Modulation of gut microbiota by soybean 7S globulin peptide that

Subscriber access provided by UNIV OF OREGON

Bioactive Constituents, Metabolites, and Functions

Modulation of gut microbiota by soybean 7S globulin peptide that involved lipopolysaccharide-peptide interaction Kaining Han, Danyang Luo, Yuan Zou, Shiyuan Dong, Zhili Wan, and Xiao-Quan Yang J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b07109 • Publication Date (Web): 05 Feb 2019 Downloaded from http://pubs.acs.org on February 5, 2019

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 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 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.

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 45

Journal of Agricultural and Food Chemistry

Modulation of gut microbiota by soybean 7S globulin peptide that involved lipopolysaccharide-peptide interaction Kaining Hana, Danyang Luoa, Yuan Zoub, Shiyuan Dongd, Zhili Wana and Xiaoquan Yang* a,c

a

Research and Development Center of Food Proteins, Department of Food Science

and Technology, South China University of Technology, Guangzhou 510640, China b

Department of Bioengineering, College of Food Science, South China Agricultural University, Guangzhou 510640, China

Guangdong Province Key Laboratory for Green Processing of Natural Products and

c

Product Safety, Guangzhou 510640, China d

College of Food Science and Engineering, Ocean University of China, Qingdao 266003, China

Corresponding Author * Tel: +86-20-87114262. Fax: +86-20-87114263. E-mail: [email protected].

1

ACS Paragon Plus Environment


1

Abstract

2

Soybean protein exhibiting nutritional significance for the control of metabolic

3

syndrome, and evidence suggests that gut microbiota is implicated in the control of

4

metabolic disorders. This study aimed to investigate the modulation of

5

pepsin-released peptides of soybean 7S globulin on gut microbiota, and possible

6

association between changes of gut microbiota composition and lipopolysaccharide

7

(LPS)-peptide interaction. In vitro fermentation experiments showed that the

8

extension region (ER) fragments of soybean 7S globulin selectively suppressed

9

pro-inflammatory Gram-negative bacteria. ER peptides also promoted the highest

10

production of short chain fatty acids (SCFAs), which was associated with increase of

11

Lachnospiraceae and Lactobacillaceae relative abundance. Isothermal titration

12

calorimetry (ITC) and Langmuir monolayer studies demonstrated that ER peptides

13

exhibited highly affinity to LPS in presence of Ca2+ and developed into β-sheet rich

14

aggregates structures, thus weakened the stability of LPS monolayers. This finding

15

supplies a possible explanation for improvement effects of soybean 7S globulin on

16

metabolic disease.

17 18

Keywords: soybean 7S globulin, extension region, peptides, gut microbiota,

19

lipopolysaccharide, interaction

20 21 22 2


Page 2 of 45

Page 3 of 45


23

Introduction

24

The worldwide prevalence of metabolic syndrome (MetS) imposes a tremendous

25

burden on economic and health care systems. The MetS is a cluster of coexisting

26

cardiovascular risk factors that include obesity, dyslipidaemia, hypertension and

27

hyperglycaemia or type 2 diabetes mellitus (T2DM), in association with insulin

28

resistance and systemic inflammation.1 It has been established that gut microbiota

29

plays key roles in host metabolism and health, and the association between the

30

changes in the gut microbiota and the obesity and related metabolic disorders.2 Gut

31

microbiota can also produce signaling molecules involved in regulating energy

32

metabolism of host, such as short-chain fatty acids (SCFAs).3 Interestingly, soy

33

foods have always been exhibited the nutritional significance in the control of

34

chronic diseases, including the ability to alleviate obesity and related cardiovascular

35

diseases outcome, and improve insulin resistance and mitigate diabetes.4, 5 Recently,

36

Huang et al. reviewed the response of gut microbiota to soy foods and their

37

components, summarized that consumption of soy foods can modify Firmicutes to

38

Bacteroidetes ratio and reduce the gut pathogenic bacteria populations, thus lowering

39

the risk of diseases and leading to beneficial effects on human health.6 Moreover,

40

Cross et al. reported soy significantly shifted the cecal microbial community and

