Identification and Characterization of Bacillus megaterium as Probiotic Bacteria in Poultry Feed
Keywords:
gastrointestinal resistance, in vitro properties, probioticAbstract
The present study was planned to identify the Bacillus probiotic characteristics from a strain knows as Bacillus spp. in
with the aim of use as a probiotic candidate in broiler chickens feed. The strain was identified phenotypically and
evaluated for their viability through total plate count method, colony morphology, catalase test, hemolysis activity, pH
(2 and 3) and bile salts (0.3% w/v) tolerance under simulated gastrointestinal tract (GIT) conditions, rate of
survivability (%), spores resistance at high temperature and antibiotics susceptibility tests. Registered under the code
IBNA 66, the strain was identified by API 50CHB (ID 99.9%) and ABIS online (91.8% similarity) as Bacillus
megaterium. The strain presented a viable count of 3 x 1011 CFU/mL after 22±2h of incubation at 37°C, 120 rpm with
positive catalase, and non-hemolytic activity results. Also, Bacillus megaterium showed a significant resistance and
survivability at pH 2 (P <0.0001, 62.09%) and pH 3 (P <0.0001, 77.53%), bile salts (P <0.0001, 84.27%), with a high
ability to produce spores (after 120 min. at 80°C show 9.47 Log CFU/mL, P <0.0001). The antibiotic susceptibility
test showed 100.00% resistance of strain to oxacillin (1 μg/mL), with multiple antibiotic resistance indices above 0.5.
In conclusion, Bacillus megaterium can be an ideal probiotic candidate that can potentially be formulated and applied
in the poultry feed for improving performance and modulated GIT microflora.
References
Vazquez, A. P. Bacillus species are superior
probiotic Feed-Additives for Poultry. Journal of
Bacteriology & Mycology: Open Access, 2016, 2(3).
doi:10.15406/jbmoa.2016.02.00023.
Jadhav, K., Sharma, K. S., Katoch, S., Sharma, V. K.
and Mane, B. G. Probiotics in broiler poultry feeds: A
review. J. Anim. Nutr. Physiol, 2015, 1, pp. 4-16.
Chaucheyras-Durand, F., Durand, H. Probiotics in
animal nutrition and health. Benef Microbes. 2010, 1(1),
pp. 3-9. doi: 10.3920/BM2008.1002. PMID: 21840795.
Ciurescu, C., Dumitru, M., Gheorghe, A., Untea, A.
E. Effect of Bacillus subtilis on growth performance,
bone mineralization, and bacterial population of broilers
fed with different protein sources. Poultry Science,
, 99(11), pp. 5960-5971.
Al-Thubiani, A. S. A., Maher, Y. A., Fathi, A.,
Abourehab, M. A. S., Alarjah, M., Khan, M. S. A., AlGhamdi, S. B. Identification and characterization of a
novel antimicrobial peptide compound produced by
Bacillus megaterium strain isolated from oral
microflora. Saudi Pharmaceutical Journal, 2018, 26(8),
pp. 1089–1097. doi:10.1016/j.jsps.2018.05.019.
Dumitru, G., Ciurescu, G., Sorescu, I. In vitro
evaluation of some probiotic properties of Lactobacillus
strains isolated from chickens’ gut. Scientific Papers:
Animal Science and Biotechnologies, 2020, 53 (1), pp.
-51.
EFSA. Guidance on the assessment of the toxigenic
potential of Bacillus species used in animal nutrition.
EFSA Journal, 2014, 12, 3665.
EFSA. Introduction of a Qualified Presumption of
Safety (QPS) approach for assessment of selected
microorganisms referred to EFSA. EFSA Journal, 2007,
, pp. 1–16.
Ciurescu, C., Dumitru, M., Sorescu, I. Effect of
chickpea and probiotics on broiler chicks’ performance
and gut microflora populations. Scientific Papers:
Animal Science and Biotechnologies, 2020, 53 (2), pp.
