Scientific Developments in the Fields of Eubiotic and Phytogenic Ingredients

Leon Broom, Ph.D.

Senior Manager, Gut Health Consultancy and Visiting Research Fellow, University of Leeds

www.guthealthconsultancy.com

Presentation

1. Introduction

2. Challenges

3. Current state/latest developments

4. Summary

Introduction

Main reasons for interest:

1. Increasing global human population and demand for animal protein.

2. Resultant resource pressures require improved production efficiency.

3. Consumers demand more ‘natural’ production methods, which includes both reduced antibiotic use and risk of foodborne illness/pathogens.

Definitions

Eubiosis (eubiotic) – (promotes) optimum microbial balance in the intestine.

Phytogenic - derived from plants.

Importance of the intestine

Home to major populations of bacteria, viruses, fungi and bacteriophages –more cells and genes than host!

Major immune organ – home to 70% of immune cells.

Nutrient digestion and absorption.

Gastrointestinal disorders are very costly to the animal production industry, for example:

Necrotic enteritis = $6 billionCoccidiosis = $3 billionDysb(acter)iosis = $2 billion?

Gut microbiome is the centre of the…..body!

Gut microbiome

Immune/vaccine responses

Metabolism/body composition

Behaviour/psychology

Muscle myopathies

Cancer

Autoimmune & metabolic diseases

??

(Young, 2017)

Gut microbiota simplified

(Diagram taken from Bailey, 2010; Stanley et al., 2013. Oakley et al., 2014)

Ileum>109 cells per gram of contentsLactobacillusCandidatus Arthromitus, EnterococcusEscherichia coli/ShigellaClostridium XI Caeca1011 – 1012 cells per gram of contentsClostridiaceaeLachnospiraceaeRuminococcaceaeLactobacillaceaeUnknown & unclassified

Proximal<109 cells per gram of contentsLactobacillus dominant (up to 99%)EnterococcusClostridiaceaeEnterobacteriaceaeStreptococcus

Gut microbiota establishment and stability

(Taken from Pourabedin and Zhao, 2015)

Eubiosis involves multiple components

External agent(s) (e.g. nutrients, drugs, environment/bedding material, invading microbes, mycotoxins)

Microbiota Gut barrier/Immune system

Due to interactions, factors that contribute to each component can help achieve (or not) eubiosis.

Gut barrierMucusHost defense peptidesAntibodies (IgA)Epithelial cellsGut-associated immune cells

Gut health - definition‘the ability of the gut to perform normal physiological functions and to maintain

homeostasis, thereby supporting its ability to withstand infections and non-infectious

stressors’ (Kogut et al., 2017)

(Kogut et al., 2017)

Challenges

What to target?What is an optimal gut microbiota?

Gut microbiota is complex (still much not well identified/characterised).➢ Who is really ‘good’ or ‘bad’?

Studies highlight diverse microbiota among ‘similar’ individuals.➢ significant inter-individual variation.

However, despite this variation, metabolic function appears largely conserved.

(e.g. Stanley et al., 2013; Oakley et al., 2014)

What to target?E.g. sub-(MIC*)inhibitory effects

MIC often considered lowest concentration of an antimicrobial with any effect on bacteria.

MIC defined by lowest concentration that prevents visible growth, thus ignoring effects on:

➢ adherence➢ host defence susceptibility➢ virulence expression ➢ growth dynamics

What more can we learn from AGPs**? Seek ‘immuno-cooperation’?

(Broom, 2017) (*minimum inhibitory concentration; **antimicrobial growth promoters)

What to target?E.g. inflammation?

Inflammation is an essential immune response to protect and repair.

Inflammatory responses are nutrient demanding.

However, studies show increased pro-inflammatory responses better protect against an array of pig and poultry pathogens.

Interest in promoting anti-inflammatory processes, which favour Th2 (extracellular) responses at the expense of Th1 (intracellular).

What’s really the optimum in commercial production?

Seek ‘immuno-cooperation’?(Broom and Kogut, accepted)

Where to target?E.g. antimicrobial effects of organic acids

(Diagram taken from Bailey, 2010; Broom, 2015)

>pH 6.5All OAs effectively dissociated.Microbial penetration?Effective pH reduction?

<pH 5.5Largely undissociated*.>Microbial penetration?Less pH reduction potential

(*Higher pKa = less dissociated at given pH)

Gut microbiota/health manipulation challenges

Complexity of gut microbiome (still unknown community members of unknown influence) and interaction with immune system/responses.

Knowing what to influence, when and where?➢ Earliest possible intervention seems most effective.

