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Isolation and characterization of Bacillus spp. strains as potential probiotics for poultry

Journal: Canadian Journal of Microbiology

Manuscript ID cjm-2019-0019.R1

Manuscript Type: Article

Date Submitted by the Author: 18-Apr-2019

Complete List of Authors: Peñaloza Vázquez, Alejandro; Oklahoma State University, Biochemistry & Mol. Biol.Ma, Li; Oklahoma State University, National Institute for Microbial Forensics & Food and Agricultural Biosecurity, Department of Entomology and Plant PathologyRayas-Duarte, Patricia; Oklahoma State University, Department of Biochemistry and Molecular Biology and Robert M Kerr Food & Agricultural Products Center

Keyword: Poultry, probiotics, Bacillus, Salmonella, Exoenzymes

Is the invited manuscript for consideration in a Special

Issue? :Not applicable (regular submission)

https://mc06.manuscriptcentral.com/cjm-pubs

Canadian Journal of Microbiology

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Isolation and characterization of Bacillus spp. strains as potential probiotics for poultry

Alejandro Penaloza-Vazquez, Li Maria Ma, and Patricia Rayas-Duarte

Alejandro Penaloza-Vazquez; Department of Biochemistry and Molecular Biology, Oklahoma

State University, Stillwater, OK 74078 USA, [email protected]

Li Maria Ma; National Institute for Microbial Forensics & Food and Agricultural Biosecurity,

Department of Entomology and Plant Pathology, Oklahoma State University, Stillwater, OK

74078 USA, [email protected]

Patricia Rayas-Duarte; Department of Biochemistry and Molecular Biology and Robert M Kerr

Food & Agricultural Products Center, Oklahoma State University, Stillwater, OK 74078 USA,

[email protected]

Corresponding author: Alejandro Penaloza-Vazquez, Department of Biochemistry and Molecular Biology, Oklahoma State University. Stillwater, OK 74078 Room 107 FAPC USA. [email protected]

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ABSTRACT

Probiotics have become one of the potential solutions to global restriction on antibiotic

use in food animal production. Bacillus species have been attractive probiotics partially due to

their long-term stability during storage. In this study, 200 endospore-forming bacteria isolates

were recovered from sourdough and the gastrointestinal tract (GIT) of young broiler chicks.

Based on the production of a series of exoenzymes and survivability under stress conditions

similar to those in the poultry gastrointestinal tract (GIT), 42 isolates were selected and identified

by 16S rRNA gene sequencing. Seven strains with a profile of high enzymatic activities were

further evaluated for sporulation efficiency, biofilm formation, compatibility among themselves

(Bacillus spp.) and antagonistic effects against three pathogenic bacteria to poultry and human

including Enterococcus cecorum, Salmonella enterica, and Shiga Toxin-Producing-Escherichia

coli. The strains from sourdough were identified as Bacillus amyloliquefaciens while the ones

from the chicks’ GIT were Bacillus subtilis. These strains demonstrated remarkable potential as

probiotic for poultry.

KEYWORDS

Poultry, probiotics, Bacillus spp., Enterococcus cecorum, Salmonella Muenchen, Shiga toxin-

producing E. coli [STEC], exoenzymes, phytase, antagonisms

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Introduction

Increasing antibiotic resistance in animal and human pathogens has led to the ban on the

use of antibiotics as growth promoters (AGPs) by the European Union (EU) in 2006

(http://europa.eu/rapid/press-release_IP-05-1687_en.htm). In the United States, although the use

of AGPs are not banned, the FDA issued guidelines for the industry to voluntarily withdraw

medically important antibiotics for animal growth promotion (Teillant and Laxminarayan 2015).

To eliminate the use of AGPs in the poultry industry, producers are taking several approaches

including 1) the incorporation of live microbial feed supplements (probiotics), which benefit the

host animal by improving its intestinal microbial balance and 2) management changes to

maintain animal productivity (Teillant and Laxminarayan 2015; Vila et al. 2010).

The microbes most frequently used as probiotics include lactic acid bacteria (such as

Lactobacillus spp., Bifidobacterium spp.) and Saccharomyces boulardii which are isolated from

traditional fermented products, fruits, gut, feces and breast milk of human subjects (Angmo et al.

2016; Fontana et al. 2013; Torres-Maravilla et al. 2016). The 2006 EU’s ban on the use of

antibiotics as growth promoters has expended the interest in using probiotics as alternative for

antibiotics in animal feed (Blajman et al. 2015; Chaucheyras-Durand and Durand 2010; Fallah et

al. 2013; Khan 2013; Yamazaki et al. 2012). Some of the Bacillus spp. has also been used as

probiotics (Galagarza et al. 2018; Yan et al. 2016; Zhou et al. 2019).

