ISSN 1519-6984 (Print)

ISSN 1678-4375 (Online)

THE INTERNATIONAL JOURNAL ON NEOTROPICAL BIOLOGY

THE INTERNATIONAL JOURNAL ON GLOBAL BIODIVERSITY AND ENVIRONMENT

Original Article

Isolation and identification of keratinolytic probiotic

Bucillus licheniformis bacteria from the soil below poultry
slaughterhouse waste
Isolamento e identificação de bactérias probióticas queratinolíticas
Bacillus licheniformis do solo sob resíduos de abatedouro de aves

P. Vanakia , F. Zabolia , H. Kaboosia* , R. Izadi. Amolia  and F. Savadkoohib 

Department of Microbiology, Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran
a

b
Department of Biology, Ayatollah Amoli Branch, Islamic Azad University, Amol, Iran

Abstract
Feathers make up 7% of the total weight of adult chickens and keratin protein makes up 85% of the feathers. Today,
the keratinase enzymes of some Bacillus strains are used to degrade and process raw keratin waste for animal and
poultry feed. According to various studies, the probiotic properties of some spore-shaped Bacillus have also been
proven. The study aimed to isolation of the keratinolytic Bacillus bacteria that they have probiotic properties for
using in the livestock and poultry feed industry. We were able to isolate 8 strains of Bacillus licheniformis with
kreatin degrading properties from the soil of Baharan chicken slaughterhouse (Qom city, Iran) applying heat shock,
alcohol- and keratin-rich culture medium, and after microscopic and biochemical analysis, 16S rDNA gene was
isolated. The measurement results of keratinase activity showed that the three strains of Bacillus licheniformis pvkr6,
pvkr 15, and pvkr41 had the highest activity with 124.08, 101.1, and 100.18 U/ml. The results of probiotic properties
evaluation also revealed that among all the isolates, only Bacillus licheniformis pvkr15 and Bacillus licheniformis
PTCC 1595 (positive control) were γ-hemolytic strains. The percentage of surface hydrophobicity of the strains was
obtained from 3.27 to 30.57. It was also shown that, on average, all the strains had acceptable susceptibility to the
tested antibiotics except penicillin G. Bacillus licheniformis pvkr15 with highest keratinase activity (101.1U/ml) was
considered an optional probiotics due to its abilities such as (biofilm formation, being safe cause of γ-hemolytic
activity, high susceptibility to antibiotics such as streptomycin, gentamicin, cefixime, amoxicillin, tetracycline,
vancomycin, erythromycin and having a moderate hydrophilic (hydrophobicity: 19.09%), high survivability in pH
2, 2.5 and 3, strong resistance to bile salts and moderate antagonistic activity against pathogenic bacterium like
Proteus mirabilis and the ability to grow under anaerobic conditions). By using this strain, after hydrolysis of keratin
protein in the feather structure, to replace part of the protein of livestock and poultry feed, not only is no need to
separate bacteria from the feed, but also the strain play role of an useful and effective additive in animal growth.
Keywords: bacillus, keratinase, probiotic activity, keratinolytic bacteria.

Resumo
As penas representam 7% do peso total das galinhas adultas e a proteína de queratina compõe 85% das penas.
Hoje, as enzimas queratinase de algumas cepas de Bacillus são usadas para degradar e processar resíduos de
queratina brutos para alimentação de animais e aves. De acordo com vários estudos, as propriedades probióticas
de alguns Bacillus em forma de esporos também foram comprovadas. O estudo teve como objetivo o isolamento das
bactérias queratinolíticas Bacillus que possuem propriedades probióticas para uso na indústria de ração animal e
avícola. Conseguimos isolar 8 cepas de Bacillus licheniformis com propriedades degradantes de creatina do solo do
abatedouro de frangos de Baharan (cidade de Qom, Irã) aplicando choque térmico, meio de cultura rico em álcool
e queratina e, após análise microscópica e bioquímica, o gene 16S rDNA foi isolado. Os resultados da medição da
atividade da queratinase mostraram que as três cepas de Bacillus licheniformis pvkr6, pvkr15 e pvkr41 tiveram a
maior atividade com 124,08, 101,1 e 100,18 U/ml. Os resultados da avaliação das propriedades probióticas também
revelaram que dentre todos os isolados apenas Bacillus licheniformis pvkr15 e Bacillus licheniformis PTCC 1595
(controle positivo) eram cepas γ-hemolíticas. A porcentagem de hidrofobicidade superficial das cepas foi obtida de
3,27 a 30,57. Também foi demonstrado que, em média, todas as cepas apresentaram suscetibilidade aceitável aos
antibióticos testados, exceto penicilina G. Bacillus licheniformis pvkr15 com maior atividade de queratinase (101,1U/
ml) foi considerado um probiótico opcional devido às suas habilidades como formação de biofilme, sendo causa
segura de atividade γ-hemolítica, alta suscetibilidade a antibióticos como estreptomicina, gentamicina, cefixima,
amoxicilina, tetraciclina, vancomicina, eritromicina e ter uma hidrofílica moderada (hidrofobicidade: 19,09%), alta

*e-mail: h.kaboosi@iauamol.ac.ir
Received: October 19, 2021 – Accepted: March 8, 2022
This is an Open Access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use,
distribution, and reproduction in any medium, provided the original work is properly cited.

Brazilian Journal of Biology, 2024, vol. 84, e257473  |  https://doi.org/10.1590/1519-6984.257473 1/15

Vanaki, P. et al.

capacidade de sobrevivência em pH 2, 2,5 e 3, forte resistência aos sais biliares e atividade antagonista moderada
contra bactérias patogênicas como Proteus mirabilis e a capacidade de crescer em condições anaeróbicas. Ao utilizar
esta cepa, após a hidrólise da proteína queratina na estrutura da pena, para substituir parte da proteína da ração
de gado e aves, não só não há necessidade de separar as bactérias da ração, mas também a cepa desempenha um
papel útil e eficaz aditivo no crescimento animal.
Palavras-chave: bacilo, queratinase, atividade probiótica, bactérias queratinolíticas.

