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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
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Journal of Agricultural and Food Chemistry
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Dietary Clostridium Butyricum induces a phased shift in fecal microbiota structure and
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increases the acetic acid-producing bacteria in a weaned piglet model
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Jie Zhang1,2,†, Xiyue Chen1,†, Ping Liu1, Jinbiao Zhao1, Jian Sun1,2, Wenyi Guan2, Lee J.
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Johnston3, Crystal Levesque4, Peixin Fan5,6, Ting He1, Guolong Zhang 7*, and Xi Ma1,8,9
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*
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1
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China Agricultural University, Beijing 100193, People's Republic of China
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2
State Key Laboratory of Animal Nutrition, College of Animal Science and Technology,
Department of Animal Husbandry and Veterinary, Beijing Vocational College of Agriculture,
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Beijing 102442, People's Republic of China
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3
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56267, USA
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Dakota State University, Brookings, South Dakota 57007, USA
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Emerging Pathogens Institute, University of Florida, Gainesville, Florida 32608, USA
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Department of Animal Sciences, Institute of Food and Agricultural Sciences, University of
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Florida, Gainesville, Florida 32608, USA
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7
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USA
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8
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266109, People's Republic of China
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9
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Southwestern Medical Center, Dallas, Texas 75230, USA
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†
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* 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.
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Phone: 86-13811794292. Fax: 86-10-62733688.
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ORCID
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Xi Ma: 0000-0003-4562-9331
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Jie Zhang: 0000-0001-5010-0833
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Ping Liu: 0000-0002-8298-6576
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Ting He：0000-0002-2856-9501
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Journal of Agricultural and Food Chemistry
Abstract
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Clostridium butyricum (C. butyricum) is known as a butyrate producer and a regulator of
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gut health，but whether it exerts beneficial effect as a dietary supplement via modulating the
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intestinal microbiota remains elusive. This study investigated the impact of C. butyricum on
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the fecal microbiota composition and their metabolites on d 14 and d 28 after weaned with 10
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g/kg dietary supplementation of C. butyricum. Dynamic changes of microbial compositions
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showed dramatically increasing Selenomonadales and decreasing Clostridiales on d 14 and d
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28. Within Selenomonadales, Megasphaera became the main responder by increasing from
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3.79% to 11.31%. Following a prevalent of some acetate producers (Magasphaera) and
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utilizers (Eubacterium_hallii) at the genus level and while a significant decrease in fecal
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acetate on d 28, the present data suggested that C. butyricum influenced microbial
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metabolism by optimizing the structure of microbiota and enhancing acetate production and
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utilization for butyrate production.
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Keywords: Clostridium butyricum, fecal microbiota, short chain fatty acid, phased shift,
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acetate production, weaned piglet
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Introduction
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The probiotics, intestinal microbiota, and microbial metabolites are all involved in intestinal
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ecosystem and have many crucial functions in animal health, such as enhancing growth
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performance and reducing weaned stress. Weaned stress is often accompanied by intestinal
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dysbiosis, with some abrupt changes in the gut microbiota composition of young animals
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(including infants), resulting in diarrhea, growth retardation, even mortality.1
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A diet added probiotics, Clostridium butyricum (C. butyricum) is one of the important
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factors for benefiting health.2,3 C. butyricum is an anaerobic, gram-positive butyric
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acid-producing bacillus. C. butyricum resides in the gastrointestinal tract and has a protective
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role against pathogenic bacteria and intestinal injury by modulating gut microbial
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metabolites,3,4,5 such as short-chain fatty acids (SCFAs).6 Our previous study has confirmed
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the beneficial effects of butyrate, a main metabolite of C. butyricum, in the control of
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weanling diarrhea.7 The anti-inflammatory and probiotics traits of certain strains of C.
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butyricum has been reported with in vitro experiments.3
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However, the oral approach of C. butyricum to affect intestinal microbial composition
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and butyrate production in weanling animal remains unclear and whether it acts like butyrate
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to modulate the intestinal dysbacteriosis in vivo is unknown. Considering the complex and
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diverse community of intestinal microbiota, the practical effectiveness of C. butyricum in the
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intestine partly depends on the interaction with intestinal microbiota. Thus, it is important to
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discover the modified intestinal microbiota and their metabolites in vivo with
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supplementation of C. Butyricum. This manuscript investigates the effect of dietary C.
