Simulated Digestion and Fermentation in vitro by Human Gut

Subscriber access provided by READING UNIV

Article

Simulated Digestion and Fermentation in vitro by Human Gut Microbiota of Polysaccharides from Bee Collected Pollen of Chinese Wolfberry Wangting Zhou, Yamei Yan, Jia Mi, hongcheng zhang, Lu Lu, Qing Luo, Xiaoying Li, Xiaoxiong Zeng, and Youlong Cao J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.7b05546 • Publication Date (Web): 09 Jan 2018 Downloaded from http://pubs.acs.org on January 9, 2018

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 37

Journal of Agricultural and Food Chemistry

Simulated Digestion and Fermentation in vitro by Human Gut Microbiota of Polysaccharides from Bee Collected Pollen of Chinese Wolfberry

Wangting Zhou,† Yamei Yan,‡ Jia Mi,‡ Hongcheng Zhang,§ Lu Lu,‡ Qing Luo,‡ Xiaoying Li,‡ Xiaoxiong Zeng,†,* Youlong Cao‡,* †

College of Food Science and Technology, Nanjing Agricultural University, Nanjing

210095, People’s Republic of China ‡

National Wolfberry Engineering Research Center, Yinchuan 750002, People’s

Republic of China §

Institute of Apicultural Research, Chinese Academy of Agricultural Sciences,

Beijing 100093, People’s Republic of China

*To whom correspondence should be addressed. Tel & Fax: +86 25 84396791, E-mail: [email protected] (X. Zeng); +86 951 6886783, E-mail address: [email protected] (Y. Cao)

1

ACS Paragon Plus Environment


Page 2 of 37

1

ABSTRACT

2

Digestion and fermentation in vitro of polysaccharides from bee collected pollen of

3

Chinese wolfberry (WBPPS) were investigated in the present study. It was found that

4

WBPPS was mainly consisted of mannose, ribose, rhamnose, galacturonic acid, glucose,

5

galactose, xylose and arabinose in a molar ratio of 0.38: 0.09: 0.17: 0.64: 0.22: 0.67:

6

0.08: 1.03, respectively. WBPPS was not affected by human saliva. The fraction A

7

(molecular weight 1340 kDa) of WBPPS was not broken down in simulated gastric and

8

small intestinal juices, while the small fraction B (molecular weight 523 kDa) of

9

WBPPS was degraded. Moreover, fermentation in vitro revealed that WBPPS could

10

significantly enhance the production of short-chain fatty acids and modulate gut

11

microbiota composition, via increasing the relative abundances of genera Prevotella,

12

Dialister, Megamonas, Faecalibacterium, Alloprevotella and decreasing the numbers of

13

genera

14

Phascolarctobacterium, Parasutterella, Clostridium sensu stricto and Fusobacterium.

15

KEYWORDS: Bee collected pollen from Chinese Wolfberry; Polysaccharides;

16

Simulated digestion; Fermentation; Short-chain fatty acids; Gut microbiota

Bacteroides,

Clostridium

XlVa,

Parabacteroides,

2


Escherichia/Shigella,

Page 3 of 37


17

INTRODUCTION

18

Bee collected pollen, as the male gametophyte of angiosperms and gymnosperms, is

19

made up of pollen grain, a small amount of saliva secreted by bees and nectar in plant.1

20

It contains abundant nutrients,2,3 such as carbohydrates, proteins, amino acids, nucleic

21

acids, phenols, fatty acids, minerals and vitamins. Therefore, bee collected pollen is used

22

as a dietary supplement for human in USA.4 In past fewer years, there are some reports

23

on polysaccharides from bee collected pollen and their biological activities. The

24

polysaccharides from bee collected pollen are reported to have immunomodulatory,

25

antioxidant and antitumor activities.5-7 In recent years, much attention has been paid on

26

the prebiotic activity of polysaccharides, for a growing number of works proved that

27

plant polysaccharides could be degraded by gut microbiota and thereby influence the

28

host health by their metabolic products.8 For example, the short-chain fatty acids

29

(SCFAs) could lower the pH of colon and prevent various diseases.9 Meanwhile, it has

30

been reported that polysaccharides could alter the composition of gut community. For

31

example, the polysaccharides from Fuzhuan brick tea could significantly simulated the

32

growth of Bacteroides and Prevotella10 which are reported to be the key members of gut

33

microbiota for hydrolysis of dietary fiber.11 The polysaccharides from mycelial

34

Ganoderma lucidum could reduce high fat diet-induced gut dysbiosis which is

35

accompanied by the decreased level of endotoxin-bearing Proteobacteria and the ratio of

36

Firmicutes/Bacteroidetes (F/B).12 Pleurotus eryngii polysaccharides could contribute to

37

the host immune response by increasing the abundances of Bacteroidaceae and

38

Lactobacillaceae which are able to promote the secretion of immune factors.13 However,

3



39

the effects of bee collected pollen polysaccharides on gut health have seldom been

40

reported. From this standpoint, we aimed to investigate the digestion and fermentation of

41

polysaccharides from bee collected pollen of Chinese wolfberry (WBPPS) in the present

42

study. Moreover, the possible effects of WBPPS fermentation on production of SCFAs

43

and composition of gut microbiota were evaluated.

44

As we known, dietary fiber can be unaltered to transit into the large intestine without

45

digestion in oral and gastrointestinal tract of human.14 It is still unknown whether

46

WBPPS can be digested by human saliva and simulated gastrointestinal fluids. Thus, the

47

first should be done is to provide some information about the digestion of WBPPS. The

48

study of carbohydrate digestion in vivo is time-consuming and costly, but there exists

49

several in vitro methods for analyzing polysaccharides digestion. Hu et al.15 reported an

50

in vitro model to explore the digestion of polysaccharides from the seeds of Plantago

51

asiatica L. It was found that the polysaccharide was not influenced in saliva, but

52

degraded in gastrointestinal tract. Carnachaned et al.16 applied an appropriated method

53

to investigate the effects of simulated gastric and intestinal juices on polysaccharides

54

from golden and green kiwifruits. The results elucidated that chemical composition and

55

structure of the polysaccharides in insoluble fiber fraction from golden and green

56

kiwifruits were largely unchanged. In the present study, therefore, simulated digestion

57

model was used to examine whether WBPPS is being broken down in the

58

digestive system. Furthermore, the effects of WBPPS on gut microbiota and production

59

of SCFAs were investigated via in vitro fermentation by human intestinal microbiota in

60

mixed cultures.

4


Page 4 of 37

Page 5 of 37


61

MATERIALS AND METHODS

62

Materials and Chemicals. Bee collected pollens from Chinese wolfberry were kindly

63

provided by the National Engineering Research Center of Wolfberry (Yinchuan, China).

64

3-Methyl-1-phenyl-2-pyrazolin-5-one (PMP) and monosaccharide standards (arabinose,

65

fucose, galactose, galacturonic acid, glucose, glucuronic acid, mannose, rhamnose,

66

ribose and xylose) were purchased from Sigma Chemical Co., Ltd. (St. Louis, MO,

67

USA). HPLC grade of acetonitrile was purchased from TEDIA Co., Inc. (Fairfield,

68

USA). Gastric lipase, pepsin, pancreatin, trypsin and bile salt were purchased from

69

Solarbio Science & Technology Co., Ltd. (Beijing, China). Acetic, propionic, n-butyric,

70

i-butyric, n-valeric, i-valeric and 2-ethylbutyric acids were obtained from Aladdin

71

Industrial Inc. (Shanghai, China). Inulin was purchased from Nanjing Oddfoni

72

Biological Technology Co., Ltd. (Nanjing, China). 3,5-Dinitrosalicylic acid (DNS) and

73

other analytical reagents were obtained from Sinopharm Chemical Reagent Co., Ltd.

