Biological Optimization and Characterization of Phytase from New Local Isolate Bacillus megaterium EBD 9-1 as Poultry Feed Additive

Abstract

This study was carried out to determine the efficiency of various biological wastes, nutrients, some metal ions, temperature, pH on thermostable phytase production. The new isolate Bacillus megaterium EBD 9-1 phytase was used to study the biochemical characteristics. The phytase activity increased markedly at the late stationary and the highest phytase activity after the fourth day of culture. A new growth medium was developed, resulting in a 2.34-fold increase in enzyme production. The crude phytase was optimally active at pH 5.5-7.0 at 37^oC and retained its activity at temperatures up to 80^oC. The enzyme exhibited thermostability, retaining ~50% activity at 70^oC and were fairly stable up to pH 10. KCl was determined as the best metal ion (1437 FTU/mL), but in the studies conducted, enzyme production in 1% Wheat bran and 0.5% Na-Phytate medium was 1615 FTU/mL and 2989 FTU/mL in the presence of KCl and CaCl₂, respectively. Large-scale production and lyophilization of the phytase were performed. It remained stable in lyophilized form at +4^oC for 1 year. Lyophilized phytase was optimally active. Its effectiveness in the presence of feed additives was also compared with the commercial enzyme at extreme conditions such as high temperature, pH and long incubation time and it was retained its activity and also found to be more efficient than the commercial enzymes. These properties indicated that the B. megaterium EBD 9-1 phytase appeared to be suitable for animal feed additive for poultry to improve the phosphorus bioavailability.

Keywords

• Bacillus megaterium
• Feed additive
• Optimization
• Phosphorus
• Phytase

Supporting Institution

TUBITAK

Project Number

217O127

Ethical Statement

No ethical committee approval is required as no humans or animals were used in the research.

Thanks

This work forms part of the project supported by the Scientific and Technological Research Council of Türkiye (TÜBİTAK) (TOVAG-Project Number: 217O127).

References

1. Hernandez, J.R., Gulizia, J.P., Adkins, J.B., Rueda, M.S., Haruna, S.I., Pacheco, W.J., Downs, K.M., Effect of phytase level and form on broiler performance, tibia characteristics, and residual fecal phytate phosphorus in broilers from 1 to 21 days of age. Animals (Basel), 12 (15) (2022), 1952. https://doi.org/10.3390/ani12151952
2. Woyengo, T., Nyachoti, C., Anti-nutritional effects of phytic acid in diets for pigs and poultry-current knowledge and directions for future research. Canadian Journal of Animal Science, 93 (2013), 9–21. https://doi.org/10.4141/cjas2012-017
3. Coudert, E., Corrales, N. L., Juanchich, A., Poujol, M., Ribeiro, B., Chalvon-Demersay, T., Fondevila, G., The Role of phosphorus sources and phytase in growth performance and feed digestibility in broilers. Animals (Basel), 15 (14) (2025), 2111. https://doi.org/10.3390/ani15142111
4. Singh, P.K., Utilisation of phytic acid in poultry diets. Poultry Planner, 3 (11) (2002), 17-19.
5. Singh, P.K., Significance of phytic acid and supplemental phytase in chicken nutrition: A review. World Poultry Science Journal, 64 (2008), 553–580. https://doi.org/10.1017/s0043933908000202
6. Walk, C.L., Rama Rao, S.V., Increasing dietary phytate has a significant anti-nutrient effect on apparent ileal amino acid digestibility and digestible amino acid intake requiring increasing doses of phytase as evidenced by prediction equations in broilers. Poultry Science, 99 (2020), 290–300. https://doi.org/10.3382/ps/pez489
7. Nelson, T.S., Sheih, T.R., Wodzinski, R.J., Ware, J.H., Effect of supplemental phytase on the utilization of phytate phosphorus by chicks. Journal of Nutrients, 101 (1971), 1289-1294. https://doi.org/10.1093/jn/101.10.1289
8. Cowieson, AJ., Hruby, M., Pierson, E.E.M., Evolving enzyme technology: impact on commercial poultry nutrition. Nutrients Research Reviews, 19 (1) (2006), 90-103. https://doi.org/10.1079/NRR2006121