41

improved cardiometabolic health in female low-fit rats.7 However, due to the

42

complexity of soy components, it is difficult to pinpoint precisely which are the main

43

components responsible for the particular health-promoting effects, also the

44

mechanism of action exerted by soy components. 3



45

Among multiple bioactive properties of the different soy components, the

46

remarkable plasma cholesterol-lowering function8 and the improvement effect of

47

obesity-induced metabolic abnormalities9 of soybean 7S globulin (β-conglycinin)

48

have been highlighted. Soybean 7S globulin and 11S globulin are major storage

49

protein in soybean seed. Fernandez-Raudales et al. revealed that the higher 7S

50

globulin/11S globulin ratio in soy milk may preferentially promote the growth of

51

Bacteroides-Prevotella group in overweight and obese men.10 The pepsin

52

hydrolysates of soybean 7S globulin exhibited an antibacterial activity against

53

Escherichia coli (E. coli) and maintained a relatively healthy gut microbial

54

community in mice even after infection of E. coli.11 In addition, Butteiger et al.

55

observed an increased gut microbial diversity in soybean protein-fed Golden Syrian

56

hamsters than milk protein-fed group.12 A latest research indicated that dietary

57

soybean protein exerted protective effects against high-fat diet-induced obesity by

58

means of gut microbiota-driven biotransformation of bile acids.13 However, there is

59

currently no consensus on the impacts of dietary soybean protein on gut microbiota.

60

Also, the impacts of gastrointestinal (GI) digestive peptides of dietary soybean

61

protein on the gut microbiota and related metabolic dysfunctions are still lacking and

62

remained to be elucidated.

63

Soybean 7S globulin has a trimeric structure consisting of α (∼67 kDa), α' (∼71

64

kDa) and β (∼50 kDa) subunits.14, 15 The α and α' subunits contain an N-terminal

65

extension region (α, 125 residues, ∼15 kDa; α', 141 residues, ∼17 kDa, respectively)

66

and a common core region, while β subunit contains only core region (~439 residues, 4


Page 4 of 45

Page 5 of 45


68

∼47kDa).14,

69

structure linear epitopes, which were essential for their allergenic capacity.17 Our

70

previous research had revealed that pepsin hydrolysis actually cleaved or separated

71

the N-terminal extension region of α and α' subunits from common core region of α,

72

α' and β subunits in soybean 7S globulin,18 and resulted fragments exhibited strong

73

resistance to pepsin digestion may ascribe to the formation of worm-like fibril

74

aggregates.19

75

triglycerides-lowering properties of α' subunit of soybean 7S globulin in vivo.20

76

Especially the ER fragment of the α' subunit has been implicated in regulating the

77

low-density lipoproteins uptake and degradation of cell in dose-dependent manner.21

78

And we also developed a novel strategy to fractionate N-terminal extension region

79

(ER) from core region (CR) of soybean 7S globulin subunits by using pepsin

80

digestion combined with ultrafiltration fractionation method.16 Furthermore, the

81

digestibility of soybean protein was affected by many factors, such as heat treatment,

82

age and antinutritional factors.22 Particularly, up to 20% of the Bowman-Birk

83

inhibitor of chymotrypsin and trypsin and the Kunitz inhibitor of trypsin were still

84

existence in most commercially heated meals.23 Therefore, these ER and CR

85

fragments of soybean 7S globulin may be abundant in gastrointestinal tract.

67

16

Soybean 7S globulin showed a significant resistance to the GI

digestion, and released large GI resistant fragments retained in their primary

It

has

been

evidenced

that

the

plasma

cholesterol

and

86

Considering the important implications of gut microbiota on host physiology, we

87

hope to know whether soybean 7S globulin peptides contribute to shape the gut

88

microbial community and the way of the protein-gut microbiota interaction. In this 5



89

study, we investigated the responses of gut microbiota and SCFAs production to

90

soybean 7S globulin and their pepsin digestive fragments (CR fragments and ER

91

peptides). More recently, the key cues of LPS from gut Gram-negative bacteria for

92

triggering metabolic syndrome have raised attention. LPS makes up approximately

93

75% of the Gram-negative bacterial outer membrane surface, involved activation of

94

host inflammation and stimulation of inner immune response system, could be

95

considered a major risk factor that provides an unifying mechanism to explain

96

obesity related metabolic syndrome.24 From this view, we further studied the

97

interactions between ER peptides of soybean 7S globulin and LPS by using

98

isothermal titration calorimetry (ITC) and Langmuir monolayer method, which

99

might provide a more comprehensive picture for the improvement effect of

100

metabolic disorders by soybean 7S globulin at the molecular level.