74

(Shanghai, China).

75

Preparation of WBPPS. The preparation of WBPPS was carried out according the

76

reported method17 with slight modification. Briefly, the powder of bee collected pollens

77

from Chinese wolfberry was extracted three times with distilled water (the ratio of water

78

to material 20 mL/g) at 85 ℃ for 2 h each, the extract was centrifuged at 4000 rpm for

79

20 min. The supernatants were combined and concentrated, the resulting solution was

80

mixed with a quadruple volume of absolute ethanol and kept overnight at 4 ℃. The

81

precipitates were collected, dissolved in distilled water, dialyzed for 48 h with distilled

82

water and lyophilized, affording crude WBPPS.

5



83

Characterization of WBPPS. The contents of total carbohydrates, protein and uronic

84

acid were measured by phenol-sulphuric acid, Bradford and m-hydroxydiphenyl assays,

85

respectively, according to the reported methods.18 The molecular weight of WBPPS was

86

determined by high-performance gel permeation chromatography (HPGPC) with a

87

Shimadzu LC-2A series apparatus (Shimadzu Corp., Kyoto, Japan) equipped with a TSK

88

G4000PWXL column (7.8 × 300 mm, Tosoh Crop., Tokyo, Japan) and evaporative

89

light-scattering detector (ELSD). Moreover, distilled water was used as the mobile phase

90

at a flow rate of 0.8 mL/min and the temperature of column oven was set at 35 ℃. The

91

monosaccharide composition of WBPPS was analyzed by HPLC according to the

92

reported method of pre-column derivatization with PMP.19

93

Simulated Saliva Digestion. The simulated saliva digestion was performed according

94

to the reported method15 with slight modification. Firstly, the fresh human saliva was

95

collected from a healthy donor who did not take antibiotics last three months. Attentively,

96

the donor should rinse his mouth and discard the initial 30 s saliva before collection. The

97

collected saliva was centrifuged (4000 rpm, 10 min) immediately and the supernatant

98

was used for simulated saliva digestion. The amylase activity of saliva was measured by

99

the reported method of van Ruth and Roozen.20 Secondly, WBPPS was dissolved in

100

deionized water at a concentration of 8.0 mg/mL. Tube A was the mixture of 4.0 mL

101

WBPPS solution with 4.0 mL of saliva, tube B was the mixture of 4.0 mL deionized

102

water and 4.0 mL saliva, and tube C was the mixture of WBPPS solution and 4.0 mL

103

deionized water. Thirdly, all the test tubes were kept at water bath oscillator (37 ℃, 100

104

rpm). During the incubation, sample (2.0 mL) for further analysis (content of reducing

6


Page 6 of 37

Page 7 of 37


105

sugars, content of released monosaccharides, molecular weight of polysaccharide) was

106

taken out at 0, 2 and 4 h, respectively, and immersed immediately into a boiling water

107

bath for 10 min to inactivate enzyme activity. Each experiment was repeated three times.

108

Simulated Gastric Digestion. The simulated gastric digestion was investigated based

109

on the reported method15,21 with some modifications. Firstly, gastric electrolyte solution

110

(GES, 250 mL) consisting of 775 mg NaCl, 37.5 mg CaCl2·2H2O, 275 mg KCl and 150

111

mg NaHCO3 was prepared. Secondly, 37.5 mg gastric lipase, 35.4 mg pepsin and 1.5 mL

112

CH3COONa were added to 150 mL GES and the mixture was gently mixed by magnetic

113

stirrer for 10 min. The pH of the resulting solution was adjusted to 3.0 by using 0.1 M

114

HCl solution, affording the simulated gastric juice. Thirdly, 16.0 mL of 8.0 mg/mL

115

WBPPS solution was mixed with 16.0 mL of simulated gastric juice for digestion in

116

water bath oscillator (37 ℃, 100 rpm), and the mixture of 16.0 mL distilled water and

117

16.0 mL simulated gastric juice was used as blank control. During the digestion, sample

118

(4.0 mL) for further analysis (content of reducing sugars, content of released

119

monosaccharides, molecular weight of polysaccharide) was taken out at 0, 2, 4 and 6 h,

120

respectively, and immersed immediately into a boiling water bath for 10 min to

121

inactivate enzyme activity. Each experiment was performed in triplicate.

122

Simulated Small Intestinal Digestion. The simulated small intestinal juice was

123

assembled according to the method described in the literature15,21 with little modification.

124

Firstly, intestinal electrolyte solution (IES, 250 mL), consisting of 1.35 g NaCl, 162.5

125

mg KCl and 82.5 mg CaCl2·2H2O, was prepared and the pH of IES was adjusted to 7

126

with 0.1 M NaOH solution. Secondly, 20 g of IES, 20 g of pancreatin solution (7%,

7



127

w/w), 40 g of bile salt solution (4%, w/w) and 2.6 mg of trypsin were mixed and the pH

128

of mixture was adjusted to 7.5 by 0.2 M NaOH solution, affording the simulated small

129

intestinal juice. Thirdly, the pH of gastric digested solution at 6 h of digestion was

130

adjusted to 7.0 and mixed with simulated small intestinal juice at a ratio of 10:3. Then,

131

the simulated small intestinal digestion was carried out as described above for simulated

132

gastric digestion.

133

In vitro Fermentation of WBPPS. In vitro fermentation of WBPPS by fresh human

134

feces was carried out according to the reported method22,23 with minor modification.

135

Firstly, 1.0 g WBPPS or inulin (positive control) was dissolved in 100 mL autoclaved

136

basal nutrient growth medium (2.0 g/L yeast extract, 2.0 g/L peptone, 0.1 g/L NaCl, 0.04

137

g/L KH2PO4, 0.04 g/L K2HPO4, 0.01 g/L MgSO4·7H2O, 0.01 g/L CaCl2, 2 g/L NaHCO3,

138

0.02 g/L hemin, 0.5 g/L cysteine-HCl, 0.5 g/L bile salts, 2.0 mL/L Tween 80, 1,0 mL/L

139

1% resazurin solution and 10 µL/L vitamin K) to afford a final concentration of 10.0

140

mg/mL (w/v). Additionally, basal nutrient growth medium without any other carbon

141

source was used as blank control. Secondly, the fresh human feces were obtained from 3

142

healthy volunteers who did not take any antibiotics over 3 months and the fecal slurry

143

(10%, w/v) was prepared by suspending feces in sterilized phosphate buffered saline

144

(0.1 M, pH 7.2). Thirdly, 1.0 mL 10% fecal slurry was added to 9.0 mL nutrient growth

145

medium containing WBPPS or inulin in 25 mL flask, and the fermentation was then

146

carried out in an Anaero Pack system (Mitsubishi Gas Chemical Co., Inc., Tokyo, Japan)

147

at 37 ℃. The samples at fermentation of 0, 6, 12 and 24 h were taken out for further

148

study. Each experiment was performed in triplicate.

8


Page 8 of 37

Page 9 of 37


149

Determination of Content of Reducing Sugar. The content of reducing sugar was

150

measured by the method of DNS with little modification.24 Briefly, 0.5 mL of sample

151

solution was mixed with 1.5 mL DNS reagent, and the mixture was heated at 100 ℃

152

for 15 min. After cooling, 8.0 mL distilled water was added and the absorbance of

153

mixture was determined at 550 nm by spectrophotometer (Shanghai Meipuda Instrument

154

Co., Ltd, Shanghai, China). In addition, the calibration curve was created by using

155

glucose as standard.

