Bacterial Fermentation of Water-Soluble Cellulose Acetate Raises

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Bioactive Constituents, Metabolites, and Functions

Bacterial Fermentation of Water-Soluble Cellulose Acetate Raises Large-Bowel Acetate and Propionate and Decreases Plasma Cholesterol Concentrations in Rats Tomomi Genda, Takashi Kondo, Shunsaku Sugiura, Shingo Hino, Shu Shimamoto, Toshikazu Nakamura, Shizuka Ukita, and Tatsuya Morita J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b04093 • Publication Date (Web): 25 Oct 2018 Downloaded from http://pubs.acs.org on October 30, 2018

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Journal of Agricultural and Food Chemistry

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Bacterial Fermentation of Water-Soluble Cellulose Acetate Raises Large-Bowel Acetate and Propionate and Decreases Plasma Cholesterol Concentrations in Rats

Tomomi Genda † , Takashi Kondo † , Shunsaku Sugiura ‡ , Shingo Hino § , Shu Shimamoto / / , Toshikazu Nakamura / / , Shizuka Ukita / / and Tatsuya Morita § *

†

Department of Bioscience, Graduate School of Science and Technology,

Shizuoka University, Shizuoka 422 -8529, Japan. ‡

Department of Agriculture, Graduate School of Integrated Science and

Technology, Shizuoka University, Shizuoka 422 -8529, Japan. §

College of Agriculture, Academic Institute, Shizuoka University, Shizuoka

422-8529, Japan. //

Daicel Corporation, Konan 2-18-1, Minatoku, Tokyo 108-8230, Japan.

*Corresponding author, Tel/Fax: +81-54-238-5132; E-mail: [email protected]

ACS Paragon Plus Environment


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ABSTRACT: We hypothesized that water-soluble cellulose acetate (WSCA)

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could be useful tool for the delivery of short-chain fatty acids to the large

3

intestine. Rats were fed a control diet or a diet containing graded levels of

4

WSCA for up to 21 d. Consuming WSCA dose-dependently increased large-

5

bowel acetate and propionate concentrations through the bacterial

6

fermentation. When WSCA was used as substrate, acetyl esterase activity in

7

the cecal bacteria was detected solely in rats fed WSCA, in which the

8

activity increased over time accompanying with the increased number of

9

Bacteroides xylanisolvens. Consuming WSCA at 4% level increased the

10

goblet cell numbers and mucin contents in the cecum and lowered plasma

11

cholesterol concentrations, which tended to correlate with the portal plasma

12

concentrations of propionate. The results suggest that bacterial fermentation

13

of WSCA is characterized by the greater production of acetate and

14

propionate, which may contribute to the physiologic alterations.

15 16

KEYWORDS: water-soluble cellulose acetate, Bacteroides xylanisolvens,

17

acetate, propionate, plasma cholesterol

18 19 20


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21 22

INTRODUCTION Short-chain fatty acids (SCFAs) are the primary products of bacterial

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fermentation of dietary fibers in the large bowel of humans and other

24

omnivores. SCFAs act to maintain the normal physiologic function of the

25

large bowel. 1 Butyrate is the major fuel supplied to colonocytes . In contrast,

26

the majority of propionate is absorbed into the liver via the portal vein,

27

where propionate may inhibit cholesterol synthesis. 2 Recent studies revealed

28

that SCFAs stimulate the release of anorexic gut hormones, peptide YY

29

(PYY) and glucagon like peptide-1 (GLP-1) from the enteroendocrine L

30

cells via activation of the G protein coupled receptors 41 and 43 ( GPCR-41,

31

43). 3, 4 Intriguingly, propionate is the most potent ligand for GPCR41 and

32

equipotent with acetate for GPCR43. 4 Acetate is the most abundant end-

33

product of the large bowel fermentation. Thus, both acetate and propionate

34

could function to increase satiety-enhancing properties of food.