9. Olukosi, O.A., Cowieson, A.J., Adeola, O., Age-related influence of a cocktail of xylanase, amylase, and protease or phytase individually or in combination in broilers. Poultry Science, 86 (2007), 77-86. https://doi.org/10.1093/ps/86.1.77
10. Williams, C.L., Evaluation of Phytate and Phytase Interactions and Phytase Phase-Feeding on Bird Performance, Bone Characteristics and Meat Quality in Young Growing Broilers. Graduate Theses, University of Arkansas, 2014.
11. Walk, C.L., Santos, T.T., Bedford, M.R., Influence of superdoses of a novel microbial phytase on growth performance, tibia ash, and gizzard phytate and inositol in young broilers. Poultry Science, 93 (2014), 1172–1177. https://doi.org/10.3382/ps.2013-03571
12. Ptak, A., Bedford, M.R., Swiatkiewicz, S., Zyła, K., Józefiak, D., Phytase modulates ileal microbiota and enhances growth performance of the broiler chickens. PLoS One, 10 (2015), e0119770. https://doi.org/10.1371/journal.pone.0119770
13. Wu, D., Wu, S.B., Choct, M., Swick, R.A., Comparison of 3 phytases on energy utilization of a nutritionally marginal wheat-soybean meal broiler diet. Poultry Science, 94 (2015), 2670–2676. https://doi.org/10.3382/ps/pev222
14. Gonzalez De La Huebra, M., Robouch, P., Emteborg, H., Bellorini, S., Cizek-Stroh, A., Von Holst, C., Application of the CEN/ISO standard for phytase activity measurements to a new phytase product: determination of a robust conversion factor by an ınterlaboratory study for screening feed samples, Journal AOAC International, 102 (6) (2019), 1808-1813. https://doi.org/10.5740/jaoacint.19-0107
15. Tous, N., Tarradas, J., Francesch, M., Font-i-Furnols, M., Ader, P., Torrallardona, D., Effects of exogenous 6-phytase (Ec 3.1. 3.26) supplementation on performance, calcium and phosphorous digestibility, and bone mineralisation and density in weaned piglets. Animals (Basel), 11 (2021), 1787. https://doi.org/10.3390/ani11061787
16. Demirkan, E., Baygın, E., Usta, A., Screening of phytate hydrolysis Bacillus sp. isolated from soil and optimization of the certain nutritional and physical parameters on the production of phytase. Turkish Journal of Biochemistry, 39 (2) (2014), 206–214. https://doi.org/10.5505/tjb.2014.26817
17. Demirkan, E., Sevgi, T., Akcakoca, D., Ersoy, F., Partial purification, characterization and wheat bran degradation studies of a new phytase from the Bacillus megaterium EBD 9-1 strain. Turkish Journal of Biochemistry, 42 (3) (2017), 329–337. https://doi.org/10.1515/tjb-2016-0265
18. Choi, Y.M., Suh, H.J., Kim, J.M., Purification and properties of extracellular phytase from Bacillus sp. KHU-10. Journal of Protein Chemistry, 20 (2001), 287-292. https://doi.org/10.1023/a:1010945416862
19. Lan, G.Q., Abdullah, N., Jalaludin, S., Ho, Y.W., Efficacy of supplementation of a phytase-producing bacterial culture on the performance and nutrient use of broiler chickens fed corn-soybean meal diets. Poultry Science, 81 (10) (2002), 1522-1532. https://doi.org/10.1093/ps/81.10.1522
20. Shirley, R.B., Edwards, H.M., Graded levels of phytase past industry standards improves broiler performance. Poultry Science, 82 (2003), 671-680. https://doi.org/10.1093/ps/82.4.671
21. Dilger, R.N., Onyango, E.M., Sands, J.S., Adeola, O., Evaluation of microbial phytase in broiler diets. Poultry Science, 83 (2004), 962-970. https://doi.org/10.1093/ps/83.6.962
22. Gürbüz, Y., Cetin, M., Sengul, T., Effect of phytase supplementation on performance, tibia ash and serum p in broilers fed diets with different levels of phosphorus. Journal of Applied Animal Research, 36 (2009), 137-140. https://doi.org/10.1080/09712119.2009.9707048
23. Pieniazek, J., Smith, K.A., Williams, M.P., Manangi, M.K., VazquezAnon, M., Solbak, A., Miller, M., Lee, J.T. (2017). Evaluation of increasing levels of a microbial phytase in phosphorus deficient broiler diets via live broiler performance, tibia bone ash, apparent metabolizable energy, and amino acid digestibility. Poultry Science, 96, 370-382. https://doi.org/10.3382/ps/pew225
24. Ciftci, M., Dalkilic, B., Ali Azman, M., Effects of microbial phytase supplementation on feed consumption and egg production of laying hens. International Journal of Poultry Science, 4 (2005), 758-760. https://doi.org/10.3923/ijps.2005.758.760
25. Âmin, M.R., Hamidi, N.E., Effect of phytase supplementation on the performance of babcock-380-layer hens. Journals of Tropical Resources and Sustainable Science, 1 (1) (2013), 36-41. https://doi.org/10.47253/jtrss.v1i1.668
26. AOAC., Official methods of analysis. 17th ed. Phytase Activity in Feed. Association of Official Analytical Chemists: Washington, USA, 2000.
