (LD) group fed an HFD containing low-dose (10%)

112

WGQ, and high-dose (HD) group fed an HFD containing high-dose (49%) WGQ. The

113

animals were fed the abovementioned experimental diets for 8 weeks; the

114

composition of the experimental diets is shown in Table 1. Food intake was recorded.

115

The experiment design was approved by the Animal Care and Use Committee of

116

Southwest University (Permit SYXK2009-0002) and strictly conducted in accordance

117

with the guidelines for animal care of the National Institute of Health.23

118

Sample Collection. After treatment, the rats were weighed, fasted overnight

119

(12−14 h), and lightly anesthetized with ethyl ether.4 The tail and body distance

120

(anal-to-nasal length) were measured.9 After decapitation, blood was collected from

121

the neck of each rat into a blood collection tube (Vacutainer, Liuyang City Medical

122

Instrument Factory, Hunan, China) containing heparin as an anticoagulant. The

123

plasma was centrifuged at 1400 ×g for 15 min at 4 °C (5810 centrifuge, Eppendorf 6

ACS Paragon Plus Environment

Page 6 of 36

Page 7 of 36

Journal of Agricultural and Food Chemistry

124

China Ltd., Shanghai, China), and the obtained serum was stored at −80 °C until

125

analysis.24 The cecum of each rat was removed and weighed together with the

126

contents. Approximately 0.2–0.4 g of fresh cecal contents of each rat was placed in 10

127

mL test tubes to determine the pH; 0.2–1.0 g of fresh cecal contents of each rat was

128

placed in weighing bottles to determine the water content. Up to 0.2 g of cecal

129

contents of each rat was placed in 2 mL micro-centrifuge tubes and then stored at

130

−20 °C for SCFA determination. To determine the microbiota, the cecal contents of

131

three rats per cage were pooled, and 0.2 g samples were stored at −80 °C for DNA

132

extraction. Finally, the cecum of each rat was washed, dried, weighed, and then stored

133

at 4 °C for surface area determination.

134

Serum Lipid Analysis. Feed efficiency ratio was evaluated as follows: total weight

135

gain (g)/total feed intake (g) ×100.25 Lee’s index, which reflects body fat percentage,

136

was calculated from the following equation: body weight (g)1/3 × 1000/ naso-anal

137

length (cm).26 The levels of TG, TC, LDL-C, and HDL-C in the serum were analyzed

138

using assay kits, and measurements were performed using a 7020 Automatic Analyzer

139

(Hitachi, Tokyo, Japan) in accordance with the manufacturer’s instructions.

140

Non-high-density lipoprotein cholesterol (non-HDL-C) was evaluated as TC –

141

(HDL-C),27

142

(non-HDL-C)/(HDL-C).5

and

the

atherogenic

index

(AI)

was

calculated

as

143

Analysis of Intestinal Environment Factors. The pH and water content of fresh

144

cecal content were measured as described by Shen et al.12 The surface area of the

145

cecum was determined as described by Loeschke et al.28 with some modifications. In 7

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

146

brief, the caeca were spread and delineated on A3 papers. The profiles were copied to

147

new papers and then dried to constant weights (Ws, accurate to 0.001 g).

148

Simultaneously, the per unit area (1cm2) weights (Wu, accurate to 0.001 g) for each

149

paper were determined to calculate the surface area as follows: surface area of cecum

150

(cm2) = Ws/Wu. The SCFA concentrations of cecal contents were analyzed using a

151

7890A gas chromatograph (GC, Agilent Technologies, California, USA) equipped

152

with a DB-WAX capillary column (122-7032, 30 m × 0.25 µm × 0.25 mm, Agilent

153

Technologies) as previously described.15 The initial oven temperature (90 °C) was

154

maintained for 30 s, raised to 150 °C at 5 °C/min, and then held for 3.0 min.

