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. Vanaki F. Zaboli H. Kaboosi R. Izadi Amoli F. Savadkoohi About the authors

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 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

According to research, millions tons of feathers are produced annually as a by-product of the factory poultry trade worldwide (Tesfaye et al., 2017TESFAYE, T., SITHOLE, B. and RAMJUGERNATH, D., 2017. Valorisation of chicken feathers: a review on recycling and recovery route—current status and future prospects. Clean Technologies and Environmental Policy, vol. 19, no. 10, pp. 2363-2378. http://dx.doi.org/10.1007/s10098-017-1443-9.
http://dx.doi.org/10.1007/s10098-017-144...
). Chicken feathers are utilized for a variety of products, including fertilizers, feed, and biofilms. Feather is composed of approximately 90% hard keratin (Ramakrishnan et al., 2018RAMAKRISHNAN, N., SHARMA, S., GUPTA, A. and ALASHWAL, B.Y., 2018. Keratin based bioplastic film from chicken feathers and its characterization. International Journal of Biological Macromolecules, vol. 111, pp. 352-358. http://dx.doi.org/10.1016/j.ijbiomac.2018.01.037. PMid:29320725.
http://dx.doi.org/10.1016/j.ijbiomac.201...
). The use of current added value for feathers is the conversion to feather powder as a digestible dietary protein for animal feed, applying physical and chemical treatments (Tiwary and Gupta, 2012TIWARY, E. and GUPTA, R., 2012. Rapid conversion of chicken feather to feather meal using dimeric keratinase from Bacillus licheniformis ER-15. Journal of Bioprocessing & Biotechniques, vol. 2, no. 4, pp. 1000123. http://dx.doi.org/10.4172/2155-9821.1000123.
http://dx.doi.org/10.4172/2155-9821.1000...
). These methods can eliminate the keratin protein amino acids and reduce the quality and digestibility of the protein (Elkin et al., 1996ELKIN, R.G., FREED, M.B., HAMAKER, B.R., ZHANG, Y. and PARSONS, C.M., 1996. Condensed tannins are only partially responsible for variations in nutrient digestibilities of sorghum grain cultivars. Journal of Agricultural and Food Chemistry, vol. 44, no. 3, pp. 848-853. http://dx.doi.org/10.1021/jf950489t.
http://dx.doi.org/10.1021/jf950489t...
). Due to this issue, the use of keratin degrading enzymes produced by microorganisms producing amino acids and peptides to compensate for the lack of protein for animal feed has become an important and attractive issue in biotechnological activities (Cai et al., 2008CAI, C., LOU, B. and ZHENG, X., 2008. Keratinase production and keratin degradation by a mutant strain of Bacillus subtilis. Journal of Zhejiang University. Science. B., vol. 9, no. 1, pp. 60-67. http://dx.doi.org/10.1631/jzus.B061620. PMid:18196614.
http://dx.doi.org/10.1631/jzus.B061620...
; Zabihi et al., 2011ZABIHI, O., MOSTAFAVI, S.M., RAVARI, F., KHODABANDEH, A., HOOSHAFZA, A., ZARE, K. and SHAHIZADEH, M., 2011. The effect of zinc oxide nanoparticles on thermo-physical properties of diglycidyl ether of bisphenol A/2, 2′-Diamino-1, 1′-binaphthalene nanocomposites. Thermochimica Acta, vol. 521, no. 1-2, pp. 49-58. http://dx.doi.org/10.1016/j.tca.2011.04.003.
http://dx.doi.org/10.1016/j.tca.2011.04....
). Alkaline proteases are a group of proteolytic enzymes able to degrade proteins, such as keratin, which are difficult to dissolve more efficiently than other proteases, such as trypsin, pepsin, and papain. Previous studies have indicated that some Bacillus species are able to produce proteases for feather digestion (Shamsipur et al., 2012SHAMSIPUR, M., MIRAN BEIGI, A.A., TEYMOURI, M., POURSABERI, T., MOSTAFAVI, S.M., SOLEIMANI, P., CHITSAZIAN, F. and TASH, S.A., 2012. Biotransformation of methyl tert-butyl ether by human cytochrome P450 2A6. Biodegradation, vol. 23, no. 2, pp. 311-318. http://dx.doi.org/10.1007/s10532-011-9510-0. PMid:21915685.
http://dx.doi.org/10.1007/s10532-011-951...
; Sabir et al., 2021SABIR, S.M., ZEB, A., MAHMOOD, M., ABBAS, S.R., AHMAD, Z. and IQBAL, N., 2021. Phytochemical analysis and biological activities of ethanolic extract of Curcuma longa rhizome. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 81, no. 3, pp. 737-740. http://dx.doi.org/10.1590/1519-6984.230628. PMid:32965334.
http://dx.doi.org/10.1590/1519-6984.2306...
; Pustokhina et al., 2021PUSTOKHINA, I., SERAJ, A., HAFSAN, H., MOSTAFAVI, S.M. and ALIZADEH, S.M., 2021. Developing a Robust Model Based on the Gaussian Process Regression Approach to Predict Biodiesel Properties. International Journal of Chemical Engineering, vol. 2021, pp. 5650499. http://dx.doi.org/10.1155/2021/5650499.
http://dx.doi.org/10.1155/2021/5650499...
). Keratin is an insoluble protein with a stable structure that forms protective levels in various animals and is a structural component of skin, wool, hair, feathers, and nails (Sharma and Gupta, 2016SHARMA, S. and GUPTA, A., 2016. Sustainable management of keratin waste biomass: applications and future perspectives. Brazilian Archives of Biology and Technology, vol. 2016, no. 0, pp. 59. http://dx.doi.org/10.1590/1678-4324-2016150684.
http://dx.doi.org/10.1590/1678-4324-2016...
). Keratinase is able to hydrolyze keratin and degrade feathers. Microorganisms, such as bacteria, fungi, and actinomycetes have been reported to produce keratinase for use as energy and carbon sources. Resistance to proteolytic enzymes has been attributed to the complex structure of beta-keratin filaments. In addition, disulfide cross-links produce a compact three-dimensional network as a result of the intermolecular bonds of disulfide between the rod and the terminal domains of the constituent molecules (Riffel and Brandelli, 2006RIFFEL, A. and BRANDELLI, A., 2006. Keratinolytic bacteria isolated from feather waste. Brazilian Journal of Microbiology, vol. 37, no. 3, pp. 395-399. http://dx.doi.org/10.1590/S1517-83822006000300036.
http://dx.doi.org/10.1590/S1517-83822006...
).

Keratinolytic microorganisms may play an important role in industrial processes and the biotechnology and pharmaceutical industries (Vidmar and Vodovnik, 2018VIDMAR, B. and VODOVNIK, M., 2018. Microbial keratinases: enzymes with promising biotechnological applications. Food Technology and Biotechnology, vol. 56, no. 3, pp. 312-328. http://dx.doi.org/10.17113/ftb.56.03.18.5658. PMid:30510475.
http://dx.doi.org/10.17113/ftb.56.03.18....
). Some bacteria, such as Bacillus licheniformis and Bacillus subtilis produce keratinase (Mousavi et al., 2013MOUSAVI, S., SALOUTI, M., SHAPOURY, R. and HEIDARI, Z., 2013. Optimization of keratinase production for feather degradation by Bacillus subtilis. Jundishapur Journal of Microbiology, vol. 6, no. 8, pp. 1G. http://dx.doi.org/10.5812/jjm.7160.
http://dx.doi.org/10.5812/jjm.7160...
). The keratinase produced by B. licheniformis increases the total digestibility of crude feather amino acids and commercial feather powder. This enzyme can replace up to 7% of dietary protein for growing chickens (Garbeva et al., 2003GARBEVA, P., VAN VEEN, J. and VAN ELSAS, J., 2003. Predominant Bacillus spp. in agricultural soil under different management regimes detected via PCR-DGGE. Microbial Ecology, vol. 45, no. 3, pp. 302-316. http://dx.doi.org/10.1007/s00248-002-2034-8. PMid:12632212.
http://dx.doi.org/10.1007/s00248-002-203...
).