101

Materials and Methods

102

Materials

103

Soybean 7S globulin was isolated according to the method of Nagano et al.25

104

The protein content of soybean 7S globulin lyophilized powder was 89.31±0.06%,

105

which was determined by Dumas combustion method (N×6.25). Porcine pepsin

106

(P7000, ≥250 units/mg), bile salts, Ra rough mutant strain lipopolysaccharide

107 108

(RaLPS, molecular weight ∼4.2 kDa) from Escherichia coli (E. coli) EH100, and

109

infusion (BHI) broth medium, tryptone and yeast extract were purchased from Oxoid

110

(Basingstoke, UK). All other chemicals used were of analytical grade or better.

Thioflavin T (Th T) were purchased from Sigma (St. Louis, MO, USA). Brain heart

6


Page 6 of 45

Page 7 of 45

111


Fractionation of Pepsin Digested Peptides from Soybean 7S Globulin

112

The pepsin digested peptides of soybean 7S globulin were fractionated

113

according to the method we described before.16 Briefly, soybean 7S globulin was

114

dissolved in distilled water and adjusted to pH 3.5. Porcine pepsin was added to final

115

concentration of 400 units/mL. The hydrolysis reaction was performed at 37 °C for 6

116

h and terminated by readjust pH to 7.0. Then the hydrolysate was centrifuged at

117

9690 g at 25 °C for 20 min and the obtained supernatant was fractionated using a

118

Labscale™ TFF membrane system (Millipore, USA) with a 10 kDa cut-off

119

membrane. The permeate fraction was concentrated and freeze-dried, which was

120

designed as extension region (ER) peptides, while the retentate fraction was also

121

concentrated and freeze-dried, which was designed as core region (CR) fragments.

122

The composition of the soybean 7S globulin, CR fragments and ER peptides was

123

analyzed by Tricine-SDS-PAGE method (Supporting Information Figure S1). It was

124

found that the ER peptides consisted of three large fragments (about 6, 10, and 11

125

kDa), while CR fragments only contained the about 47 kDa fragment. From the

126

result of peptide sequence identification analyzed by HPLC-MS/MS, it had

127

demonstrated that ER peptides mainly derived from the N-terminal extension regions

128

in α and α' subunits of soybean 7S globulin.26

129

Fermentation in vitro Using Human Fecal Inoculum

130

Fecal samples were obtained from three healthy male donors who were free of

131

known metabolic or GI disorders and had avoided antibiotic treatment for at least 3

132

months prior to the study. All donors were fully aware of the scope of our study and 7



133

signed an informed consent form. And the study was approved by Ethics Committee

134

of South China University of Technology (Approval No. FP2017-0912). The fresh

135

fecal samples were collected in sterile sample collection vessels and kept at 4 °C

136

before further treating. Then an equal amount of collected fresh fecal samples from

137

each donor was mixed immediately. The mixed fecal sample was diluted with sterile

138

BHI broth medium, then the fecal slurry was centrifuged (300 g, 4 °C, 5 min) after 5

139

min of shaking. The supernatant was collected and mixed with an equal volume of

140

40% sterile glycerol, then preserved at −80 °C for further inoculation. The procedure

141

of human fresh fecal sampling and further treatment was accomplished in 2 h. The

142

fecal inoculum and basic fermentation medium were prepared according to the

143

method of our previous study.27 For the inoculation, the fecal inoculum was

144

inoculated into sterile fermentation medium containing 1% and 2% (w/v) of soybean

145

7S globulin, CR fragments and ER peptides, and named as 7S1, 7S2, CR1, CR2,

146

ER1 and ER2, respectively. Fermentation in vitro was conducted at 37 °C in the

147

tubes which were equipped with rubber plugs and screw caps to maintain an

148

anaerobic environment. After a period of 24 h of fermentation, the cultures were

149

separated at 16 000 g at 4 °C for 10 min. The pellets and supernatants were

150

preserved at −80 °C for further analysis. All inoculation steps were conducted in the

151

anaerobic workstation (Electrotek workstation, Electrotek, UK) containing 10% H2,

152

10% CO2 and 80% N2.