1. Introduction innate immune and resistance to pathogens. The use of

probiotics in the feed, poultry, and aquaculture industries
According to research, millions tons of feathers
is important (Munir et al., 2019). However, removing
are produced annually as a by-product of the factory
antibiotics from poultry feed can predispose them to
poultry trade worldwide (Tesfaye et al., 2017). Chicken
infections caused by certain pathogens, such as Escherichia
feathers are utilized for a variety of products, including
coli, Clostridium perfringens, and Salmonella species, leading
fertilizers, feed, and biofilms. Feather is composed of
to low yields and serious damage to the poultry industry
approximately 90% hard keratin (Ramakrishnan et al.,
(Al-Sultan, 2003; Marins et al., 2021; Wekhe et al., 2007).
2018). The use of current added value for feathers is
Probiotic products as live microbial foods are beneficial
the conversion to feather powder as a digestible dietary
to animal health as a viable alternative to antibiotics to
protein for animal feed, applying physical and chemical
prevent disease and stimulate animals (Jin et al., 1998;
treatments (Tiwary and Gupta, 2012). These methods can
Teo and Tan, 2006). The adhesion of probiotics can be
eliminate the keratin protein amino acids and reduce the
quality and digestibility of the protein (Elkin et al., 1996). related to the characteristics of their cell surface, namely
Due to this issue, the use of keratin degrading enzymes hydrophobicity, which can result in a strong interaction
produced by microorganisms producing amino acids and with the mucosa if high enough. However, not only is
peptides to compensate for the lack of protein for animal the hydrophobicity of probiotic cell surfaces responsible
feed has become an important and attractive issue in for their attachment to epithelial cells, but more specific
biotechnological activities (Cai et al., 2008; Zabihi et al., mechanisms include lipoteichoic acid, extracellular
2011). Alkaline proteases are a group of proteolytic enzymes components (exopolysaccharides or proteins), or surface
able to degrade proteins, such as keratin, which are difficult proteins (Grigoryan et al., 2018; Mohanty et al., 2019).
to dissolve more efficiently than other proteases, such as The aim of this study was to isolate and investigate the
trypsin, pepsin, and papain. Previous studies have indicated characteristics of Bacillus probiotic bacteria producing
that some Bacillus species are able to produce proteases the enzyme keratinase for use in feather degrading and
for feather digestion (Shamsipur et al., 2012; Sabir et al., to provide a protein source for animal feed.
2021; Pustokhina et al., 2021). Keratin is an insoluble
protein with a stable structure that forms protective
levels in various animals and is a structural component 2. Material and Methods
of skin, wool, hair, feathers, and nails (Sharma and Gupta,
2016). Keratinase is able to hydrolyze keratin and degrade 2.1. Sample collection
feathers. Microorganisms, such as bacteria, fungi, and The soil samples were randomly collected from different
actinomycetes have been reported to produce keratinase for areas of the bed soil of Baharan Poultry slaughterhouse
use as energy and carbon sources. Resistance to proteolytic (Qom, Iran). A total of 100 samples were collected so that
enzymes has been attributed to the complex structure of
each soil sample was 5 g and was poured sterilely into a
beta-keratin filaments. In addition, disulfide cross-links
separate sterile falcon and delivered to the microbiological
produce a compact three-dimensional network as a result
laboratory (Islamic Azad University, Ayatollah Amoli
of the intermolecular bonds of disulfide between the rod
Branch), then 1 g of each sample was weighed below
and the terminal domains of the constituent molecules
the microbial hood (Sarvo Tajhiz Sakoo Company, Iran)
(Riffel and Brandelli, 2006).
and the microbial suspension of the unit was prepared
Keratinolytic microorganisms may play an important
and homogenized by the shaker (MICRO-SPIN – KIAGEN
role in industrial processes and the biotechnology and
company, Iran).
pharmaceutical industries (Vidmar and Vodovnik,
2018). Some bacteria, such as Bacillus licheniformis and
2.2. Isolation and identification of bacterial strains
Bacillus subtilis produce keratinase (Mousavi et al., 2013).
The keratinase produced by B. licheniformis increases Briefly, one gram of the soil sample was diluted
the total digestibility of crude feather amino acids and 1:10 (w/v) in sterile saline (0.9%) (Samen pharmaceutical
commercial feather powder. This enzyme can replace up to company, Iran). Afterwards, the suspensions were allowed
7% of dietary protein for growing chickens (Garbeva et al., to settle in the soil particles for 3 min and 25 ml of the
2003). microbial suspension supernatant then reached 50 ml at a
Bacillus species are gram-positive bacteria that can be ratio of 1:1 with 25 ml of buffered peptone water (QUELAB,
found widely in soil and plants (Elshaghabee et al., 2017). Cat No:39-9587, Canada). This solution was divided into
Bacillus species have found a variety of food products, two equal parts and treated individually with heat and
such as probiotics. Probiotics are living microorganisms alcohol to separate Bacillus bacteria (Mohamadkhani
giving the host health benefits. Probiotics can increase and Shishegaran, 2020). In this way, for heat treatment,