35

Clearly, raising large-bowel SCFA concentrations has the potential to

36

improve colonic health and modulate cholesterol metabolism and an energy

37

intake. Dietary strategies to raise SCFAs in the large bowel, therefore, may

38

be of public health and clinical benefit. For example, starch -butyrate ester

39

and inulin-propionate ester were developed to increase the respective SCFA

40

production aiming at colorectal cancer protection and appetite regulation in



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humans. 5-7

42

Water-soluble cellulose acetate (WSCA) could be another candidate for

43

the targeted delivery of SCFAs to the human colon. A limited introduction

44

of acetyl moiety into the cellulose molecule increases the water solubility of

45

cellulose derivatives and the accessibility of cellulolytic enzymes. 8 On

46

recent findings, acetyl esterases have been identified as a prerequisite for

47

endoglucanase-catalyzed cellulose acetate depolymerization. 9 More recently,

48

Bacteroides xylanisolvens (BX) and Bacteroides ovatus (BO) has been

49

isolated from human feces as xylan-degrading bacteria 10-12 , which possess a

50

potent acetyl esterase activity in the periplasmic space and/or on the cell

51

surface. 12,

52

by BX and BO, and further degraded and fermented by large bowel bacteria.

53

Large-bowel concentrations of acetate and other SCFAs would be increased

54

by both of the release from the esterified acetate and the colonic

55

fermentation of the liberated cellulose from WSCA.

56

13

Collectively, we envisaged that WSCA is de -acetylated mainly

Unlike traditional cellulosic additives, WSCA is water-soluble, colorless

57

and odorless. Its water solution exhibits relatively low viscosity 1 4 ; it is

58

considered easy to incorporate the material into beverages, baked goods and

59

the likes to modify dispersions of other food materials and textures. In the

60

study, therefore, we aimed to determine whether WSCA raises the large -


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bowel amounts of acetate and other SCFAs through the bacterial

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fermentation when fed to rats. Also, we examined the effects of WSCA on

63

the plasma cholesterol concentrations, mucosal architecture in the cecum

64

and the release of gut hormones.

65 66

MATERIALS AND METHODS

67

Materials. WSCA was provided from Daicel Corporation. (Osaka, Japan).

68

Briefly, WSCA with a degree of substitution of 0.78 2 (DS, 0.782) and with a

69

viscosity average degree of polymerization of 128 measured in dimethyl

70

sulfoxide at 25 C was prepared from commercially available cellulose

71

acetate (L-50 of Daicel Corporation.) by a partial de-acetylation under

72

acidic conditions. 15 Fructo-oligosaccharides (FOS, Meioligo P®) and

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resistant maltodextrin (RM, Fibersol 2®) were purchased from Meiji Seika

74

(Tokyo, Japan) and Matsutani Chemical Industry (Kobe, Japan),

75

respectively.

76

Care of Animals. All aspects of animal care were under the oversight of the

77

institutional animal ethics committee of Shizuoka University under accepted

78

guidelines (approval no. 26-14, 27-14). Male Wistar rats (7wk-old) were

79

purchased from Shizuoka Laboratory Animal Center (Shizuoka, Japan) and

80

housed individually in screen-bottomed stainless-steel cages in a



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temperature- (23 ± 1 C) and light- (light on from 07:00 to 19:00) controlled

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room. For adaptation, rats were fed a control diet for 7 d. The control diet

83

was formulated from 250 g /kg casein, 602.25 g /kg cornstarch (Cornstarch

84

y, Nihon Shokuhin Kako, Shizuoka, Japan), 50 g /kg cellulose powder

85

(Cellulose Powder (crystalline cellulose), Oriental Yeast Co., Ltd, Tokyo,

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Japan), and 50 g /kg corn oil. The remainder of the diet consisted of

87

vitamins (10 g/kg), minerals (35 g/kg), and choline bitartrate (2.5 g/kg). The

88

compositions of vitamins and minerals were based on AIN -76. 16 Rats were

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allocated to groups on the basis of body weight and allowed free access to

90

experimental diets and water. Supplementation of WSCA, FOS, or RM was

91

performed by replacement of an equal amount of cornstarch in the control

92

diet.