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butyricum on maintaining the homeostasis of intestinal microbiota in weaned piglets and
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deepens the understanding of how C. butyricum exerts a positive influence on gut microbiota.
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Additionally, the anatomical and physiological similarities between pigs and humans in size
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and polyphagia indicate the weaned piglets as an ideal alterative model for human gut 4
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microbiota.8 It will benefit to understand the microbiota maturation in the weaned period of
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humans.
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Materials and methods
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Animals and experimental design
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This experiment followed the recommendations of "Laboratory Animals-Guideline of
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Welfare and Ethics of China (ICS 65.020.30), approved by the Institutional Animal Care and
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Use Committee of China Agricultural University.
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48 crossbred piglets (Duroc × Landrace × Large White) with an age of 26 ± 1 day were
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selected from a pool of 24 litters (body weight: 7.0 ± 0.5 kg) and randomly assigned to two
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groups including control and C. butyricum treatment with 6 animals per pen and 4 replicate
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pens per group. A non-medicated corn-soybean basal diet in mash form used in the control
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group was formulated to satisfy the NRC (2012) nutrient requirements for 11 to 20 kg body
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weight pigs (Table 1). 10 g/kg of C. butyricum (China General Microbiological Culture
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Collection Center, Strain No. 1.336) was added in basal diet for treatment group with the
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quantity of C. butyricum in spore state at a minimum of 1 × 108 CFU/g.
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During the trial, piglets were allowed free access to water and diets. Body weight of
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each piglet was recorded on d 0, 14, and 28, and feed intake was recorded on a weekly basis.
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Growth performance indices including average daily feed intake (ADFI), average daily gain
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(ADG), and feed conversion ratio (F/G) were collected. Freshly voided feces were collected
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on d 14 and d 28 from 8 pigs (2 pigs/pen, randomly) and immediately frozen in liquid
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nitrogen for future isolation of bacterial genomic DNA and analysis of SCFAs. On d 28,
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heparin-anticoagulated blood samples from the jugular vein were acquired from the same
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pigs used for fecal collection. Plasma samples were obtained by centrifugation at 3,000 × g
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for 10 min at 4°C and analyzed for hormones including peptide tyrosine-tyrosine (PYY),
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glucagon-like peptide 1 (GLP-1), and serotonin (5-HT). 5
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Extraction of fecal DNA
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The E.Z.N.A Stool DNA Kit (Omega Bio-tek, Norcross, GA, USA) was used for the
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extraction of total bacterial DNA with the manufacturer’s protocols. DNA was quantified
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with NanoDrop 2000 Spectrophotometer (Thermo Scientific) and further assessed by running
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on 1% agarose gel electrophoresis prior to Illumina Miseq sequencing analysis.
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PCR amplification
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PCR amplification was performed on the V3-V4 region of 16S rRNA gene using TransStart
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Fastpfu® DNA Polymerase (Takara) for initial denaturation at 95°C for 3 min, then at 95°C
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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.
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Forward
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(5’-GGACTACHVGGGTWTCTAAT-3’) were used for the primers with an 8-bp unique
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sequence for each sample. PCR reactions were conducted in 20 μL reactions including 4 μL
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of 5 × Fastpfu buffer, 2 μL of 2.5 mM dNTPs, 0.4 μL of Fastpfu polymerase, 0.8 μL of 5 μM
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primers and 10 ng of template DNA for three replications.
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Illumina MiSeq sequencing
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PCR products were purified with AxyPrep DNA Purification kit (Axygen Biosciences, Union
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City, USA) after running at 2% agarose gels electrophoresis. The visualized PCR products on
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agarose gels were quantified on QuantiFluor-ST Fluorimeter (Promega, Wisconsin, USA)
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using PicoGreen dsDNA Quantitation Kit (Invitrogen, Carlsbad, USA). Purified amplicons
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were gathered in equimolar for sequencing (2 × 300 bp) on Illumina MiSeq by Allwegene
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(Beijing, China) according to standard protocols. Raw sequencing data were deposited in the
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NCBI SRA Database with an accession NO. PRJNA383295 (SRA).
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Bioinformatics analysis of sequencing data
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QIIME (version 1.17) was used for demultiplexing and quality-filtering raw fastq files with
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the following criteria: (i) Sequencing reads were clipped with an average quality score of