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 Keratinolytic probiotic Bucillus licheniformis bacteria

from the initial microbial suspension, with a volume 1595 as positive control and Bacillus licheniformis PTCC
ratio of 1:10, and with 18 ml of peptone water, it reached 1525 as negative control were considered as standard
to 20 ml and was placed in a 65 ° C water bath for one strain (National Center of Genetic and Biological Resources,
hour. Then, ten serial dilutions with peptone water were Iran). The plates were incubated at 37 °C for 1 to 5 days
prepared from the suspension treated (from 10-1 to 10-10) and keratinolytic strains were identified and isolated based
after homogenization of the last dilution was cultured on the clear keratinolysis area around them.
100 ml on the LB agar (two replications per dilution)
and were incubated for 24 hours at 37 ° C. For alcoholic 2.5. Biomolecular identification of bacteria
treatment, the initial microbial suspension was reduced For biomolecular identification of isolated bacteria, the
to a volume of 20 ml with a volume ratio of 1:1, and with isolated bacteria were cultured with keratinase activity in
10 ml of 96% ethanol, so that the final concentration LBA (Luria_Bertani Agar, Merck Company, Germany, Cat
of alcohol reached 50%. The sample was treated with No: 110283) medium and incubated for 24 hrs at 37 °C.
alcohol for one hour. The next steps were the same as Genomic DNA of the selected isolates was extracted with
heat treatment (Barbosa et al., 2005). Serial dilution was Peqlab, Cat No: 51304 kit (Dena Zist Company, Iran).
performed on each treated sample and mass and linear The 16S rDNA was utilized to amplify a segment of DNA
cultures were then prepared from their final concentration; through the use of the following universal primers of
hence, the refined colonies were stored at 70 °C for 24 h 27F(5’AGAGTTGATCATGGCTCAG-3’) and 1492R(5’TACGG
(Barbosa et al., 2005). To identify the isolated bacteria, ATACCTTGTTACGACTT-3’) (Takapouzist Company, Tehran,
biochemical tests, including catalase, gelatinase, amylase, Iran) (Batisson et al., 2009). PCR was performed on a total
lecithinase, Simmons citrate agar, urease, caseinase, O/F volume of 25 μl with 190 ng of template DNA, 10 pmol
(obligate or facultative aerobes) and TSI (Triple Sugar Iron) of each primer, 10 mM dNTP (Thermo Fisher Scientific
were employed according to the instructions described Company, USA, Cat No: 18427088), 60 mM MgSO4 (Millipore
in Bergey’s manual systematic bacteriology (Vos et al., Sigma Company, Germany, Cat No: 105886), 10X Taq buffer
2011). Finally, a typical colony of each subculture sample (Thermo Fisher Scientific Company, USA, Cat No: B38), and
was cultured and the bacteria were identified with colony 10 U/μl of Taq polymerase (Roche Company, Swiss). The PCR
morphology, gram staining compared to standard species reaction was as follows: 94 °C for 10 min, 35 cycles 94 °C
(Bacillus licheniformis PTCC 1595 and Bacillus licheniformis for 1 min, 54 °C for 1 min, 72 °C for 1 min, and the final
PTCC 1525 as positive control). length at 72 °C for 10 min. In addition, Bacillus licheniformis
PTCC 1595 as positive control was used in this process.
2.3. Feather keratin substrate The amplicon of 16S rDNA was determined applying
The method of preparing keratin powder is described agarose gel electrophoresis. The obtained sequences were
by (Wawrzkiewicz et al., 1987). The feathers obtained from read and processed through three bioinformatics programs
the slaughterhouse were washed with distilled water and Chromas, Edit Seq, and Seq Man. The sequences of genes
detergent (Dishwashing Liquid, Prile Company, Iran) to processed at the NCBI were BLAST (Lin et al., 1995).
degrease and remove slaughterhouse contaminants from
feathers. Chloroform: ethanol solution (v/v) was then 2.6. Keratinase enzyme production
applied to separate the fat from the feathers. Afterwards, The keratinase enzyme was extracted by the use of the
500 ml of DMSO (Dimethyl sulfoxide, Merck Milipore, modification method of Sangali and Brandelli (Andersen,
Germany, Cat: 102950) was added to the lipid-free feathers 2001). The isolated keratinolytic Bacillus bacteria were
and they were heated at 100 °C for 120 min. Following the cultured in keratin broth medium (keratin (10 g/l), NaCl
removal of the precipitate, one liter of acetone (-70°C) was (0.5 g/l) (Merck Company, Germany, Cat No: 106404),
added dropwise to the supernatant at 4 °C to precipitate K2HPO4 (0.3 g/l) (Sigma- Aldrich, USA, Cat No: 1551128),
the keratin protein and separate it from the solvent and and KH2PO4 (0.4 g/l) (Merck Company, Germany, Cat No:
acetone. Lastly, the keratin precipitate was separated and 105104)) at 37 °C/150 rpm and were incubated for 5 days.
dried. Keratin powder was employed as a substrate for the The supernatant was separated via centrifugation at
keratin culture medium. 12,000 rpm for 30 min at 4 °C and utilized for keratinolytic
assay and enzyme analysis.
2.4. Isolation of keratin degrading bacteria
To identify keratin-degrading Bacillus spp, all 42 isolated 2.7. Keratinase assay
strains were cultured separately on culture medium According to the method described by Senegal and
containing 1.5% keratin (as the only carbon source) and Brandelli with a little contradiction (Sangali & Brandelli,
incubated at 37 °C for 5 days. The degradability of keratin 2000), briefly, 300 μl of medium containing extracted
substrates in culture medium was evaluated on the basis of keratin enzyme as explained in the previous step, was
keratin containing NaCl (0.5 g/l) (Merck Company, Germany, prepared. Afterwards, 2700 μl containing 10 g/l keratin
Cat No: 106404), K2HPO4 (0.3 g/l) (Sigma-Aldrich Company, in Tris buffer (Sigma-Aldrich, USA, Cat No: T1503) with
USA, Cat No: 1551128), KH2PO4 (0.4 g/l) (Merck Company, final concentration 50 mM and pH was added and placed
Germany, Cat No: 105104), agar (15 g/l) (Merck Company, in a water bath at 50 °C for 15 min. After this period, to
Germany), and keratin (15 g/l) (keratin is obtained by the prevent the activity of keratinase enzymes from continuing,
method described above (keratin substrate)) (Sangali 10% Trichloroacetic acid (Merck Company, Germany,
and Brandelli, 2000). Also, Bacillus licheniformis PTCC Cat No: 100807) was added to each vial. Subsequently,

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Vanaki, P. et al.

