The Modulatory Effect of Anthocyanins from Purple ... - ACS Publications

Mar 15, 2016 - Department of Food Science and Engineering, School of Marine ... short-chain fatty acid (SCFA) concentrations at different time points...
0 downloads 0 Views 855KB Size
Subscriber access provided by Purdue University Libraries

Article

The Modulatory Effect of Anthocyanins from Purple Sweet Potato on Human Intestinal Microbiota in Vitro Xin Zhang, Yang Yang, Zufang Wu, and Peifang Weng J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.6b00586 • Publication Date (Web): 15 Mar 2016 Downloaded from http://pubs.acs.org on March 15, 2016

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

The Modulatory Effect of Anthocyanins from Purple Sweet Potato on Human

2

Intestinal Microbiota in Vitro

3

Xin Zhang* , Yang Yang, Zufang Wu, and Peifang Weng

4

Running header: Modulatory Effect of Anthocyanins on Intestinal Microbiota

5

Department of Food Science and Engineering, School of Marine Sciences, Ningbo

6

University, Ningbo 315211, P.R. China



Corresponding author: Xin Zhang; E-mail address: [email protected]

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 2 of 36

7

ABSTRACT: In order to investigate the modulatory effect of purple sweet potato

8

anthocyanins (PSPAs) on human intestinal microbiota, PSPAs were prepared by

9

column chromatography and their influence on intestinal microbiota was analyzed by

10

monitoring the bacterial populations and analyzing short-chain fatty acid (SCFA)

11

concentrations at different time points. The numbers (log10 cell/mL) of

12

Bifidobacterium

13

Clostridium histolyticum and total bacteria after 24 h of culture in anaerobic

14

fermentation broth containing PSPAs were 8.44 ± 0.02, 8.30 ± 0.01, 7.80 ± 0.03, 7.60

15

± 0.03 and 9.00 ± 0.02, respectively, compared with 8.21 ± 0.03, 8.12 ± 0.02, 7.95 ±

16

0.02, 7.77 ± 0.02 and 9.01 ± 0.03, respectively, in the controls. The results showed

17

that

18

Lactobacillus/Enterococcus spp., inhibited the growth of Bacteroides-Prevotella and

19

Clostridium histolyticum, and did not affect the total bacteria number. Total SCFA

20

concentrations in the cultures with PSPAs were significantly higher than in the

21

controls (P90%) were purchased from Shanghai Qibang Biological Co. (Shanghai,

81

China). The AB-8 resin was purchased from Anhui Sanxing Chemical Co. (Bengbu,

82

China). All solvents used for chromatography were of HPLC grade.

(M3G),

cyanidin-3,5-β-D-O-diglucoside

(C35G)

83

To identify the peaks, solutions of C3G, M3G, C35G and C3R were prepared in a

84

2% formic acid solution. Serial dilutions were generated in a 2% formic acid solution

85

to produce following standard solutions for the standard curves: 100 µg/mL to 0.25

86

µg/mL.

87

PSP Samples. PSPs were obtained from a local market in Ningbo, China. Fresh

88

samples were selected randomly, washed with running tap water, cut into pieces of

89

approximately 0.5 cm, lyophilized, ground into powder, and sifted through a 100

90

mesh sieve. The prepared samples were stored at -80 °C until use.

91

Preparation of PSPAs. PSPAs were prepared from PSPs using a reported method,

92

with some modifications.14 Briefly, 50.0 g of PSP powder were extracted with 1,600

93

mL of acid-ethanol (HCl, 1.5 mol/L) at 80 °C for 60 min. Then, the extract was

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

94

centrifuged at 4,500 g for 15 min, and the resulting insoluble residue was treated

95

again. The supernatants were combined and concentrated. The resulting residue was

96

dissolved with deionized water, filtered through a 0.45 µm cellulose filter and applied

97

to a column (30 × 1.6 cm) of wet-packed AB-8 resin according to our previously

98

established method. After reaching adsorptive saturation, the column was washed with

99

2 bed volumes (BVs) of distilled water and then eluted with 4 BVs of an ethanol

100

solution (60:40, v/v). The effluent of the ethanol solution was collected and

101

concentrated, affording the PSPA extract.

102

HPLC-ESI-MS/MS Analysis and Characterization of PSPAs. PSPA samples

103

were analyzed on an Agilent Technologies 1200 series HPLC (Agilent Technologies,

104

CA, USA) using a reported method, with a slight modification.15 Separation was

105

achieved on a Zorbax Stablebond Analytical SB-C18 column (4.6 mm × 250 mm, 5

106

µm, Agilent Technologies, USA). Elution was performed using the following gradient

107

of mobile phase A (aqueous 2% formic acid solution) and mobile phase B (methanol):

108

0-2 min, 10-20% B; 2-40 min, 20-55% B; 40-41 min, 55-80% B; and 41-45 min, 80%

109

B. The quantification of different anthocyanins was based on the peak areas and

110

calculated as equivalents of the standard compounds; all contents were expressed as

111

milligrams per gram dry weight. The temperature of the column oven was set at 40 °C,

112

the flow rate was set at 0.6 mL/min, and the injection volume was 5 µL.