156

Determination of pH and SCFAs. The pH value of sample of in vitro fermentation

157

was measured by pH meter. The contents of SCFAs were determined by reported

158

method25 of gas chromatography (GC) with some modifications. Briefly, Agilent 6890 N

159

GC system equipped with a flame ionization detector (FID) and HP-INNOWax column

160

(30 m × 0.25 mm × 0.25 µm) was used. The operation conditions of GC were as follows:

161

the flow rate of carrier gas (nitrogen), 19 mL/min; initial column temperature, 100 ℃

162

for 1 min, then programmed to 180 ℃ at a rate of 4 ℃/min and held for 4 min; the

163

temperature of detector and injection port, 250 ℃; the flow rates of hydrogen, air and

164

nitrogen makeup gas in detector, 30, 260 and 30 mL/min, respectively. In the present

165

study, the content of lactic acid was determined by lactic acid test Kit (Nanjing

166

Jiancheng Bioengineering Institute, Nanjing, China). Each measurement was performed

167

for three times.

168

Analysis of Gut Microbiota. In the present study, it included 4 groups of in vitro

169

fermentation: original fecal slurry group (OR group), fermentation in vitro for 24 h

170

without addition any carbon source group (BLK group), fermentation in vitro for 24 h

9



Page 10 of 37

171

with addition of WBPPS (WBPPS group) and fermentation in vitro for 24 h with

172

addition of inulin (INL group). The total bacterial DNA of each group was extracted by a

173

TIANamp Stool DNA Kit (Tiangen Biotech Co., Ltd., Beijing, China) and all the

174

extracted DNA samples were sent to Shanghai GeneSky Biological Technology Co., Ltd

175

for analysis of microbiota composition. For high-throughput sequencing, the V4 region

176

of 16S rDNA was chosen for amplification and sequenced by Illumina Miseq.

177

Furthermore, all the results were based on sequenced reads and operational taxonomic

178

units (OTUs). Alpha diversity was analyzed by Mothur. The bar blot of gut community

179

composition, principal component analysis (PCA) and cluster analysis were performed

180

by R packages (V2.15.3, http://www.r-project.org/). Heatmap was performed using

181

HemI 1.0.26

182

Statistical Analysis. The experimental data are expressed as mean ± standard deviation

183

(SD) of triplicates. The data were evaluated by using SPSS and any difference was

184

analyzed by one way analysis of variance (ANOVA) followed by Duncan’s

185

multiple-range test. P < 0.05 was regarded as statistically significant difference.

186

RESULTS AND DISCUSSION

187

WBPPS was prepared from bee collected pollen of Chinese wolfberry through hot water

188

extraction, ethanol precipitation and freeze-drying. The contents of total carbohydrates,

189

uronic acid and protein for WBPPS were 61.29 ± 1.59%, 23.98 ± 1.13% and 14.22 ±

190

0.26%, respectively. WBPPS was mainly composed of mannose, ribose, rhamnose,

191

galacturonic acid, glucose, galactose, xylose and arabinose in a molar ratio of 0.38: 0.09:

192

0.17:

0.64:

0.22:

0.67:

0.08:

1.03,

respectively,

10


indicating

that

it

was

Page 11 of 37


193

heteropolysaccharide. Moreover, two peaks were observed in the HPGPC chromatogram

194

of WBPPS (Figure 1a), and the molecular weights for fractions A (major fraction,

195

87.02%) and B (13.98%) were 1340.1 ± 45.8 and 523.2 ± 13.7 kDa, respectively.

196

Digestion of WBPPS by Saliva. Saliva is the first digestive juice contacting with

197

sample and its main content is salivary amylase which can hydrolyze α-(1→4) linkages

198

in starch and other carbohydrate.21 In this study, the amylase activity of the collected

199

saliva was determined to be 105 ± 4 D units/mL, which is in the range of normal value

200

of salivary amylase activity (18-208 D units/mL) reported by van Ruth and Roozen.20 As

201

shown in Figure 1b, the retention times and areas of the peaks for fractions A and B did

202

not change after treatment by saliva, indicating that the human saliva had no effect on

203

digestion of WBPPS. Furthermore, there was not significant difference in the contents of

204

reducing sugars before and after treatment by saliva (Table 1), illustrating that WBPPS

205

could not be degraded by saliva.

206

Digestion of WBPPS under Simulated Gastric and Small Intestinal Conditions. As

207

illustrated in Figure 1c, the retention time and area of the peak for fraction A was not

208

changed within 6 h under the simulated gastric condition, indicating that fraction A was

209

not influenced by simulated gastric juice. However, it was found that fraction B was

210

gradually degraded under the simulated gastric condition, and a new peak (fraction C)

211

was observed. The molecular weight of fraction C, one degraded product from fraction

212

B of WBPPS, was determined to be 2.68 ± 0.36 kDa. Besides, the content of reducing

213

sugars was significantly increased due to the simulated gastric digestion (Table 1).

214

However, no free monosaccharide was found to be generated from WBPPS during the

11



215

whole process of gastric digestion (Figure 2a). This result is consistent with a previous

216

report that the polysaccharide from G. atrum exhibited a little extent of change but no

217

free monosaccharide generated throughout the simulated gastric digestion.27 Therefore,

218

it was speculated that the glycosidic bonds of fraction B was probably broken under the

219

simulated gastric condition. So that the degradation of fraction B did not generate free

220

monosaccharide and induce the increment of reducing sugars.

221

As presented in Figure 1d, the retention time area of the peak for fraction A did not

222

change after the simulated small intestinal digestion, demonstrating that fraction A was

223

also not affected by simulated small intestinal juice. But it was found that fraction C was

224

shifted to fraction D (molecular weight, 1.27 ± 0.03 kDa) due to simulated small

225

intestinal digestion. Furthermore, the data from Figure 2b and Table 1 suggested that

226

there was no free monosaccharide released from WBPPS during the simulated small

227

intestinal digestion. It provided similar information in accordance with WBPPS

228

digestion in simulated gastric juice. So it was concluded that both simulated gastric and

229

small intestinal juices had little effect on WBPPS degradation.

230

According to the results mentioned above, it was considered that WBPPS could be

231

delivered to the colon in a relatively unaltered state, and it was expected that gut

232

microbiota could utilize WBPPS. Thus, the fermentation in vitro of WBPPS by fresh

233

human feces was investigated in the present study to evaluate possible effects on

234

production of SCFAs and gut microbiota due to the fermentation of WBPPS.

235

Effects of WBPPS Fermentation in vitro on SCFAs Production. As shown in Figure

236

3a, WBPPS was gradually degraded during the fermentation in vitro, indicating that

12


Page 12 of 37

Page 13 of 37


237

WBPPS could be utilized by intestinal microbiota. Meanwhile, as illustrated in Figure

238

3b, the pH values of BLK, WBPPS and INL groups significantly declined from 8.93 to

239

7.59 (△pH = 1.34), 7.95 to 5.49 (△pH = 2.46) and 8.90 to 4.15 (△pH = 4.75),

240

respectively, after 24 h fermentation. The results demonstrated that the addition of

241

WBPPS had a greater impact on the pH value of gut environment compared with BLK

242

group. In addition, the initial pH value of WBPPS group showed significant difference

243

compared with that of blank or inulin group, this was probably associated with the high

244

content of uronic acid in WBPPS (23.98 ± 1.13%). Furthermore, the pH value of

245

WBPPS group was lower than that of BLK group at the same fermentation time,

246

whereas it was higher than that of INL group. It was speculated that the pH value was

247

correlated to the level of SCFAs produced during the in vitro fermentation.

