4. Introduction I
Why Bacillus subtilis and subspecies are a keystone in Biotechnology?
A) Bacillus ssp. Have a diverse metabolism producing exoenzymes some of
them are thermostable, antibiotics, biosurfactants, etc. All these compounds
have great value in the industry and economical. One example is the
production of vitamin B12 by Bacillus megaterium that has shifted from
chemical synthesis to exclusive biotechnological synthesis in less than 15
years. The underlying extraordinary achievement in metabolic engineering
and bioprocess engineering important industrial producers Bacillus subtilis.
5. Introduction II
Why Bacillus subtilis and subspecies are a keystone in Biotechnology?
B) In addition of the great metabolic capacity these bacteria are able to
produce spores, which can be incorporated in the formulation of the
products and conferring long stable shelf life. Some examples of
products are: Detergents, Shampoos, deodorants, carpet cleaners, water
treatment tanks, etc.
6. Introduction III
Why Bacillus subtilis and subspecies are a keystone in Biotechnology?
C) During the last 17 years Bacillus spp. have been studied as
Probiotic microorganisms
7. 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
9. 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
10. §The increase of productivity in the poultry industry has been accompanied by
various impacts, including emergence of a large variety of pathogens and
bacterial resistance (Chicken is a major protein food source for humans).
§These impacts are in part due to the indiscriminate use of chemotherapeutic
agents as a result of management practices in rearing cycles.
§Regulatory and consumer pressure on the livestock industry to reduce antibiotic
usage has increased and direct-fed microbials (DFMs) may have a part to
play in programs to lower dependence on antibiotics, especially where the
poultry sector tends toward more natural feed additives.
Description of the Problem:
11. The Role of Probiotics in the Poultry Industry
§The Healthy gut microbiota in chickens is maintained with the use of probiotics
and anti-inflammatory ingredients via competitive exclusion, protection of the
epithelium in the gut, and interactions with the commensal microbiota.
§Sales of probiotic-fed chicken products in the United States have increased 34
percent in the last year due to the demand for antibiotic-free poultry.
§According to the U.S. Department of Agriculture, Tyson Foods, the country’s
largest processor of chicken, announced it would use probiotic-fed chickens in its
operations by September 2017.
12. Profitability
Major source of concern in the poultry industry
Feed cost accounts for up to 70 % of total expenses during production
Feed needs to be affordable
Challenges producers as cheap sources of nutrients poorly impact growth and
performance
Grower paid on weight not per bird
◦ Added Incentive
16. Project Description (Concept):
•The main objective of this project is the isolation and characterization of
sporulate Bacillus spp. strains with the aim to be used as probiotics for
prevention of bacterial diseases in poultry, as well as demonstrating the
potential role of probiotics in the growth performance and immune response of
poultry, safety and wholesomeness of dressed poultry meat evidencing
consumer’s protection.
•The proposed business model is for OSU to license Bacillus spp. strains to the
broiler poultry industry in the U.S. for inclusion in formulations to eliminate the
use of human antibiotics growth promoters (AGPs).
22. Technical Overview Isolation
10-1 10-2 10-3 10-4
Dilutions were continued until 10-9 and
plated each dilution on LB + Ox Bile at 2%
10-5
10-6
10-7
Isolated colonies with
different morphology were
picked up and re-streaked on
selective agar medium
Isolated colonies were
analyzed by Gram stained
28. One Day 42 days
Evaluation of Growth
Performances in broilers
supplemented with
Probiotics (FCR) & Body
Composition.
29. 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
30. 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
(Lara and Rostagno, 2013)
33. 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.
35. 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).
36. Inhibitory effect of probiotic selected strains against E. coli and Salmonella
strain muenchen both isolated Poultry Pathogens at ODDL
A) B)
Inhibitory effect of probiotic strains (BO3; B019; BO24; BC9; BC12 and BC21). Where: A) is showing the effect of
the probiotic strains on E. coli at 107 CFU per ml and B) is also showing the effect on Salmonella muenchen also at
107 CFU per ml. Red arrow points out to the clearest inhibitory effect.
37. 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; BC12
and BC21 & the three strains Mix) on E. cecorum also at 108 CFU per ml. Red arrow points out to the clearest
inhibitory effect.
A) B)
38. 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
40. 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).
41. 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