113

Low-resolution electrospray mass spectrometry was performed with a solariX ion

114

trap mass spectrometer (Bruker Daltonics, MA, USA). The experimental conditions

115

were as follows: ESI interface and nebulizer, 50 psi; dry gas, 15.0 psi; dry temperature,

ACS Paragon Plus Environment

Page 6 of 36

Page 7 of 36

Journal of Agricultural and Food Chemistry

116

320 °C; MS/MS, scan from m/z 200 to 1500; ion trap, scan from m/z 200 to 1,500;

117

source accumulation, 50 ms; ion accumulation time, 300 ms; flight time to acquisition

118

cell, 1 ms; smart parameter setting (SPS) and compound stability, 50%; and trap drive

119

level, 60%.

120

In Vitro Fermentation of PSPAs. The effects of the PSPAs on human intestinal

121

microbiota were investigated using our previously reported method, with slight

122

modifications.16 Fecal samples were obtained from 8 healthy volunteers (4 females

123

and 4 males, 25-30 years old) who did not have any history of gastrointestinal

124

disorders and had not been treated with antibiotics for the last 6 months. Fecal slurries

125

were prepared by mixing fresh fecal samples with autoclaved phosphate-buffered

126

saline to yield 10% (w/v) suspensions. Anthocyanin samples were mixed with

127

autoclaved nutrient basal growth medium at a final concentration of 1% (w/v). The

128

basal nutrient medium (pH 7.0) contained peptone, 2.00 g/L; yeast extract, 2.00 g/L;

129

NaCl, 0.10 g/L; K2HPO4, 0.04 g/L; KH2PO4, 0.04 g/L; MgSO4·7H2O, 0.01 g/L;

130

CaCl2·6H2O, 0.01 g/L; NaHCO3, 2.00 g/L; hemin, 0.02 g/L; cysteine HCl, 0.50 g/L;

131

bile salts, 0.50 g/L; resazurin, 1.00 mg/L; Tween 80, 2.00 mL/L; vitamin K1, 10.00 µL;

132

and distilled water. The samples were then inoculated with 150 µL of fecal slurry

133

(10%, w/v) with a manual homogenizer in an anaerobic atmosphere of 10% H2, 10%

134

CO2 and 80% N2 at 37 °C (anaerobic incubator made in Shanghai, China). All

135

experiments were repeated in triplicate.

136

Enumeration of Bacteria by Fluorescent in Situ Hybridization. The

137

enumeration of the bacteria was conducted using fluorescent in situ hybridization

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

138

(FISH) as described in our previous report, with minor modifications.16 Briefly,

139

culture samples (100 µL) were taken at 0, 6, 12 and 24 h, added to 300 µL of a filtered

140

paraformaldehyde solution (4%, w/v), and fixed overnight at 4 °C to enumerate the

141

bacteria. The fixed bacterial cells were washed twice with 400 µL of PBS,

142

re-suspended in 600 µL of PBS/ethanol (1:1, v/v) and stored at -20 °C. Hybridization

143

was performed using 16S rRNA-targeted oligonucleotide probes labeled with cyanine

144

dye-3 (Cy3) fluorescent dye to enumerate specific bacterial groups, whereas DAPI

145

was used to count the total number of bacteria. The probes used in our study were

146

Bif164, Lab158, Bac303 and His150, and the probe names, target genera or species

147

and probe sequences were listed in Table 1. An Axio Imager A1 epifluorescence

148

microscope (Carl Zeiss, Göttingen, Germany) was used to count the bacterial cells. At

149

least six random fields were counted on each slide, and the bacterial numbers were

150

expressed as log10 cells per milliliter ± standard deviation (SD).

151

Determination of SCFA and Lactic Acid Content. The analysis of SCFA and

152

lactic acid content was performed using an Agilent 1100 series HPLC system with a

153

Beckman Ultrasphere column (4.6 × 250 mm, 5 µm, Beckman Instruments Inc., CA,

154

USA).17 Fermentation samples (50 µL) were collected after 24 h, centrifuged at 5,000

155

g for 15 min, and then filtered through a 0.22 µm cellulose membrane. The mobile

156

phase consisted of a 20 mM KH2PO4 solution (pH 2.5, A) and methanol (B) in the

157

following gradient system: 0-16 min, 5% B; 16-30 min, 5 to 30% B; and 30-40 min,

158

30% B. The flow rate was 0.8 mL/min, and the detector was set at 210 nm. The

159

temperature of the column oven was set at 30 °C, and the injection volume was 20 µL.

ACS Paragon Plus Environment

Page 8 of 36

Page 9 of 36

Journal of Agricultural and Food Chemistry

160

SCFA and lactic acid concentrations were calculated according to calibration curves

161

of the respective authentic compounds, including formic, acetic, propionic, butyric

162

and lactic acids. The analysis was conducted three times.

163

Statistical Analysis. The obtained results were analyzed using SPSS version 16.0

164

(SPSS Inc., Chicago, IL, USA). Any significant difference was determined by

165

one-way analysis of variance (ANOVA) followed by Tukey test for multiple

166

comparisons; a difference was considered statistically significant at P