Bacillus species are gram-positive bacteria that can be found widely in soil and plants (Elshaghabee et al., 2017ELSHAGHABEE, F.M., ROKANA, N., GULHANE, R.D., SHARMA, C. and PANWAR, H., 2017. Bacillus as potential probiotics: status, concerns, and future perspectives. Frontiers in Microbiology, vol. 8, pp. 1490. http://dx.doi.org/10.3389/fmicb.2017.01490. PMid:28848511.
http://dx.doi.org/10.3389/fmicb.2017.014...
). Bacillus species have found a variety of food products, such as probiotics. Probiotics are living microorganisms giving the host health benefits. Probiotics can increase innate immune and resistance to pathogens. The use of probiotics in the feed, poultry, and aquaculture industries is important (Munir et al., 2019MUNIR, I., BANO, A. and FAISAL, M., 2019. Impact of phosphate solubilizing bacteria on wheat (Triticum aestivum) in the presence of pesticides. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 79, no. 1, pp. 29-37. http://dx.doi.org/10.1590/1519-6984.172213. PMid:30726453.
http://dx.doi.org/10.1590/1519-6984.1722...
). However, removing antibiotics from poultry feed can predispose them to infections caused by certain pathogens, such as Escherichia coli, Clostridium perfringens, and Salmonella species, leading to low yields and serious damage to the poultry industry (Al-Sultan, 2003AL-SULTAN, S., 2003. The effect of Curcuma longa (turmeric) on overall performance of broiler chickens. International Journal of Poultry Science, vol. 2, no. 5, pp. 351-353. http://dx.doi.org/10.3923/ijps.2003.351.353.
http://dx.doi.org/10.3923/ijps.2003.351....
; Marins et al., 2021MARINS, A., CRISTALDO, P.F., PAIVA, L.R., MIRAMONTES, O. and DESOUZA, O.A., 2021. new approach to mark termites (Cornitermes cumulans (Kollar) Blattodea: Isoptera) for laboratory bioassays. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 83, pp. 83. PMid:34730713.; Wekhe et al., 2007WEKHE, S., OGBAMGBA, K. and OBOH, C., 2007. Preliminary investigation of the effect of Rhizophora racemosa (mangrove) feed additive on broiler performance. African Journal of Biotechnology, vol. 6, no. 16, pp. 103-111.). Probiotic products as live microbial foods are beneficial to animal health as a viable alternative to antibiotics to prevent disease and stimulate animals (Jin et al., 1998JIN, L.Z., HO, Y.W., ABDULLAH, N. and JALALUDIN, S., 1998. Growth performance, intestinal microbial populations, and serum cholesterol of broilers fed diets containing Lactobacillus cultures. Poultry Science, vol. 77, no. 9, pp. 1259-1265. http://dx.doi.org/10.1093/ps/77.9.1259. PMid:9733111.
http://dx.doi.org/10.1093/ps/77.9.1259...
; Teo and Tan, 2006TEO, A.-L. and TAN, H.-M., 2006. Effect of Bacillus subtilis PB6 (CloSTAT) on broilers infected with a pathogenic strain of Escherichia coli. Journal of Applied Poultry Research, vol. 15, no. 2, pp. 229-235. http://dx.doi.org/10.1093/japr/15.2.229.
http://dx.doi.org/10.1093/japr/15.2.229...
). The adhesion of probiotics can be related to the characteristics of their cell surface, namely hydrophobicity, which can result in a strong interaction with the mucosa if high enough. However, not only is the hydrophobicity of probiotic cell surfaces responsible for their attachment to epithelial cells, but more specific mechanisms include lipoteichoic acid, extracellular components (exopolysaccharides or proteins), or surface proteins (Grigoryan et al., 2018GRIGORYAN, S., BAZUKYAN, I. and TRCHOUNIAN, A., 2018. Aggregation and adhesion activity of lactobacilli isolated from fermented products in vitro and in vivo: a potential probiotic strain. Probiotics and Antimicrobial Proteins, vol. 10, no. 2, pp. 269-276. http://dx.doi.org/10.1007/s12602-017-9283-9. PMid:28455689.
http://dx.doi.org/10.1007/s12602-017-928...
; Mohanty et al., 2019MOHANTY, D., PANDA, S., KUMAR, S. and RAY, P., 2019. In vitro evaluation of adherence and anti-infective property of probiotic Lactobacillus plantarum DM 69 against Salmonella enterica. Microbial Pathogenesis, vol. 126, pp. 212-217. http://dx.doi.org/10.1016/j.micpath.2018.11.014. PMid:30423346.
http://dx.doi.org/10.1016/j.micpath.2018...
). The aim of this study was to isolate and investigate the characteristics of Bacillus probiotic bacteria producing the enzyme keratinase for use in feather degrading and to provide a protein source for animal feed.

2. Material and Methods

2.1. Sample collection

The soil samples were randomly collected from different areas of the bed soil of Baharan Poultry slaughterhouse (Qom, Iran). A total of 100 samples were collected so that each soil sample was 5 g and was poured sterilely into a separate sterile falcon and delivered to the microbiological laboratory (Islamic Azad University, Ayatollah Amoli Branch), then 1 g of each sample was weighed below the microbial hood (Sarvo Tajhiz Sakoo Company, Iran) and the microbial suspension of the unit was prepared and homogenized by the shaker (MICRO-SPIN – KIAGEN company, Iran).

