Dietary Clostridium Butyricum induces a phased shift in fecal

1 day ago - Clostridium butyricum (C. butyricum) is known as a butyrate producer and a regulator of gut health,but whether it exerts beneficial effe...
2 downloads 5 Views 1MB Size
Subscriber access provided by UNIV OF NEW ENGLAND ARMIDALE

Bioactive Constituents, Metabolites, and Functions

Dietary Clostridium Butyricum induces a phased shift in fecal microbiota structure and increases the acetic acid-producing bacteria in a weaned piglet model Jie Zhang, Xiyue Chen, Ping Liu, Jinbiao Zhao, Jian Sun, Wenyi Guan, Lee J. Johnston, Crystal L. Levesque, Peixin Fan, Ting He, Guolong Zhang, and Xi Ma J. Agric. Food Chem., Just Accepted Manuscript • DOI: 10.1021/acs.jafc.8b01253 • Publication Date (Web): 23 Apr 2018 Downloaded from http://pubs.acs.org on April 23, 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 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 34

Journal of Agricultural and Food Chemistry

1

Dietary Clostridium Butyricum induces a phased shift in fecal microbiota structure and

2

increases the acetic acid-producing bacteria in a weaned piglet model

3 4

Jie Zhang1,2,†, Xiyue Chen1,†, Ping Liu1, Jinbiao Zhao1, Jian Sun1,2, Wenyi Guan2, Lee J.

5

Johnston3, Crystal Levesque4, Peixin Fan5,6, Ting He1, Guolong Zhang 7*, and Xi Ma1,8,9

6

*

7

1

8

China Agricultural University, Beijing 100193, People's Republic of China

9

2

State Key Laboratory of Animal Nutrition, College of Animal Science and Technology,

Department of Animal Husbandry and Veterinary, Beijing Vocational College of Agriculture,

10

Beijing 102442, People's Republic of China

11

3

12

56267, USA

13

4

14

Dakota State University, Brookings, South Dakota 57007, USA

15

5

Emerging Pathogens Institute, University of Florida, Gainesville, Florida 32608, USA

16

6

Department of Animal Sciences, Institute of Food and Agricultural Sciences, University of

17

Florida, Gainesville, Florida 32608, USA

18

7

19

USA

20

8

21

266109, People's Republic of China

22

9

23

Southwestern Medical Center, Dallas, Texas 75230, USA

24



25

* Correspondence: E-mail: [email protected] (G. Zhang) or [email protected] (X. Ma).

West Central Research & Outreach Center, University of Minnesota, Morris, Minnesota

Department of Animal Science, College of Agriculture and Biological Sciences, South

Department of Animal Science, Oklahoma State University, Stillwater, Oklahoma 74078,

College of Animal Science and Technology, Qingdao Agricultural University, Qingdao,

Department of Internal Medicine, Department of Biochemistry, University of Texas

These authors contributed equally to this work.

1

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

26

Phone: 86-13811794292. Fax: 86-10-62733688.

27

ORCID

28

Xi Ma: 0000-0003-4562-9331

29

Jie Zhang: 0000-0001-5010-0833

30

Ping Liu: 0000-0002-8298-6576

31

Ting He:0000-0002-2856-9501

32

2

ACS Paragon Plus Environment

Page 2 of 34

Page 3 of 34

33

Journal of Agricultural and Food Chemistry

Abstract

34

Clostridium butyricum (C. butyricum) is known as a butyrate producer and a regulator of

35

gut health,but whether it exerts beneficial effect as a dietary supplement via modulating the

36

intestinal microbiota remains elusive. This study investigated the impact of C. butyricum on

37

the fecal microbiota composition and their metabolites on d 14 and d 28 after weaned with 10

38

g/kg dietary supplementation of C. butyricum. Dynamic changes of microbial compositions

39

showed dramatically increasing Selenomonadales and decreasing Clostridiales on d 14 and d

40

28. Within Selenomonadales, Megasphaera became the main responder by increasing from

41

3.79% to 11.31%. Following a prevalent of some acetate producers (Magasphaera) and

42

utilizers (Eubacterium_hallii) at the genus level and while a significant decrease in fecal

43

acetate on d 28, the present data suggested that C. butyricum influenced microbial

44

metabolism by optimizing the structure of microbiota and enhancing acetate production and

45

utilization for butyrate production.

46 47

Keywords: Clostridium butyricum, fecal microbiota, short chain fatty acid, phased shift,

48

acetate production, weaned piglet

49

3

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

50

Introduction

51

The probiotics, intestinal microbiota, and microbial metabolites are all involved in intestinal

52

ecosystem and have many crucial functions in animal health, such as enhancing growth

53

performance and reducing weaned stress. Weaned stress is often accompanied by intestinal

54

dysbiosis, with some abrupt changes in the gut microbiota composition of young animals

55

(including infants), resulting in diarrhea, growth retardation, even mortality.1

56

A diet added probiotics, Clostridium butyricum (C. butyricum) is one of the important

57

factors for benefiting health.2,3 C. butyricum is an anaerobic, gram-positive butyric

58

acid-producing bacillus. C. butyricum resides in the gastrointestinal tract and has a protective

59

role against pathogenic bacteria and intestinal injury by modulating gut microbial

60

metabolites,3,4,5 such as short-chain fatty acids (SCFAs).6 Our previous study has confirmed

61

the beneficial effects of butyrate, a main metabolite of C. butyricum, in the control of

62

weanling diarrhea.7 The anti-inflammatory and probiotics traits of certain strains of C.