-7.
Flores, C. A., T., Duong, N., Augspurger, Lee., J. T.
Efficacy of Bacillus subtilis administered as a direct-fed
microorganism in comparison to an antibiotic growth
promoter and in diets with low and high DDGS inclusion
levels in broiler chickens. J. Appl. Poult. Res., 2019, 28,
pp. 902–911.
Barbosa, T. M., Serra, C. R., La Ragione R. M,
Woodward, M J., Henriques, A. O. Screening for
Bacillusisolates in the broiler gastrointestinal tract. Appl
Environ Microbiol., 2005, 71(2), pp. 968-78. doi:
1128/AEM.71.2.968-978.2005. PMID: 15691955;
PMCID: PMC546680.
Nguyen, A. T. V., Nguyen, D. V., Tran, M. T.,
Nguyen, L. T., Nguyen, A. H., Phan, T. N. Isolation and
characterization of Bacillus subtilis CH16 strain from
chicken gastrointestinal tracts for use as a feed
supplement to promote weight gain in broilers. Letter in
Applied Microbiology, 2015, 60, pp. 580-588.
Holt, J. G., Krieg, N. R., Sneath, P. H. A., Staley, J.
T., William S. T. Bergey's Manual of Determinative
Bacteriology Williams and Walkins, Baltimore, 1994.
Stoica, C., Sorescu, I. ABIS online – Advanced
Bacterial Identification Software, an original tool for
phenotypic bacterial identification. Regnum
Prokaryotae, 2017, www.tgw1916.net.
Dumitru, M., Sorescu, I., Hăbeanu, M., Tabuc, C.,
Idriceanu, L., Jurcoane, Ș. Preliminary characterisation
of Bacillus subtilis strain use as dietary probiotic bioadditive in weaning piglet. J. Food and Feed Research,
, 45 (2), pp. 203-211.
Sorescu, I., Dumitru, M., Ciurescu, G. Lactobacillus
spp. and Enterococcus faecium strains isolation,
identification, preservation and quantitative
determinations from turkey gut content. Rom.
Biotechnol. Lett., 2019, 24(1), pp. 41-49.
Lee, J., Park, I., Choi, Y., Cho, J. Bacillus strain as
feed additive: in vitro evaluation of its potential
probiotic properties. Rev. Colombiana de Ciencias
Pecuarias, 2012, 25, pp. 577‒585.
Dumitru, M., Hăbeanu, M., Tabuc, C., Jurcoane, Ș.
Preliminary characterization of the probiotic properties
of a bacterial strain for used in monogastric nutrition.
Bulletin of the University of Agricultural Sciences and
Veterinary Medicine, Animal Science and
Biotechnologies, 2019, 76(2), pp. 102-108.
Ritter, A. C., Paula, A., Correa, F., Veras, F. F.,
Brandelli, A. Characterization of Bacillus subtilis
available as probiotics. Journal of Microbiology
Research, 2018, 8(2), pp. 23–32. https://
doi.org/10.5923/j.microbiology.20180802.01.
Chaiyawan, N., Taveeteptaikul, P., Wannissorn, B.
Characterization and probiotic properties of Bacillus
strains isolated from broiler. Thai J. Vet. Med., 2010,
(2), pp. 207–214.
Zulkhairi Amin, F. A., Sabri, S., Ismail, M., Chan,
K. W., Ismail, N., Mohd Esa, N., Zawawi, N. Probiotic
properties of Bacillus strains isolated from Stingless Bee
(Heterotrigona itama) Honey collected across Malaysia.
International Journal of Environmental Research and
Public Health, 2019, 17(1), pp. 278.
doi:10.3390/ijerph17010278
Ramlucken, U., Lalloo, R., Roets, Y., Moonsamy,
G., van Rensburg, C. J., Thantsha, M. S. Advantages of
Bacillus-based probiotics in poultry production.