2.2. Isolation and identification of bacterial strains

Briefly, one gram of the soil sample was diluted 1:10 (w/v) in sterile saline (0.9%) (Samen pharmaceutical company, Iran). Afterwards, the suspensions were allowed to settle in the soil particles for 3 min and 25 ml of the microbial suspension supernatant then reached 50 ml at a ratio of 1:1 with 25 ml of buffered peptone water (QUELAB, Cat No:39-9587, Canada). This solution was divided into two equal parts and treated individually with heat and alcohol to separate Bacillus bacteria (Mohamadkhani and Shishegaran, 2020MOHAMADKHANI, M.K. and SHISHEGARAN, A., 2020. Identifying Associations between Local Drought and Global Sea Surface Temperature. Journal of Research in Science, Engineering and Technology, vol. 8, no. 3, pp. 1-4.). In this way, for heat treatment, from the initial microbial suspension, with a volume ratio of 1:10, and with 18 ml of peptone water, it reached to 20 ml and was placed in a 65 ° C water bath for one hour. Then, ten serial dilutions with peptone water were prepared from the suspension treated (from 10-1 to 10-10) after homogenization of the last dilution was cultured 100 ml on the LB agar (two replications per dilution) and were incubated for 24 hours at 37 ° C. For alcoholic treatment, the initial microbial suspension was reduced to a volume of 20 ml with a volume ratio of 1:1, and with 10 ml of 96% ethanol, so that the final concentration of alcohol reached 50%. The sample was treated with alcohol for one hour. The next steps were the same as heat treatment (Barbosa et al., 2005BARBOSA, T.M., SERRA, C.R., LA RAGIONE, R.M., WOODWARD, M.J. and HENRIQUES, A.O., 2005. Screening for Bacillus isolates in the broiler gastrointestinal tract. Applied and Environmental Microbiology, vol. 71, no. 2, pp. 968-978. http://dx.doi.org/10.1128/AEM.71.2.968-978.2005. PMid:15691955.
http://dx.doi.org/10.1128/AEM.71.2.968-9...
). Serial dilution was performed on each treated sample and mass and linear cultures were then prepared from their final concentration; hence, the refined colonies were stored at 70 °C for 24 h (Barbosa et al., 2005BARBOSA, T.M., SERRA, C.R., LA RAGIONE, R.M., WOODWARD, M.J. and HENRIQUES, A.O., 2005. Screening for Bacillus isolates in the broiler gastrointestinal tract. Applied and Environmental Microbiology, vol. 71, no. 2, pp. 968-978. http://dx.doi.org/10.1128/AEM.71.2.968-978.2005. PMid:15691955.
http://dx.doi.org/10.1128/AEM.71.2.968-9...
). To identify the isolated bacteria, biochemical tests, including catalase, gelatinase, amylase, lecithinase, Simmons citrate agar, urease, caseinase, O/F (obligate or facultative aerobes) and TSI (Triple Sugar Iron) were employed according to the instructions described in Bergey’s manual systematic bacteriology (Vos et al., 2011VOS, P., GARRITY, G., JONES, D., KRIEG, N.R., LUDWIG, W. and RAINEY, F.A., 2011. Bergey's manual of systematic bacteriology: the firmicutes. USA: Springer Science & Business Media, pp. 56-67.). Finally, a typical colony of each subculture sample was cultured and the bacteria were identified with colony morphology, gram staining compared to standard species (Bacillus licheniformis PTCC 1595 and Bacillus licheniformis PTCC 1525 as positive control).

2.3. Feather keratin substrate

The method of preparing keratin powder is described by (Wawrzkiewicz et al., 1987WAWRZKIEWICZ, K., ŁOBARZEWSKI, J. and WOLSKI, T., 1987. Intracellular keratinase of Trichophyton gallinae. Journal of Medical and Veterinary Mycology, vol. 25, no. 4, pp. 261-268. http://dx.doi.org/10.1080/02681218780000601. PMid:2444691.
http://dx.doi.org/10.1080/02681218780000...
). The feathers obtained from the slaughterhouse were washed with distilled water and detergent (Dishwashing Liquid, Prile Company, Iran) to degrease and remove slaughterhouse contaminants from feathers. Chloroform: ethanol solution (v/v) was then applied to separate the fat from the feathers. Afterwards, 500 ml of DMSO (Dimethyl sulfoxide, Merck Milipore, Germany, Cat: 102950) was added to the lipid-free feathers and they were heated at 100 °C for 120 min. Following the removal of the precipitate, one liter of acetone (-70°C) was added dropwise to the supernatant at 4 °C to precipitate the keratin protein and separate it from the solvent and acetone. Lastly, the keratin precipitate was separated and dried. Keratin powder was employed as a substrate for the keratin culture medium.

2.4. Isolation of keratin degrading bacteria

To identify keratin-degrading Bacillus spp, all 42 isolated strains were cultured separately on culture medium containing 1.5% keratin (as the only carbon source) and incubated at 37 °C for 5 days. The degradability of keratin substrates in culture medium was evaluated on the basis of keratin containing NaCl (0.5 g/l) (Merck Company, Germany, Cat No: 106404), K2HPO4 (0.3 g/l) (Sigma-Aldrich Company, USA, Cat No: 1551128), KH2PO4 (0.4 g/l) (Merck Company, Germany, Cat No: 105104), agar (15 g/l) (Merck Company, Germany), and keratin (15 g/l) (keratin is obtained by the method described above (keratin substrate)) (Sangali and Brandelli, 2000SANGALI, S. and BRANDELLI, A., 2000. Feather keratin hydrolysis by a Vibrio sp. strain kr2. Journal of Applied Microbiology, vol. 89, no. 5, pp. 735-743. http://dx.doi.org/10.1046/j.1365-2672.2000.01173.x. PMid:11119146.). Also, Bacillus licheniformis PTCC 1595 as positive control and Bacillus licheniformis PTCC 1525 as negative control were considered as standard strain (National Center of Genetic and Biological Resources, Iran). The plates were incubated at 37 °C for 1 to 5 days and keratinolytic strains were identified and isolated based on the clear keratinolysis area around them.

2.5. Biomolecular identification of bacteria

For biomolecular identification of isolated bacteria, the isolated bacteria were cultured with keratinase activity in LBA (Luria_Bertani Agar, Merck Company, Germany, Cat No: 110283) medium and incubated for 24 hrs at 37 °C. Genomic DNA of the selected isolates was extracted with Peqlab, Cat No: 51304 kit (Dena Zist Company, Iran). The 16S rDNA was utilized to amplify a segment of DNA through the use of the following universal primers of 27F(5'AGAGTTGATCATGGCTCAG-3') and 1492R(5'TACGGATACCTTGTTACGACTT-3') (Takapouzist Company, Tehran, Iran) (Batisson et al., 2009BATISSON, I., CROUZET, O., BESSE-HOGGAN, P., SANCELME, M., MANGOT, J.-F., MALLET, C. and BOHATIER, J., 2009. Isolation and characterization of mesotrione-degrading Bacillus sp. from soil. Environmental Pollution, vol. 157, no. 4, pp. 1195-1201. http://dx.doi.org/10.1016/j.envpol.2008.12.009. PMid:19121884.
http://dx.doi.org/10.1016/j.envpol.2008....
). PCR was performed on a total volume of 25 μl with 190 ng of template DNA, 10 pmol of each primer, 10 mM dNTP (Thermo Fisher Scientific Company, USA, Cat No: 18427088), 60 mM MgSO4 (Millipore Sigma Company, Germany, Cat No: 105886), 10X Taq buffer (Thermo Fisher Scientific Company, USA, Cat No: B38), and 10 U/μl of Taq polymerase (Roche Company, Swiss). The PCR reaction was as follows: 94 °C for 10 min, 35 cycles 94 °C for 1 min, 54 °C for 1 min, 72 °C for 1 min, and the final length at 72 °C for 10 min. In addition, Bacillus licheniformis PTCC 1595 as positive control was used in this process. The amplicon of 16S rDNA was determined applying agarose gel electrophoresis. The obtained sequences were read and processed through three bioinformatics programs Chromas, Edit Seq, and Seq Man. The sequences of genes processed at the NCBI were BLAST (Lin et al., 1995LIN, X., KELEMEN, D.W., MILLER, E.S. and SHIH, J., 1995. Nucleotide sequence and expression of kerA, the gene encoding a keratinolytic protease of Bacillus licheniformis PWD-1. Applied and Environmental Microbiology, vol. 61, no. 4, pp. 1469-1474. http://dx.doi.org/10.1128/aem.61.4.1469-1474.1995. PMid:7747965.
http://dx.doi.org/10.1128/aem.61.4.1469-...
).