to separate the substrate from the enzyme suspension, 24 hs. After transferring the contents to glass tubes, the
the vials were centrifuged at 10000 g for 5 min. After cells were incubated at 37 °C for 24 hrs without shaking
separation, the absorbance of the supernatant fluid was (Wolfe et al., 2004). The tubes were stained with 1% crystal
measured at 440 nm. violet and washed with distilled water. The biofilm layer,
In the next step, to deposit the native protein (enzyme), if present, appears purple between the air and the liquid.
76 g of ammonium sulfate (Merck Millipore Company,
Germany, Cat No: 101209) was dissolved in 100 ml of 2.8.4. Cell surface hydrophobicity
distilled water. Through a special decanter (Novin Pyrex The percentage of surface adhesion to solvent (xylene)
Company, Iran), 70 ml of the above solution (at -20 °C) in was measured and calculated based on the method of
drops of 30 ml crud enzyme obtained from pvkr15 strain Xu et al. (2009). The adhesion to xylene (apolar solvent)
was added as overnight in cold room at -20 °C. It was then indicates surface hydrophobicity. Briefly, Bacillus was
centrifuged at 5000 RPM for 10 min. The sediment was cultured overnight in LB broth medium, the bacteria
dissolved in 200 μl of Tris buffer with pH 7.5-8. Finally, were centrifuged at 3000 rpm for 5 min. The bacterial cell
the dialysis was performed using a microfuge dialysis bag deposition were suspended in PBS solution. Afterwards,
with at 10,000 RPM and using the soup obtained from 1 ml of xylene solvent was added to 3 ml of bacterial
the final sediment, the enzymatic activity (U/ml) was suspension and vortexed (Vortex Spin, Micro-Spin Model,
measured and calculated (Andersen, 2001). Kiagen Company, Iran) for 1 min and kept for 5 min
to separate in two phases. We measured the aqueous
2.8. Safety assessment phases with a spectrophotometer (Beckman Company,
DU530 Model, USA) at a wavelength of 600 nm (A5min).
2.8.1. Hemolytic activity The experiment was repeated twice. The values ​​less
The keratinolytic Bacillus strains were analyzed for than 30% (30%>) were considered as (low), between 30-
their hemolytic activity on nutrient agar containing 7% 60% (30% ≤, 60%>) as (average), and values ​​greater than
(v/v) defibrinated sheep blood (Quelab Company, Tehran, 60% (60%≤) were considered as (high). Strains with low
Iran) and incubated at 37°C for 96 hrs. The plates were solvent adhesion are considered as potential probiotics.
checked every 12 hrs. Also, Bacillus licheniformis PTCC The affinities of Bacillus to solvents was calculated with
1525 and Bacillus licheniformis PTCC 1595 (purchased from the following Formula 1:
National Center of Genetic and Biological Resources, Iran)
respectively were positive and negative controls for this A5 min
experiment (AlGburi et al., 2016). Adhesion % = (1 - ) × 100 (1)
A0 min

2.8.2. Resistance to antibiotics

Müller-Hinton Agar (Merck, Germany) were employed 2.8.5. Bile salt tolerance
to evaluate antibiotic susceptibility of what please clarify To evaluate bile salt tolerance, as in the previous stage,
via disk diffusion method (Maričević and Žganjar, 2022). an 18-hour pre-culture was prepared separately in LB
Nine antimicrobials were chosen to test for susceptibility to broth in terms of sterile conditions. For the experiment,
antibiotics so that if there is any antibiotic resistance and the falcons containing 10 ml of LB broth (for preparation
the possibility of transmission from probiotic Bacillus to of control samples) and the falcons containing 10 ml of LB
livestock and poultry and finally humans, to avoid the use broth with a concentration of 0.3% Oxgall were prepared.
of these Bacillus in livestock and poultry feed as a probiotic The desired colony was removed from the plate and cultured
bacterium; these antibiotics are including streptomycin in all media. The falcons were incubated in a shaker
(10 μg), gentamicin (10 μg), erythromycin (10 μg), cefixime incubator at 37 °C and 180 rpm for 6 hrs. The turbidity of
(5 μg), penicillin G (10 μg), amoxicillin (25 μg), tetracycline the incubated environments under these conditions was
(30 μg), rifampin (30 μg), and vancomycin (30 μg). All the then read every 15 min with a spectrophotometer at a
Müller-Hinton plates were incubated for 24 hrs at 37 °C. wavelength of 600 nm to delay the growth generated with
In addition, the standard strain were including Bacillus Oxgall for each strain compared to the control sample for
licheniformis PTCC 1595 and Bacillus licheniformis PT1525 as the strain until reaching a turbidity of 0.3 was determined
the positive and negative control strains, respectively. (Gilliland et al., 1980).
Due to the size of growth inhibition zone, the results
were interpreted as resistant, intermediate susceptible, 2.8.6. Resistance to acid
and susceptible to the Clinical and Laboratory Standards To evaluate resistance to acid, we employed the
Institute table (CLSI, 2016). pre-cultures on LB broth prepared for overnight, after
homogenizing them by vortex, 1 ml aliquots of each active
2.8.3. Biofilm formation culture was poured into falcons containing LB culture
Tube adhesion method was applied to detect the ability medium with HCL acid 37% (Merck Millipore Company,
to form biofilms (Wolfe et al., 2004). The keratinolytic Germany, Cat No: 100317) was reached to pHs 2, 2.5, and
strains were inoculated into the Subwoofer wireless 3 using pH Meter device (Beckman Company, Model of
transmitter (SWT) broth and incubated at 37 °C with 72, USA). Next, the inoculated media were incubated in a
incubator shaker (GFL Company, 3031 Model, India) for shaker incubator at 37 °C for 3-6 hrs. After the first three

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 Keratinolytic probiotic Bucillus licheniformis bacteria

hours, the10-μl samples (two samples from the vortex positions were removed for each sequence pair (pairwise
falcons) were cultured as spots in LB agar (two replicates deletion option). There were a total of 1392 positions in
from each medium) and the inoculated media were the final dataset. Evolutionary analyses were conducted
incubated at 37 °C for 24 hrs. All the experiments were in MEGA11 (Tamura et al., 2021; Stecher et al., 2020;
repeated three times (Singhal et al., 2010). Sabir et al., 2021).