153

16S rRNA Gene Sequencing and Bioinformatic Analysis

154

Extractions of microbial genomic DNA from each sample were carried out using 8


Page 8 of 45

Page 9 of 45


155

the method of Yu and Morrison.28 The integrity and concentration of extracted DNA

156

were analyzed by agarose gel electrophoresis. The V4 hypervariable region of 16S

157

rRNA gene was amplified using the primers of 515F and 806R. Amplicons were

158

extracted from agarose gels and purified with the GeneJET Gel Extraction kit

159

(Thermo Scientific). Sequencing library was constructed using the Ion Plus

160

Fragment Library Kit 48 rxns (Thermo Scientific) and sequencing was performed on

161

the Ion S5TMXL platform at Novogene Bioinformatics Technology Co., Ltd (Beijing,

162

China). Cutadapt29 was used to remove low-quality regions of reads. Then the reads

163

were classified to individual samples according to unique barcode sequences, and

164

primers and barcode sequences were removed from reads to generate raw reads. And

165

chimeras were filtered out using UCHIME Algorithm30 from raw reads to generate

166

clean reads. Operational Taxonomic Units (OTUs) were clustered based on 97%

167

identity threshold using UPARSE.31 The taxonomic annotation of each OTU was

168

performed with Mothur and SILVA SSU rRNA database.32,

169

taxonomic annotation results, the top 10 species (at the phylum level and family

170

level) or top 12 species (at the genera level) with the highest abundance in

171

fermentation samples were presented in terms of relative abundance. QIIME34 and R

172

software were employed to analyze the α-diversity and β-diversity of microflora. The

173

biomarkers with statistically significant differences among samples were screened

174

using LEfSe analysis.35

175

Short Chain Fatty Acids (SCFAs) Production

176

33

Based on the

SCFAs (acetate, propionate and butyrate) of fermentation samples were 9



177

analyzed according to the method of Tian et al.36 with some modifications. Five

178

hundred microliters of supernatant of fermentation samples or standards (acetic acid,

179

propionic acid and butyric acid, 0.05~0.6 mg/mL of each standard) were mixed with

180

0.5 mL of 0.3 mg/mL 2-ethylbutyric acid (internal standard) which was dissolved in

181

0.2 M HCl, then 200 μL of 0.15 M oxalic acid was added and the mixture was

182

centrifuged (16 000 g, 4 °C, 10 min) after 30 min of standing. Analysis was

183

performed on an Agilent 7890B gas chromatography system (Agilent Technologies,

184

Palo Alto, CA, USA) equipped with a FID detector and a HP-INNOWax column (30

185

m×0.320 mm×0.25 μm). One microliter of sample was analyzed with the following

186

temperature procedure: 100 °C of initial temperature, increased to 170 °C at 5 °C

187

/min. The carrier gas was nitrogen at a flow rate of 1 mL/min.

188

Interactions of ER Peptides with RaLPS

189

Isothermal Titration Calorimetry (ITC) Measurement

190

ITC measurements were performed on a MicroCal PEAQ-ITC instrument

191

(Malvern Instruments Ltd., Worcestershire, UK) at 25 °C. RaLPS was dissolved in

192

10 mM HEPES buffer (pH 7.0) at a concentration of 50 μM. ER peptides were

193

dissolved in 10 mM HEPES buffer (pH 7.0) or 10 mM HEPES buffer containing 2.5

194

mM CaCl2 (pH 7.0) at a concentration of 0.5 mM. Two hundred and fifty microlitres

195

of RaLPS solution were injected into the sample cell, and 50 μL of ER peptides

196

solution were filled into the syringe. Titrations were performed with the procedure of