155

DNA Extraction and Barcoded Pyrosequencing. Approximately 0.2 g of pooled

156

cecal contents of three rats per cage was subjected to DNA extraction by using a

157

TIANamp Stool DNA Kit following the manufacturer’s instructions. The extracted

158

DNA was dissolved in 50 µL of elution buffer. Concentration and quality were

159

checked using a NanoDrop 2000 spectrophotometer (Thermo Scientific, Wilmington,

160

USA). Universal primers 341F (5′- CCT ACG GGN GGC WGC AG -3′) and 805R

161

(5′- GAC TAC HVG GGT ATC TAA TCC -3′) were used to amplify the hypervariable

162

V3–V4 regions of the 16S rRNA gene.29 The reverse primer contained a 6 bp

163

error-correcting barcode unique to each sample.30 Qubit 2.0 DNA Assay Kits were

164

used to measure the concentrations of amplification products. Pyrosequencing was

165

performed on Illumina MiSeq platforms following the manufacturer’s manuals at

166

Sangon Biotech Co., Ltd., Shanghai, China. Raw sequence data were deposited into

167

the NCBI sequence read archive database (https://www.ncbi.nlm.nih.gov/sra) under 8

ACS Paragon Plus Environment

Page 8 of 36

Page 9 of 36

Journal of Agricultural and Food Chemistry

168

accession no. SRP071820.

169

Bioinformatics and Statistical Analysis. Raw pyrosequencing reads were

170

assigned to each sample according to the unique barcode. Reads with low-quality

171

scores and short lengths, along with reads that did not contain exact matches with the

172

primer

173

http://prinseq.sourceforge.net/).15 Pairs of reads from the original DNA fragments

174

were merged by FLASH (Version 1.2.3, http://sourceforge.net/projects/flashpage/).30

175

The quality filtering of reads was analyzed by using MOTHUR (Version 1.31,

176

http://mothur.org/) and QIIME software (Version, 1.7.0, http://qiime.org/).31 The

177

remaining high-quality 16S rRNA sequences were clustered into operational

178

taxonomic units (OTUs) with 97% identity by UCLUST (Version 1.1.579,

179

http://www.drive5.com/uclust/downloads1_1_579.html).32 Taxonomy was assigned

180

using the RDP classifier (Version 2.2, http://rdp.cme.msu.edu/).33 The taxonomy of all

181

high-quality sequences at the phylum and genus levels was selected to recalculate the

182

proportion with the R software package (http://cran.r-project.org/).30 We created

183

histograms at the phylum level and major genus composition of dominant phyla by

184

using Microsoft Excel 2010 (Microsoft, Washington, USA). Subsequently, a heat map

185

at the genus level was generated using custom R scripts. Alpha and beta diversities of

186

intestinal microbiota on the basis of the microbial OTUs were analyzed using

187

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

190

component analysis (PCA) was conducted on basis of weighted UniFrac distance

191

matrices to compare the beta diversities.29 Data are presented as mean values with

192

their standard errors. Statistical analysis was conducted through one-way ANOVA

193

using SPSS 20.0 software (IBM, New York, USA). Significant differences between

194

groups were determined through Duncan’s multiple range tests. Statistical

195

significance was considered at P < 0.05.

196

RESULTS

197

Composition of WGQ and Its Effect on Body Weight. The moisture content of

198

WGQ was 8.71%±0.03%. The ash, fat, protein, β-glucan, and TDF contents (on a dry

199

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

200

g, 5.77±0.28 g/100 g, and 19.01±0.54 g/100 g, respectively. On this basis, detailed

201

characterizations of the fat, protein, dietary fiber, and β-glucan composition of diets

202

are listed in Table 1.

203

The effects of WGQ administration on the body weight, feed efficiency ratio, and

204

Lee’s index of the rats are presented in Table 2. All HFD groups, including the BC,

205

LD, and HD groups, gained higher body weight, feed efficiency ratios, and Lee’s

206

index than the NC group. The body weight gain in both LD and HD groups was

207

higher than that in the BC group, with that of the HD group being significantly higher

208

(P < 0.05). Given that higher feed efficiency ratio corresponds to increased growth,25

209

the ratios of the HD group were significantly higher (P < 0.05) than those of the other

210

groups. The Lee’s indexes of the HD group were significantly lower (P < 0.05) than

211

those of the BC and LD groups. 10

ACS Paragon Plus Environment

Page 10 of 36

Page 11 of 36

Journal of Agricultural and Food Chemistry

212

Serum Lipid Concentration. Changes in serum lipid levels are shown in Table 2.

