Small molecular weight metabolites regulating growth and ... › alternativestoantibiotics...Body weight gain of chickens Body weight gain = final body weight – initial body weight

Small molecular weight metabolites regulating growth and immunity as

postbiotic antibiotic alternatives

Inkyung Park

Animal Bioscience and Biotechnology Laboratory, Beltsville Agricultural Research Center, Agricultural Research Service, USDA, MD 20705

Introduction: antibiotic alternatives

• Avian coccidiosis with Emeria spp

• Necrotic enteritis

• With AGP withdrawal, there is an urgent need to develop antibiotic alternatives. Gallucci and Matzinger, 2001; Shirley and Lillehoj, 2012

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Market age and weight since 1925 (chickens)

• Bacillus subtilis as probiotics in chicken feed

- Shows growth-promoting effect (Gadde et al. 2017)

- Shows a protective role against chicken pathogen (Park et al. 2019)

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CON BMD PB1 PB2 PB3

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IL6 in ileum

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CON BMD PB1 PB2 PB3

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Occludin in ileum

Bacillus subtilis by Nano Creative/Science Source

(Gadde et al. 2018)

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CON BMD PB1 PB2 PB3

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Body weight (D 14)

― Con: basal diet ― BMD: antibiotics ― PB1: B. subtilis 1781 ― PB2: B. subtilis 747 ― PB3: B. subtilis 1781+747

Introduction: antibiotic alternatives

• What is the mechanisms of dietary Bacillus subtilis supplementation?

Introduction: gut microbiota and metabolites

“Postbiotics” novel materials promoting gut health as functional additive of diet

Objective of this study

• Therefore, the current study was undertaken to characterize the metabolic alterations in the chicken gut following dietary supplementation with B. subtilis DFMs with the goal of identifying potential chemical compounds that might be directly used to improve poultry growth performance without the use of AGPs.

Materials and Methods

84 male day-old Ross 708 broilers

d 0

Normal feed

d 14 d 21

• Initial body weight • Allocation to 3 treatments

Measurements: • Metabolomic profiling of

the ileal contents by mass spectrometry (Metabolon, Durham, NC)

ileal content from euthanized chciekn (2 chickens/pen = total 8 chickens/treatment)

• Body weight • Feed intake

Experimental feed

Treatments Supplements Dose Chickens/cage Replication

CON Basal diet 7 4

B. subtilis 1781 CON+B. subtilis 1781 1.5 × 105 CFU/g feed 7 4

B. subtilis 747 CON+B. subtilis 747 1.5 × 105 CFU/g feed 7 4

• B. subtilis strains were obtained from Church & Dwight Co. Inc. (Waukesha, WI).

Metabolic pathways

• Statistical Analysis

• Growth performance

- PROC MIXED in SAS (SAS Inst. Inc., Cary NC)

- P values < 0.05

- PDIFF option

• Ileal biochemicals

- Array Studio software (OmicSoft, Cary, NC)

- the programs R (R Foundation for Statistical Computing, Vienna, Austria)

- JMP (SAS Institute)

- P values < 0.05

- Random Forest Analysis (RFA) by computing the Mean Decrease Accuracy

Materials and Methods

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b b

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Control B. subtilis 1781 B. subtilis 747

Bo

dy

wei

gh g

ain

, g

Body weight gain of chickens

Body weight gain = final body weight – initial body weight

Control

B. subtilis 1781

B. Subtilis 747

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Comp.1 [24.43%]

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-10

0

10

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30

Co

mp

.2 [11

.99

%]

Co

mp

2 (

11.9

9)

Comp 1 (24.43)

Principal component analysis of ileal biochemicals

Result: growth effect and biochemical distribution

Result: hierarchical clustering heatmap

Control

B. subtilis 1781

B. subtilis 747

-4.00 4.00

• Total 674 biochemicals

• Con vs B. subtilis 1781 - Increased 209 (25; P < 0.05) - Decreased 461 (58; P < 0.05)

• Con vs B. subtilis 747 - Increased 265 (12; P < 0.05) - Decreased 402 (38; P < 0.05)

Bio

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mic

al p

rofi

les

Result: after B. subtilis 1781 and 747 treatment

• 193 amino acid pathways

• 32 carbohydrate pathways

• 263 lipid pathways

• 42 cofactors and vitamins pathways

• 51 nucleotide pathways

• 12 energy pathways

• 81 unknown pathways

• 498 Human Metabolome Database (HMDB)