The genetics and physiology of the genus Bacillus are remarkable (Diomande et al. 2015;

Hong et al. 2009), offering a great diversity pool with potential future uses in humans and

animals as probiotics (Peng et al. 2019; Tarnecki et al. 2019). Several studies show that Bacillus

spores are present in the intestinal tract of animals suggesting that they are able to survive in

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http://europa.eu/rapid/press-release_IP-05-1687_en.htm

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such environment (Ambas et al. 2015; Barbosa et al. 2005; Khan 2013; Nguyen et al. 2015;

Ozkan et al. 2013; Parova et al. 1994; Wolfenden et al. 2011). The essential features of Bacillus

spp. include their ability to survive and germinate in the gut, form biofilms, and secrete

antimicrobials (Barbosa et al. 2005; Chaiyawan et al. 2010; Elshaghabee et al. 2017; Hong et al.

2009; Tam et al. 2006).

The major advantages of spores over vegetative cells are their heat stability and extended

shelf life without losing viability. The survival of spores during the baking process

(Permpoonpattana et al., 2012) offers the possibility of using spores as probiotic supplements

during feed pelleting. Bacillus spp. have been found in feces and ileal biopsies of diverse

mammals, suggesting that they may colonize rather than transit the intestinal tract (Barbosa et al.

2005; Fakhry et al. 2008; Guo et al. 2006). In pigs fed with Bacillus spp. (Cai et al. 2015), spores

accounted for 72% of the total counts after 4–6 h in the stomach and proximal section of the

small intestine. After 24 h, spores constituted only 12% of the total counts in the stomach,

caecum, and mid-colon. Less spores and more vegetative cells were detected after 24 h, but total

counts increased only 2.1-fold compared to time zero (Leser et al. 2008). In the EU usage of

Bacillus spp. in animal nutrition is regulated by the European Food Safety Authority (EFSA

2015). The species B. subtilis, B. amyloliquefaciens and B. licheniformis received a Qualified

Presumed Safety (QPS) status, provided that they are proved to be non-toxigenic (EFSA 2015).

Bacillus species are on the Food and Drug Administration’s GRAS (generally regarded as safe)

list. Commercial preparations are mainly based on three Bacillus spp., e.g., B. subtilis, B.

licheniformis and B. cereus, whereas the probiotic potential of other Bacillus spp. remains poorly

investigated.

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The objective of this study was to isolate Bacillus spp. from two different sources,

including fermented food products (sourdough) and the contents of gastrointestinal track (GIT)

of healthy 1-day-old chicks, with the aim of identifying potential probiotic strains for use in

poultry production. Initially, endospore-forming bacteria were isolated from both sources after

enrichment with the criteria of growth at 39oC (a temperature close to chicken body temperature)

and sporulation in less than 3 days. These isolates were sequentially screened for bile and acid

tolerance, and enzymatic activities that are related to feed digestion, e.g., phytase, cellulose,

protease and α-amylase. Isolates with a profile of high enzymatic activities were further

evaluated for sporulation efficiency, biofilm formation, and antagonistic activities against

common poultry and foodborne pathogens. These selected Bacillus isolates have the potential as

probiotics for poultry.

Materials and methods

Strains and media

All the media purchased were from Neogen Corporation (Lansing, MI, USA) and

prepared according to the instructions of the manufacturer. Chicken basal starter feed (CBSF)

medium was prepared as follows: chicken basal starter feed was ground in a coffee grinder

(Hamilton Beach Model 80335, Southern Pines, NC, USA) and sieved to pass US Standard

Testing Sieve No. 30 (opening 600 μm). Ten grams of the sieved CBS feed were resuspended in

500 ml of reverse osmosis (RO) water, mixed well, and made up to 1 liter (pH 6.6). The

suspension was autoclaved at 121oC for 20 min and used as medium broth. For agar plates of

CBSF medium, 15 g of bacteriological agar per liter were added. The pathogens used in this

study included Shiga toxin-producing E. coli [STEC], Salmonella Muenchen, and Enterococcus

cecorum isolated from chickens at Oklahoma Animal Disease Diagnostic Laboratory. All

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Bacillus cultures and the pathogenic strains were maintained in 20% glycerol (v/v) and stored at

-20°C. Bacillus strains were grown in Luria-Bertani (LB) broth and the pathogens in Tryptic Soy

Broth (TSB) at 39°C prior to their use in the experiments. Whole wheat flour, wheat bran and

rice bran were purchased from a local supermarket.

Origin of Bacillus ssp. strains

(A) Sourdough starter, the starter was prepared utilizing the protocol of (Suas 2009) with

modifications. Briefly, 100 g of whole wheat flour was added to 100 g of tap water, mixed to get

a smooth paste and incubated at 28°C. The starter was maintained by adding fresh flour for three

days until CO2 production was visible, and then stored at 4°C. (B) GIT, ten Cobb 500 broilers 1-

day-old (Cobb-Vantress Inc., Siloam Springs, AK, USA) were harvested and the contents from

different parts of the GIT were collected and transported to the laboratory at 4oC. The sourdough

starter as well as the contents from the crop, gizzard, small intestine, cecum and large intestine

were subsequently used in the enrichment for endospore forming bacteria that not only can

sporulate in relative short time (<3 days) but also have the ability to digest phytate.