213

The serum levels of TC, TG, HDL-C, LDL-C, and non-HDL-C were significantly

214

higher (P < 0.05) in the BC, LD, and HD groups than in the NC group. Compared

215

with the BC group, the HD group showed significantly lower (P < 0.05) TC, LDL-C,

216

and non-HDL-C levels while significantly higher (P < 0.05) HDL-C levels. The AIs

217

of the BC, LD, and HD groups were significantly higher (P < 0.05) than those of the

218

NC group. Moreover, the AI levels were significantly lower (P < 0.05) in the LD and

219

HD groups than in the BC group.

220

SCFA Concentration. The generation of SCFAs in the cecal content was examined

221

by measuring the concentrations of acetate, propionate, and butyrate. As shown in

222

Table 3, the concentrations of the total SCFAs and each acid were significantly lower

223

(P < 0.05) in the BC group than in the NC group. The propionate and butyrate

224

concentrations were significantly higher (P < 0.05) in the LD group than in the BC

225

group. The acetate, propionate, butyrate, and total SCFA concentrations were

226

significantly higher (P < 0.05) in the HD group than in the BC and LD groups.

227

Cecal Indexes. As indexes of indigestible residues and fermentative activity,34 the

228

cecal weight, surface area, and content weight were measured (Table 3). No

229

significant difference in these indexes was observed between NC and BC groups.

230

These indexes increased in the LD group compared with the BC group, but the

231

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

233

than those in the other groups. The water contents of each group showed no 11

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

234

significant difference (Table 3). The pH values of the cecal contents were significantly

235

higher (P < 0.05) in the BC and LD groups than in the NC and HD groups. In addition,

236

the pH values of the HD group showed no significant difference compared with those

237

of the NC group.

238

Alpha Diversity of Microbial 16S rRNA Genes. Twelve samples from the four

239

groups were evaluated. After the sequence optimization process, a total of 834,517

240

reads were generated, corresponding to an average of 208,629 reads per group. After

241

quality filtering, the average length of each read was more than 420 bp. Sequences

242

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

245

Chao indexes, of the HD group were significantly lower (P < 0.05) than those of the

246

other groups. However, the NC, BC, and LD groups showed no significant differences.

247

These results indicate that HFD did not influence the alpha diversity within the

248

microbial community, whereas high doses of WGQ reduced this diversity.

249

Taxonomic Analyzes of Bacterial Communities. A total of 23 bacteria phyla were

250

identified in all samples. Bacteroidetes and Firmicutes were the two most dominant

251

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

253

(44.52%) was lower than that of Firmicutes (50.22%). However, the average values of

254

Bacteroidetes in the BC, LD, and HD groups were 49.43%, 50.50%, and 55.78%,

255

respectively, which are higher than those of Firmicutes (46.67%, 45.47%, and 41.37%, 12

ACS Paragon Plus Environment

Page 12 of 36

Page 13 of 36

Journal of Agricultural and Food Chemistry

256

correspondingly). At the genus level, all 204 detected genera were shared by all

257

samples. Bacteroidetes in all samples mainly consists of Prevotella, unclassified

258

Prevotellaceae, Alloprevotella, Bacteroides, unclassified Porphyromonadaceae, and

259

Alistipes (Figure 1B).35 Prevotella is the most dominant genus in all groups, and its

260

average relative abundance values in the NC, BC, LD, and HD groups were 19.04%,

261

21.20%, 32.30%, and 39.38%, respectively. These results suggest that the relative

262

abundance of Prevotella increased remarkably with increasing WGQ dosage. In

263

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,

266

Streptococcus, Anaerostipes, unclassified Christensenellaceae, and unclassified

267

Lachnospiraceae

268

Ruminococcaceae decreased in the HD group. The relative abundance of

269

Anaerovibrio increased with increasing WGQ dosage. By contrast, Ruminococcus,

270

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 14 of 36

278

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

280

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

283

HFD or normal diet.