248

As shown in Figure S1 (supplementary material), 6 kinds of SCFAs were effectively

249

separated by GC with HP-INNOWax column and all were detected in WBPPS

250

fermentation liquor at 24 h. Through fermentation, the concentration of total SCFAs in

251

WBPPS group increased from 6.40 ± 0.32 (Table 2) to 44.97 ± 1.30 mM (24 h), which

252

was significantly higher than that of BLK group. The results suggested that WBPPS

253

could enhance the production of SCFAs. Compared with BLK group, WBPPS

254

significantly enhanced the production of acetic and propionic acids. It has been reported

255

that acetic and propionic acids are beneficial for host health. For example, acetic acid

256

can be used as the energy source for gut microbiota28 and peripheral tissues.29 Propionic

257

acid can lower serum cholesterol level30 and protect against diet-induced obesity.31

258

It is interesting to note that the concentration of lactic acid in WBPPS fermentation

13



259

increased from 0.13 ± 0.03 (0 h) to 1.69 ± 0.08 mM (6 h), but it then gradually

260

decreased to 0.48 ± 0.05 mM (24 h). It might be that lactic acid produced was converted

261

into other SCFAs by human gut microbiota. It has been reported that lactate could be

262

used as a precursor for butyrate biosynthesis in colon.32 Moreover, it was found that the

263

concentrations of i-valeric and n-valeric acids in BLK group were nearly 3.5- and

264

1.5-folds higher than those of WBPPS group at 24 h fermentation, respectively. It is

265

easy to understand that several amino acids released from proteins can be precursors for

266

branched-chain fatty acids synthesis, such as valerate.33 Therefore, the higher

267

concentration of valeric acid in BLK group might be resulted from the metabolism of

268

protein (without addition of carbon source in BLK group). For INL group, it exhibited

269

the most abundant of lactic and acetic acids and lowest of i-butyric, n-butyric, i-valeric

270

and n-valeric acids at each fermentation time point. In a word, WBPPS could promote

271

the generation of SCFAs with similar effect as inulin.

272

Effects of WBPPS Fermentation in vitro on Gut Microbiota. In the present study,

273

the effects of WBPPS on gut microbiota were investigated by in vitro fermentation with

274

fresh human feces and high throughput sequencing analysis. The sequencing analyses

275

included 12 samples (4 groups, each groups repeated 3 times) and a total of 565851

276

usable raw reads were obtained. After the raw reads got filtered and clustered with 97%

277

similarity, 153 ± 29 OTUs per sample were obtained. As exhibited in Figure S2, both

278

sample Rarefaction curve and Shannon curve gradually reached to a stable state,

279

indicating that data size was big enough to reflect mostly biological information in each

280

sample and more reads only produced fewer OTUs. Moreover, the coverage rates for the

14


Page 14 of 37

Page 15 of 37


281

samples all reached over 99.89%, indicating that the data for analysis were reasonable.

282

The alpha diversity of each group is shown in Table 3. For Chao 1 index and ACE

283

index, it could estimate the community richness in samples. For Shannon index and

284

Simpson index, it could estimate the community diversity in samples. According to the

285

data in Table 3, no significant differences existed among BLK, WBPPS and INL groups

286

for community richness. However, the community diversities for INL and WBPPS

287

groups were lower than that for BLK group. The similar result on gut community

288

diversity of Lentinula edodes-derived polysaccharide34 or Chinese yam polysaccharide35

289

was observed. It might be correlated with the fact that WBPPS and inulin could promote

290

the growth of beneficial gut microbiota and inhibit bacterial pathogens.

291

PCA and cluster analysis were used to identify the overall difference of gut

292

microbial community among different treatment groups. As illustrated in Figure 3c, it

293

showed a distinct separation for 4 groups and the first two axes could explain 89.18%

294

variation for different groups. Horizontal axis represents the first principal component,

295

which elucidated 70.08% influence on gut community structure by different treatments.

296

On PC1, the distances for OR, WBPPS and INL groups were close, manifesting that the

297

three groups had relative similar community structure compared with BLK group. In

298

addition, WBPPS and INL groups were dispersed in the second principal component,

299

demonstrating that WBPPS and inulin exhibited different impacts on gut microbiota.

300

Moreover, cluster analytical results (Figure 3d) that built by using the Bray-Curtis

301

distance are consistent with the results of PCA, further explaining that WBPPS had great

302

influence on gut microbiota community.

15



303

As shown in Figure 4a, all the groups were mainly consisted of Firmicutes,

304

Bacteroidetes, Proteobacteria at the phylum level. Notably, the relative abundances of

305

Firmicutes and Bacteroidetes changed greatly after 24 h fermentation in vitro. It has

306

been reported that the ratio of F/B is closely associated with obesity, because the two

307

bacteria have different efficiency on energy extraction from diet and possess different

308

effects on metabolism of absorbed calories to host.36 In addition, F/B ratio is found to be

309

in high level in obese individuals37 although opposite trend reported.38 As displayed in

310

Figure 4b, WBPPS and inulin reduced greatly the ratio of F/B compared with BLK,

311

which suggested WBPPS and inulin could influence host health by decreasing the ratio

312

of F/B. Besides, Fusobacteria and Actinobacteria were more abundant in BLK and INL

313

groups, respectively. The results indicated that Fusobacteria would be excessive growth

314

without carbon source (in BLK group) and Actinobacteria could be promoted by inulin.

315

As it can be seen from Figure 4c, 45 kinds of main genera were found in human gut

316

feces. Among them, Prevotella, Bacteroides, Faecalibacterium, Dialister, Clostridium

317

XIVa, Megamonas, Alloprevotella, Lachnospiracea

318

Parasutterella, Phascolarctobacterium, Escheriachia/Shigella and Bifidobacterium

319

were the dominating genera in gut. In order to find the primary differences of

320

community composition among OR, BLK, WBPPS and INL groups, those genera that

321

relative abundance below 1% were excluded from the groups. As a result, 16 varieties of

322

genus were selected for comparison. Compared with BLK group, 5 genera (Prevotella,

323

Dialister, Megamonas, Faecalibacterium and Alloprevotella) in WBPPS group were

324

significantly higher (Table 4), while 8 genera (Bacteroides, Clostridium XlVa,

incertae sedis, Parabacteroides,

16


Page 16 of 37

Page 17 of 37


325

Parabacteroides,


Phascolarctobacterium,

326

Clostridium sensu stricto and Fusobacterium) were significantly lower.

Parasutterella,

327

It is obvious that Prevotella in WBPPS and INL groups was far more abundant than

328

that in BLK group, indicating that the addition of WBPPS and inulin could largely

329

promote the growth of Prevotella during fermentation in vitro. It has been reported that

330

high-fiber diet was strongly related to an increase in Prevotella.39 Prevotella potentially

331

contains a set of genes or enzymes for polysaccharide degradation and then the products

332

produced are utilized for the growth of Prevotella. For example, Filippo et al.40 found

333

that genera of Prevotella and Xylanibacter with some certain genes for the hydrolysis of

334

cellulose and xylan were exclusively present in gut microbiota of rural Africa children

335

whose diet rich-in starch, fiber and plant polysaccharide. Fuse et al.41 reported that

336

fructanase, sugar transporter and fructokinase proteins were involved in Fructan

337

utilization by Prevotella intermedia. In addition, the increase of Prevotella influences

338

host health. Kovatcheva-Datchary et al.42 found that the increase of Prevotella could

339

protect against Bacteroides-induced glucose intolerance and increase glycogen storage

340

in some certain individuals. Thus, the increase of Prevotella might regulate blood

341

glucose and reduces the risk of diabetes.