2.6. Keratinase enzyme production

The keratinase enzyme was extracted by the use of the modification method of Sangali and Brandelli (Andersen, 2001ANDERSEN, N.H., 2001. Protein structure, stability, and folding. In: K.P. MURPHY. Methods in molecular biology. Totowa, New Jersey: Humana Press, vol. 168, 252 pp.). The isolated keratinolytic Bacillus bacteria were cultured in keratin broth medium (keratin (10 g/l), NaCl (0.5 g/l) (Merck Company, Germany, Cat No: 106404), K2HPO4 (0.3 g/l) (Sigma- Aldrich, USA, Cat No: 1551128), and KH2PO4 (0.4 g/l) (Merck Company, Germany, Cat No: 105104)) at 37 °C/150 rpm and were incubated for 5 days. The supernatant was separated via centrifugation at 12,000 rpm for 30 min at 4 °C and utilized for keratinolytic assay and enzyme analysis.

2.7. Keratinase assay

According to the method described by Senegal and Brandelli with a little contradiction (Sangali & Brandelli, 2000SANGALI, S. and BRANDELLI, A., 2000. Feather keratin hydrolysis by a Vibrio sp. strain kr2. Journal of Applied Microbiology, vol. 89, no. 5, pp. 735-743. http://dx.doi.org/10.1046/j.1365-2672.2000.01173.x. PMid:11119146.), briefly, 300 μl of medium containing extracted keratin enzyme as explained in the previous step, was prepared. Afterwards, 2700 μl containing 10 g/l keratin in Tris buffer (Sigma-Aldrich, USA, Cat No: T1503) with final concentration 50 mM and pH was added and placed in a water bath at 50 °C for 15 min. After this period, to prevent the activity of keratinase enzymes from continuing, 10% Trichloroacetic acid (Merck Company, Germany, Cat No: 100807) was added to each vial. Subsequently, to separate the substrate from the enzyme suspension, the vials were centrifuged at 10000 g for 5 min. After separation, the absorbance of the supernatant fluid was measured at 440 nm.

In the next step, to deposit the native protein (enzyme), 76 g of ammonium sulfate (Merck Millipore Company, Germany, Cat No: 101209) was dissolved in 100 ml of distilled water. Through a special decanter (Novin Pyrex Company, Iran), 70 ml of the above solution (at -20 °C) in drops of 30 ml crud enzyme obtained from pvkr15 strain was added as overnight in cold room at -20 °C. It was then centrifuged at 5000 RPM for 10 min. The sediment was dissolved in 200 μl of Tris buffer with pH 7.5-8. Finally, the dialysis was performed using a microfuge dialysis bag with at 10,000 RPM and using the soup obtained from the final sediment, the enzymatic activity (U/ml) was measured and calculated (Andersen, 2001ANDERSEN, N.H., 2001. Protein structure, stability, and folding. In: K.P. MURPHY. Methods in molecular biology. Totowa, New Jersey: Humana Press, vol. 168, 252 pp.).

2.8. Safety assessment

2.8.1. Hemolytic activity

The keratinolytic Bacillus strains were analyzed for their hemolytic activity on nutrient agar containing 7% (v/v) defibrinated sheep blood (Quelab Company, Tehran, Iran) and incubated at 37°C for 96 hrs. The plates were checked every 12 hrs. Also, Bacillus licheniformis PTCC 1525 and Bacillus licheniformis PTCC 1595 (purchased from National Center of Genetic and Biological Resources, Iran) respectively were positive and negative controls for this experiment (AlGburi et al., 2016ALGBURI, A., VOLSKI, A., CUGINI, C., WALSH, E.M., CHISTYAKOV, V.A., MAZANKO, M.S., BREN, A.B., DICKS, L.M.T. and CHIKINDAS, M.L., 2016. Safety properties and probiotic potential of Bacillus subtilis KATMIRA1933 and Bacillus amyloliquefaciens B-1895. Advances in Microbiology, vol. 6, no. 6, pp. 432-452. http://dx.doi.org/10.4236/aim.2016.66043.
http://dx.doi.org/10.4236/aim.2016.66043...
).

2.8.2. Resistance to antibiotics

Müller-Hinton Agar (Merck, Germany) were employed to evaluate antibiotic susceptibility of what please clarify via disk diffusion method (Maričević and Žganjar, 2022MARIČEVIĆ, M., & ŽGANJAR, I., 2022. Recognition model of counterfeiting digital records of biometric photographic image. Tehnički Glasnik, vol. 16, no. 1, pp. 67-73.). Nine antimicrobials were chosen to test for susceptibility to antibiotics so that if there is any antibiotic resistance and the possibility of transmission from probiotic Bacillus to livestock and poultry and finally humans, to avoid the use of these Bacillus in livestock and poultry feed as a probiotic bacterium; these antibiotics are including streptomycin (10 μg), gentamicin (10 μg), erythromycin (10 μg), cefixime (5 μg), penicillin G (10 μg), amoxicillin (25 μg), tetracycline (30 μg), rifampin (30 μg), and vancomycin (30 μg). All the Müller-Hinton plates were incubated for 24 hrs at 37 °C. In addition, the standard strain were including Bacillus licheniformis PTCC 1595 and Bacillus licheniformis PT1525 as the positive and negative control strains, respectively. Due to the size of growth inhibition zone, the results were interpreted as resistant, intermediate susceptible, and susceptible to the Clinical and Laboratory Standards Institute table (CLSI, 2016CLINICAL LAB STANDARDS INSTITUTE – CLSI., 2016. Performance standards for antimicrobial susceptibility testing. Clinical Lab Standards Institute, vol. 35, no. 3, pp. 16-38.).

2.8.3. Biofilm formation

Tube adhesion method was applied to detect the ability to form biofilms (Wolfe et al., 2004WOLFE, A.J., MILLIKAN, D.S., CAMPBELL, J.M. and VISICK, K.L., 2004. Vibrio fischeri σ54 controls motility, biofilm formation, luminescence, and colonization. Applied and Environmental Microbiology, vol. 70, no. 4, pp. 2520-2524. http://dx.doi.org/10.1128/AEM.70.4.2520-2524.2004. PMid:15066853.
http://dx.doi.org/10.1128/AEM.70.4.2520-...
). The keratinolytic strains were inoculated into the Subwoofer wireless transmitter (SWT) broth and incubated at 37 °C with incubator shaker (GFL Company, 3031 Model, India) for 24 hs. After transferring the contents to glass tubes, the cells were incubated at 37 °C for 24 hrs without shaking (Wolfe et al., 2004WOLFE, A.J., MILLIKAN, D.S., CAMPBELL, J.M. and VISICK, K.L., 2004. Vibrio fischeri σ54 controls motility, biofilm formation, luminescence, and colonization. Applied and Environmental Microbiology, vol. 70, no. 4, pp. 2520-2524. http://dx.doi.org/10.1128/AEM.70.4.2520-2524.2004. PMid:15066853.
http://dx.doi.org/10.1128/AEM.70.4.2520-...
). The tubes were stained with 1% crystal violet and washed with distilled water. The biofilm layer, if present, appears purple between the air and the liquid.