63

butyricum has been reported with in vitro experiments.3

64

However, the oral approach of C. butyricum to affect intestinal microbial composition

65

and butyrate production in weanling animal remains unclear and whether it acts like butyrate

66

to modulate the intestinal dysbacteriosis in vivo is unknown. Considering the complex and

67

diverse community of intestinal microbiota, the practical effectiveness of C. butyricum in the

68

intestine partly depends on the interaction with intestinal microbiota. Thus, it is important to

69

discover the modified intestinal microbiota and their metabolites in vivo with

70

supplementation of C. Butyricum. This manuscript investigates the effect of dietary C.

71

butyricum on maintaining the homeostasis of intestinal microbiota in weaned piglets and

72

deepens the understanding of how C. butyricum exerts a positive influence on gut microbiota.

73

Additionally, the anatomical and physiological similarities between pigs and humans in size

74

and polyphagia indicate the weaned piglets as an ideal alterative model for human gut 4

ACS Paragon Plus Environment

Page 4 of 34

Page 5 of 34

Journal of Agricultural and Food Chemistry

75

microbiota.8 It will benefit to understand the microbiota maturation in the weaned period of

76

humans.

77

Materials and methods

78

Animals and experimental design

79

This experiment followed the recommendations of "Laboratory Animals-Guideline of

80

Welfare and Ethics of China (ICS 65.020.30), approved by the Institutional Animal Care and

81

Use Committee of China Agricultural University.

82

48 crossbred piglets (Duroc × Landrace × Large White) with an age of 26 ± 1 day were

83

selected from a pool of 24 litters (body weight: 7.0 ± 0.5 kg) and randomly assigned to two

84

groups including control and C. butyricum treatment with 6 animals per pen and 4 replicate

85

pens per group. A non-medicated corn-soybean basal diet in mash form used in the control

86

group was formulated to satisfy the NRC (2012) nutrient requirements for 11 to 20 kg body

87

weight pigs (Table 1). 10 g/kg of C. butyricum (China General Microbiological Culture

88

Collection Center, Strain No. 1.336) was added in basal diet for treatment group with the

89

quantity of C. butyricum in spore state at a minimum of 1 × 108 CFU/g.

90

During the trial, piglets were allowed free access to water and diets. Body weight of

91

each piglet was recorded on d 0, 14, and 28, and feed intake was recorded on a weekly basis.

92

Growth performance indices including average daily feed intake (ADFI), average daily gain

93

(ADG), and feed conversion ratio (F/G) were collected. Freshly voided feces were collected

94

on d 14 and d 28 from 8 pigs (2 pigs/pen, randomly) and immediately frozen in liquid

95

nitrogen for future isolation of bacterial genomic DNA and analysis of SCFAs. On d 28,

96

heparin-anticoagulated blood samples from the jugular vein were acquired from the same

97

pigs used for fecal collection. Plasma samples were obtained by centrifugation at 3,000 × g

98

for 10 min at 4°C and analyzed for hormones including peptide tyrosine-tyrosine (PYY),

99

glucagon-like peptide 1 (GLP-1), and serotonin (5-HT). 5

ACS Paragon Plus Environment

Journal of Agricultural and Food Chemistry

Page 6 of 34

100

Extraction of fecal DNA

101

The E.Z.N.A Stool DNA Kit (Omega Bio-tek, Norcross, GA, USA) was used for the

102

extraction of total bacterial DNA with the manufacturer’s protocols. DNA was quantified

103

with NanoDrop 2000 Spectrophotometer (Thermo Scientific) and further assessed by running

104

on 1% agarose gel electrophoresis prior to Illumina Miseq sequencing analysis.

105

PCR amplification

106

PCR amplification was performed on the V3-V4 region of 16S rRNA gene using TransStart

107

Fastpfu® DNA Polymerase (Takara) for initial denaturation at 95°C for 3 min, then at 95°C

108

for 30 s by 26 cycles, 55°C for 30 s, and 72°C for 45 s and an extension at 72°C for 10 min.

109

Forward

110

(5’-GGACTACHVGGGTWTCTAAT-3’) were used for the primers with an 8-bp unique

111

sequence for each sample. PCR reactions were conducted in 20 μL reactions including 4 μL

112

of 5 × Fastpfu buffer, 2 μL of 2.5 mM dNTPs, 0.4 μL of Fastpfu polymerase, 0.8 μL of 5 μM

113

primers and 10 ng of template DNA for three replications.

114

Illumina MiSeq sequencing

115

PCR products were purified with AxyPrep DNA Purification kit (Axygen Biosciences, Union

116

City, USA) after running at 2% agarose gels electrophoresis. The visualized PCR products on

117

agarose gels were quantified on QuantiFluor-ST Fluorimeter (Promega, Wisconsin, USA)

118

using PicoGreen dsDNA Quantitation Kit (Invitrogen, Carlsbad, USA). Purified amplicons

119

were gathered in equimolar for sequencing (2 × 300 bp) on Illumina MiSeq by Allwegene

120

(Beijing, China) according to standard protocols. Raw sequencing data were deposited in the

121

NCBI SRA Database with an accession NO. PRJNA383295 (SRA).

122

Bioinformatics analysis of sequencing data

123

QIIME (version 1.17) was used for demultiplexing and quality-filtering raw fastq files with

124

the following criteria: (i) Sequencing reads were clipped with an average quality score of