“Bacillus ssp.”KeystoneinBiotechnology

ALEJANDROPEÑALOZA VAZQUEZ

BIOCHEMISTRY&MOLECULARBIOLOGY

ROBERTM.KERRFOOD&AG.PRODUCTSCENTER

Contents:

þ Introduction

¨ Project Description (concept)

¨ Technical overview

¨ Results

¨ Pathway to follow

Introduction IWhy Bacillus subtilis and subspecies are a keystone in Biotechnology?

A) Bacillus ssp. Have a diverse metabolism producing exoenzymes some ofthem are thermostable, antibiotics, biosurfactants, etc. All these compoundshave great value in the industry and economical. One example is theproduction of vitamin B12 by Bacillus megaterium that has shifted fromchemical synthesis to exclusive biotechnological synthesis in less than 15years. The underlying extraordinary achievement in metabolic engineeringand bioprocess engineering important industrial producers Bacillus subtilis.

Introduction IIWhy Bacillus subtilis and subspecies are a keystone in Biotechnology?

B) In addition of the great metabolic capacity these bacteria are able toproduce spores, which can be incorporated in the formulation of theproducts and conferring long stable shelf life. Some examples ofproducts are: Detergents, Shampoos, deodorants, carpet cleaners, watertreatment tanks, etc.

Introduction IIIWhy Bacillus subtilis and subspecies are a keystone in Biotechnology?

C) During the last 17 years Bacillus spp. have been studied asProbiotic microorganisms

Probiotics are live microorganisms that, when ingested in adequate amounts, provide health benefits to the host. The strains most frequently used as probiotics include lactic acid bacteria like Lactobacillus spp., Bifidobacterium spp. and Saccharomyces boulardii which are isolated from traditional fermented products, fruits, gut, feces and breast milk of human subjects.

Probiotics

Probiotic Mechanisms of Action

Background Information•Broiler is a bird raised to produce meat for consumption

•Commercial houses typically hold around 30,000 birds

•Broiler industry has very small margins

•Therefore, management wants to be economical but also improve the profit

§The increase of productivity in the poultry industry has been accompanied byvarious impacts, including emergence of a large variety of pathogens andbacterial resistance (Chicken is a major protein food source for humans).

§These impacts are in part due to the indiscriminate use of chemotherapeuticagents as a result of management practices in rearing cycles.

§Regulatory and consumer pressure on the livestock industry to reduce antibioticusage has increased and direct-fed microbials (DFMs) may have a part toplay in programs to lower dependence on antibiotics, especially where thepoultry sector tends toward more natural feed additives.

Description of the Problem:

The Role of Probiotics in the Poultry Industry

§The Healthy gut microbiota in chickens is maintained with the use of probioticsand anti-inflammatory ingredients via competitive exclusion, protection of theepithelium in the gut, and interactions with the commensal microbiota.

§Sales of probiotic-fed chicken products in the United States have increased 34percent in the last year due to the demand for antibiotic-free poultry.

§According to the U.S. Department of Agriculture, Tyson Foods, the country’slargest processor of chicken, announced it would use probiotic-fed chickens in itsoperations by September 2017.

Profitability Major source of concern in the poultry industryFeed cost accounts for up to 70 % of total expenses during productionFeed needs to be affordableChallenges producers as cheap sources of nutrients poorly impact growth and performanceGrower paid on weight not per bird ◦ Added Incentive

Contents:

¨ Background

þ Project Description (concept)

¨ Technical overview

¨ Results

¨ Pathway to follow

Project Description (Concept):•The main objective of this project is the isolation and characterization ofsporulate Bacillus spp. strains with the aim to be used as probiotics forprevention of bacterial diseases in poultry, as well as demonstrating thepotential role of probiotics in the growth performance and immune response ofpoultry, safety and wholesomeness of dressed poultry meat evidencingconsumer’s protection.

•The proposed business model is for OSU to license Bacillus spp. strains to thebroiler poultry industry in the U.S. for inclusion in formulations to eliminate theuse of human antibiotics growth promoters (AGPs).