93

Small-Intestinal Resistance of WSCA (experiment 1). Small-intestinal

94

resistance of acetyl ester on WSCA was tested using a brush boarder

95

membrane vesicle (BBMV) and a pancreatic lipase. The BBMVs were

96

prepared with the use of the rat small intestine according to Kessler et al. 17 ,

97

and were suspended in phosphate buffer (50 mM, pH 8.0) to have a protein

98

concentration of 4 mg /mL. WSCA (7.5 mg) or triacetin (2.3 mg ) was

99

dissolved in 2 mL of phosphate buffer. One mL of BBMV solution was

100

added to 2 mL of substrate solution, and incubated for 8 h at 37 C. The


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liberated acetate in the supernatant was measured by an HPLC, as described

102

previously. 18 As for the measurement of lipase activity, saturated amounts of

103

triacetin (135.3 mg) or WSCA (60.5 mg) were dispersed in Tris-HCl buffer

104

(150 mM, pH 7.4) using Polytron. Enzyme solution (0.5 mL) in the same

105

buffer containing 450 units of lipase (Sigma -Aldrich) was added to 2 mL of

106

substrate suspension, and incubated for 8 h at 37 C. The liberated acetate

107

was similarly measured as above.

108 109

Dose-dependent Effects of WSCA on the Cecal Fermentation, Plasma

110

Cholesterol, and Microbiota Composition in Rats (experiment 2).

111

acclimatization, the rats (n = 6 / each group) were allowed free access to

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water and the control diet or a diet containing 1, 2, or 4% WSCA, or 4%

113

FOS for 21 d. Finally, the diet was withdrawn from 7:00-15:00, and the rats

114

were anesthetized with isoflurane inhalation and killed by collecting blood

115

from the adbominal aorta with a heparin -coated syringe. Then, the cecum

116

was removed and weighed, and its contents were homogenized and used for

117

analyses of pH, mucins, organic acids, and bacterial 16S rRNA genes. The

118

cecal tissue was washed in an ice -cold saline and blotted on filter paper. The

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tissue was sectioned longitudinall y and divided into two portions. One

120

portion was used for mucosa preparation for the isolation of total RNA and


After


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successive gene expression analyses. The other portion was placed in 10%

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buffered formalin and used for tissue examination. The colonic contents

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were used for analyses of pH and organic acids. Fresh feces were collected

124

for the last 2 d of the experimental period and treated in the same manner as

125

the colonic contents. Plasma was separated from the blood and used for the

126

measurements of triglyceride and cholesterol concentrations.

127

Acetyl Esterase Activity and the Numbers of BX and BO in the Cecal

128

Contents in Rats (experiment 3).

129

given free access to water and the control diet or a diet containing 4 %

130

WSCA for 3, 7 or 21 d. At each period, 6 rats from each group were killed

131

by decapitation under isoflurane inhalation, and the cecum was removed and

132

weighed. Cecal contents were homogenized and divided into two portions:

133

one ( 150 mg) was used for analyses of pH and the numbers of BX and BO,

134

and the remained was used for analysis of acetyl esterase activity.

135

Effects of WSCA on the Portal SCFAs, Plasma Cholesterol, and Gut-

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Hormones in Rats (experiment 4). After acclimatization, the rats (n = 6 /

137

each group) were given free access to water and the control diet or a diet

138

containing 4% of WSCA, FOS or RM for 21 d. Finally, the diet was

139

withdrawn from 7:00-15:00 and the rats were anesthetized with isoflurane

140

inhalation. Portal blood ( 0.5 mL) was collected into syringe containing

After acclimatization, the rats were


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heparin, aprotinin and DPP-IV inhibitors (final concentrations were 50

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IU/mL, 500 KIU/mL, 50 µmol/L, respectively) , and then the portal vein was

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clamped. The rats were killed by collecting blood from the adbominal aorta

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with heparin-coated syringe. The cecum was removed and weighed. The

145

cecal contents were used for analyses of pH and organic acids. Portal

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plasma was separated and used for the measurements of gut hormones and

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SCFAs. Aorta plasma was separated from the blood and used for the

148

measurements of triglyceride and cholesterol conce ntrations.

149

Plasma Lipids. Plasma lipids were determined with commercially available

150

kits (Cholesterol C-test Wako, and Triglyceride G-test Wako, Wako

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Chemical Industries, Osaka, Japan).

152

Luminal pH and Cecal Organic Acids. Cecal, colonic and fecal pH values

153

were measured with a compact pH meter (Model C -1, Horiba, Tokyo,

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Japan). Cecal, colonic and fecal organic acids were measured by the internal

155

standard method using a HPLC as previously described. 19

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Bacterial DNA Extraction, 16S rRNA Gene Sequencing. Bacterial DNA

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was extracted from cecal contents (approx. 100 mg) as described

158

previously. 20 The V3–V4 region of the bacterial 16S rRNA gene was

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amplified and indexed by following the Illumina’s protocol. The details of

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16S rRNA sequencing were shown in Supplementary Method 1.