• 336 Kyoto Encyclopedia of Genes and Genomes (KEGG) codes

Result: pathways in this study B

ioch

em

ical

pro

file

s

CON vs B. subtilis 1781

Result: random forest analysis and plot

• 8 amino acids (26.7%) - 2-hydroxy-4-

(methylthio)butanoic acid - 3-(4-hydroyphenyl)lactate - beta-hydroxycholate - creatine - cystein s-sulfate - N-methylproline - glutamate gamma-methyl

ester - dimethylglycine

• 8 lipids (26.7%) - 2,4-dihydroxybutyrate - glycerophosphoethanolamine - 2R 3R-dihydroxybutyrate - chenodeoxycholte - 16-hydroxypalmitate - beta-sitosterol - 1-palmitoyl-

digalactosylglycerol (16:0) - octadecenedioate

• 5 vitamins and cofactors (16.7%) - carotene diol - alpha-tocopherol acetate - beta-cryptoxanthin - nicotinamide adenine dinucleotide (NAD+) - alpha-tocopherol

• 3 nucleotides (10.0%) - 2’deoxyguanosine - N6-methyladenosine - N1-methyladenosine

Result: random forest analysis and plot

CON vs B. subtilis 747 • 6 amino acids (20.0%) - glutamate gamma-methyl ester - betaine - taurine - dimethylglycine - 5-hydroxyindoleacetate - methylsuccinate

• 6 peptides (20.0%)

- glutaminylleucine - alanylleucine - valylleucine - leucylglycine - leucylalanine - valylglycine

• 10 lipids (33.0%) - 6-oxolithocholate - oleoylcholine - glycerophosphoglycerol - sebacate (C10-DC) - 2-hydroxyglutarate - palmitoylcholine - linoleoylcholine - 3-dehydrodeoxycholate - chenodeoxycholate - stearoylcarnitine (C18)

• 2 nucleotides (0.1 %) - uridine 5’-monophosphate (UMP) - guanine

Alanylleucine

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lite

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(Med

ium

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Control B. subtilis B. subtilis 1781 747

Glutaminylleucine



Glycylisoleucine

Valylleucine

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lite

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(Med

ium

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nsi

ty)


Phenylalanylalanine


Valylglutamine


Result: amino acids (dipeptides)

Chenodeoxycholate


3-dehydrodeoxycholate


Cholesterol


Sebacate (C10-DC)


Linoleoylcholine


Valerylglycine (C5)


Met

abo

lite

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(Med

ium

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led

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nsi

ty)

M

etab

olit

e Si

gnal

(M

ediu

m S

cale

d S

ign

al In

ten

sity

)

Result: lipids

Miyamoto et al., 2015

Met

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lite

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(Med

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N1-methyladenosine N6-methyladenosine


Guanine



Met

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lite

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(Med

ium

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led

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nal

Inte

nsi

ty)

2-deoxyguanosine Uridine 5-monophosphate



Cytidine

Result: nucleotides

N1-methyladenosine

N6-methyladenosine

Guanine

2-deoxyguanosine

Donia and Fischbach, Science, 2015

2-(4-hydroxyphenyl)propionate

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lite

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)


Salicylate-glucoside


Nicotinamide ribonucleotide (NMN)

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Nicotinamide adenine dinucleotide (NAD+)

Fructose Lactate

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Result: others

Polyphenol

Fructose

Fructose feeding negatively affects antioxidant

capacity in the blood of hypertensive rats but

improves this capacity in the liver (Girard et al., 2006, Nutrition).

Lactate ion may prevent lipid peroxidation by

scavenging free radicals such as O2- and -OH

but not lipid radicals (Groussard et al., 2000, J Appl Physiol).

epithelial barrier

Strong antioxidant

Antioxidant effects in the gut or

organs

Conclusions

• Dietary supplementation with B. subtilis has profound effects on the levels of a wide variety of chemical metabolites in the chicken gut.

• These altered metabolite levels provide a biochemical signature unique to each B. subtilis supplementation group.

• Future studies are warranted to assess the growth promoting properties, if any, of the identified chemical compounds in lieu of antibiotics.

Further study: extra data

Chicken macrophage cells (HD11)

40 metabolites of Up-regulated common metabolites between B. subtilis 1781 and 747

Proinflammatory responses

Chicken epithelial cells (8E11)

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IL6

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Mea

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N-acetylglucosamine Syringic acid

2'-deoxyadenosine β-alanine

Acknowledgment

USDA-ARS • Dr. Hyun S. Lillehoj

Church & Dwight • Dr. N. P. Zimmerman • Dr. A. H. Smith • Dr. T. Rehberger

Thank you !

Documents

Small molecular weight metabolites regulating growth and ... › alternativestoantibiotics...Body weight gain of chickens Body weight gain = final body weight – initial body weight