342

Faecalibacterium, like Faecalibacterium prausnitzii, is generally considered as one

343

of the most important bacteria in a healthy individual gut. Many surveys demonstrated

344

that Faecalibacterium prausnitzii was associated with anti-inflammatory activity.43

345

Moreover, Faecalibacterium prausnitzii could produce butyrate which had great benefits

346

for host, such as providing energy for colonic epithelium cells, inhibiting inflammation

17



Page 18 of 37

347

and modulating oxidative stress.44 Compared with BLK group, WBPPS group showed

348

the highest relative abundance of Faecalibacterium prausnitzii. However, it is strange

349

that the concentration of n-butyric acid in WBPPS and BLK groups had no significant

350

difference but i-butyric acid was significantly higher in BLK group. It has been reported

351

that

352

butyryl-CoA:acetate CoA-transferase route.45 Fusobacterium, especially Fusobacterium

353

varium, also could synthesize butyrate with amino acids released from protein.46

354

Therefore, the higher concentration of butyric acid in BLK group was probably

355

associated with the increase of the two bacteria. In addition, Dialister is a producer of

356

propionic acid47 and the higher concentration of propionate in WBPPS group might be

357

the consequence of growth of this genus.

Clostridium

XIVa

had

the

ability

to

produce

butyrate

through

the

358

Bacteroides fragilis, belonging to genus Bacteroides, was the most species in BLK

359

group. It could protect against and curve intestinal inflammation.48 However, some other

360

researches reported that enterotoxigenic Bacteroides fragilis was associated with

361

colorectal cancer and inflammatory diarrhea.49,50 Besides, Clostridium XlVa like

362

Clostridium clostridioforme,51 Escherichia/Shigella like Escherichia coli,52 Clostridium

363

sensu stricto like Clostridium perfringens,53 Fusobacterium like Fusobacterium

364

varium54 are usually regarded as opportunistic pathogen. Compared with BLK group,

365

the four species were much less in WBPPS group. Therefore, it concluded that WBPPS

366

could improve the composition of gut microbiota by preventing the growth of pathogen.

367

In conclusion, WBPPS was not digested by saliva, but one small fraction of WBPPS

368

(fraction B) was degraded under simulated gastric and small intestinal conditions. Most

18


Page 19 of 37


369

part of WBPPS could pass through the digestive system without being broken down and

370

reach the large intestine safely. Through fermentation, WBPPS affected the ecosystem of

371

the intestinal tract by promoting the production of SCFAs, especially acetic and

372

propionic acids. Furthermore, it was found that WBPPS could shape the gut microbiota,

373

mainly increasing the genus of Prevotella, Dialister, Megamonas, Faecalibacterium,

374

Alloprevotella and decreasing the genus of Bacteroides, Clostridium XlVa,

375

Parabacteroides,

376

Clostridium sensu stricto and Fusobacterium. The results suggested that WBPPS had the

377

potential to be developed as functional ingredients to improve human health and prevent

378

disease through promoting the gut health.

379

ASSOCIATED CONTENT

380

Supporting information

381

Gas chromatograms of short-chain fatty acids for standard solution and WBPPS after

382

fermentation, samples Rarefaction curves and Shannon curves for each treatment groups

383

AUTHOR INFORMATION

384

Corresponding Authors

385

*Phone: +86-25-84396791. E-mail: [email protected] (X Zeng).

386

*E-mail: [email protected] (Y Cao).

387

ORCID

388

Xiaoxiong Zeng: 0000-0003-2954-3896

389

Funding


Phascolarctobacterium,

19


Parasutterella,


390

This work was supported by Primary, Secondary and Tertiary Industry Integration

391

Project of Ningxia Hui Autonomous Region (YES-16-0506), Pilot Project by Ningxia

392

Academy of Agriculture and Forestry Science (NKYZ-16-05 and JLC01) and a project

393

funded by the Priority Academic Program Development of Jiangsu Higher Education

394

Institutions (PAPD).

395

Notes

396

The authors declare no competing financial interest.

397

REFERENCE

398

1.

399

Kubina, R.; Moździerz, A.; Buszman, E. Polyphenols from bee pollen: Structure,

400

absorption, metabolism and biological activity. Molecules 2015, 20, 21732-21749.

401

2.

402

pollen: A review. J. Sci. Food Agric. 2016, 96, 4303-4309.

403

3. Yang, K.; Wu, D.; Ye, X.; Liu, D.; Chen, J.; Sun, P. Characterization of chemical

404

composition of bee pollen in China. J. Agric. Food Chem. 2013, 61, 708-718.

405

4.

406

of a new water-soluble bee pollen polysaccharide from Crataegus pinnatifida Bge.

407

Carbohydr. Polym. 2009, 78, 80-88.

408

5.

409

activity of Sonoran Desert bee pollen. Food Chem. 2009, 115, 1299-1305.

410

6.

411

polysaccharides of pine pollen on mouse macrophages. Int. J. Biol. Macromol. 2016, 91,

Rzepeckastojko, A.; Stojko, J.; Kurekgórecka, A.; Górecki, M.; Kabaładzik, A.;

Denisow, B.; Denisow-Pietrzyk, M. Biological and therapeutic properties of bee

Li, F.; Yuan, Q. P.; Rashid, F. Isolation, purification and immunobiological activity

Blaisew, L. B.; Owenk, D.; Stephen, B.; Anthony, D. L.; Thomas, D. Antioxidant

Geng, Y.; Xing, L.; Sun, M. M.; Su, F. C. Immunomodulatory effects of sulfated

20


Page 20 of 37

Page 21 of 37


412

846-855.

413

7.

414

of bee pollen polysaccharides from Rosa rugosa. Mol. Med. Rep. 2013, 7, 1555-1558.

415

8.

416

polysaccharides. Biotechnol. Adv. 2013, 31, 318-337.

417

9. Chen, C.; Huang, Q.; Fu, X.; Liu, R. H. In vitro fermentation of mulberry fruit

418

polysaccharides by human fecal inocula and impact on microbiota. Food Funct. 2016, 7,

419

4637-4643.

420

10. Chen, G. J.; Xie, M. H.; Wan, P.; Chen, D.; Ye, H.; Chen, L. G.; Zeng, X. X.; Liu, Z.

421

H. Digestion under saliva, simulated gastric and small intestinal conditions and

422

fermentation in vitro by human intestinal microbiota of polysaccharides from Fuzhuan

423

brick tea. Food Chem. 2018, 244, 331-339.

424

11. Chen, T. T.; Long, W. M.; Zhang, C. H.; Liu, S.; Zhao, L. P.; Hamaker, B. R.

425

Fiber-utilizing capacity varies in Prevotella- versus Bacteroides-dominated gut

426

microbiota. Sci. Rep. 2017, 7, 2594 (Doi: 10.1038/s41598-017-02995-4).

427

12. Chang, C. J.; Lin, C. S.; Lu, C. C.; Martel, J.; Ko, Y. F.; Ojcius, D. M.; Tseng, S. F.;

428

Wu, T. R.; Chen, Y. Y. M.; Young, J. D.; Lai, H. C. Ganoderma lucidum reduces

429

obesity in mice by modulating the composition of the gut microbiota. Nat. Commun.