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BX and BO Quantification by real -time PCR. The numbers of BX and BO

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in the cecal contents were quantified by real-time PCR. The details were

163

shown in Supplementary Method 2.

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Histochemical Analyses.

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was prepared from paraffin-embedded samples. After periodic acid Schiff

166

staining, crypt column length and the numbers of goblet cells per crypt

167

column were determined at the light microscopic level as described

168

previously. 21

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RNA Isolation and Quantitative RT-PCR. Total RNA isolation and

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quantitative RT-PCR were performed as previously described. 22 The primer

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pairs and protocols for PCR of Muc2 23 , Muc3 23 and 18S rRNA 24 have been

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previously reported. 18S rRNA was used as a reference gene. The data were

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expressed relative to the control group.

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Mucin Analysis. Mucin fraction in the cecal contents was isolated and O-

175

linked oligosaccharide chain was measured as mucin by the method as

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described previously. 2 1

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Bacterial Esterase Activity. Cell-free extract of bacteria was prepared from

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the cecal contents as described previously. 1 8 Finally, the cell-free extract

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was suspended in phosphate buffer (50mM, pH8.0) and diluted with the

180

same buffer to have a protein concentration of 1.0 mg/mL for an enzyme

Five-m-thick cross-section for each staining


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source of bacterial acetyl esterase. WSCA (7.5 mg) was dissolved in 2 mL of

182

50 mmol /L phosphate buffer (pH 8.0) as a substrate solution, and 0.5 mL of

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cell-free extract was added to the substrate solution . Then, reaction medium

184

was incubated at 37 C for 4 h. The liberated acetate in the supernatant was

185

measured by a HPLC as described previously 18 .

186

Portal GLP-1 and PYY. Plasma active GLP-1 and PYY concentrations were

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measured using the GLP-1 (Active) ELISA (EGLP-35K, Millipore), and

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Mouse/Rat PYY ELISA kit (Wako Chemical Industries, Osaka, Japan) ,

189

respectively.

190

Portal SCFAs. SCFAs in portal plasma were measured by using liquid

191

chromatography-tandem mass spectrometry (LC -MS/MS). The details were

192

shown in Supplementary Method 3.

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Statistical analyses. Data are expressed as means ± SEMs or pooled SEMs

194

(Tables). Statistical analyses were carried out using JMP8.0.1 software

195

(SAS Institute). Variance homogeneity was examined with the Bartlett test.

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Data were analyzed by one-way ANOVA and significant differences among

197

means were identified by the Tukey-Kramer test. When variances were not

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homogenous, the data were analyzed by Kruskal -Wallis ANOVA, and

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followed by Steel Dwass test. Data of -diversity was further analyzed by

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repeated measures ANOVA. As for -diversity, analysis of similarity



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(ANOSIM) was used to detect statistical significances between microbial

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compositions in different dietary groups. Acetyl esterase activity and

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number of BX in the cecal contents were analyzed by one-way ANOVA

204

within the 4% WSCA group, because the control values were not detected.

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Regression analyses were performed using the Stat Cel 2 program (Tokyo

206

Shoseki). Statistical significance was accepted at p < 0.05.

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RESULTS

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Small-Intestinal Resistance of WSCA (experiment 1).

210

esterase and pancreatic lipase efficiently liberated acetate into the reaction

211

media when triacetin was used as substrate, but the acetyl ester on WSCA

212

was resistant in the small-intestine digestion (Figure S1).

213

Dose-dependent Effects of WSCA on the Cecal Fermentation, Plasma

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Cholesterol, and Microbiota Composition in Rats (experiment 2).

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were no significant differences in food intake and body weight gain among

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the groups (Table 1). Plasma triglyceride concentrations did not differ, but

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plasma cholesterol concentrations in the 4% WSCA showed 22% reduction

218

compared with those in the control, and the differences were significant.