Enrichment of endospore forming bacteria

For enrichment, LB medium amended with 5% of bran wheat (LBBW) or 5% of bran rice

(LBBR) was used for sourdough starters and CBSF for GIT contents. These broth media were

inoculated at 1% with the fermented sourdough (starter) or GIT contents and incubated for 3

days at 39°C with shaking at 200 rpm. After incubation, the cultures were heated in a water bath

at 80°C for 45 min to kill the vegetative cells before cooled down to room temperature and used

for inoculation of fresh broth at 1% (v/v). The inoculated fresh broths were incubated at 39°C for

3 days. The entire enrichment procedure was repeated five times before isolation. The 39°C

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incubation temperature was used to select for spore former bacteria that can grow well at

chicken’s body temperature (ca. 40-42oC) (Bolzani et al. 1979)

Isolation of endospore forming bacteria

After the fifth transfer and incubation of the cultures, serial dilutions (1:10, v/v) were done and

100 l from each dilution was plated on LB or CBSF agar medium. The agar plates were

incubated at 39°C for 12 h. Based on colony morphology and Gram-stains, isolates of endospore

forming bacteria were identified from sourdough and GIT contents.

Screening of bacterial spore formers

The isolates of endospore forming bacteria were first screened for bile tolerance,

followed by acid tolerance, and activity of extracellular enzymes including phytase, α-amylase,

protease, and cellulolytic activity. Only isolates meeting bile and acid tolerance thresholds

(described below) were screened for identified by 16S rRNA gene sequencing (Tamura et al.

2013). The 16S rDNA was amplified by polymerase chain reaction (PCR) using the prokaryotic

16S rDNA universal primers 8F (5’-AGAGTTTGATCCTGGCTCAG-3’) forward primer and

1542R (5’-ACAAAGGAGGTGATC-3’) reverse primer. The PCR was performed with an

automated DNA thermal cycler (Perkin-Elmer 2720, Applied Biosystems Inc. Foster City, CA

USA). The amplification cycle profile was as follows: an initial denaturation step at 94°C for 5

min; 35 cycles of denaturation at 94°C for 60 s, primer annealing at 60°C for 60 s, and primer

extension at 72°C for 120 s. Final extension step at 72°C for 10 min PCR products were purified

with the ExoSAP-IT PCR Product Cleanup Kit (Thermo Fisher Sci., Waltham, MA, USA) and

sequenced with the BigDye® Terminator v3.1 Cycle Sequencing Kit (Applied Biosystems™

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Corp., Foster City, CA, USA) according to manufacturer's instructions. Sequence data from

selected 16S rDNA from selected strains were used to construct a phylogenetic tree using the

statistical analysis maximum likelihood method, test of phylogenic Bootstrap method with 1000

replicates. All these analyses were performed utilizing the software Molecular Evolutionary

Genetic Analysis version 6.06 Mac OSX (Tamura et al. 2013).

Bile tolerance test

Growth at different bile concentrations was evaluated by agar gradient-plate technique as

described before (Weinberg 1959). Briefly, the first layer was 10 ml of LB agar without ox-bile;

the second layer was prepared with LB agar amended with ox-bile at 5.0% (w/v). The gradient

plates were inoculated with 100 l of overnight culture (incubated for 12 h at 39°C). After 12 h

of incubation at 39°C, the growth on the plates was monitored by measuring growth line (mm) of

cultures from 0% bile concentration to 5%.

Acid Tolerance

Tolerance to low pH was tested for the bacterial cultures as described before (Conway et al.

1987; Nithya and Halami 2013). Cells from active cultures (incubated for 16 h) were harvested

by centrifugation for 15 min at 6800 xg and 4°C (Sorvall RC-5C Plus, Thermo Fisher Scientific,

Waltham, MA, USA). Pellets were washed once with phosphate-saline buffer (PBS at pH 7.2),

resuspended in PBS (pH 3) and incubated at 39°C. The gizzard of male broilers has a pH 3 (Reis

et al. 2017). Surviving microorganisms were enumerated at 0, 1, 2, 3 and 4 h by plating in LB

agar and the count was expressed in colony-forming units (CFU) per milliliter. The survival rate

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was calculated using the formula (Nithya and Halami 2013): Survival (%) = Log number of cells

survived (CFU ml-1) x 100/Log number of initial cells inoculated (CFUml-1).

Extracellular enzymes activity

Overnight cultures of the 42 isolates were individually prepared by incubation at 39°C in

LB for 16 h with shaking at 250 rpm and then the cells were harvested by centrifugation for 15

min at 6800 xg and 4°C. The pellets were washed with sterile water and resuspended in water to

an optical density of 0.1 at 600 nm (OD600). The suspensions were used to inoculate plates by

streaking on enzyme-specific agar plates. After incubation, the extracellular enzymatic activities

were evaluated by measuring the diameter in mm of clear zones around the isolated colonies

utilizing a Dial Caliper with metric scale (Bel-Art Products, Wayne, NJ). The strains were scored

according to diameter size as follows: 1, low (6-10 mm); 2, moderate (11–20 mm); 3, high (21–

25 mm); 4, very high (≥26 mm).