430

2015, 6, 7489 (Doi: 10.1038/ncomms8489).

431

13. Ma, G. X.; Kimatu, B. M.; Zhao, L. Y.; Yang, W. J.; Pei, F.; Hu, Q. H. In vivo

432

fermentation of a Pleurotus eryngii polysaccharide and its effects on fecal microbiota

433

composition and immune response. Food Funct. 2017, 8, 1810-1821.

Wang, B.; Diao, Q.; Zhang, Z.; Liu, Y.; Gao, Q.; Zhou, Y.; Li, S. Antitumor activity

Xu, X.; Xu, P.; Ma, C.; Tang, J.; Zhang, X. Gut microbiota, host health, and

21



434

14. Cockburn, D. W.; Koropatkin, N. M. Polysaccharide degradation by the intestinal

435

microbiota and its influence on human health and disease. J. Mol. Biol. 2016, 428,

436

3230-3252.

437

15. Hu, J. L.; Nie, S. P.; Min, F. F.; Xie, M. Y. Artificial simulated saliva, gastric and

438

intestinal digestion of polysaccharide from the seeds of Plantago asiatica L. Carbohydr.

439

Polym. 2013, 92, 1143-1150.

440

16. Carnachan, S. M.; Bootten, T. J.; Mishra, S.; Monro, J. A.; Simsa, I. M. Effects of

441

simulated digestion in vitro on cell wall polysaccharides from kiwifruit (Actinidia spp.).

442

Food Chem. 2012, 133, 132-139.

443

17. Fan, J.; Wu, Z.; Zhao, T.; Sun, Y.; Ye, H.; Xu, R.; Zeng, X. X. Characterization,

444

antioxidant and hepatoprotective activities of polysaccharides from Ilex latifolia Thunb.

445

Carbohydr. Polym. 2014, 101, 990-997.

446

18. Wang, M.; Jiang, C.; Ma, L.; Zhang, Z.; Cao, L.; Liu, J.; Zeng, X. X. Preparation,

447

preliminary characterization and immunostimulatory activity of polysaccharide fractions

448

from the peduncles of Hovenia dulcis. Food Chem. 2013, 138, 41-47.

449

19. Yuan, Q.; Xie, Y.; Wang, W.; Yan, Y.; Ye, H.; Jabbar, S.; Zeng, X. X. Extraction

450

optimization, characterization and antioxidant activity in vitro of polysaccharides from

451

mulberry (Morus alba L.) leaves. Carbohydr. Polym. 2015, 128, 52-62.

452

20. van Ruth, S. M.; Roozen, J. P. Influence of mastication and saliva on aroma release

453

in a model mouth system. Food Chem. 2000, 71, 339-345.

454

21. Chen, C.; Zhang, B.; Fu, X.; You, L. J.; Abbasi, A. M.; Liu, R. H. The digestibility

455

of mulberry fruit polysaccharides and its impact on lipolysis under simulated saliva,

22


Page 22 of 37

Page 23 of 37


456

gastric and intestinal conditions. Food Hydrocolloids 2016, 58, 171-178.

457

22. Li, W.; Wang, K.; Sun, Y.; Ye, H.; Hu, B.; Zeng, X. X. Influences of structures of

458

galactooligosaccharides and fructooligosaccharides on the fermentation in vitro by

459

human intestinal microbiota. J. Funct. Foods 2015, 13, 158-168.

460

23. Xie, M.; Chen, G.; Hu, B.; Zhou, L.; Ou, S.; Zeng, X. X.; Sun, Y. Hydrolysis of

461

dicaffeoylquinic acids from Ilex kudingcha happens in the colon by intestinal

462

microbiota. J. Agric. Food Chem. 2016, 64, 9624-9630.

463

24. Miller, G. L. Use of dinitrosalicylic acid reagent for determination of reducing

464

sugar. Anal. Chem. 1959, 31, 426-428.

465

25. Hu, J. L.; Nie, S. P.; Li, C.; Xie, M. Y. In vitro fermentation of polysaccharide from

466

the seeds of Plantago asiatica L. by human fecal microbiota. Food Hydrocolloids 2013,

467

33, 384-392.

468

26. Deng, W.; Wang, Y.; Liu, Z.; Cheng, H.; Xue, Y. HemI: A toolkit for illustrating

469

heatmaps. PLoS One 2014, 9, 11 (Doi: 10.1371/journal.pone.0111988).

470

27. Ding, Q.; Nie, S.; Hu, J.; Zong, X.; Li, Q.; Xie, M. In vitro and in vivo

471

gastrointestinal digestion and fermentation of the polysaccharide from Ganoderma

472

atrum. Food Hydrocolloids 2017, 63, 646-655.

473

28. Peng, X.; Li, S.; Luo, J.; Wu, X.; Liu, L. Effects of dietary fibers and their mixtures

474

on short chain fatty acids and microbiota in mice guts. Food Funct. 2013, 4, 932-938.

475

29. Wong, J. M.; de Souza, R.; Kendall, C. W.; Emam, A.; Jenkins, D. J. Colonic

476

health: Fermentation and short chain fatty acids. J. Clin. Gastroenterol. 2006, 40,

477

235-243.

23



Page 24 of 37

478

30. Hosseini, E.; Grootaert, C.; Verstraete, W.; Van de Wiele, T. Propionate as a

479

health-promoting microbial metabolite in the human gut. Nutr. Rev. 2011, 69, 245-258.

480

31. Lin, H. V.; Frassetto, A.; Kowalik, E. J.; Nawrocki, A. R.; Lu, M. F. M.; Kosinski,

481

J. R.; Hubert, J. A.; Szeto, D.; Yao, X. R.; Forrest, G.; Marsh, D. J. Butyrate and

482

propionate protect against diet-induced obesity and regulate gut hormones via free fatty

483

acid

484

(doi:10.1371/journal.pone.0035240).

485

32. Bourriaud, C.; Robins, R. J.; Martin, L.; Kozlowski, F.; Tenailleau, E.; Cherbut, C.;

486

Michel, C. Lactate is mainly fermented to butyrate by human intestinal microfloras but

487

inter-individual variation is evident. J. Appl. Microbiol. 2005, 99, 201-212.

488

33. Davila, A. M.; Blachier, F.; Gotteland, M.; Andriamihaja, M.; Benetti, P. H.; Sanz,

489

Y.; Tome, D. Intestinal luminal nitrogen metabolism: Role of the gut microbiota and

490

consequences for the host. Pharmacol. Res. 2013, 68, 95-107.

491

34. Xu, X.; Zhang, X. Lentinula edodes-derived polysaccharide alters the spatial

492

structure of gut microbiota in mice. PLoS One 2015, 10, e0115037 (Doi:

493

10.1371/journal.pone.0115037).

494

35. Kong, X. F.; Zhang, Y. Z.; Wu, X.; Yin, Y. L.; Tan, Z. L.; Feng, Y.; Yan, F. Y.; Bo,

495

M. J.; Huang, R. L.; Li, T. J. Fermentation characterization of Chinese yam

496

polysaccharide and its effects on the gut microbiota of rats. Int. J. Microbiol. 2009,

497

598152 (Doi:10.1155/2009/598152).

498

36. Million, M.; Lagier, J. C.; Yahav, D.; Paul, M. Gut bacterial microbiota and obesity.

499

Clin. Microbiol. Infect. 2013, 19, 305-313.

receptor

3-independent

mechanisms.