284

DISCUSSION

285

Qingke accounts for more than 97.7% of the total varieties of Tibetan barley. The

286

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

290

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

ACS Paragon Plus Environment

in

Qingke

exhibits

Page 15 of 36

Journal of Agricultural and Food Chemistry

300

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Page 16 of 36

Page 17 of 36

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Page 18 of 36

Page 19 of 36

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

410

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

411

units

412

20

ACS Paragon Plus Environment

Page 20 of 36

Page 21 of 36

Journal of Agricultural and Food Chemistry

413

References

414

(1) Andersson, A. A. M.; Dimberg, L.; Åman, P.; Landberg, R. Recent findings on

415

certain bioactive components in whole grain wheat and rye. J. Cereal Sci. 2014,

416

59, 294–311.

417

(2) Zhong, Y. D.; Marungruang, N.; Fåk, F.; Nyman, M. Effects of two whole-grain

418

barley varieties on caecal SCFA, gut microbiota and plasma inflammatory

419

markers in rats consuming low- and high-fat diets. Br. J. Nutr. 2015, 113, 1558–

420

157.

421 422 423 424 425 426

(3) Aman, P.; Graham, H. Analysis of total and insoluble mixed-linked (1→3), (1→4)-β-D-glucans in barley and oats. J. Agric. Food Chem. 1987, 35, 704–709. (4) Park, S. O.; Park, B. S. Bifidogenic effect of grain larvae extract on serum lipid, glucose and intestinal microflora in rats. J. Biosci. 2015, 40, 513–520. (5) Zhu, F. M.; Du, B.; Xu, B. J. A critical review on production and industrial applications of beta-glucans. Food Hydrocoll. 2016, 52, 275–288.

427

(6) Kim, H.; Turowski, M.; Anderson, W. H. K.; Young, S. A.; Kim, Y.; Yokoyama, W.

428

Supplementation of Hydroxypropyl Methylcellulose into Yeast Leavened

429

All-Whole Grain Barley Bread Potentiates Cholesterol-Lowering Effect. J. Agric.

430

Food Chem. 2011, 59, 7672–7678.

431

(7) Knutsen, S. H.; Holtekjølen, A. K. Preparation and analysis of dietary fibre

432

constituents in whole grain from hulled and hull-less barley. Food Chem.

433

2007,102, 707–715.

434

(8) Zhu, F. M.; Du, B.; Xu, B. J. Superfine grinding improves functional properties 21

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

435

and antioxidant capacities of bran dietary fibre from Qingke (hull-less barley)

436

grown in Qinghai-Tibet Plateau, China. J. Cereal Sci. 2015, 65, 43–47.

437

(9) Wang, H. S.; Tang, X.; Cheserek, M. J.; Shi, Y. H.; Li, G. W. Obesity prevention

438

of synthetic polysaccharides in high-fat diet fed C57BL/6 mice. J. Funct. Foods

439

2015, 17, 563–574.

440 441

(10) Scott, K. P.; Duncan, S. H.; Flint, H. J. Dietary fibre and the gut microbiota. Nutr. Bull. 2008, 33, 201–211.

442

(11) Schwiertz, A.; Taras, D.; Schäfer, K.; Beijer, S.; Bos, N. A.; Donus, C.; Hardt, P.

443

D. Microbiota and SCFA in lean and overweight healthy subjects. Obesity, 2010,

444

18, 190–195.

445

(12) Shen, R. L.; Dang, X. Y.; Dong, J. L.; Hu, X. Z. Effects of Oat β-Glucan and

446

Barley β-Glucan on Fecal Characteristics, Intestinal Microflora, and Intestinal

447

Bacterial Metabolites in Rats. J. Agric. Food Chem. 2012, 60, 11301–11308

448

(13) De Angelis, M.; Eustacchio, M.; Lucia, V.; Carmela, C.; Noemi, C.; Giorgia, G.

449

Effect of whole-grain barley on the human fecal microbiota and metabolome. Appl.

450

Environ. Microbiol. 2015, 81, 7945–7956.

451

(14) Foerster, J.; Gertraud, M.; Nicole, R.; Adrian, T.; Michael, B.; Heiner, B. The

452

influence of whole grain products and red meat on intestinal microbiota

453

composition in normal weight adults: a randomized crossover intervention trial.