Phytase activity

Phytase degradation plate assay was performed as described before (Nithya and Halami

2013) with a modified LB agar medium containing 6.25 g per liter of sodium phytate (LBPH).

Overnight cultures of Bacillus ssp. resuspended in water at OD600 = 0.1 were streaked onto the

surface of LBPH plates and incubated for 16 h at 39°C. After incubation, the plates were flooded

with 2% (w/v) aqueous cobalt chloride solution. After 5 min of incubation at 27°C, the cobalt

chloride solution was replaced with a freshly prepared solution containing equal volumes of

6.25% aqueous ammonium molybdate solution and 0.42% ammonium metavanadate solution.

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After 5 min of incubation, the ammonium molybdate/ammonium metavanadate solution was

removed and the plates were examined for zones of phytate hydrolysis.

-amylase activity

The medium used for evaluation of -amylase activity (Mendu et al. 2005), contained the

following ingredients (per liter): 10 g corn starch, 2 g yeast extract, 5 g peptone, 0.5 g

MgSO4(7H2O), 0.15 g CaCl2, 1 g (NH4)2HPO4 and 15 g agar. Plates containing this medium

were inoculated with a suspension of Bacillus ssp. at OD600 = 0.1. After incubation plates were

flooded with Iodine/Potassium Iodide solution (Fisher Scientific Co. LLC, Fairlawn, NJ, USA)

for 5 min at room temperature, after which the iodine solution was removed and the plates were

evaluated for clear zones.

Cellulolytic activity

Cellulolytic activity was evaluated with the following medium (Gomaa 2013): CMC agar

containing (g/L) KH2PO4 1.0, MgSO4 (7H2O) 0.5, NaCl 0.5, FeSO4 (7H2O) 0.01, MnSO4 (H2O)

0.01, NH4NO3 0.3, carboxyl methyl cellulose 10.0, agar 20.0. Plates were inoculated and

incubated as described for the phytase and -amylase activity. After incubation, the plates were

flooded with 0.1% solution of Congo red for 5 min and the stain discarded. The formation of a

clear zone indicated cellulose degradation.

Protease activity

Production of protease was tested in a medium containing the following ingredients

(g/L): glucose, 1.0; yeast extract, 0.5; CaCl2 0.1; K2HPO4, 0.5 and MgSO4, 0.2; skim milk, 10.0;

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casein, 10.0; and agar, 20.0 (Gomaa 2013). Plates containing this medium were inoculated and

incubated as described previously for other extracellular enzymes. After incubation, the plates

were flooded with protein stain Coomassie Blue G-250. Clear zones indicate protease activity.

Sporulation efficiencies

Seven isolates scored “very high” and “high” on three or more of the enzymatic activities

were evaluated for their ability to produce high yield in spores. Single well-isolated colony from

each plate was transferred to a flask of 250 ml carrying 100 ml of LB medium and incubated at

39°C for 12 h with shaking at 250 rpm (first incubation). Immediately the flasks were transferred

to a water bath for heat-shock treatment at 80°C for 40 min before cooled down to room

temperature. The cultures from heat-shock were used for inoculation at 1% (v/v) fresh LB

medium, incubated at 39°C for 12 h. With the aim to synchronize the population of spores of the

strains, the procedure was repeated three times. The spores from the third culture were used as

the working stock and used for evaluating the growth curves and spore formation. Optical

density was used to measure growth, utilizing a spectrophotometer (Spectronic 20, Bausch &

Lomb, Rochester, NY, USA) at 600 nm (OD 600 nm).

Biofilm formation

The selected strains of Bacillus ssp. were re-streaked onto LB agar medium and the plates

were incubated at 39°C for 12 h. After incubation, one colony from each plate was transferred to

50 ml polypropylene sterile tubes containing 20 ml of LB medium and incubated at 39°C for 16

h at 150 rpm. The sterile tubes containing 20 ml of LB without any inoculum were incubated

under same condition and were used as controls. Biofilm formation in each tube was measured in

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these tubes after incubation by crystal violet assay (CV) as described before (Penaloza-Vazquez

et al. 2010). Briefly, five ml of a 1% solution of CV was added to each tube and the tubes were

incubated at room temperature for 15 min before drained and rinsed thoroughly with water and

air-dried. The CV-stained biofilm in each tube was solubilized in 20 ml of 95% ethanol, of

which 10 ml was transferred to a new polypropylene tube (WRR), and the absorbance was

determined with a Spectronic 20 Spectrophotometer (Thermo Fisher Scientific) at 600 nm.