219

Cecal tissue weights and cecal contents were greater in the 2% and 4%

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WSCA, and 4% FOS than in the control. Crypt column height, goblet cell


Both BBMV-

There

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numbers, and mRNA expression of Muc3 in the cecal mucosa were

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significantly greater in 4% WSCA and 4% FOS than in the control. Cecal

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mucin contents were also greater in the 4% WSCA and 4% FOS. Cecal and

224

colonic pH declined significantly in 2% and 4% WSCA, and 4% FOS

225

compared with those in the control. Fecal pH was lower in the 2% and 4%

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WSCA than in the control. SCFA concentrations at the different regions of

227

the large intestine are illustrated in Figure 1. Acetate concentrations in the

228

WSCA groups were higher in the cecum, colon and feces than in the control,

229

while 4% FOS showed higher acetate concentrations only in the cecum and

230

feces (Figure 1A). Propionate concentrations in the 4% WSCA were the

231

highest among the groups throughout the large intestine (Figure 1B). Cecal

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n-butyrate concentrations were higher only in the 4% FOS than in the

233

control (Figure 1C). Succinate concentrations in the 2% and 4% WSCA, and

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4% FOS were higher in the cecum and colon than in the control (Figure

235

1D).

236

In relation to the microbiota, the vast majority of sequences in each group

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(98 – 99%) was assigned to five phyla: Firmicutes, Bacteroidetes,

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Proteobacteria, Verrucomicrobia, and Actinobacteria (Figure 2A).

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Firmicutes was the most abundant phylum in control, 1% and 2%WSCA, and

240

4%FOS, whereas Bacteroidetes was the most abundant phylum in 4%WSCA.



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Actinobacteria have higher population in 4% FOS than in the other groups.

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Among the WSCA groups, a reciprocal change in abundance between

243

Firmicutes and Bacteroidetes was dose-dependently observed. Alpha

244

diversity analysis of entire OTUs was performed for estimation of species

245

richness (Figure 2B). Repeated measures ANOVA on rarefaction curves

246

showed that the diet factor affected the species richness. Comparison

247

between the groups at each sampling size of sequence indicated that species

248

richness in 4% FOS was significantly lower than in the cont rol, 1% and 2%

249

WSCA. In weighted UniFrac PCoA (Figure 2C), the difference among the

250

dietary groups was significant. The top 10 bacterial species with the highest

251

relative abundance were extracted from each dietary group and comparisons

252

were made among the groups (Table 2). Typically, Bacteroides sartorii was

253

the most abundant in the control group, whereas BX and BO became

254

dominant in the 4% WSCA with low abundance of Bacteroides sartorii.

255

Bifidobacteria animalis was the most abundant in the 4% FOS. The top 10

256

bacterial family and genus with the highest relative abundance were

257

extracted from each dietary group and comparisons were made among the

258

groups (Tables S1 and S2).

259

Acetyl Esterase Activitiy and the Number of BX and BO in the Cecal

260

Contents in Rats (experiment 3).

Food intake, body weight gain and


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cecal variables were shown in Table S3. Acetyl esterase activities of cell-

262

free extract from the cecal contents in the 4% WSCA were detected at d 3,

263

and increased over time up to d 21 (Figure 3A), whereas no activities were

264

detected in the control during the experimental period. BX was not detected

265

in the control, but was alwa ys detected in the 4% WSCA in which the

266

number of BX tended to increase over time ( p = 0.068) (Figure 3B). We

267

failed to detect BO with the current primer pairs.

268

Effects of WSCA on the Portal SCFAs, Plasma Cholesterol, and Gut-

269

Hormone in Rats (experiment 4).

270

lower in the 4% WSCA than in the control. No significant differences were

271

observed in plasma triglyceride among the groups, but the plasma

272

cholesterol concentrations in the 4% WSCA showed a lower tendency (22%

273

reduction) compared with those in the control ( p = 0.07). Cecal tissue

274

weight and cecal contents were higher in the 4% WSCA, 4% FOS and 4%

275

RM than in the control, and cecal pH was lower only in the 4% WSCA than

276

in the others (Table 3). In the cecum, acetate concentrations were higher in

277

the 4% WSCA than in the 4% RM. Propionate concentrations were higher in

278

the 4% WSCA than in the others. In the portal plasma, active GLP-1

279

concentrations were higher in the 4% WSCA and 4% FOS than in the control

280

and 4% RM, while PYY concentrations were higher solely in the 4% FOS

Food intake and body weight gain were



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than in the control. Portal concentrations of acetate and propionate were

282

higher in the 4% WSCA, 4% FOS and 4% RM than in the control. Only the

283

portal propionate concentrations showed a tendency to correlate with plasma

284

cholesterol concentrations (r = - 0.99, p = 0.07) (Figure 4).