454

PLoS One, 2014, 9, e109606.

455

(15) Murphy, E. F.; Cotter, P. D.; Healy, S.; Marques, T. M.; O'Sullivan, O.; Fouhy, F.,

456

Clarke, S. F.; O'Toole, P. W.; Quigley, E. M.; Stanton, C.; Ross, P. R.; O'Doherty, 22

ACS Paragon Plus Environment

Page 22 of 36

Page 23 of 36

Journal of Agricultural and Food Chemistry

457

R. M.; Shanahan, F. Composition and energy harvesting capacity of the gut

458

microbiota: relationship to diet, obesity and time in mouse models. Gut, 2010, 59,

459

1635–1642.

460

(16) Wu, G. D.; Chen, J.; Hoffmann, C.; Bittinger, K.; Chen, Y. Y.; Keilbaugh, S. A.;

461

Bewtra, M.; Knights, D.; Walters, W. A.; Knight, R.; Sinha, R.; Gilroy, E.; Gupta,

462

K.; Baldassano, R.; Nessel, L.; Li, H. Z.; Bushman, F. D.; Lewis, J. D. Linking

463

Long-Term Dietary Patterns with Gut Microbial Enterotypes. Science, 2011, 333,

464

105–108.

465

(17) Vetrani, C.; Costabile, G.; Luongo, D.; Naviglio, D.; Rivellese, A. A.; Riccardi,

466

G.; Giacco, R. Effects of whole-grain cereal foods on plasma short chain fatty acid

467

concentrations in individuals with the metabolic syndrome. Nutrition, 2016, 32,

468

217–221.

469 470 471 472 473 474

(18) AACC. Approved Method of the AACC. 10th ed. American Association of Cereal Chemists, St. Paul, MN, USA 2003. (19) Ragaee, S.; Abdel-Aal, E. M.; Noaman, M. Antioxidant activity and nutrient composition of selected cereals for food use. Food Chem. 2006, 98, 32–38. (20) AOAC. Official Method of Analysis of AOAC Intl. 13th ed. Association of Official Analytical Chemists, Arlington, VA, USA 1980.

475

(21) Reeves, P. G.; Nielsen, F. H.; Fahey, G. C. AIN-93 purified diets for laboratory

476

rodents: final report of the American Institute of Nutrition ad hoc writing

477

committee on the reformulation of the AIN-76A rodent diet. J. Nutr. 1993, 123,

478

1939–1951. 23

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

479

(22) He, W. S.; Wang, M. G.; Pan, X. X.; Li, J. J.; Jia, C. S.; Zhang, X. M.; Feng, B.

480

Role of plant stanol derivatives in the modulation of cholesterol metabolism and

481

liver gene expression in mice. Food Chem. 2013, 140, 9–16.

482

(23) Institute of Laboratory Animal Resources, National Research Council (US)

483

Institute for Laboratory. Guide for the Care and Use of Laboratory Animals;

484

National Academies, 1985.

485

(24) Hoang, M. H.; Houng, S. J.; Jun, H. J.; Lee, J. H.; Choi, J. W.; Kim, S. H.; Kim,

486

Y. R.; Lee, S. J. Barley Intake Induces Bile Acid Excretion by Reduced

487

Expression of Intestinal ASBT and NPC1L1 in C57BL/6J Mice. J. Agric. Food

488

Chem. 2011, 59, 6798–6805.

489

(25) Baloi, M.; Carvalho, C. V. A.; Sterzelecki, F. C.; Passini, G.; Cerqueira, V. R.

490

Effects of feeding frequency on growth, feed efficiency and body composition of

491

juveniles Brazilian sardine, Sardinella brasiliensis (Steindacher 1879). Aquac. Res.

492

2016, 47, 554–560.

493

(26) Hioki, C.; Yoshida, T.; Kogure, A.; Yoshimoto, K.; Shimatsu, A. Growth

494

Hormone Administration Controls Body Composition Associated with Changes

495

of Thermogenesis in Obese KK-Ay Mice. Open Endocrinol. J. 2010, 4, 3–8.

496

(27) Kühnast, S.; Fiocco, M.; Hoorn, J. W. A.; Princen, H. M. G.; Jukema, W.