Antagonistic effect of selected strains against poultry and human pathogens

The selected Bacillus isolates and pathogen strains were cultivated in tryptic soy broth

(TSB) at 250 rpm with the exception of Enterococcus cecorum that was incubated under static

conditions at 37°C for 16 h. After incubation, the probiotic strains were centrifuged at 6800 xg at

4°C and resuspended in TSB at 108 CFUml-1 (Bacillus suspension). The pathogenic strains were

diluted at 108 CFUml-1 in tryptic soy agar (Chaiyawan et al. 2010) incubated at 45°C and

overlaid on already solidified TSA agar plates. After solidification of the second layer of TSA-

pathogen, wells were made utilizing a sterile cork borer. Each well was filled with 150 l of

Bacillus suspension. The plates were incubated at 37°C during 16 h. Plates were observed for the

formation of zone of inhibition around each well and were measured.

Compatibility test of selected strains

The selected probiotic strains were cross streaked on LB plates and incubated at 39°C

overnight. At the crossing point, growth inhibition of the antagonistic strain was evaluated.

Results and Discussion

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Isolation of endospore forming bacteria

In total, 200 endospore-forming bacteria were isolated from sourdough (SD) and

chickens’ GIT. Among these, 55 showed the ability to grow on 5% bile and 42 of these isolates

survived at pH 3. Therefore, a total of 42 endospore-forming bacteria that met the bile and acid

tolerant criteria were selected for further characterization and identified by 16S rRNA gene

sequencing and phylogenetic analysis (Table 1 and Fig. 1). Among these selected isolates,

seventeen were from SD and 25 from chicken GIT, with all the chicken GIT isolates from the

cecum and small intestine (only from these parts were isolated endospore-forming bacteria).

Phylogenetic analysis

The size of the generated fragments was in the range of 1.4–1.5 kb. PCR was followed by

DNA sequence analysis of the resulting PCR product. The 16S rDNA nucleotide sequences were

determined for all thirty-eight isolates, and a database search was conducted. Sequencing of the

16S rRNA gene was sufficient to provide reliable identification of the isolates. The BLAST

search demonstrated that these isolates were closely related to B. subtilis, with sequence

similarity > 99% to the 16S rRNA gene of B. subtilis (Table 1). The constructed phylogenetic

tree using the 16S rRNA gene sequences of the related members from the GIT isolates

demonstrated that all of them belong to B. subtilis similar to reference strains from NCBI

database (Fig. 1). Interestingly, all the B. subtilis strains isolated are located in single clade. But

inside of this big clade there is a group of eight strains that hit three reference strains belonging

to GIT of insect (KX879798.1 Bacillus sp. strain SAUFO39 and two from chicken GIT

KM492822.1_B. subtilis strain CH13, and KM492825_B. subtilis strain CH16).

The SD strains were identified as B. subtilis and B. amyloliquefaciens and these are

located across all the big clade (Fig. 1). Interestingly, all B. amyloliquefaciens strains from SD

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located in clade A are associated with reference strains involved in fermentation and production

of extracellular enzymes.

Growth at different bile concentrations

Bile tolerance plays an important role during selection of probiotic strains (Gilliland et al.

1984). The results from the ox-bile gradient plates showed that 60% (17/30) of isolated strains

from sourdough were able to grow on LB medium containing ox-bile at 5.0% (w/v). However,

all the isolated strains from GIT were able to growth in presence of 5.0% presence of ox-bile

(Nithya and Halami 2013) (Table 1).

Acid tolerance of isolates

An important characteristic of the strains for probiotic use is the ability to remain alive

during both ingestion and in the harsh environment of the GIT. In birds, the proventriculus and

gizzard (true stomach) is the glandular stomach where digestion begins. In the gizzard,

hydrochloric acid and digestive enzymes, such as pepsin, begin to break down the feed more

significantly than the enzymes secreted by the salivary glands (Svihus 2014). For this reason,

acid tolerance was evaluated in our strains. The percentage of strains that survive for 3 h at pH 3

isolated from GIT was 90% (23/25), whereas isolated from sourdough was 60% (17/30). This

difference in tolerance to acid conditions could be intrinsic to the origin of the strains. The pH of

gastric juices of birds has been reported to be in average of 3.5 (Nithya and Halami 2013; Svihus

2014), pH in sourdoughs range from 4.0 to 4.5 (Baye et al. 2013).

Extracellular enzymes activity

The different enzymatic activities are shown in Table 1 for both sets of strains

(sourdough and GIT) and examples of plates illustrated in Fig. 2. The enzymatic activities were

different among strains and between sources of origin.

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Phytase activity

There was clear difference on phytase activity associated with the origins of the strains.