Overcoming inter-individual variation.

Technologies.➢ Evolving techniques (e.g. high throughput sequencing) & resultant

knowledge will be invaluable.

Still much to learn!

Current state/latest developments

Eubiotics:

Conventional OtherProbiotics BacteriophagesPrebiotics Antimicrobial peptides Fatty acids (varying chain length) BacteriocinsEnzymes Antibodies(Essential oils) Toll-like receptor agonists

LactoferrinMinerals (e.g. Zn, Cu)Vaccines (mucosal)

Phytogenic:

Various parts of plants (sometimes purified) in various forms, notably essential oils.

…anything that influences microbial and (thus) intestinal balance.

Too many for individual consideration here!

1. Better appreciation of the importance of (strain) specificity.....

7 Lactobacillus casei strains given once daily (108 CFU/day/mouse) for 7 days by oral gavage – caecal microbiota.

(Taken from Aktas et al., 2016)

Clustered treatments not different (p≤0.05). Genera comprising >5% of the total microbiota in at least one treatment are presented.

1. Better appreciation of the importance of (strain) specificity.....

Lactobacillus paracasei F8 (□), Lactobacillus plantarum F44 (■) and Lactobacillus rhamnosusLGG (□) growth in MRS basal broth with prebiotic substrates (1% v/v)

(Taken from Ambalam et al., 2015)

FOS (fructo-oligosaccharides) GOS (galacto-oligosaccharides)IMOS (isomalto-oligosaccharides)LAC (lactulose)XOS (xylo-oligosaccharides)Glu (glucose)MRS-BB (de Man Rogosa Sharpe basal broth)Significance **P < 0·01 vs. MRS-BB

2. Combinations.....

Probiotic (0.250 kg/ton*), prebiotic (MOS product; 2, 1 and 0.5 kg/ton in starter, grower, and finisher phase, respectively) and synbiotic (½ probiotic + ½ prebiotic doses) to broilers fed ad-libitum.

(Abdel-Hafeez et al., 2017)

*>5×1010 CFU of both Bacillus licheniformisand Bacillus subtilis per gram of product.Significance abcdP < 0·05

Pro, pre & syn reportedly reduced cost per kg gain by

13, 4 and 15%, respectively.

Others

1. Combinations within eubiotic ‘class’E.g. optimised combinations of probiotic

organisms, prebiotics, etc.

2. Combinations across eubiotic ‘classes’E.g. OA(s) + phytogenic(s), enzyme(s) +

probiotic(s), etc.

3. Newer ‘classes’.....e.g. antimicrobial peptides

Antimicrobial peptides (AMP) fed (2.0 g/kg feed) to weaned pigs (24-32 days of age) on 5 commercial farms.

(Xiong et al., 2014) Significance abP < 0·05

4. Targeted delivery/protection.....

7.2 mg of either encapsulated* or unencapsulated citral mixed in 100 mg of starter feed - oral gavage (into crop) of 8 birds (12 days of age) per timepoint -digesta then incubated with C. perfringens (107 CFU/g digesta).

(Taken from Yang et al., 2016)

*soy-derived polymer called soy protein–soy polysaccharide Maillard reaction product (SPPMP)

5. Endogenous/in-situ stimulation.....

Broiler chickens challenged with a subclinical dose of E. coli K88 at 7 days of age and fed formic and propionic acid-based feed additive (1-35 days of age).

(Khodambashi Emami et al., 2017; Broom et al., submitted) Significance abP < 0·05

6. Early intervention.....

In-ovo (day 18) probiotic administration then challenged with Salmonella enteritidis (day of hatch).

(Teague et al., 2017)

Probiotic

Summary

Current and future developments

1. Latest technologies - greater interrogation and understanding of multiple components of ‘gut health’, including influence of less well studied microbiota members (e.g. viruses). Establish most relevant biomarkers!

2. Greater appreciation of individual strain, or substrate compositional, effects.3. Combinations – potential for more compatible, synergistic effects (e.g.

synbiotics, phytogenic + organic acid, enzyme + probiotic, etc.).4. Targeted delivery/protection – where to release?5. Optimised dosing/duration.6. Endogenous/in-situ stimulation (organisms optimised to produce a range of

enzymes, antimicrobial compounds, etc.).7. Early intervention (parent stock, in-ovo, etc.).8. Extra-intestinal benefits.9. ‘Immuno-cooperation’.10. Additives that are responsive to different conditions.