497

Innovative pharmaceutical interventions in cardiovascular disease: Focusing on

498

the contribution of non-HDL-C/LDL-C-lowering versus HDL-C-raising A

499

systematic review and meta-analysis of relevant preclinical studies and clinical

500

trials. Eur. J. Pharmacol. 2015, 763, 48–63. 24

ACS Paragon Plus Environment

Page 24 of 36

Page 25 of 36

Journal of Agricultural and Food Chemistry

501

(28) Loeschke, D. K.; Kautz, U.; Löhrs, U. Effects of antibiotics on caecal electrolyte

502

transport and morphology in rats contribution to the pathogenesis of

503

antibiotic-associated diarrhea. J. Mol. Med. 1980, 58, 383–385.

504

(29) Li, G. N.; Xia, X. J.; Tang, W. C.; Zhu, Y. Intestinal microecology associated

505

with fluoride resistance capability of the silkworm (Bombyx mori L.). Appl.

506

Microbiol. Biotechnol. 2016, 100, 6715–6724.

507

(30) Sun, J.; Zhang, Q.; Zhou, J.; Wei, Q. Illumina Amplicon Sequencing of 16S

508

rRNA Tag Reveals Bacterial Community Development in the Rhizosphere of

509

Apple Nurseries at a Replant Disease Site and a New Planting Site. PLoS One,

510

2014, 9, e111744.

511

(31) Caporaso, J. G.; Kuczynski, J.; Stombaugh, J.; Bittinger, K.; Bushman, F. D.;

512

Costello, E. K.; Fierer, N.; Peña, A. G.; Goodrich, J. K.; Gordon, J. I.; Huttley, G.

513

A.; Kelley, S. T.; Knights, D.; Koenig, J. E.; Ley, R. E.; Lozupone, C. A.;

514

McDonald, D.; Muegge, B. D.; Pirrung, M.; Reeder, J.; Sevinsky, J. R.;

515

Turnbaugh, P. J.; Walters, W. A.; Widmann, J.; Yatsunenko, T.; Zaneveld, J.

516

Knight R QIIME allows analysis of high-throughput community sequencing data.

517

Nat. Methods, 2010, 7, 335–336.

518 519

(32) Lindgreen, S.; Adair, K. L.; Gardner, P. P. An evaluation of the accuracy and speed of metagenome analysis tools. Sci. Rep. 2016, 6, 19233.

520

(33) Cheng, W. X.; Chen, H.; Yan, S. H.; Su, J. Q. Illumina sequencing-based

521

analyses of bacterial communities during short-chain fatty-acid production from

522

food waste and sewage sludge fermentation at different pH values. World J. 25

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

523

Page 26 of 36

Microbiol. Biotechnol. 2014, 30, 2387–2395.

524

(34) Mcintosh, G. H.; Leu, R. K. L.; Royle, P. J.; Young, G. P. A comparative study

525

of the influence of differing barley brans on DMH-induced intestinal tumours in

526

male Sprague-Dawley rats. J. Gastroenterol. Hepatol. 1996, 11, 113–119.

527 528 529 530 531 532 533

(35) Garrity, G. M.; Bell, J. A.; Lilburn, T. G. Bergey’s Manual of Systematic Bacteriology 2nd (eds Garrity, G. M. et al.), Springer, 2004. (36) Gong, L. X.; Jin, C.; Wu, X. Q; Zhang, Y. Determination of Arabinoxylans in Tibetan Hull-less Barley Bran. Procedia Eng. 2012, 37, 218–222. (37) Zhang, G. P.; Wang, J. M.; Chen, J. X. Analysis of beta-glucan content in barley cultivars from different locations of China. Food Chem. 2002, 79, 251–254. (38) Tong, L. T.; Zhong, K.; Liu, L. Y.; Zhou, X. R.; Qiu, J.; Zhou, S. M. Effects of

534

dietary

hull-less

barley

535

hypercholesterolemic hamsters. Food Chem. 2015, 169, 344–349.