285 286

DISCUSSION

287

WSCA resisted the small-intestine esterase and lipase and entered the

288

cecum, dose-dependently increased acetate concentrations over the whole

289

regions of the large intestine. Significant differences in the acetate

290

concentrations to the control were achieved at 1% dietary level of WSCA.

291

Intriguingly, higher concentrations of propionate we re manifest in the 4%

292

WSCA throughout the large intestine th an the control and 4% FOS. This

293

simply means that the liberated cellulose from WSCA after the removal of

294

acetyl moiety should be further degraded and fermented by l arge bowel

295

bacteria. Not like crystalline cellulose, that is hardly fermented and only

296

acts as inert bulk-forming fiber in rats,

297

average molecular weight is approximately 24,940 (DP, c.a., 128)

298

far smaller than those of crystalline cellulose. Therefore, we think it is

299

reasonable to hypothesize that the liberated cellulose from WSCA might be

300

fermented by bacteria as in the case of native cellulose in plant ce ll wall

25

WSCA is water-soluble and its


15

being

26,

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301

27

302

concentrations of acetate and propionate.

303

. Thus, the fermentation profile of WSCA is characterized by higher

Relative abundance of BX and BO, determined on the Illmina platform, in

304

the cecal microbiota significantly increased in rats fed 4% WSCA, and the

305

sum of them exceeded the relative abundance of 30% in 4% WSCA. This is

306

presumably because BX and BO are the limited bacteria in the rat large

307

intestine that possess acetyl esterase activities 12, 13 and the removal of

308

acetyl ester from WSCA is essential for cellulose utilization. 9 In fact, the

309

acetyl esterase activities in rats fed 4% WSCA increased over time, and

310

showed the tendency to correspond with the increase in the cecal numbers of

311

BX. However, the elevation of BX and BO was not found in 1 and 2%

312

WSCA groups with higher cecal acetate concentrations than the control.

313

Regarding this, we do not have any direct explanation at present. The

314

previous studies showed that Neisseria sp., originally isolated from soil, had

315

the potential to hydrolyze water-soluble cellulose acetate (DS, 0.88) by

316

cooperative reactions of endo - 1, 4-glucanase and acetyl esterase. 28, 29 In

317

the present study, the relative abundance of Neisseria sp. was low, but

318

increased dose-dependently in rats fed 1 – 4% WSCA diets (Figure S2).

319

This may at least in part explain the reason why the cecal acetate

320

concentrations were higher even in 1 and 2% WSCA groups.



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Recent knowledge of mixed-linkage -glucan catabolism by the human

322

gut microbiota suggested that some members of Bacteroidetes including BO,

323

BX, Bacteroides uniformis, and Bacteroides cellulosilyticus could process

324

complex glucans by the concerted action of the mixed-linkage glucan

325

utilization locus machinery. 26 In this system, glucan-binding protein, endo-

326

acting glucosidase, and transporter for the released oligosaccharides

327

expressed on the cell surface function as a unit, and the released

328

oligosaccharides are immediately sequestered into the periplasm, where they

329

are further degraded into monosaccharide. Such machinery in Bacteroidetes

330

sp. could be induced in the highest WSCA level in diet, and may permit BX

331

and BO a preferential usage of the residual cellulose after de -acetylation.

332

Moreover, Bacteroides sp. produces mainly acetate, propionate and

333

succinate during cellulose metabolism . 27 In the present study, the higher

334

concentrations of propionate were corresponding to the higher

335

concentrations of succinate, which is an intermediate product of bacteria

336

fermentation to yield propionate. 30 These findings may partly explain the

337

reason why the fermentation pattern of 4% WSCA group is characterized by

338

higher concentrations of propionate.

339 340

Dose-dependent elongation of crypt column height and increase in the number of goblet cells were observed in rats fed 1 -4% WSCA diets.