PLoS

24


One

2012,

7,

e35240

Page 25 of 37


500

37. Ismail, N. A.; Ragab, S. H.; Abd ElBaky, A.; Shoeib, A. R. S.; Alhosary, Y.; Fekry,

501

D. Frequency of Firmicutes and Bacteroidetes in gut microbiota in obese and normal

502

weight Egyptian children and adults. Arch. Med. Sci. 2011, 7, 501-507.

503

38. Schwiertz, A.; Taras, D.; Schafer, K.; Beijer, S.; Bos, N. A.; Donus, C.; Hardt, P. D.

504

Microbiota and SCFA in Lean and Overweight Healthy Subjects. Obesity 2010, 18,

505

190-195.

506

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

507

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

508

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

509

dietary patterns with gut microbial enterotypes. Science 2011, 334, 105-108.

510

40. Filippo, C. D.; Cavalieri, D.; Paola, M. D.; Ramazzotti, M.; Poullet, J. B.; Massart,

511

S.; Collini, S.; Pieraccini, G.; Lionetti, P. Impact of diet in shaping gut microbiota

512

revealed by a comparative study in children from Europe and rural Africa. Proc. Natl.

513

Acad. Sci. USA 2010, 107, 14691-14696.

514

41. Fuse, H.; Fukamachi, H.; Inoue, M.; Igarashi, T. Identification and functional

515

analysis of the gene cluster for fructan utilization in Prevotella intermedia. Gene 2013,

516

515, 291-297.

517

42. Kovatcheva-Datchary, P.; Nilsson, A.; Akrami, R.; Lee, Y. S.; Devadder, F.; Arora,

518

T.; Hallen, A.; Martens, E.; Björck, I.; Bäckhed, F. Dietary fiber-induced improvement

519

in glucose metabolism is associated with increased abundance of Prevotella. Cell Metab.

520

2015, 22, 971-982.

521

43. Martín, R.; Miquel, S.; Chain, F.; Natividad, J. M.; Jury, J.; Lu, J.; Sokol, H.;

25



522

Theodorou, V.; Bercik, P.; Verdu, E. F. Faecalibacterium prausnitzii prevents

523

physiological damages in a chronic low-grade inflammation murine model. BMC

524

Microbiol. 2015, 15, 67 (Doi:10.1186/s12866-015-0400-1).

525

44. Hamer, H. M.; Jonkers, D.; Venema, K.; Vanhoutvin, S.; Troost, F. J.; Brummer, R.

526

J. Review article: the role of butyrate on colonic function. Aliment. Pharmacol. Ther.

527

2008, 27, 104-119.

528

45. Hippe, B.; Zwielehner, J.; Liszt, K.; Lassl, C.; Unger, F.; Haslberger, A. G.

529

Quantification of butyryl CoA:acetate CoA-transferase genes reveals different butyrate

530

production capacity in individuals according to diet and age. FEMS Microbiol. Lett.

531

2011, 316, 130-135.

532

46. Potrykus, J.; White, R. L.; Bearne, S. L. Proteomic investigation of amino acid

533

catabolism in the indigenous gut anaerobe Fusobacterium varium. Proteomics 2008, 8,

534

2691-2703.

535

47. Fernández, J.; Redondo-Blanco, S.; Gutiérrez-Del-Río, I.; Miguélez, E. M.; Villar,

536

C. J.; Lombó, F. Colon microbiota fermentation of dietary prebiotics towards

537

short-chain fatty acids and their roles as anti-inflammatory and antitumour agents: A

538

review. J. Funct. Foods 2016, 25, 511-522.

539

48. Chiu, C. C.; Ching, Y. H.; Wang, Y. C.; Liu, J. Y.; Li, Y. P.; Huang, Y. T.; Chuang,

540

H. L. Monocolonization of germ-free mice with Bacteroides fragilis protects against

541

dextran sulfate sodium-induced acute colitis. BioMed Res. Int. 2014, 2014, 675786

542

(Doi:org/10.1155/2014/675786).

543

49. Ulger, T. N.; Yagci, A.; Gulluoglu, B. M.; Akin, M. L.; Demirkalem, P.; Celenk, T.;

26


Page 26 of 37

Page 27 of 37


544

Soyletir, G. A possible role of Bacteroides fragilis enterotoxin in the aetiology of

545

colorectal cancer. Clin. Microbiol. Infect. 2006, 12, 782-786.

546

50. Chen, L. A.; Meerbeke, S. V.; Albesiano, E.; Goodwin, A.; Wu, S.; Yu, H.; Carroll,

547

K.; Sears, C. Fecal detection of enterotoxigenic Bacteroides fragilis. Eur. J. Clin.

548

Microbiol. Infect. Dis. 2015, 34, 1871-1877.

549

51. Finegold, S. M.; Song, Y.; Liu, C.; Hecht, D. W.; Summanen, P.; Könönen, E.;

550

Allen, S. D. Clostridium clostridioforme: a mixture of three clinically important species.

551

Eur. J. Clin. Microbiol. Infect. Dis. 2005, 24, 319-324.

552

52. Martinez-Medina, M.; Aldeguer, X.; Lopez-Siles, M.; Gonzalez-Huix, F.;

553

Lopez-Oliu, C.; Dahbi, G.; Blanco, J. E.; Blanco, J.; Garcia-Gil, L. J.;

554

Darfeuille-Michaud, A. Molecular diversity of Escherichia coli in the human gut: New

555

ecological evidence supporting the role of adherent-invasive E. coli (AIEC) in Crohn's

556

disease. Inflamm. Bowel Dis. 2009, 15, 872-882.

557

53. Ohtani, K.; Hirakawa, H.; Paredessabja, D.; Tashiro, K.; Kuhara, S.; Sarker, M. R.;

558

Shimizu, T. Unique regulatory mechanism of sporulation and enterotoxin production in

559

clostridium perfringens. J. Bacteriol. 2013, 195, 2931-2936.

560

54. Ohkusa, T.; Okayasu, I.; Ogihara, T.; Morita, K.; Ogawa, M.; Sato, N. Induction of

561

experimental ulcerative colitis by Fusobacterium varium isolated from colonic mucosa

562

of patients with ulcerative colitis. Gut 2003, 52, 79-83.

27



Figure Captions Figure 1.

High-performance gel permeation chromatograms of WBPPS (a) and

WBPPS after digestion in saliva (b), simulated gastric juice (c) and simulated small intestinal juice (d). Figure 2. High performance liquid chromatograms of PMP derivatives of released free monosaccharides from WBPPS after digestion in simulated gastric juice (a) and simulated small intestinal juice (b). 1, mannose; 2, ribose; 3, rhamnose; 4, glucuronic acid; 5, galacturonic acid; 6, glucose; 7, galactose; 8, xylose; 9, arabinose; 10, fucose. Figure 3. High-performance gel permeation chromatogram of WBPPS after fermented in vitro at different time points (a); pH value fermented at 0, 6, 12 and 24 h, different letters indicating significant difference (P < 0.05) for different group (b); comparison of overall bacterial composition between each treatment groups by PCA (c) and cluster analysis (d). Figure 4. Gut microbial composition at phylum level (a), ratio of Firmicutes to Bacteroidetes (b) and Heat map of gut microbial composition at genus level (c).