2.8.7. Growth rate of acid resistant strains at acidic pH 2.8.10. Statistical analysis
For evaluation, as in the previous stage, an 18-hour pre- SPSS software Ver-20 was used to perform statistical
culture was prepared separately in LB broth under sterile analysis. The data are expressed as a mean ± standard
conditions. Then, liquid LB medium with pHs of 2, 2.5 and deviation (SD) calculated over three independent
3 were prepared and distributed in the sterile falcons, experiments performed in triplicate. Analysis of statistical
then 10 μl of each sample were inoculated in the falcons. significance was carried out via two-way ANOVA (at a
Afterward, the falcons incubated in a shaker incubator significance level of P<0.05) with t-test.
at 37 °C and 180 rpm for 4 hours. The turbidity of all the
samples was read at 600 nm with a spectrophotometer set.
3. Results
2.8.8. Antimicrobial assessment
To measure the antibacterial activity of the Bacillus 3.1. Identification of keratin degrading bacteria
isolates, 18-hour pre-cultures on LB broth (Merck After culturing 42 isolates on culture medium
Company, Germany) were applied. From all of 8 strains in containing 1.5% keratin, 8 strains formed a clear zone
separate falcons and in a shaker incubator (GFL Company, around their colonies after 72 hrs (Figure 1), revealing the
model 3031, India) was prepared at 37 °C. Then, it was production of the enzyme keratinase to degrade and use
centrifuged at 3000 rpm using refrigerated centrifuge the only carbon source of the culture medium (keratin).
(Kendro Company, D37520 Model, United Kingdom) and There were no clear zones around the other strains; thus,
20 μl of supernatant containing possible bacteriocin of they were not keratinolytic and were excluded from the
all 8 Bacillus licheniformis strains was cultured in LB Agar study at this stage. According to the results, the isolates
plate wells and purified of any bacterium. Afterwards, a pvkr6, pvkr8, pvkr9, and pvkr41 produced the highest
colony was removed from the each plate of purchased amount of clear zone during 72 hrs of incubation at
pathogenic bacteria (the National Center of Genetic
37 °C. As a result, these strains were classified as strong
and Biological Resources, Iran) including Bacillus cereus
keratin-degrading bacteria, followed by the pvkr15 and
(CCM2010, DSMZ31, ATCC14579, LMG6923, NCBI9373 and
pvkr26 strains, which produced a clear zone with a
NCTC2599), Escherichia coli (ATCC11775, DSM30083, CCM
smaller diameter than the mentioned strains and were
5172), Klebsiella pneumonia subsp pneumonia (ATTCC700603,
classified as moderately potent degrading bacteria. Finally,
CCUG45421 and LMG20218), Salmonella Enteritidis
strains pvkr1 and pvkr10 produced the lowest levels of
(ATCC1307603, DMS 17420, LMG 10395) and Proteus
the keratinase enzyme and a clear zone with a very small
vulgaris (ATCC25933, CCUG34294) and cultured through
diameter. These strains were classified as weak in terms
pour plate. At this stage, four wells were installed in each
of keratin degrading activity.
plate and the ends of the wells were blocked with 10 μl
of molten agar. The medium with inoculated wells were
3.2. Biochemical identification of bacteria strains
incubated at 37 °C for 24 hrs. The above experiment was
repeated once more. In addition, the pre-cultured strains The bacterial strains were isolated from soil designed as
were centrifuged at 3000 rpm after 18 hrs of incubation pvkr1 to pvkr41. The results of biochemical experiments of
and the supernatant was utilized for loading in the wells. Bacillus spp isolates are shown in Table 1. All of 10 isolates
Inhibitory zone of the strains were checked after 24 hrs (8 isolates and B. L1525 and B. L1595 as negative and
of incubation at 37 °C (Nourouzi et al., 2004). positive control, respectively) were gram-positive,
positive catalase, gelatinase, and α-amylase as positive
2.8.9. Phylogenetic analysis of 16S rRNA gene reaction and were identified as Bacillus licheniformis.
The microscopic shapes of Bacillus spp are depicted in
First, we sent PCR product to Gen Fanavaran Company
Figure 2. In addition the B. L1525 and B. L1595 were used
(Tehran, Iran) for sequencing using Sanger sequencing
as standard Bacillus lichenifomis strain and positive control
method. In sequencing, the primers concentration was
(purchased from National Center of Genetic and Biological
10 pmol and the amount of primers was 5 μmol per reading.
Resources, Iran).
The evolutionary history was inferred using the Neighbor-
Joining method (Saitou and Nei, 1987). The optimal tree
is shown. The tree is drawn to scale, with branch lengths 3.3. Biomolecular identification of isolated bacteria
in the same units as those of the evolutionary distances Indicating the Bacillus detection, 16S rDNA sequence
used to infer the phylogenetic tree. The evolutionary analysis confirmed the biochemical results and PCR
distances were computed using the Maximum Composite was performed on randomly selected colonies of
Likelihood method (Tamura et al., 2004) and are in the keratinolytic bacteria (8 isolated strains). The PCR results
units of the number of base substitutions per site. This demonstrated that several colonies were positive for
analysis involved 8 nucleotide sequences. All ambiguous Bacillus (Figure 3). All the 8 isolates were different strains

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Vanaki, P. et al.

Figure 1. Creating a clear zone with keratin-degrading bacteria. Bacillus licheniformis PTCC 1525 and Bacillus licheniformis PTCC 1595
were employed as control Bacillus strains.

Table 1. Biochemical test of Bacillus spp (n=10).