β-glucan

on

the

cholesterol

metabolism

of

536

(39) Andersson, A. A. M.; Andersson, R.; Piironen, V.; Lampi, A. M.; Nyström, L.;

537

Boros, D. Contents of dietary fibre components and their relation to associated

538

bioactive components in whole grain wheat samples from the HEALTHGRAIN

539

diversity screen. Food Chem. 2013, 136, 1243–1248.

540

(40) Damiran, D.; Yu, P. Q. Metabolic characteristics in ruminants of the proteins in

541

newly developed hull-less barley varieties with altered starch traits. J. Cereal Sci.

542

2012, 55, 351–360.

543 544

(41) Karl, J. P.; Saltzman, E. The role of whole grains in body weight regulation. Adv. Nutr. 2012, 3, 697–707. 26

ACS Paragon Plus Environment

Page 27 of 36

Journal of Agricultural and Food Chemistry

545

(42) Zhou, A. L.; Hergert, N.; Rompato, G.; Lefevre, M. Whole Grain Oats Improve

546

Insulin Sensitivity and Plasma Cholesterol Profile and Modify Gut Microbiota

547

Composition in C57BL/6J Mice. J Nutr. 2015, 145, 222–230.

548

(43) Hollænder, P. L. B.; Ross, A. B.; Kristensen, M. Whole-grain and blood lipid

549

changes in apparently healthy adults: a systematic review and meta-analysis of

550

randomized controlled studies. Am. J. Clin. Nutr. 2015, 102, 556–572.

551

(44) Tighe, P.; Duthie, G.; Brittenden, J.; Vaughan, N.; Mutch, W.; Simpson, W. G.;

552

Duthie, S.; Horgan, G. W.; Thies, Frank. Effects of Wheat and Oat-Based Whole

553

Grain Foods on Serum Lipoprotein Size and Distribution in Overweight Middle

554

Aged People: A Randomised Controlled Trial. PLoS One, 2013, 8, e70436.

555

(45) Hosseini, E.; Grootaert, C.; Verstraete, W.; dWiele, T. V. Propionate as a

556

health-promoting microbial metabolite in the human gut. Nutr. Rev. 2011, 69, 245–

557

258.

558

(46) Hu, H. W.; Zhang, L. M.; Dai, Y.; Di, H. J.; He, J. Z. pH-dependent distribution of

559

soil ammonia oxidizers across a large geographical scale as revealed by

560

high-throughput pyrosequencing. J. Soils Sediments, 2013, 13, 1439–1449.

561

(47) Zhang, J.; Guo, Z.; Xue, Z.; Sun, Z.; Zhang, M.; Wang, L.; Wang, G.; Wang, F.;

562

Xu, J.; Cao, H.; Xu, H.; Lv, Q.; Zhong, Z.; Chen, Y.; Qimuge, S.; Menghe, B.;

563

Zheng, Y.; Zhao, L.; Chen, W.; Zhang, H. A phylo-functional core of gut

564

microbiota in healthy young Chinese cohorts across lifestyles, geography and

565

ethnicities. ISME J. 2015, 9, 1979–1990.

566

(48) Kovatcheva-Datchary, P.; Nilsson, A.; Akrami, R.; Lee, Y. S.; De Vadder, F.; 27

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

567

Arora, T.; Hallen, A.; Martens, E.; Björck, I.; Bäckhed, F. Dietary Fiber-Induced

568

Improvement in Glucose Metabolism Is Associated with Increased Abundance of

569

Prevotella. Cell Metab. 2015, 22, 971–982.

570 571

(49) Ley, R. E. Gut microbiota in 2015: Prevotella in the gut: choose carefully. Nat. Rev. Gastroenterol. Hepatol. 2016, 13, 69–70.

572

28

ACS Paragon Plus Environment

Page 28 of 36

Page 29 of 36

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Page 31 of 36

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Page 33 of 36

Journal of Agricultural and Food Chemistry

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

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Figure 2

34

ACS Paragon Plus Environment

Page 34 of 36

Page 35 of 36

Journal of Agricultural and Food Chemistry

Figure 3

35

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

For Table of Contents Only:

Serum lipid WGQ

High-fat diet

Cecum

SCFA Volume

Prevotella Anaerovibrio

36

ACS Paragon Plus Environment

Page 36 of 36