28


Page 28 of 37

Page 29 of 37


Table 1 Contents of Reducing Sugars in Digested Solutions of WBPPS at Different Time Points under Saliva, Simulated Gastric and Small Intestinal Conditions. Content of reducing sugars a (mg/mL) Samples 0h

2h

4h

6h

Saliva

0.062 ± 0.003 a

0.068 ± 0.004 a

0.066 ± 0.003 a

/

Gastric juice

0.165 ± 0.004 a

0.179 ± 0.008 b

0.183 ± 0.006 b

0.190 ± 0.009 b

0.245 ± 0.003 a

0.264 ± 0.007 b

0.271 ± 0.012 b

0.268 ± 0.013 b

Small intestine juice a

Different letter means significant difference was observed in the simulated digestion process at

different duration.

29



Page 30 of 37

Table 2 Concentrations of SCFAs in Fermentation Solutions at Different Time Points of Fermentation in vitro. SCFAs (mM)

Sample

Acetic acid

Blank

Propionic acid

i-Butyric acid

n-Butyric acid

i-Valeric acid

n-Valeric acid

Lactic acid

Total

a

Anaerobic fermentation time a (h) 0

6

12

24

4.10 ± 0.25 A

7.40 ± 0.24 aB

11.14 ± 0.13 aC

14.81 ± 0.08 aD

WBPPS

12.20 ± 0.53 bB

17.63 ± 0.12 bC

24.13 ± 1.08 bD

Inulin

15.79 ± 0.46 cB

20.15 ± 0.60 cC

26.12 ± 0.83 cD

3.52 ± 0.22 aB

5.24 ± 0.22 aC

6.48 ± 0.09 aD

WBPPS

6.17 ± 0.07 bB

10.86 ± 0.33 cC

16.73 ± 0.29 cD

Inulin

6.52 ± 0.43 bB

9.04 ± 0.36 bC

12.60 ± 0.71 bD

0.36 ± 0.03 bA

0.50 ± 0.03 bB

0.58 ± 0.08 bB

WBPPS

0.12 ± 0.01 aA

0.13 ± 0.01 abA

0.16 ± 0.02 aA

Inulin

ND

ND

ND

1.09 ± 0.02 bB

1.35 ± 0.05 bC

2.84 ± 0.11 bD

WBPPS

0.92 ± 0.04 aB

1.00 ± 0.04 aB

2.78 ± 0.04 bC

Inulin

0.90 ± 0.03 aB

0.94 ± 0.02 aB

1.02 ± 0.05 aC

0.46 ± 0.03 bA

0.93 ± 0.09 bB

1.64 ± 0.11 cC

WBPPS

0.14 ± 0.03 aA

0.21 ± 0.04 aB

0.47 ± 0.02 bC

Inulin

0.10 ± 0.01 aA

0.11 ± 0.02 aA

0.11 ± 0.01 aA

0.20 ± 0.02 A

0.29 ± 0.02 B

0.58 ± 0.04 cC

WBPPS

ND

ND

0.38 ± 0.03 bA

Inulin

ND

ND

0.23 ± 0.04 aA

0.28 ± 0.02 aB

0.07 ± 0.02 aC

ND

WBPPS

1.69 ± 0.08 bD

1.39 ± 0.07 bC

0.48 ± 0.05 aB

Inulin

5.34 ± 0.12 cB

6.64 ± 0.10 cC

7.29 ± 0.23 bD

13.31 ± 0.39 aB

19.53 ± 0.18 aC

26.93 ± 0.12 aD

WBPPS

21.12 ± 0.62 bB

31.08 ± 0.48 bC

44.97 ± 1.30 bD

Inulin

28.66 ± 0.23 cB

36.88 ± 0.32 cC

47.36 ± 1.46 cD

Blank

Blank

Blank

Blank

Blank

Blank

Blank

1.33 ± 0.06 A

ND

0.85 ± 0.04 A

ND

ND

0.13 ± 0.03 A

6.40 ± 0.32 A

Different lowercase letters indicate significant differences (P < 0.05) for each SCFA among different groups. Different

capital letters indicate significant differences (P < 0.05) for each SCFA among different time points. ND is not detected.

30


Page 31 of 37


Table 3 Alpha Diversity of Samples among Different Treatment groups. Indexa Groups

a

Chao 1

ACE

Shannon

Simpson

OR

224.9 ± 6.6 b

225.8 ± 9.0 b

2.90 ± 0.04 c

0.139 ± 0.006 b

BLK

180.5 ± 17.2 a

177.7 ± 15.3 a

3.28 ± 0.06 d

0.066 ± 0.006 a

WBPPS

160.7 ± 22.2 a

161.2 ± 18.3 a

2.57 ± 0.03 b

0.163 ± 0.007 b

INL

162.8 ± 5.4 a

160.5 ± 7.9 a

2.14 ± 0.05 a

0.275 ± 0.024 c

The same lowercase letter means no significant difference existed for different treatment groups.

31



Page 32 of 37

Table 4 Relative Abundances of 15 Genera in OR, BLK, WBPPS and INL Groups Relative abundance a (%) Genus OR

BLK

WBPPS

INL

Prevotella

38.74 ± 0.93 b

5.42 ± 0.26 a

40.17 ± 1.27 b

56.40 ± 2.87 c

Dialister

4.89 ± 0.11 c

1.51 ± 0.06 a

7.96 ± 0.24 d

2.09 ± 0.07 b

Megamonas

3.00 ± 0.17 a

4.05 ± 0.64 a

6.81 ± 0.53 b

10.96 ± 1.57 c

Faecalibacterium

14.13 ± 0.59 d

0.80 ± 0.21 a

3.64 ± 0.29 c

2.07 ± 0.04 b

Alloprevotella

2.22 ± 0.14 d

0.84 ± 0.03 a

1.12 ± 0.08 b

1.33 ± 0.07 c

Blautia

0.27 ± 0.02 a

0.73 ± 0.26 b

0.36 ± 0.01 ab

2.94 ± 0.31 c

Bifidobacterium

0.08 ± 0.01 a

0.04 ± 0.01 a

0.11 ± 0.02 a

2.92 ± 0.18 b

Bacteroides

18.38 ± 0.62 b

34.04 ± 3.06 d

25.31 ± 0.35 c

11.15 ± 1.97 a

Clostridium XlVa

3.02 ± 0.15 b

11.27 ± 2.24 c

2.96 ± 0.43 b

0.31 ± 0.04 a

Parabacteroides

1.49 ± 0.07 b

7.82 ± 0.99 c

1.89 ± 0.11 b

0.47 ± 0.10 a

Escherichia/Shigella

0.09 ± 0.02 a

6.48 ± 0.30 d

1.88 ± 0.08 c

0.57 ± 0.06 b

Phascolarctobacterium

0.95 ± 0.04 b

2.24 ± 0.29 c

0.89 ± 0.11 b

0.09 ± 0.02 a

Parasutterella

1.00 ± 0.03 b

2.72 ± 0.23 c

0.83 ± 0.03 b

0.37 ± 0.04 a

Clostridium sensu stricto

0.06 ± 0.01 a

4.35 ± 1.50 b

0.09 ± 0.01 a

0.05 ± 0.01 a

Fusobacterium

0.15 ± 0.02 a

7.24 ± 0.30b

0.07 ± 0.01 a

0.04 ± 0.01 a

Lachnospiracea incertae sedis

2.31 ± 0.16 b

0.48 ± 0.06 a

0.63 ± 0.09 a

0.59 ± 0.05 a

a

Different letters indicate significant differences (P < 0.05) among different treatment groups.

32


Page 33 of 37


Figure 1

33



Figure 2

34


Page 34 of 37

Page 35 of 37


Figure 3

35



Figure 4

36


Page 36 of 37

Page 37 of 37


Graphic for table of contents

37


Simulated Digestion and Fermentation in vitro by Human Gut

Recommend Documents