2.8.4. Cell surface hydrophobicity

The percentage of surface adhesion to solvent (xylene) was measured and calculated based on the method of Xu et al. (2009)XU, H., JEONG, H., LEE, H. and AHN, J., 2009. Assessment of cell surface properties and adhesion potential of selected probiotic strains. Letters in Applied Microbiology, vol. 49, no. 4, pp. 434-442. http://dx.doi.org/10.1111/j.1472-765X.2009.02684.x. PMid:19725886.
http://dx.doi.org/10.1111/j.1472-765X.20...
. The adhesion to xylene (apolar solvent) indicates surface hydrophobicity. Briefly, Bacillus was cultured overnight in LB broth medium, the bacteria were centrifuged at 3000 rpm for 5 min. The bacterial cell deposition were suspended in PBS solution. Afterwards, 1 ml of xylene solvent was added to 3 ml of bacterial suspension and vortexed (Vortex Spin, Micro-Spin Model, Kiagen Company, Iran) for 1 min and kept for 5 min to separate in two phases. We measured the aqueous phases with a spectrophotometer (Beckman Company, DU530 Model, USA) at a wavelength of 600 nm (A5min). The experiment was repeated twice. The values ​​less than 30% (30%>) were considered as (low), between 30-60% (30% ≤, 60%>) as (average), and values ​​greater than 60% (60%≤) were considered as (high). Strains with low solvent adhesion are considered as potential probiotics. The affinities of Bacillus to solvents was calculated with the following Formula 1:

Adhesion % = (1 - A5 min A0 min ) × 100 (1)

2.8.5. Bile salt tolerance

To evaluate bile salt tolerance, as in the previous stage, an 18-hour pre-culture was prepared separately in LB broth in terms of sterile conditions. For the experiment, the falcons containing 10 ml of LB broth (for preparation of control samples) and the falcons containing 10 ml of LB broth with a concentration of 0.3% Oxgall were prepared. The desired colony was removed from the plate and cultured in all media. The falcons were incubated in a shaker incubator at 37 °C and 180 rpm for 6 hrs. The turbidity of the incubated environments under these conditions was then read every 15 min with a spectrophotometer at a wavelength of 600 nm to delay the growth generated with Oxgall for each strain compared to the control sample for the strain until reaching a turbidity of 0.3 was determined (Gilliland et al., 1980GILLILAND, S., BRUCE, B., BUSH, L. and STALEY, T., 1980. Comparisons of two strains of Lactobacillus acidophilus as dietary adjuncts for young calves. Journal of Dairy Science, vol. 63, no. 6, pp. 964-972. http://dx.doi.org/10.3168/jds.S0022-0302(80)83033-5.
http://dx.doi.org/10.3168/jds.S0022-0302...
).

2.8.6. Resistance to acid

To evaluate resistance to acid, we employed the pre-cultures on LB broth prepared for overnight, after homogenizing them by vortex, 1 ml aliquots of each active culture was poured into falcons containing LB culture medium with HCL acid 37% (Merck Millipore Company, Germany, Cat No: 100317) was reached to pHs 2, 2.5, and 3 using pH Meter device (Beckman Company, Model of 72, USA). Next, the inoculated media were incubated in a shaker incubator at 37 °C for 3-6 hrs. After the first three hours, the10-μl samples (two samples from the vortex falcons) were cultured as spots in LB agar (two replicates from each medium) and the inoculated media were incubated at 37 °C for 24 hrs. All the experiments were repeated three times (Singhal et al., 2010SINGHAL, K., JOSHI, H. and CHAUDHARY, B., 2010. Bile and acid tolerance ability of probiotic Lactobacillus strains. Journal of Global Pharma Technology, vol. 2, no. 12, pp. 17-25.).

2.8.7. Growth rate of acid resistant strains at acidic pH

For evaluation, as in the previous stage, an 18-hour pre-culture was prepared separately in LB broth under sterile conditions. Then, liquid LB medium with pHs of 2, 2.5 and 3 were prepared and distributed in the sterile falcons, then 10 μl of each sample were inoculated in the falcons. Afterward, the falcons incubated in a shaker incubator at 37 °C and 180 rpm for 4 hours. The turbidity of all the samples was read at 600 nm with a spectrophotometer set.

2.8.8. Antimicrobial assessment

To measure the antibacterial activity of the Bacillus isolates, 18-hour pre-cultures on LB broth (Merck Company, Germany) were applied. From all of 8 strains in separate falcons and in a shaker incubator (GFL Company, model 3031, India) was prepared at 37 °C. Then, it was centrifuged at 3000 rpm using refrigerated centrifuge (Kendro Company, D37520 Model, United Kingdom) and 20 μl of supernatant containing possible bacteriocin of all 8 Bacillus licheniformis strains was cultured in LB Agar plate wells and purified of any bacterium. Afterwards, a colony was removed from the each plate of purchased pathogenic bacteria (the National Center of Genetic and Biological Resources, Iran) including Bacillus cereus (CCM2010, DSMZ31, ATCC14579, LMG6923, NCBI9373 and NCTC2599), Escherichia coli (ATCC11775, DSM30083, CCM 5172), Klebsiella pneumonia subsp pneumonia (ATTCC700603, CCUG45421 and LMG20218), Salmonella Enteritidis (ATCC1307603, DMS 17420, LMG 10395) and Proteus vulgaris (ATCC25933, CCUG34294) and cultured through pour plate. At this stage, four wells were installed in each plate and the ends of the wells were blocked with 10 μl of molten agar. The medium with inoculated wells were incubated at 37 °C for 24 hrs. The above experiment was repeated once more. In addition, the pre-cultured strains were centrifuged at 3000 rpm after 18 hrs of incubation and the supernatant was utilized for loading in the wells. Inhibitory zone of the strains were checked after 24 hrs of incubation at 37 °C (Nourouzi et al., 2004NOUROUZI, J., MIRZAII, M. and NOROUZI, M. (2004). Study of Lactobacillus as probiotic bacteria. London, UK: Intech Publication.).