27. Dersjant-Li, Y., Awati, A., Schulze, H., Partridge, G., Phytase innon-ruminantanimalnutrition: a critical review on phytase activities in the gastrointestinal tract and influencing factors. Journal of Science and Food Agriculture, 95 (2015), 878–896. https://doi.org/10.1002/jsfa.6998
28. Taheri, H.R., Heidari, A., Shahir, M.H., Effect of high-dose phytase supplementation in broilers from 22 to 42 days post-hatch given diets severely limited in available phosphorus. Brazilian Poultry Science, 56 (2015), 330-336. https://doi.org/10.1080/00071668.2015.1020480
29. Gautier, A.E., Walk, C.L., Dilger, R.N., Effects of a high level of phytase on broiler performance bone ash phosphorus utilization and phytate dephosphorylation to inositol. Poultry Science, 97 (2018), 211–218. https://doi.org/10.3382/ps/pex291
30. Peretti, D.C., Callegari, M.A., Dias, C.P., Bergamo, G.S.R., Vasanthakumari, B.L., Costa, C.R.M., Carvalho, R.H., Silva, C.A., Enhanced farrowing efficiency and sow performance with Escherichia coli-derived 6-phytase supplementation during late gestation and lactation. Animals (Basel), 15 (21) (2025), 3090. https://doi.org/10.3390/ani15213090
31. Hirvonen, K., Bai, Y., Headey, D., Masters W.A., Affordability of the EAT–Lancet reference diet: a global analysis. Lancet Global Health, 8 (1) (2019), e59-e66. https://doi.org/10.1016/s2214-109x(19)30447-4
32. Singh, P.K., Kumar, G., Kumar, V., Singh, D., Enhanced phytase production by Bacillus subtilis subsp. subtilis in solid state fermentation and its utility in ımproving food Nutrition. Protein Peptide Letters, 28 (10) (2021), 1083-1089. https://doi.org/10.2174/0929866528666210720142359
33. Widawati, S., Suliasih, A., Sugiharto, S., Sudiana, I.M., Characterization of plant growth promoting bacteria isolated from water in mangrove ecosystem. IOP Conference Series: Earth Environment Science, 976 (2022), 012039. https://doi.org/10.1088/1755-1315/976/1/012039
34. Farhat, A., Chouayekh, H., Farhat, M., Bouchaala, K., Bejar, S., Gene cloning and characterization of a thermostable phytase from Bacillus subtilis US417 and assessment of its potential as a feed additive in comparison with a commercial enzyme. Molecular Biotechnology, 40 (2008), 127–135. https://doi.org/10.1007/s12033-008-9068-1
35. Purnamasari, L., Phytase enzyme characteristic of Bacillus subtilis to increase the quality of poultry feed. Journal of Livestock Science and Production, 2 (2) (2018), 93-98.
36. Lamid, M., Al-Arif, A., Asmarani, O., Warsito, S.H., Characterization of phytase enzymes as feed additive for poultry and feed. IOP Conference Series: Earth and Environmental Science, 137 (2018), 1-4. https://doi.org/10.1088/1755-1315/137/1/012009
37. Lei, X.G., Porres, J.M., Phytase enzymology, applications, and biotechnology. Biotechnology Letters, 25 (2003), 1787–1794. https://doi.org/10.1023/a:1026224101580
38. Saleh, A.A., Elsawee, M., Soliman, M.M., Elkon, R.Y., Alzawqari, M.H., Shukry, M., Abdel Moneim, A.M.E., Eltahan, H., Effect of bacterial or fungal phytase supplementation on the performance, egg quality, plasma biochemical parameters, and reproductive morphology of laying hens. Animals (Basel), 11 (2) (2021), 540. https://doi.org/10.3390/ani11020540
39. Chondrou, T., Adamidi, N., Lygouras, D., Hirota, S.A., Androutsos, O., Svolos, V., Dietary phytic acid, dephytinization, and phytase supplementation alter trace element bioavailability-a narrative review of human ınterventions. Nutrients, 16 (23) (2024), 4069. https://doi.org/10.3390/nu16234069
40. Lambré, C., Barat Baviera, J.M., Bolognesi, C., Cocconcelli, P.S., Crebelli, R., Gott, D.M., Grob, K., Lampi, E., Mengelers, M., Mortensen, A., Rivière, G., Steffensen, I.L., Tlustos, C., Loveren, H.V., Vernis, L., Zorn, H., Glandorf, B., Roos, Y., Andryszkiewicz, M., Kovalkovicova, N., Liu, Y., Lunardi, S., Chesson, A., Safety evaluation of the food enzyme 3-phytase from the non-genetically modified Aspergillus niger strain PHY93-08, EFSA Journal, 22 (7) (2024), e8876. https://doi.org/10.2903/j.efsa.2024.8876
41. Huang H., Luo H., Yang P., Meng K., Wang Y., Yuan T., Bai Y., Yao B., A novel phytase with preferable characteristics from Yersinia intermedia. Biochemistry and Biophysics Research Community, 350 (2006), 884-889. https://doi.org/10.1016/j.bbrc.2006.09.118
42. Khan, K., Zaneb, H., Rehman, Z., Maris, H., Rehman, H., Effect of phytase supplementation on growth performance in broiler chickens. Pakistan Journal of Zoology, 51 (2) (2019), 731-735. http://dx.doi.org/10.17582/journal.pjz/2019.51.2.531.535