Sample Citratase
Catalase Lecithinase caseinase Urease Gelatinase α-Amylase Glucose H2S O/F
number (Simmons citrate)

Pvkr1 3+ 1+ 3+ - 1+ - 1+ - 1+ Obligate
aerobes

Pvkr6 3+ - 2+ - 1+ 1+ 1+ - - Facultative
anaerobes

Pvkr8 1+ - 2+ - 1+ - 1+ - - Obligate
aerobes

Pvkr9 1+ 1+ 1+ - 1+ 2+ 1+ - 1+ Facultative
anaerobes

Pvkr10 1+ - 1+ - 1+ 1+ 1+ - - Facultative
anaerobes

Pvkr15 3+ 1+ 3+ - 1+ 3+ 1+ - - Facultative
anaerobes

Pvkr26 2+ - 2+ - 1+ 1+ 1+ - - Obligate
aerobes

Pvkr41 3+ 1+ 2+ - 1+ - 1+ - - Obligate
aerobes

B. L1525 2+ - 3+ - 1+ 2+ 1+ - - Obligate
aerobes

Obligate
B. L1595 3+ 1+ 1+ - 1+ - 1+ - -
aerobes

+: sign of the existence; -: sign of none existence; O: Obligate; F: Facultative.

of Bacillus licheniformis and were recorded in ENA as activity (63.74±0.24 U/ml). The pvkr1, pvkr6, and
represented in supplemental section. pvkr15 strains formed yellow colonies and the pvkr8,
pvkr9, pvkr10, pvkr26, and pvkr41 strains formed white
3.4. Keratinase activity colonies on feather meal agar plate. In addition, the effect
of enzyme concentration on keratinase activity (Figure 5)
We examined 10 Bacillus species for keratinase
implied that maximum keratinase activity belonged
activity before and after sedimentation and concentration
the pvkr6 strain (specific activity 54.83±1.61 and total
of keratin enzyme. All the strains (8 strains and B.
activity 372.25±0.81 U/ml) and the pvkr15 strain (specific
L1595 as positive control) produced keratinase except
activity 50.45±2.6 and total activity 303.29±1.35 U/ml)
the negative control (B. L1525), isolated and allowed to was recorded at 0.07 M. Minimum keratinase activity also
grow on feathers meal powder as the source of nitrogen belonged the pvkr1 strain (specific activity 62.16±2.3 and
and carbon. This result showed that the pvkr6 and total activity 191.22±0.72 U/ml) at 0.03 M and the
pvkr15 strains could degrade the feathers completely B. L1595 strain (specific activity 23.28±0.53 and total
after 7 days at 37 °C. As shown in Figure 4, the highest activity 146.73±1.9 U/ml) at 0.07 M.
level of keratinase activity belonged to the pvkr6 strain Following the enzyme deposition, keratinase activity
(124.08±0.27 U/ml) and pvkr15 strain (101.1±0.45 U/ml). levels of the isolated pvkr15 strain and B. L1595 as
The pvkr1 strain produced the lowest level of keratinase positive control were also measured. As shown Figure 6,

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 Keratinolytic probiotic Bucillus licheniformis bacteria

Figure 2. Microscopic shape of Bacillus spp. As can be seen in the figure, the Pvkr10, Pvkr41 and B. L 1595 strains have the longest length
in terms of bacilli elongation and the Pvkr1, Pvkr8, Pvkr9, Pvkr15 and B. L 1525 have medium length and the Pvkr6 and Pvkr26 have very
short bacilli. Bacillus licheniformis PTCC 1525 (negative control), during growth and reproduction, produces a large amount of mucous
secretions, which turn red in staining, but rod and gram-positive bacilli (purple) are seen among the red secretions.

Figure 3. PCR results of 16S rDNA gene (10000 bp). (A) Lane 1: pvkr1; Lanes 2-5: pvkr2, pvkr6, pvkr8, and pvkr9, respectively; Lane M:
ladder marker (10k bp); (B) Lanes 1-4: pvkr10, pvkr15, pvkr26, and pvkr41, respectively; Lane 5: B. L 1595 as positive control; Lane M:
ladder marker (10k bp).

both of the strains produced keratinase and keratinase activity in the selected strains (Figure 7) showed that
activity of the pvkr15 strain (160.36±0.85 U/ml) was keratinase activity of the pvkr15 strain was recorded
more than that of B. L1595 (92.34±0.94 U/ml). Moreover, at 0.119 M (specific activity 45.73±3.9 and total activity
the effect of enzyme concentration on keratinase 481.07±2.5 U/ml).

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Vanaki, P. et al.

3.5. Hemolytic activity on bacterial pathogens, including Bacillus cereus,

Hemolytic activity of the Bacillus strains was determined Escherichia coli, Klebsiella pneumoniae while pvkr8,
using blood agar medium, supplemented with defibrinated pvkr10 and both control groups (Bacillus licheniformis
blood. After 96 hrs of incubation at 37 °C, the strains of 1525 and Bacillus licheniformis 1595) had an inhibitory effect
pvkr1, pvkr8, and pvkr10 produced α -hemolysis and the on the growth of Salmonella enterica. Additionally, pvkr1,
pvkr6, pvkr9, pvkr26, and pvkr41 strains demonstrated pvkr6, pvkr8, pvkr9, pvkr10, pvkr15, and pvkr26 inhibited
α -hemolysis weakly while the pvkr15 strain showed no the growth of Proteus mirabilis. In other words, these strains
hemolytic activity during 96 hrs (Figure 8); thus, they produced bacteriocin against the mentioned pathogenic
are γ -hemolytic strains. Therefore, one strain (pvkr15) bacteria and stopped their growth (Figure 9).
was selected as γ-hemolytic with the probiotic potential.
3.8. Hydrophobicity
3.6. Antibiotic susceptibility of the Bacillus licheniformis The adhesion of Bacillus strains to xylene are represented
strains in supplemental section (Figure 10), showing the most
Selected Bacillus strains displayed various levels of adhesive bacteria to pvkr8 and pvkr15 with a 22.44% and
antibiotic resistance, ranging from resistance to susceptibility 19.58% range of hydrophobicity percentage, respectively.
(Table 2). It was found that all the strains were sensitive to In addition, the relative range of hydrophobicity was 3.27 to
streptomycin, gentamycin, amoxicillin, tetracycline, rifampin, 30.57. The highest level of hydrophobicity belonged to
vancomycin, erythromycin, and cefixime. The highest B. licheniformis PTCC 1595 that was 30.57%.
resistance belonged to penicillin G and the only strains
sensitive to penicillin G were pvkr8 and pvkr9. 3.9. Biofilm formation with the Bacillus licheniformis
strains
3.7. Antagonistic properties of the Bacillus licheniformis Given the importance of the ability of probiotic bacteria
strains against pathogenic bacteria to form biofilms to maintain survival, herein, the ability
According to the data obtained from this experiment, to form biofilms was examined for each of the strains.
none of the isolated strains had an inhibitory effect Based on the results, it can be concluded that all the
strains, including isolated strains and control strains, have
biofilm formation properties. This result implied that 100%
of isolates as biofilm formers were detected (Figure  11).