Livestock Science, 2020, 241 (104215), pp. 1-15.
doi:10.1016/j.livsci.2020.104215.
Nemutanzhela, M. E., Roets, Y., Gardiner, N.,
Lalloo, R. The use and benefits of Bacillus based
biological agents in aquaculture. Sustainable
aquaculture techniques. IntechOpen. Rijeka, Croatia,
UK. 33, 2014.
Kavitha, M., Raja, M., Perumal, P. Evaluation of
probiotic potential of Bacillus spp. isolated from the
digestive tract of freshwater fish Labeo calbasu
(Hamilton, 1822). Aquaculture Reports, 11, pp. 59–69.
doi:10.1016/j.aqrep.2018.07.001.
Ramluckena, U., Lallooa, R., Roetsa, Y.,
Moonsamya, G., Jansen van Rensburg, C., Thantsha,
M.S. Advantages of Bacillus-based probiotics in poultry
production. Livestock Science, 2020, 241, pp. 1-15.
https://doi.org/10.1016/j.livsci.2020.104215.
Nithya, V., Halami, P. M. Evaluation of the probiotic
characteristics of Bacillus species isolated from different
food sources. Ann. Microbiol., 2013, 63, pp. 129-137.
Fontana, L., Bermudez-Brito, M., Plaza-Diaz, J.,
Munoz-Quezada, S., Gil, A. Sources, isolation,
characterisation and evaluation of probiotics. British
Journal of nutrition, 2013, 109(S2), pp. S35-S50.
Sharma, K., Sharma, N., Sharma, R. Identification
and evaluation of in vitro probiotic attributes of novel
and potential strains of lactic acid bacteria isolated from
traditional dairy products of north-west himalayas.
Journal of Clinical Microbiology and Biochemical Clin
Microbiol Biochem Technol 2(1), pp. 018-025.
doi:10.17352/jcmbt.000011.
Mingmongkolchai, S., Panbangred, W. In vitro
evaluation of candidate Bacillus spp. for animal feed.
The Journal of General and Applied Microbiology,
, 63(2), pp. 147–156.
doi:10.2323/jgam.2016.09.002.
Setlow, P. Spores of Bacillus subtilis: their resistance
to and killing by radiation, heat and chemicals. Journal of Applied Microbiology, 2006, 101(3), pp. 514–525.
doi:10.1111/j.1365-2672.2005.02736.x.
Grant, A., Gay, C. G., Lillehoj, H. S. Bacillus spp. as
direct-fed microbial antibiotic alternatives to enhance
growth, immunity, and gut health in poultry. Avian
Pathology, 2018, 47(4), pp. 339–351.
doi:10.1080/03079457.2018.1464117.
Galarza-Seeber, R., Latorre, J. D., HernandezVelasco, X., Wolfenden, A. D., Bielke, L. R., Menconi,
A., Tellez, G. Isolation, screening and identification of
Bacillus spp. as direct-fed microbial candidates for
aflatoxin B1 biodegradation. Asian Pacific Journal of
Tropical Biomedicine, 2015, 5(9), pp. 702-706.
Dumitru, M, Habeanu, M, Lefter, N. A., Gheorghe,
A. The effect of Bacillus licheniformis as direct-fed
microbial product on growth performance,
gastrointestinal disorders and microflora population in
weaning piglets. Rom Biotechnol Lett. 2020, 25(6), pp.
-2069. doi: 10.25083/rbl/25.6/2060.2069.
Knarreborg, A., Brockmann, E., Høybye, K., Knap,
I., Lund, B., Milora, N., Leser, T.D. Bacillus subtilis
(DSM17299) modulates the ileal microbial communities
and improves growth performance in broilers. Int. J.
Prebiotic Probiotic, (2008), 3, pp. 83-88.
Glasscock, J. M. Evaluation of different probiotic
strains supplemented in commercial broiler rations and
their influences on performance, yield, and intestinal
microbiota. Electronic theses and dissertations, 2017,