Only 23% (7/30) of the isolates from sourdough show phytase activity whereas 100% (25/25)

from GIT have phytase activity. The low number of strains with phytase activity isolated from

sourdough could be explained in part from their source of origin. The whole wheat flour utilized

to make the sourdough contains 8% of iron. Therefore, microorganisms growing in sourdough

are in an environment with available iron and there is no need to produce phytase for the release

of iron (Lioger et al. 2007). In addition, it has been reported that 4.1±0.4 ppm of iron inhibits

phytase activity (Quan et al. 2001; Santos et al. 2015). Phosphorus (P) is primarily stored in the

form of phytates in plants, thus is poorly available for monogastric livestock, such as pigs and

poultry (Humer et al. 2015). One of the main sources of phytases are the microflora of the GIT

and this could explain why all of the strains isolated from GIT exhibit phytase activity which

releases inorganic phosphorus from phytic acid or its salts, the major forms of organic

phosphorus in plant-derived food and feed ingredients (Humer et al. 2015; Jain et al. 2016;

Shobirin et al. 2009). Thus, production of phytase is a critical and selective mode to survive in

the GIT of the monogastric animals.

-amylase

Sixty six percent (20/30) strains isolated from sourdough showed moderate to high -amylase

activity (Table 1). These results are in agreement with previous reports where Bacillus spp. -

amylase plays an important role during fermentation of starch (Lin et al. 1998; Scazzina et al.

2009; Schallmey et al. 2004). In addition, whole wheat flour used to prepare the sourdough

contains 74% of starch (Choct et al. 1998). This high concentration of starch may exert a

selective effect on the sourdough microflora where the -amylase is critical for surviving of the

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microorganisms. In contrast, the GIT strains only 32% (8/25) of strains isolated had -amylase

activity (Table 1). The composition of chicken feed is a mix of ground grains mostly soybean

and corn in the U.S. plus mineral and vitamin supplements. The composition of the chicken feed

for non-starch polysaccharides is lower than the whole wheat flour, with an average

concentration of 43% non-starch polysaccharides(Choct 2015). Other carbon sources in the

chicken feed can be utilized by GIT strains.

Cellulolytic activity

Similar cellulolytic activity was observed between the two sets of strains SD and GIT (60

and 56%, respectively). Interestingly, the strains isolated from sourdough showed medium

activity whereas, the GIT had from low to high activity (Table 1). These results also could be

associated with the composition in fiber of the origin of the strains. For instance, the content of

fiber in whole wheat flour is 13.3% (fns.usda.gov). In the chicken feed the content of fiber varies

according with the formulation of the feed and the stage of development of the birds e.g., in the

starter (1-21 day) feed 6% of fiber is recommended and in growing and finisher feed the value

ranges from 12 to 18% (Lumpkins et al. 2004).

Protease activity

During the first seven days after hatching, the chickens are fed with a diet very rich in

protein; the main source of protein and amino acids being soybean meal, complemented by corn

(Angmo, Kumari, Savitri, & Bhalla, 2016). The percentage of corn and soybean meal in the feed

varies according to the growth stages of the broilers. For instance, the recommended diet for

Cobb-Vantress broilers during the starting period (1-7 days) the percentage of corn is 58.3% and

the soybean meal is 38.4% (Demattê Filho et al. 2015). The amount of crude protein in average

in corn is 7.18% (Lee et al. 2016), and for soybean meal the average is 46.7% (U.S.S.E. 2016).

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The Bacillus ssp. strains were isolated from GIT (Table 1), when the content of protein and

amino acids are highest. For this reason, the GIT Bacillus ssp. strains isolated from this

environment did not necessarily require extracellular protease activity. This could explain why

only 12% (3/25) of the GIT strains showed protease activity despite the activity in these strains

being high. In contrast, whole wheat flour utilized to prepare the sourdough contains only 13%

of crude protein (fns.usda.gov). Therefore, protein and amino acids content in sourdough could

be a limiting factor for bacterial growth in sourdough. This may be the reason why 53% (16/30)

of the strains isolated from sourdough showed protease activity (Table 1). The results from

sourdough suggest that the production of extracellular protease activity in the isolated strains

could be an advantage for survival in an environment where proteins are not in abundance.

Sporulation efficiency of seven selected isolates

Because sporulation efficiency is critically important for industrial production of feed

supplements demanding high yield, in a short time and at low cost, we performed an initial

screening to assess sporulation efficiency of the isolated strains. The optical density reached

highest value between 8 and 24 h after inoculation (Fig. 3). The six strains selected had high

sporulation efficiency (more than 90%), and their spores were resistant to heat treatment at 80°C

for 40 min. The production of spores reached the highest value at 16 h post-inoculation (Fig. 3).

Biofilm formation by six selected isolates

The six strains selected to be used as probiotic strains in animal trial showed the ability to

form biofilm (Fig. 4). Such an ability would increase the potential use of these strains as

probiotics since it may prevent the adhesion of pathogenic bacteria to the GI tract membrane

surfaces (Wong et al. 2013). In some circumstances, microbial biofilms can consist of a single

species, as in infections of heart valves, catheters, and medical prosthetic devices, but biofilms

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associated with different regions of the GI tract are usually multispecies consortia whose

development is determined by environmental and nutritional factors, as well as by the chemical

composition of the substratum and host defensive mechanisms associated with the innate and

adaptive immune systems (Macfarlane and Dillon 2007; Permpoonpattana et al. 2012; Tam et al.