Contents:

¨ Background

¨Project Description (Concept)

þ Technical overview

¨ Results

¨ Pathway to follow

Poultry Research Center

Source of probiotics

Isolation ••Selective Media

Identification ••Characterization

Technical General Overview

Sourdough

&Gastrointestinal tract

(GIT)

Technical Overview Isolation

10-1 10-2 10-3 10-4

Dilutionswerecontinueduntil10-9 and

platedeachdilutiononLB+OxBileat2%

10-5 10-6 10-7

Isolatedcolonieswith

differentmorphologywere

pickedupandre-streakedon

selectiveagarmedium

Isolatedcolonieswere

analyzedbyGramstained

Technical Overview Identification

16S rRNAWhole genome

Technical Overview (Biofermentation)

••Inoculum preparation

••Erlenmeyer baffled flask

Incubated at 39oC for 20 H

••The third flask was used as working stock

Previous step was repeated 3 times •• Working stock

was used for inoculating the Fermentor

BIOFLO FERMENTOR

Technical Overview III (Biofermentation)

Chicken Feed Inoculation

One Day 42 days

Evaluation of Growth Performances in broilers supplemented with Probiotics (FCR) & Body Composition.

DEXA Scan (duel-energy X-ray absorptiometry)

The DEXA (duel-energy X-ray absorptiometry) scan provides one of the most highly accurate measurements of body composition available, registering fat and lean mass distribution throughout the entire body. In simple terms, it is capable of detailing overall, as well as regional, fat mass, lean mass, and bone mass

Genotype and Breeding◦ Genotype selection to increase breast meat yield and feed conversion ratio

◦ This has made today’s broiler more susceptible to heat stress than ever before

(LaraandRostagno,2013)

CONTENTS:

¨ Background

¨Project Description (Concept)

¨Targets and Milestones next 12 months

þ Results

¨ Pathway to follow

Oklahoma State University

Identification of selected strains:

1) Over 120 strains of Bacillus spp. have been isolated from two main sources fermented foods and gut microbiota.

2) The phylogenetic tree from these strains confirmed they are safe . It was based on 16S rRNA library preparation and 2x250 bp sequencing on Illumina MiSeq.

Phylogenetic tree of probiotic strains from fermented foods

Characteristics of Selected Strains

1. High production of exoenzymes, e.g., proteases, amylase, phytases, etc., which could improved

animal digestion.

2. Have a demonstrated beneficial effect on the host (Male broilers)

3. Be non-pathogenic, non-toxic and free of significant adverse side effects

4. Be able to survive through the gastrointestinal tract (in vitro and in vivo)

5. Be present in the product in an adequate number of viable cells to confer the health benefit. Be

compatible with product matrix, processing and storage conditions to maintain the desired

properties, and accurate labelling (thermostable strains).

Inhibitory effect of probiotic selected strains against E. coli and Salmonellastrain muenchen both isolated Poultry Pathogens at ODDLA) B)

Inhibitory effect of probiotic strains (BO3; B019; BO24; BC9; BC12 and BC21). Where: A) is showing the effect ofthe probiotic strains on E. coli at 107 CFU per ml and B) is also showing the effect on Salmonella muenchen also at107 CFU per ml. Red arrow points out to the clearest inhibitory effect.

Inhibitory Effect of selected Probiotic strains on Pathogenic strains isolated from Chickens I

Inhibitory effect of probiotic strains. Where: A) is showing the effect of the probiotic strains (BO3; B019; BO24 &Mix BO3 + BO24) on E. cecorum at 108 CFU per ml and B) is also showing the effect probiotic strains (BC9; BC12and BC21 & the three strains Mix) on E. cecorum also at 108 CFU per ml. Red arrow points out to the clearestinhibitory effect.

A) B)

Animal Trial (preliminary results)Animal Male broilers improved efficiency in Body Weight Gain (BWG) significantly (P < 0.05) during the first two weeks of growth. Same trend was observed until the end of the experiment (42 Days)

Feed Conversion Ratio (FCR)= Consumed feed/Weight Gain

CONTENTS:

¨ Background

¨Project Description (Concept)

¨Targets and Milestones next 12 months

¨Results

þ Pathway to follow

Conclusions:

1. Currently 120 strains had been isolated from fermented foods and chicken gut.

2. A preliminary animal trial was performed utilizing three strains (OSU-3, OSU-19 & OSU-24) with high production of exoenzymes, improved animal digestion. Male broilers improved efficiency in Body Weight Gain (BWG) significantly (P < 0.05).

I. Second Animal trail utilizing previous strains and new strains isolated from Chicken GIT with the addition of the Probiotics in the feed. Parameters to evaluate are:

• Growth Performances in broilers

• Intestinal microarchitecture

• Cyclic heat stress

• Metagenomics

Pathway to follow

Acknowledgments Collaborators:

Dra. Patricia Rayas-Duarte

Dr. Zorba Josué Hernández Estrada

Dr. Ali Beker

Students:

Sarah Schobert

Julio Alberto Domínguez García

Kamry Martin

Ashley Harris

Brienna Milleson

Thiago Montagner Souza

Question(s)?