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341

Previous studies demonstrated that SCFAs have a trophic effect on the

342

mucosa with a different magnitude (n-butyrate > propionate > acetate). 31 n-

343

Butyrate concentrations were low in rats fed WSCA, but higher

344

concentrations of acetate and propionate may contribute to such effects.

345

Although the mucosal mRNA expression of Muc3 was greater in 4% WSCA

346

and 4% FOS, but that of Muc2, coded a major secretory mucin in the

347

intestine 32 , was comparable among the groups. Accordingly, it appears more

348

likely that higher cecal mucin contents in 4% WSCA and 4% FOS were due

349

to a greater numbers of mucosal goblet cells.

350

One interesting feature of the physiological effects of WSCA is plasma

351

cholesterol-lowering effect, which was reproducibly observed in

352

experiments 2 and 4 to the same degree. Propionate has been shown to lower

353

plasma cholesterol concentrations when fed to rats 2 and to inhibit the

354

synthesis of cholesterol from [ 14 C] acetate in freshly isolated rat

355

hepatocytes 33, 34 in the concentration range observed in the portal blood of

356

rats fed on diet supplemented with oat bran (0.1 – 0.8 mmol/L). 35

357

Collectively, the fermentation profile of WSCA might be related to this

358

effect, because the majority of propionate produced by the bacterial

359

fermentation in the large intestine enter the portal vein without being

360

consumed by colonocytes. 2 In the present study, the portal concentra tions of



Page 20 of 40

20

361

propionate tended to negatively correlated with the plasma cholesterol

362

concentrations.

363

The portal concentrations of both active GLP -1 and PYY were

364

significantly increased in the 4% FOS, while only active GLP-1 showed a

365

significant increase in 4% WSCA, in spite of being higher cecal

366

concentrations of acetate and propionate com pared with those of 4% FOS .

367

This is totally unexpected and the reason for this is unclear, but recent

368

studies indicated that dietary FOS or inulin exceptionally increased the

369

intestinal L-cell density. 36, 37

370

To our knowledge this is the first demonstration that WSCA can be used

371

to raise large bowel levels of acetate and propionate in rats through the

372

bacterial fermentation. Apparently, higher propionate concentrations in th e

373

large intestine by WSCA consumption were related to the decreased plasma

374

cholesterol concentrations in rats. These findings suggest that WSCA should

375

be another candidate of foodstuffs to targeted delivery of acetate and

376

propionate in the large intestine.

377 378

AUTHOR INFORMATION

379

Corresponding Author

380

Tel/Fax: 81-54-238-5132, E-mail: [email protected]


Page 21 of 40


21

381

Author Contribution

382

Tomomi Genda contributed to experiments, data analysis, discussion and

383

writing manuscript. Shingo Hino , Shu Shimamoto, and Toshikazu Nakamura

384

contributed to data analysis and discussion. Takashi Kondo, Shunsaku

385

Sugiura, and Shizuka Ukita provided technical support. Tatsuya Morita

386

contributed to study design and critical revision.

387

Funding

388

The present study was supported by Daicel Corporation.

389 390

ABBREVIATIONS USED

391

BBMV, brush boarder membrane vesicle; BO, Bacteroides ovatus; BX,

392

Bacteroides xylanisolvens ; FOS, fructo-oligosaccharides; GLP-1, glucagon

393

like peptide-1; GPCR-41, G protein coupled receptors 41; GPCR -43, G

394

protein coupled receptors 43; PYY, peptide YY ; OTU, operational

395

taxonomic unit; RM, resistant maltodextrin; SCFA, short-chain fatty acids;

396

WSCA, water-soluble cellulose acetate.

397 398

SUPPORTING INFORMATION. Methods of 16S rRNA gene sequencing

399

(Supplementary method 1), BX and BO quantification by real-time PCR

400

(Supplementary method 2) and portal SCFAs (Supplementary method 3) and



Page 22 of 40

22

401

resistance of WSCA against BBMV-esterase and pancreatic lipase (Figure

402

S1), relative abundance of Neisseria (Figure S2), relative abundance of

403

cecal microbiota at the family level (Table S1) and at the genus level (Table

404

S2) in rats fed the control diet or a diet containing 1, 2, 4% WSCA or 4%

405

FOS diet for 21 d (experiment 2), Food intake, body weight gain, and cecal

406

variables in rats fed the control diet or a diet containing 4 % WSCA diet for

407

3, 7 or 21 d (experiment 3) (Table S3) are available in Supporting

408

Information.