2006).

Antagonistic effect against poultry and foodborne pathogens of selected strains

Inhibition zones were observed among these selected isolates against Salmonella

Muenchen, Shiga toxin-producing E. coli [STEC] and Enterococcus cecorum. A representative

of such assay result is shown in Figure 5. Sourdough isolate OSU1013-3 and chicken GIT

isolates OSU 1015-9 and -12 were antagonistic to STEC while chicken GIT isolates OSU1015-

12 and -21 were antagonistic to Salmonella Muenchen. All selected isolates had exhibited

antagonistic activity against the poultry pathogen E. cecorum.

Compatibility test of selected strains

The compatibility studies showed that the three selected strains isolated from GIT were

compatible. However, only two strains of the selected from sourdough were compatible.

Selected strains to be used as probiotic supplement

From sourdough three strains were selected (Table 1): (i) OSU1013-3 (SD3) due to very

high activity of phytase and α-amylase; (ii) OSU1013-19 (SD19) strain showed very high

protease activity, high -amylase and cellulolytic activities; (iii) the third selected strain was

OSU1013-24 (SD24) which showed high activity in the four enzymes activities evaluated. From

GIT also three strains were selected (Table1): (i) OSU1015-9 with very high phytase and α-

amylase activities, (ii) OSU1015-12 with very high phytase and protease activities and high

cellulolytic activity, (iii) OSO1015-21 with very high phytase and cellulolytic activities.

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Conclusions

Results obtained in the present study showed the survivability of the Bacillus cultures,

tested in conditions of high bile and low pH values. This will help the strains to reach the small

intestine and colon and contribute to the balance of intestinal microflora in the host. All the

tested cultures produced high levels of extracellular enzymes including phytase, α-amylase,

cellulolytic, and protease. In addition, all the strains were able to form biofilms, as well as

exhibited a wide spectrum of antibacterial activity against pathogens found in poultry. Based on

the results of our study, B. amyloliquefaciens strains OSU1013-3, OSU1013-19, and OSU1013-

24 isolated from sourdough plus the B. subtilis strains OSU1015-9, OSU1015-12, and OSU1015-

21 isolated from chicken GIT exhibited remarkable in vitro probiotic properties and thus can be

considered to have positive traits for use as additive feed supplements in chickens. Furthermore,

the phylogenetic studies performed show a significant correlation between the phenotype and

phylogenic group. All these strains will be further assessed using in-vivo animal trials to evaluate

performance and response to chicken pathogens. The results from these experiments will allow

us to develop a new probiotic additive for chicken feed.

Acknowledgements

The current study was funded by Oklahoma State University Technology Business Development

Program, Oklahoma Cooperative Experiment Station and United States Department of

Agriculture, National Institute of Food and Agriculture.

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Figure Legends

Figure 1. Phylogenic tree of Bacillus isolates based on 16S rRNA gene sequences. The tree was

constructed using the maximum likelihood method. The numbers at the branches are bootstrap

confidence percentage from 1000 bootstrapped trees.

Figure 2. Enzymatic activities of Bacillus isolates on agar plates. (A) Phytase, (B) α-amylase,

(C) cellulolytic activity, and (D) protease activity. Neg = Negative, Pos = Positive.

Figure 3. Sporulation kinetics of Bacillus isolates measured as growth curve (A) and sporulation

efficiencies (B). The number of spores per milliliter of culture was determined as the number of

heat-resistant (80oC for 45 min) CFU on Luria-Bertani plates.

Figure 4. Biofilm formation of selected Bacillus spp. evaluated by optical density at 600 nm

utilizing the crystal violet assay. The bars represent the average of three replicates of each strain

and the vertical lines are the standard deviation.

Figure 5. Antagonistic activity of Bacillus isolates against (A) E. coli O157: H7, (B) Salmonella

Muenchen, C) and D) Enterococcus cecorum. Where: OSU1013-3 (BO3), OSU1013-19 (BO19),

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OSU1013-24 (BO24), OSU1015-9 (BC9), OSU1015-12 (BC12), OSU1015-21 (BC21), BO3 +

BO24 (24+3) and BC9 + BC12 + BC 21 (MIX). The black arrows point to the zone of inhibition.