2.8.9. Phylogenetic analysis of 16S rRNA gene

First, we sent PCR product to Gen Fanavaran Company (Tehran, Iran) for sequencing using Sanger sequencing method. In sequencing, the primers concentration was 10 pmol and the amount of primers was 5 μmol per reading. The evolutionary history was inferred using the Neighbor-Joining method (Saitou and Nei, 1987SAITOU, N. and NEI, M.T., 1987. he neighbor-joining method: a new method for reconstructing phylogenetic trees. Molecular Biology and Evolution, vol. 4, no. 4, pp. 406-425. PMid:3447015.). The optimal tree is shown. The tree is drawn to scale, with branch lengths in the same units as those of the evolutionary distances used to infer the phylogenetic tree. The evolutionary distances were computed using the Maximum Composite Likelihood method (Tamura et al., 2004TAMURA, K., NEI, M. and KUMAR, S., 2004. Prospects for inferring very large phylogenies by using the neighbor-joining method. Proceedings of the National Academy of Sciences of the United States of America, vol. 101, no. 30, pp. 11030-11035. http://dx.doi.org/10.1073/pnas.0404206101. PMid:15258291.
http://dx.doi.org/10.1073/pnas.040420610...
) and are in the units of the number of base substitutions per site. This analysis involved 8 nucleotide sequences. All ambiguous positions were removed for each sequence pair (pairwise deletion option). There were a total of 1392 positions in the final dataset. Evolutionary analyses were conducted in MEGA11 (Tamura et al., 2021TAMURA, K., STECHER, G. and KUMAR, S., 2021. MEGA11: molecular evolutionary genetics analysis version 11. Molecular Biology and Evolution, vol. 38, no. 7, pp. 3022-3027. http://dx.doi.org/10.1093/molbev/msab120. PMid:33892491.
http://dx.doi.org/10.1093/molbev/msab120...
; Stecher et al., 2020STECHER, G., TAMURA, K. and KUMAR, S., 2020. Molecular evolutionary genetics analysis (MEGA) for macOS. Molecular Biology and Evolution, vol. 37, no. 4, pp. 1237-1239. http://dx.doi.org/10.1093/molbev/msz312. PMid:31904846.
http://dx.doi.org/10.1093/molbev/msz312...
; Sabir et al., 2021).SABIR, S.M., ZEB, A., MAHMOOD, M., ABBAS, S.R., AHMAD, Z. and IQBAL, N., 2021. Phytochemical analysis and biological activities of ethanolic extract of Curcuma longa rhizome. Brazilian Journal of Biology = Revista Brasileira de Biologia, vol. 81, no. 3, pp. 737-740. http://dx.doi.org/10.1590/1519-6984.230628. PMid:32965334.
http://dx.doi.org/10.1590/1519-6984.2306...

2.8.10. Statistical analysis

SPSS software Ver-20 was used to perform statistical analysis. The data are expressed as a mean ± standard deviation (SD) calculated over three independent experiments performed in triplicate. Analysis of statistical significance was carried out via two-way ANOVA (at a significance level of P<0.05) with t-test.

3. Results

3.1. Identification of keratin degrading bacteria

After culturing 42 isolates on culture medium containing 1.5% keratin, 8 strains formed a clear zone around their colonies after 72 hrs (Figure 1), revealing the production of the enzyme keratinase to degrade and use the only carbon source of the culture medium (keratin). There were no clear zones around the other strains; thus, they were not keratinolytic and were excluded from the study at this stage. According to the results, the isolates pvkr6, pvkr8, pvkr9, and pvkr41 produced the highest amount of clear zone during 72 hrs of incubation at 37 °C. As a result, these strains were classified as strong keratin-degrading bacteria, followed by the pvkr15 and pvkr26 strains, which produced a clear zone with a smaller diameter than the mentioned strains and were classified as moderately potent degrading bacteria. Finally, strains pvkr1 and pvkr10 produced the lowest levels of the keratinase enzyme and a clear zone with a very small diameter. These strains were classified as weak in terms of keratin degrading activity.

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.

3.2. Biochemical identification of bacteria strains

The bacterial strains were isolated from soil designed as pvkr1 to pvkr41. The results of biochemical experiments of Bacillus spp isolates are shown in Table 1. All of 10 isolates (8 isolates and B. L1525 and B. L1595 as negative and positive control, respectively) were gram-positive, positive catalase, gelatinase, and α-amylase as positive reaction and were identified as Bacillus licheniformis. The microscopic shapes of Bacillus spp are depicted in Figure 2. In addition the B. L1525 and B. L1595 were used as standard Bacillus lichenifomis strain and positive control (purchased from National Center of Genetic and Biological Resources, Iran).

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

3.3. Biomolecular identification of isolated bacteria

Indicating the Bacillus detection, 16S rDNA sequence analysis confirmed the biochemical results and PCR was performed on randomly selected colonies of keratinolytic bacteria (8 isolated strains). The PCR results demonstrated that several colonies were positive for Bacillus (Figure 3). All the 8 isolates were different strains of Bacillus licheniformis and were recorded in ENA as represented in supplemental section.

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).

3.4. Keratinase activity

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

Figure 4
Keratinase activity of Bacillus spp before sedimentation of keratin enzyme.
Figure 5
Effect of enzyme concentration on keratinase activity in the isolated strains.

Following the enzyme deposition, keratinase activity levels of the isolated pvkr15 strain and B. L1595 as positive control were also measured. As shown Figure 6, both of the strains produced keratinase and keratinase activity of the pvkr15 strain (160.36±0.85 U/ml) was more than that of B. L1595 (92.34±0.94 U/ml). Moreover, the effect of enzyme concentration on keratinase activity in the selected strains (Figure 7) showed that keratinase activity of the pvkr15 strain was recorded at 0.119 M (specific activity 45.73±3.9 and total activity 481.07±2.5 U/ml).

Figure 6
Keratinase activity of the selected strains after sedimentation of keratin enzyme.
Figure 7
The effect of enzyme concentration on keratinase activity in the selected strains.

3.5. Hemolytic activity

Hemolytic activity of the Bacillus strains was determined using blood agar medium, supplemented with defibrinated blood. After 96 hrs of incubation at 37 °C, the strains of pvkr1, pvkr8, and pvkr10 produced α-hemolysis and the pvkr6, pvkr9, pvkr26, and pvkr41 strains demonstrated α-hemolysis weakly while the pvkr15 strain showed no hemolytic activity during 96 hrs (Figure 8); thus, they are γ-hemolytic strains. Therefore, one strain (pvkr15) was selected as γ-hemolytic with the probiotic potential.

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.

3.6. Antibiotic susceptibility of the Bacillus licheniformis strains

Selected Bacillus strains displayed various levels of antibiotic resistance, ranging from resistance to susceptibility (Table 2). It was found that all the strains were sensitive to streptomycin, gentamycin, amoxicillin, tetracycline, rifampin, vancomycin, erythromycin, and cefixime. The highest resistance belonged to penicillin G and the only strains sensitive to penicillin G were pvkr8 and pvkr9.

Table 2
Antibiotic susceptibility.

3.7. Antagonistic properties of the Bacillus licheniformis strains against pathogenic bacteria

According to the data obtained from this experiment, none of the isolated strains had an inhibitory effect on bacterial pathogens, including Bacillus cereus, Escherichia coli, Klebsiella pneumoniae while pvkr8, pvkr10 and both control groups (Bacillus licheniformis 1525 and Bacillus licheniformis 1595) had an inhibitory effect on the growth of Salmonella enterica. Additionally, pvkr1, pvkr6, pvkr8, pvkr9, pvkr10, pvkr15, and pvkr26 inhibited the growth of Proteus mirabilis. In other words, these strains produced bacteriocin against the mentioned pathogenic bacteria and stopped their growth (Figure 9).

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

3.8. Hydrophobicity

The adhesion of Bacillus strains to xylene are represented in supplemental section (Figure 10), showing the most adhesive bacteria to pvkr8 and pvkr15 with a 22.44% and 19.58% range of hydrophobicity percentage, respectively. In addition, the relative range of hydrophobicity was 3.27 to 30.57. The highest level of hydrophobicity belonged to B. licheniformis PTCC 1595 that was 30.57%.

Figure 10
Hydrophobicity percentage of the isolated strains.

3.9. Biofilm formation with the Bacillus licheniformis strains

Given the importance of the ability of probiotic bacteria to form biofilms to maintain survival, herein, the ability to form biofilms was examined for each of the strains. 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).

Figure 11
Biofilm formation in the isolated strains. The right tube contains one of the isolated strains that has formed biofilm and the left tube is a culture medium

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.