References• Abdel-Hafeez, H. M., E. S. E. Saleh, S. S. Tawfeek, I. M. I. Youssef, and A. S. A. Abdel-Daim. 2017. Effects of probiotic, prebiotic, and synbiotic with and without

feed restriction on performance, hematological indices and carcass characteristics of broiler chickens. Asian-Australasian Journal of Animal Sciences, 30:672-682https://doi.org/10.5713/ajas.16.0535

• Aktas, B., T. J. De Wolfe, N. Safdar, B. J. Darien, and J. L. Steele. 2016. The impact of Lactobacillus casei on the composition of the cecal microbiota and innate immunesystem is strain specific. PLoS ONE 11(5):e0156374. doi:10.1371/journal.pone.0156374

• Ambalam, P., K. K. Kondepudi, P. Balusupati, I. Nilsson, T. Wadstrom, and A. Ljungh. 2015. Prebiotic preferences of human lactobacilli strains in co-culture withbifidobacteria and antimicrobial activity against Clostridium difficile. Journal of Applied Microbiology, doi:10.1111/jam.12953

• Bailey, R. A. 2010. Intestinal microbiota and the pathogenesis of dysbacteriosis in broiler chickens. Ph.D. thesis, University of East Anglia• Broom, L. J. 2017. The sub-inhibitory theory for antibiotic growth promoters. Poultry Science 96:3104-3108 http://dx.doi.org/10.3382/ps/pex114• Broom, L. J. 2015. Organic acids for improving intestinal health of poultry. World’s Poultry Science Journal 71:630–642 https://doi.org/10.1017/S0043933915002391• Broom, L. J., A. Daneshmand, E. N. Graystone and N. Khodambashi Emami. 2017. Effects of commercial organic acid blends on male broilers challenged with E. coli

K88: nutrient digestibility, short chain fatty acids, digesta pH, and tibia ash. Journal of Applied Poultry Research (submitted)• Broom, L. J., and M. H. Kogut. 2017. Inflammation: friend or foe for animal production? Poultry Science (accepted)• Khodambashi Emami, N., A. Daneshmand, S. Zafari Naeini, E. N. Graystone and L. J. Broom. 2017. Effects of commercial organic acid blends on male broilers

challenged with E. coli K88: Performance, microbiology, intestinal morphology and immune response. Poultry Science 96:3254-3263 http://dx.doi.org/10.3382/ps/pex106• Kogut, M. H., X. Yin, J. Yuan, and L. J. Broom. 2017. Gut Health in Poultry. CAB Reviews (accepted)• Oakley, B. B., Lillehoj, H. S., Kogut, M. H., Kim, W. K., Maurer, J. J., Pedroso, A., Lee, M. D., Collett, S. R., Johnson, T. J., and Cox, N. A. 2014. The chicken

gastrointestinal microbiome. FEMS Microbiology Letters, 360: 100–112.• Pourabedin, M., and X. Zhao. 2015. Prebiotics and gut microbiota in chickens. FEMS Microbiology Letters, 362 doi: 10.1093/femsle/fnv122• Stanley, D., Geier, M. S., Hughes, R. J., Denman, S. E., and Moore, R. J. 2013. Highly variable microbiota development in the chicken gastrointestinal tract. PLoS ONE

8:e84290. doi: 10.1371/journal.pone.0084290.• Teague, K. D., L. E. Graham, J. R. Dunn, H. H. Cheng, N. Anthony, J. D. Latorre, A. Menconi, R. E. Wolfenden, A. D. Wolfenden, B. D. Mahaffey, M. Baxter, X.

Hernandez-Velasco, R. Merino-Guzman, L. R. Bielke, B. M. Hargis, and G. Tellez. 2017. In ovo evaluation of FloraMax R -B11 on Marek´s disease HVT vaccineprotective efficacy, hatchability, microbiota composition, morphometric analysis, and Salmonella enteritidis infection in broiler chickens. Poultry Science 96:2074–2082http://dx.doi.org/10.3382/ps/pew494

• Xiong, X., H. S.Yang, L. Li, Y. F. Wang, R. L. Huang, F. N. Li, S. P. Wang, and W. Qiu. 2014. Effects of antimicrobial peptides in nursery diets on growth performance ofpigs reared on five different farms. Livestock Science, 167:206–210 http://dx.doi.org/10.1016/j.livsci.2014.04.024

• Yang, Y. Q. Wang, M. S. Diarra, H. Yu, Y. Hua, and J. Gong. 2016. Functional assessment of encapsulated citral for controlling necrotic enteritis in broiler chickens.Poultry Science 95:780–789 http://dx.doi.org/10.3382/ps/pev375

Thank you for your attention!

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