197

0.4 μL for the first injection followed by 18 injections of 2 μL each at 150 seconds

198

intervals and at a stirring rate of 750 rpm. In addition, titrations of 10 mM HEPES 10


Page 10 of 45

Page 11 of 45


199

buffer containing 2.5 mM CaCl2 (pH 7.0) into ER peptides solution (0.5 mM) (pH

200

7.0), and ER peptides solution (0.5 mM) containing 2.5 mM CaCl2 (pH 7.0) into

201

RaLPS solution (50 μM) were also performed. Data were analyzed by Malvern

202

MicroCal PEAQ-ITC software. All experiments were carried out in three replicates.

203

Langmuir Monolayers

204

The surface pressure (π)–area (A) measurements of RaLPS monolayers were

205

conducted at 25 °C using a thermostated Langmuir trough system (KSV NIMA,

206

Espoo, Finland) which equipped with a platinum Wilhelmy plate. Prior to

207

measurements, 300 mL of subphase solution were added into Langmuir trough and

208

subphase surface was compressed and cleaned by suction. RaLPS vesicles were

209

prepared in the solution of chloroform/methanol/water (6:4:1, v/v/v) at a

210

concentration of 1 mg/mL. Monolayers were formed by slowly spreading 70 μL of

211

RaLPS solution onto the subphase surface with a microsyringe. After waiting for 20

212

min to allow solvent evaporation and stabilization of the monolayers, ER peptides

213

solutions which prepared with distilled water were dropped into subphase

214

underneath the RaLPS monolayers at LPS/peptides molar ratios of 1:0, 1:0.86,

215

1:1.71 and 1:2.57. Compression was performed with a rate of 8 mm/min after 30 min

216

of equilibration. In order to study the influence of Ca2+ ions, Ca2+-free buffer (10

217

mM HEPES and 100 mM NaCl, pH 7.0) and Ca2+-load buffer (10 mM HEPES, 100

218

mM NaCl and 20 mM CaCl2, pH 7.0) were used as subphase in measurements. From

219

the obtained π−A isotherms data, the static compression modulus (Cs-1) of

220

monolayers was calculated with the following equation37: 11



Page 12 of 45

221

Cs-1= −A (∂π/ ∂A)T

222

Where A is the area per molecule at a given surface pressure, π is the corresponding

223

surface pressure and T is the temperature.

224

Confocal Laser Scanning Microscopy (CLSM) Observation

225

Clean glass slides were attached to the dipper for Langmuir–Blodgett film and

226

then immersed in buffer subphase. When the RaLPS films were compressed to a

227

surface pressure of 30 mN/m, the submerged glass slides were slowly lifted at the

228

speed of 2 mm/min and the surface pressure was maintained at 30 mN/m during

229

transfer. The LB films were observed using a Zeiss 710 Confocal Laser Scanning

230

Microscopy (CLSM) (Zeiss, Oberkochen, Germany). Th T was used for staining of

231

peptides and excited at 405 nm with an emission filter of 448-540 nm.

232

Statistical Analysis

233

Data were expressed as mean ± standard deviation (SD). Statistically significant

234

differences among groups were performed by one-way analysis of variance

235

(ANOVA) with Duncan's multiple range test (p < 0.05). And p < 0.05 was

236

considered significant in Pearson's correlation analysis. Statistical analyses were

237

completed in SPSS 19.

238

Results and Discussion

239

Overview of Gut Microbial Community

240

The 16S rRNA sequencing analysis showed that bacterial communities of

241

fermentation samples were dominated by Firmicutes, followed by Proteobacteria,

242

also

including

Bacteroidetes,

Actinobacteria, 12


Chloroflexi,

Tenericutes,

Page 13 of 45


243

Armatimonadetes, Chlorobi, Ignavibacteriae and Acidobacteria (Figure 1A).

244

Compared with CR (CR1 and CR2) samples, 7S (7S1 and 7S2) and ER (ER1 and

245

ER2) samples presented a significantly higher Firmicutes relative abundance (p

Modulation of gut microbiota by soybean 7S globulin peptide that

Recommend Documents