Table 3
Resistance of the isolated Bacillus licheniformis to bile salts.

Additionally, the results of resistance to low pH (Figure 12) demonstrated the viability of Bacillus licheniformis strains. The pvkr9 strain showed slow growth at pH 2 whereas the growth continued at pH 2.5 and 3 for 3 hours. In the pvkr26 strain, we observed weak growth at pH 2 and 2.5 for only the first 2 hours, but its growth continued at pH 3 for 3 hours. The growth of pvkr8 strain also stopped completely at different pH. The other strains had weak and slow growth compared to the control samples (growth of strains in the medium with neutral pH [7.2]). Moreover, the results of pH effect on biofilms showed that all the strains survived the acidic conditions and were able to grow on the LBB agar.

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

3.11. Phylogenetic analysis of 16S rRNA gene

The evolutionary history was inferred through the neighbor-Joining method. The analysis involved eight nucleotide sequences. As can be seen in the Figure 13, pvkr1 and pvkr9 strains have common ancestors 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).

Figure 13
Phylogenic tree illustrates the evolutionary relationships between different 16S rRNA nucleotide sequences of Bacillus licheniformis.

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 keratin waste in the nature (Qiu et al., 2020QIU, J., WILKENS, C., BARRETT, K. and MEYER, A.S., 2020. Microbial enzymes catalyzing keratin degradation: Classification, structure, function. Biotechnology Advances, vol. 44, pp. 107607. http://dx.doi.org/10.1016/j.biotechadv.2020.107607. PMid:32768519.
http://dx.doi.org/10.1016/j.biotechadv.2...
). It has been suggested to employ their keratin degradation potential in the livestock and poultry feed industry. There has also been a lot of research on the isolation of spore-shaped Bacillus strains with probiotic properties and the results reveal that many Bacillus strains have significant probiotic properties and can have very beneficial effects on the host body (Hassan et al., 2013HASSAN, M.A., HAROUN, B.M., AMARA, A.A. and SEROUR, E.A., 2013. Production and characterization of keratinolytic protease from new wool-degrading Bacillus species isolated from Egyptian ecosystem. BioMed Research International, vol. 2013, pp. 175012. http://dx.doi.org/10.1155/2013/175012. PMid:23936776.
http://dx.doi.org/10.1155/2013/175012...
). However, none of the reports examined the simultaneous evaluation of probiotic properties and the ability of these bacteria to degrade keratin. In this experiment, with modified methods and materials, our objective was to isolate Bacillus bacteria with probiotic properties and the ability to degrade keratin. Herein, the keratinase-producing bacteria isolated from poultry farm soil identified Bacillus licheniformis. In the present work, 42 strains were isolated on suspended soil samples after heat and alcohol treatment. All the isolates were selected and screened to isolate Bacillus strains with the selective method. Among all the isolated strains, only eight Bacillus strains were able to degrade keratin. The keratinolytic activity of the strains was between 48.91-124.08 U/ml and the highest keratinolytic activity belonged to Bacillus licheniformis as pvkr6, pvkr15, and pvkr41 strains with 124.08, 101.1, and 100.18 U/ml, respectively. Additionally, the highest keratinase activity was recorded at an enzyme concentration of 0.07 M, which belonged the pvkr6 with specific activity 54.83 U/ml and 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. In our paper, all of the eight Bacillus keratinolytic strains produced protease enzyme and the strain isolated from poultry soil revealed that the bacteria producing keratinase enzyme belonged to Bacillus licheniformis. Mohamed et al. reported that B. amyloliquefaciens MA20 and B. subtilis have the keratinolytic ability of wool-degrading bacillus species and the keratinolytic activity of these strains were in the range of 0.814-0.922 U/ml (Hassan et al., 2013HASSAN, M.A., HAROUN, B.M., AMARA, A.A. and SEROUR, E.A., 2013. Production and characterization of keratinolytic protease from new wool-degrading Bacillus species isolated from Egyptian ecosystem. BioMed Research International, vol. 2013, pp. 175012. http://dx.doi.org/10.1155/2013/175012. PMid:23936776.
http://dx.doi.org/10.1155/2013/175012...
). Mukesh Kumer et al. reported that the maximum keratinase 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., 2012KUMAR, D.M., PRIYA, P., BALASUNDARI, S.N., DEVI, G., REBECCA, A.I.N. and KALAICHELVAN, P., 2012. Production and optimization of feather protein hydrolysate from Bacillus sp. MPTK6 and its antioxidant potential. Middle East Journal of Scientific Research, vol. 11, no. 7, pp. 900-907.). Another study by Deivasigamani and Alagappan (2008)DEIVASIGAMANI, B. and ALAGAPPAN, K., 2008. Industrial application of keratinase and soluble proteins from feather keratins. Journal of Environmental Biology, vol. 29, no. 6, pp. 933-936. PMid:19297995. 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., 1995BÖCKLE, B., GALUNSKY, B. and MÜLLER, R., 1995. Characterization of a keratinolytic serine proteinase from Streptomyces pactum DSM 40530. Applied and Environmental Microbiology, vol. 61, no. 10, pp. 3705-3710. http://dx.doi.org/10.1128/aem.61.10.3705-3710.1995. PMid:7487006.
http://dx.doi.org/10.1128/aem.61.10.3705...
), and some bacteria, including Streptomyces pactum (Seyfi, 2017SEYFI, R., 2017. Application of artificial neural network in modeling separation of microalgae. UJRSET, vol. 5, no. 4, pp. 43-49.), Bacillus licheniformis PWD-1 (Williams et al., 1990WILLIAMS, C.M., RICHTER, C., MACKENZIE JUNIOR, J. and SHIH, J.C., 1990. Isolation, identification, and characterization of a feather-degrading bacterium. Applied and Environmental Microbiology, vol. 56, no. 6, pp. 1509-1515. http://dx.doi.org/10.1128/aem.56.6.1509-1515.1990. PMid:16348199.
http://dx.doi.org/10.1128/aem.56.6.1509-...
), Chryseobacterium sp.Kr6 and Streptomyces fradiae (Riffel et al., 2003RIFFEL, A., LUCAS, F., HEEB, P. and BRANDELLI, A., 2003. Characterization of a new keratinolytic bacterium that completely degrades native feather keratin. Archives of Microbiology, vol. 179, no. 4, pp. 258-265. http://dx.doi.org/10.1007/s00203-003-0525-8. PMid:12677362.
http://dx.doi.org/10.1007/s00203-003-052...
), Bacillus halodurans (Takami et al., 1992TAKAMI, H., NAKAMURA, S., AONO, R. and HORIKOSHI, K., 1992. Degradation of human hair by a thermostable alkaline protease from alkaliphilic Bacillus sp. no. AH-101. Bioscience, Biotechnology, and Biochemistry, vol. 56, no. 10, pp. 1667-1669. http://dx.doi.org/10.1271/bbb.56.1667.
http://dx.doi.org/10.1271/bbb.56.1667...
and 1999TAKAMI, H., NOGI, Y. and HORIKOSHI, K., 1999. Reidentification of the keratinase-producing facultatively alkaliphilic Bacillus sp. AH-101 as Bacillus halodurans. Extremophiles, vol. 3, no. 4, pp. 293-296. http://dx.doi.org/10.1007/s007920050130. PMid:10591021.
http://dx.doi.org/10.1007/s007920050130...
), Fervidobacterium species (Friedrich and Antranikian, 1996FRIEDRICH, A.B. and ANTRANIKIAN, G., 1996. Keratin degradation by Fervidobacterium pennavorans, a novel thermophilic anaerobic species of the order Thermotogales. Applied and Environmental Microbiology, vol. 62, no. 8, pp. 2875-2882. http://dx.doi.org/10.1128/aem.62.8.2875-2882.1996. PMid:16535379.
http://dx.doi.org/10.1128/aem.62.8.2875-...
), and some dermatophyte fungi (55), which secrete the enzyme keratinase. In the current research also the strains isolated from poultry soil were keratinase-producing bacteria of the Bacillus licheniformis strain.