409 410

REFERENCES

411

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412

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413

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542

GLP-1 levels in Zucker diabetic rats. Life Sci. 2011, 88, 392-9.

543 544 545 546 547 548 549 550 551 552 553 554 555 556 557 558 559 560



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561 562

FIGURE CAPTIONS

563

Figure 1 Luminal concentrations of acetate (A), propionate (B), n-butyrate

564

(C), and succinate (D) at the different regions of the large intestine in rats

565

fed the control diet or a diet containing 1, 2 , or 4% of WSCA or 4% of FOS

566

for 21d (experiment 2)

567

Values are means ± SEMs, n = 6. Values without a common superscript

568

letter differ, p < 0.05. FOS, fructo-oligosaccharides; WSCA, water-soluble

569

cellulose acetate.

570 571

Figure 2 Microbial compositions at the phyla level (A), rarefaction curves

572

(-diversity curves) (B), principal coordinate analysis plot based on

573

weighted UniFrac (-diversity) (C) in rats fed the control diet or a diet

574

containing 1, 2, or 4% of WSCA or 4% of FOS for 21d (experiment 2)

575

Values are represented as means (A) or means ± SEMs (B ), n = 6. Values

576

without a common superscript letter differ, p < 0.05. FOS, fructo-

577

oligosaccharides; WSCA, water-soluble cellulose acetate.

578 579

Figure 3 Changes in microbial esterase activity (A) and the number of BX

580

(B) of the cecal contents in rats fed 4% WSCA diet during the feeding


Page 31 of 40


31

581

period (experiment 3)

582

Values are means ± SEMs (n=6). Values without a common superscript letter

583

differ, p < 0.05.

584

BX, Bacteroides xylanisolvens; WSCA, water-soluble cellulose acetate.

585 586

Figure 4 Correlations between the plasma cholesterol concentrations and

587

the portal concentrations of acetate ( A), propionate (B), and n-butyrate (C)

588

in rats fed the control diet or a diet containing 4 % of WSCA, FOS or RM for

589

21d (experiment 4)

590

Values are represented as means ± SEMs, n = 6.

591

FOS, fructo-oligosaccharides; RM, resistant maltodextrin; WSCA, water-

592

soluble cellulose acetate.

593 594 595 596 597 598 599 600



Page 32 of 40

32

601


Page 33 of 40


33

Table 1 Food Intake, Body Weight Gain, Plasma Lipids, and Cecal, Colonic and Fecal Variables in Rats Fed the Control Diet or a Diet Containing 1, 2, or 4% WSCA or 4% FOS for 21d (experiment 2)a Food intake, g Body weight gain, g Plasma lipids, mmol/L Triglycerides Cholesterol Cecum Contents, g Tissue weight, g Crypt column, µm Goblet cells, n/ crypt Mucin, µmol/g Gene expression, relative value Muc2 Muc3 Luminal pH Cecum Colon Feces

Control 349 92

1% WSCA 341 88

2% WSCA 328 89

4% WSCA 331 82

4% FOS 323 87

Pooled SEM 14 7

2.5 3.2 a

2.6 3.0 ab

2.7 2.9 ab

2.4 2.5 b

3.3 2.7 ab

0.6 0.3

2.7 c 0.53 c 176 c c 23.3 0.37 a

2.8 c 0.58 bc 179 bc bc 24.7 0.31 a

4.0 b 0.66 b 184 bc bc 24.7 0.36 a

5.5 a 0.81a 193 ab b 25.3 0.54 b

5.2 a 0.83a 202 a a 27.5 0.82 b

0.4 0.04 8 0.9 0.19

1.0 b 1.0

1.1 ab 1.2

1.0 ab 1.3

0.9 a 1.8

7.9 a 8.0 a 8.3 a

7.7 a 7.2 b 8.3 a

7.0 b 6.6 b 7.4 b

6.4 b 6.6 b 7.0 b

a

0.9 ab 1.4 6.4 b 6.7 b 7.6 ab

0.2 0.4 0.3 0.3 0.4

Values are means, n = 6. Values with different letters are significantly different (p

Bacterial Fermentation of Water-Soluble Cellulose Acetate Raises

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