3.10. The resistance of the isolated Bacillus licheniformis to

bile salts and acidic environment
As shown in Table 3, the growth rates of pvkr9 and
pvkr15 in the medium containing bile salts were between
15 and 40 min relative to their controls and were
classified as the group of bacteria resistant to Oxgall 0.3%
concentration. However, the growth delay of pvkr8 was
between 40 and 60 min. It can be thus considered that this
strain was a bacterium with weak resistance to bile salts
while other strains showed a growth delay of more than
60 min classified as bile salt-sensitive bacteria.
Figure 4. Keratinase activity of Bacillus spp before sedimentation Additionally, the results of resistance to low pH (Figure 12)
of keratin enzyme. demonstrated the viability of Bacillus licheniformis strains.

Figure 5. Effect of enzyme concentration on keratinase activity in the isolated strains.

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 Keratinolytic probiotic Bucillus licheniformis bacteria

The pvkr9 strain showed slow growth at pH 2 whereas strains survived the acidic conditions and were able to
the growth continued at pH 2.5 and 3 for 3 hours. In the grow on the LBB agar.
pvkr26 strain, we observed weak growth at pH 2 and
2.5 for only the first 2 hours, but its growth continued at 3.11. Phylogenetic analysis of 16S rRNA gene
pH 3 for 3 hours. The growth of pvkr8 strain also stopped
The evolutionary history was inferred through the
completely at different pH. The other strains had weak and
neighbor-Joining method. The analysis involved eight
slow growth compared to the control samples (growth of
nucleotide sequences. As can be seen in the Figure 13,
strains in the medium with neutral pH [7.2]). Moreover,
pvkr1 and pvkr9 strains have common ancestors
the results of pH effect on biofilms showed that all the
and are considered as in-group, which are closer to
pvkr6 and pvkr15 strains than other strains. The pvkr26 and
pvkr41 also have common ancestors that are considered
as outgroup and are closer to the pvkr8 strain (Figure 13).

4. Discussion

To date, numerous studies have been performed on

the isolation and identification of Bacillus bacteria with
keratin degradation properties. The results of these studies
showed that several Bacillus strains have a very high
ability to produce the keratinase enzyme and in some
studies, researchers have isolated the strains from soil
samples (particularly poultry and slaughterhouse soil) or
Figure 6. Keratinase activity of the selected strains after keratin waste in the nature (Qiu et al., 2020). It has been
sedimentation of keratin enzyme. suggested to employ their keratin degradation potential

Figure 7. The effect of enzyme concentration on keratinase activity in the selected strains.

Figure 8. Hemolytic Activity of the strains; (A) pvkr15: γ -hemolysis; (B) pvkr10: α -hemolysis; (C) Bacillus licheniformis PTCC 1525:
positive control of α -hemolysis; (D) Bacillus licheniformis PTCC 1595: negative control of α -hemolysis.

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Vanaki, P. et al.

Table 2. Antibiotic susceptibility.

Strain Vancomycin Rifampin Tetracycline Amoxicillin Penicillin Cefixime Erythromycin Gentamycin Streptomycin

pvkr1 S S S I R S I S S

pvkr6 S S S S R S S S S

pvkr8 S S S S S S I S S

pvkr9 S S S S S R S S S

pvkr10 S I S S R S S S S

pvkr15 S I S S R S I S S

pvkr26 S I S S R I I S S

pvkr41 S I S S R S S S S

B.l1525 S R S S R R R I R

B.l1595 S R S S R R R S S

S: Sensitive, I: Intermediate, R: Resistance.

Figure 9. Antagonist properties of the isolated Bacillus spp against the pathogenic bacteria.

Figure 10. Hydrophobicity percentage of the isolated strains.

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 Keratinolytic probiotic Bucillus licheniformis bacteria