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Table 1. Bacillus strains preselected for potential use as probiotics in poultry

Strain ID culture

collectionOrigen

GenBank accession number and genus species with homology to isolated

strains

Phytase -amylase Cellulolytic Protease

OSU1013-2 SD* KY392897 B. thuringiensis ND High High LowOSU1013-3 SD KY392898 B. amyloliquefaciens Very high High Low LowOSU1013-5 SD KY392899 B. thuringiensis ND High High HighOSU1013-6 SD KY392900 B. subtilis ND High High HighOSU1013-7 SD KY392901 B. subtilis ND High High HighOSU1013-9 SD KY392902 B. subtilis ND High High HighOSU1013-10 SD KY392903 B. subtilis ND High High HighOSU1013-11 SD KY392904 B. amyloliquefaciens ND High High LowOSU1013-13 SD KY392905 B. subtilis ND High High LowOSU1013-19 SD KY392906 B. amyloliquefaciens ND High High Very

highOSU1013-20 SD KY392907 B. subtilis ND High High HighOSU1013-21 SD KY392908 B. subtilis ND High High HighOSU1013-23 SD KY392909 B. amyloliquefaciens ND High High HighOSU1013-24 SD KY392910 B. amyloliquefaciens High High High HighOSU1013-25 SD KY392911 B. amyloliquefaciens Very high High Low LowOSU1013-28 SD KY392912 B. amyloliquefaciens Very high High Low LowOSU1013-29 SD KY392913 B. subtilis ND High High Low

OSU1015-1 SI KY392914 B. subtilis Very high Low High LowOSU1015-2 SI KY392915 B. subtilis Very high Low Low LowOSU1015-3 SI KY392916 B. subtilis Very high Low High LowOSU1015-4 SI KY392917B. subtilis Very high Low High Very

HighOSU1015-5 SI KY392918 B. subtilis Very high Low Moderate LowOSU1015-6 SI KY392919 B. subtilis Very high Low Low LowOSU1015-7 SI KY392920 B. subtilis Moderate High Low LowOSU1015-8 SI KY392921 B. subtilis Moderate Low Moderate LowOSU1015-9 CE KY392922 B. subtilis Very high Very high High LowOSU1015-10 SI KY392923 B. subtilis High Moderate Low LowOSU1015-11 SI KY392924 B. subtilis Very high Low High LowOSU1015-12 CE KY39295 B. subtilis Very high Low High Very

HighOSU1015-13 SI KY392926 B. subtilis Moderate Low Moderate LowOSU1015-14 SI KY392927 B. subtilis High Low High LowOSU1015-15 SI KY392928 B. subtilis High Low High LowOSU1015-16 CE KY392929 B. subtilis Very High Very High Low LowOSU1015-17 SI KY392930 B. subtilis Low High Low LowOSU1015-18 SI KY392931 B. subtilis Low Very High Low LowOSU1015-19 SI KY392932 B. subtilis Moderate High Low LowOSU1015-20 SI KY392933 B. subtilis Moderate Low High LowOSU1015-21 CE KY392934 B. subtilis Very high Low Very High Very

HighOSU1015-22 SI KY392935 B. subtilis High Low Low LowOSU1015-23 SI KY392936 B. subtilis High Low High LowOSU1015-24 CE KY392937 B. subtilis Very high Low Low LowOSU1015-25 SI KY392938 Bacillus subtilis Moderate Low Low Low

*Where: Sourdough (SD); Small Intestine (SI); Cecum (CE); No detectable (ND). Strains in bold are selected as candidates for use as potential probiotic strains. The strains were scored according to diameter size of the enzymatic halo activity as follows: 1, low (6-10 mm); 2, moderate (11–20 mm); 3, high (21–25 mm); 4, very high (≥26 mm).

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GIT19

GIT20

GIT18

GIT17

GIT16

GIT15

GIT14

GIT12

GIT8

GIT7

GIT6

GIT5

GIT3

GIT2

SD29

SD28

SD25

SD24

SD23

KX870886.1 Bacillus amyloliquefaciens strain KU19 16S ribosomal RNA gene partial sequence

SD21

SD20

KY271752.1 Bacillus amyloliquefaciens strain strain SXAU001

SD19

SD11

KT003246.1 Bacillus amyloliquefaciens strain HN-26

SD10

SD9

SD7

LC155964.1 Bacillus subtilis strain: 6R3-15

SD6

KY118085.1 Bacillus amyloliquefaciens strain L4-6

SD3

KM492823.1 Bacillus subtilis strain CH14 (Chicken probiotics)

SD2

SD5

SD13

KX879798.1 Bacillus sp. strain SAUF039 (Insect gastrointestinal)

GIT1

KM492822.1 Bacillus subtilis strain CH13 (From chicken gastrointenal track)

GIT4

GIT9

GIT10

GIT11

GIT13

GIT21

KM492825.1 Bacillus subtilis strain CH16 (Chicken gastrointestinal)

gi|159162017:434247-435818_Lactobacillus_acidophilus_NCFM_chromosome_complete_genome

Clostridium_perfringens_SM101

Clostridium_botulinum_B_str._Eklund_17B

Salmonella_enterica_subsp._enterica_serovar_Typhi_str._CT18

gi|218703261:4656045-4657586_Escherichia_coli_UMN026_chromosome_complete_genome

Haloquadratum_walsbyi_C23

0.10

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DraftA

DC

B

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Neg��Pos Neg��Pos

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Draft

(A)

(B)

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Draft

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Draft

A

C�

B

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Draft - University of Toronto T-Space · probiotics (Galagarza et al. 2018; Yan et al. 2016; Zhou...

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