Kunert (1973)KUNERT, J., 1973. Keratin decomposition by dermatophytes. I. Sulfite production as a possible way of substrate denaturation. Zeitschrift für Allgemeine Mikrobiologie, vol. 13, no. 6, pp. 489-498. http://dx.doi.org/10.1002/jobm.19730130606. PMid:4748158.
http://dx.doi.org/10.1002/jobm.197301306...
reported that the isolated keratinolytic bacteria from feather with probiotic activities belonged to Burkholderia, Pseudomonas, Chryseobacterium, and Microbacterium and were selected and screened on skim milk agar plates for proteolytic activity. Studies have implied that these bacteria could grow on a variety of extract of keratin, such as chicken nail hair and wool, in which all the strains have been reported as keratinolytic protease (Wakil et al., 2011WAKIL, S., DADA, M. and ONILUDE, A., 2011. Isolation and characterization of keratinase-producing bacteria from poultry waste. Journal of Pure & Applied Microbiology, vol. 5, no. 2, pp. 567-580.). This study focused on keratinolytic protease production from Bacillus strain. The proteolytic activities of all the isolates were detected on the LB agar medium with 1.5% (w/v) skim milk. Among the isolates analyzed, eight isolates showed proteolytic activity. Herein, we investigated the keratinolytic Bacillus strains with probiotic potential for antimicrobial susceptibility testing. One of the selection factors of probiotic strains was antibiotic susceptibility of isolated strains (Gueimonde et al., 2013GUEIMONDE, M., SÁNCHEZ, B., DE LOS REYES-GAVILÁN, C.G. and MARGOLLES, A., 2013. Antibiotic resistance in probiotic bacteria. Frontiers in Microbiology, vol. 4, pp. 202. http://dx.doi.org/10.3389/fmicb.2013.00202. PMid:23882264.
http://dx.doi.org/10.3389/fmicb.2013.002...
). Therefore, the susceptibility of the strains to antibiotics was measured. The pattern of antibiotic susceptibility has revealed that all the strains were sensitive to the majority of antibiotics except penicillin G. One of the important features of probiotic bacteria is their ability to form biofilm (Salas-Jara et al., 2016SALAS-JARA, M.J., ILABACA, A., VEGA, M. and GARCÍA, A., 2016. Biofilm forming Lactobacillus: new challenges for the development of probiotics. Microorganisms, vol. 4, no. 3, pp. 35. http://dx.doi.org/10.3390/microorganisms4030035. PMid:27681929.
http://dx.doi.org/10.3390/microorganisms...
). Biofilm formation is one of the central mechanisms in insuring bacterial survival in harsh environments (Zhao et al., 2017ZHAO, X., ZHAO, F., WANG, J. and ZHONG, N., 2017. Biofilm formation and control strategies of foodborne pathogens: food safety perspectives. RSC Advances, vol. 7, no. 58, pp. 36670-36683. http://dx.doi.org/10.1039/C7RA02497E.
http://dx.doi.org/10.1039/C7RA02497E...
). Comparing with free-cells or planktonic cell, the packed bacterial presence in biofilms 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 gut, probiotic bacteria must have the ability to attach to the cells lining the inner surface of the gut and form a biofilm there. Consequently, the strains with a higher percentage of surface hydrophobicity also had a greater ability to bind to 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 pvkr15 inhibited the growth of different types of bacteria, such as Salmonella Enterica, and proteus mirabilis. Acid and bile tolerance in intestinal environment is important for effective functions in the intestines (Kim et al., 2018KIM, M., NAM, D.G., KIM, S.B., IM, P., CHOE, J.S. and CHOI, A.J., 2018. Enhancement of viability, acid, and bile tolerance and accelerated stability in lyophilized Weissella cibaria JW 15 with protective agents. Food Science & Nutrition, vol. 6, no. 7, pp. 1904-1913. http://dx.doi.org/10.1002/fsn3.762. PMid:30349680.
http://dx.doi.org/10.1002/fsn3.762...
). This study demonstrated that the high level of survival under acidic conditions belonged to all the strains except pvkr26. Bile salts have been shown to inhibit bacterial growth by disordering cell membranes. Some Bacillus spps are sensitive or weakly tolerant to bile salt concentrations (Zulkhairi Amin et al., 2019ZULKHAIRI AMIN, F.A., SABRI, S., ISMAIL, M., CHAN, K.W., ISMAIL, N., MOHD ESA, N., MOHD LILA, M.A. and ZAWAWI, N., 2019. Probiotic properties of Bacillus strains isolated from stingless bee (Heterotrigona itama) honey collected across Malaysia. International Journal of Environmental Research and Public Health, vol. 17, no. 1, pp. 278. http://dx.doi.org/10.3390/ijerph17010278. PMid:31906055.
http://dx.doi.org/10.3390/ijerph17010278...
). Although, in our study, the survival rates of Bacillus pvkr9, pvkr15 strains in 0.3% bile salt were higher than of those of other strains. This finding implied that pvkr9 and pvkr15 are resistant to bile salts in the intestines and could pass through this area and have probiotic effects. According to a study by Hironimus et al., the Bacillus Laevolacticus, Bacillus racemilacticus, and Bacillus coagulans strains were resistant to pHs 2, 2.5, and 3, and Bacillus racemilacticus and Bacillus coagulans strains were resistant to bile salts with 0.3% concentration (Hyronimus et al., 2000HYRONIMUS, B., LE MARREC, C., HADJ SASSI, A. and DESCHAMPS, A., 2000. Acid and bile tolerance of spore-forming lactic acid bacteria. International Journal of Food Microbiology, vol. 61, no. 2-3, pp. 193-197. http://dx.doi.org/10.1016/S0168-1605(00)00366-4. PMid:11078170.
http://dx.doi.org/10.1016/S0168-1605(00)...
). As described, the results of these researchers are consistent with the results of the present study. The probiotic bacteria should not lyse red blood cells of the host. Our experiments showed that only the pvkr15 strain could be classified as probiotic and all the other stains caused the lyses of red blood cells. Hemolytic 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

The results revealed that only the 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 industry. Other strains, despite having some probiotic characteristics, showed fundamental disadvantages, disqualifying them from being used for this purpose.

Acknowledgements

The authors acknowledge the Microbiology Department, Ayatollah Amoli Branch, Islamic Azad University And about the name of the last author acknowledge the Biology Department, Ayatollah Amoli Branch, Islamic Azad University.

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Publication Dates

  • Publication in this collection
    09 May 2022
  • Date of issue
    2024

History

  • Received
    19 Oct 2021
  • Accepted
    08 Mar 2022
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