Table 3. Resistance of the isolated Bacillus licheniformis to bile salts. activity belonged to Bacillus licheniformis as pvkr6, pvkr15,
and pvkr41 strains with 124.08, 101.1, and 100.18 U/ml,
Growth Delay respectively. Additionally, the highest keratinase activity
Strain Results
(Min)
was recorded at an enzyme concentration of 0.07 M, which
pvkr1 D>60 min Sensitive belonged the pvkr6 with specific activity 54.83 U/ml and
pvkr6 D>60 min Sensitive
total activity 372.25 U/ml and the pvkr15 with specific
activity 50.45 U/ml and total activity 303.29 U/ml at 0.07 M.
pvkr8 40 min <D< 60 min Weakly tolerant In our paper, all of the eight Bacillus keratinolytic strains
pvkr9 40 min <D< 15 min Tolerant produced protease enzyme and the strain isolated from
pvkr10 D>60 min Sensitive
poultry soil revealed that the bacteria producing keratinase
enzyme belonged to Bacillus licheniformis. Mohamed et al.
pvkr15 15 min <D< 40 min Tolerant reported that B. amyloliquefaciens MA20 and B. subtilis have
pvkr26 D>60 min Sensitive the keratinolytic ability of wool-degrading bacillus species
pvkr41 D>60 min Sensitive
and the keratinolytic activity of these strains were in the
range of 0.814-0.922 U/ml (Hassan et al., 2013). Mukesh
B. L1525 D>60 min Sensitive Kumer  et al. reported that the maximum keratinase
B. L1595 D>60 min Sensitive and caseinase activity of the MPTK6 strain were 2.4 and
2.1 U/ml, respectively. Also, the MPTK6 strain was able to
degrade the crude feather to FPH under optimal conditions
(30 g/l chicken feather, pH 10.0 and 72 h fermentation)
(Kumar et al., 2012). Another study by Deivasigamani and
Alagappan (2008) in line with this study indicated that the
highest keratinase activity of the isolated Bacillus species
from slaughterhouse and poultry soil samples applying
azo-keratin medium was 0.1225 U/ml).
Previous studies have shown that keratin is digested and
degraded in nature with many diverse organisms, including
some insects, such as silkworm larvae, carpet beetles,
lice (Böckle et al., 1995 ), and some bacteria, including
Streptomyces pactum (Seyfi, 2017), Bacillus licheniformis
PWD-1 (Williams et al., 1990), Chryseobacterium sp.Kr6 and
Streptomyces fradiae (Riffel et al., 2003), Bacillus halodurans
(Takami et al., 1992 and 1999), Fervidobacterium species
(Friedrich and Antranikian, 1996), and some dermatophyte
fungi (55), which secrete the enzyme keratinase.
Figure 11. Biofilm formation in the isolated strains. The right tube In the current research also the strains isolated from
contains one of the isolated strains that has formed biofilm and poultry soil were keratinase-producing bacteria of the
the left tube is a culture medium Bacillus licheniformis strain.
Kunert (1973) reported that the isolated keratinolytic
bacteria from feather with probiotic activities belonged
to Burkholderia, Pseudomonas, Chryseobacterium, and
in the livestock and poultry feed industry. There has also Microbacterium and were selected and screened on skim
been a lot of research on the isolation of spore-shaped milk agar plates for proteolytic activity. Studies have implied
Bacillus strains with probiotic properties and the results that these bacteria could grow on a variety of extract of
reveal that many Bacillus strains have significant probiotic keratin, such as chicken nail hair and wool, in which all
properties and can have very beneficial effects on the host the strains have been reported as keratinolytic protease
body (Hassan et al., 2013). However, none of the reports (Wakil et al., 2011). This study focused on keratinolytic
examined the simultaneous evaluation of probiotic protease production from Bacillus strain. The proteolytic
properties and the ability of these bacteria to degrade activities of all the isolates were detected on the LB agar
keratin. In this experiment, with modified methods and medium with 1.5% (w/v) skim milk. Among the isolates
materials, our objective was to isolate Bacillus bacteria with analyzed, eight isolates showed proteolytic activity. Herein,
probiotic properties and the ability to degrade keratin. we investigated the keratinolytic Bacillus strains with
Herein, the keratinase-producing bacteria isolated from probiotic potential for antimicrobial susceptibility testing.
poultry farm soil identified Bacillus licheniformis. In the One of the selection factors of probiotic strains was antibiotic
present work, 42 strains were isolated on suspended susceptibility of isolated strains (Gueimonde et al., 2013).
soil samples after heat and alcohol treatment. All the Therefore, the susceptibility of the strains to antibiotics
isolates were selected and screened to isolate Bacillus was measured. The pattern of antibiotic susceptibility has
strains with the selective method. Among all the isolated revealed that all the strains were sensitive to the majority
strains, only eight Bacillus strains were able to degrade of antibiotics except penicillin G. One of the important
keratin. The keratinolytic activity of the strains was features of probiotic bacteria is their ability to form biofilm
between 48.91-124.08 U/ml and the highest keratinolytic (Salas-Jara et al., 2016). Biofilm formation is one of the

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Vanaki, P. et al.

Figure 12. Resistance of the isolated Bacillus licheniformis at different pH.

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 Keratinolytic probiotic Bucillus licheniformis bacteria

activity is one of the most common assays to distinguish

pathogenic bacteria form probiotics. In this experiment,
only γ-hemolytic bacteria were Bacillus licheniformis
pvkr15 and Bacillus licheniformis PTCC 1595 (control strain).
In the isolated Bacillus licheniformis strains, only Bacillus
licheniformis pvkr15 was γ-hemolytic and the other strains
exhibited α-hemolytic activity, which could not be classifies
as probiotic bacteria. The results revealed that the pvkr8 and
pvkr15 strains respectively had a cell surface hydrophobicity
of 22.44% and 19.56%. This adherence is essential for probiotic
bacteria affecting the intestines. Thus, Bacillus licheniformis
pvkr15 as a keratinolytic spore forming probiotic Bacillus
strain is suitable as a nutritional supplement in humans,
fish, and animal.

5. Conclusion
Figure 13. Phylogenic tree illustrates the evolutionary
relationships between different 16S rRNA nucleotide sequences The results revealed that only the Bacillus licheniformis
of Bacillus licheniformis. pvkr15 produced acceptable keratinase enzyme and
demonstrated potential for surviving in the gut environment
with the ability to produce spores. Hence, this strain can
be employed as a probiotic in animal and chicken feed
central mechanisms in insuring bacterial survival in harsh
industry. Other strains, despite having some probiotic
environments (Zhao et al., 2017). Comparing with free-cells
characteristics, showed fundamental disadvantages,
or planktonic cell, the packed bacterial presence in biofilms
disqualifying them from being used for this purpose.
protects them from environmental factors and anti-microbial
treatments. In our work, 100% of isolate were able to form
thick biofilms. The range of hydrophobicity was between
3.27-19.65%. To be able to exert their beneficial effects in the Acknowledgements
gut, probiotic bacteria must have the ability to attach to the The authors acknowledge the Microbiology Department,
cells lining the inner surface of the gut and form a biofilm Ayatollah Amoli Branch, Islamic Azad University And about
there. Consequently, the strains with a higher percentage of the name of the last author acknowledge the Biology
surface hydrophobicity also had a greater ability to bind to Department, Ayatollah Amoli Branch, Islamic Azad University.
the cells on the inner surface of the intestine. In the present
study, the antibacterial activity of the strains was also
investigated against some pathogens. Bacillus licheniformis
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