Selecting β-galactosidase producing Bacillus spp. and determining condtions for lactose fermentation
Main Article Content
Abstract
The study was conducted to select Bacillus spp. strains capable of producing the enzyme β-galactosidase and determine the suitable temperature and pH for lactose fermentation. Among the 21 Bacillus spp. strains surveyed using the X-gal method, 6 strains (B6, B7, B9, B11, B17, and B18) showed the characteristic blue color of X-gal on agar plates after 72 hours, indicating the presence of β-galactosidase enzyme. Four strains (B6, B9, B17, and B18) displaying the darkest blue color were selected for further enzyme activity determination using ortho-nitrophenyl-β-galactoside (oNPG). The results revealed that strain B18 exhibited the highest enzyme activity and was identified through molecular biological techniques as Bacillus licheniformis. The optimal conditions for lactose fermentation by B. licheniformis B18 were determined to be at 30°C and pH 7.0, with the enzyme activity reaching the highest value of 533.08 U/L. Strain B. licheniformis B18 is recommended for further research to optimize other factors for β-galactosidase production.
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial 4.0 International License.
Keywords
Bacillus licheniformis, lactase, oNPG, X-gal, β-galactosidase
References
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Amin, A. A., & Ali, S. M. (2023). Characterization of an isolated lactase enzyme produced by Bacillus licheniformis ALSZ2 as a potential pharmaceutical supplement for lactose intolerance. Frontiers in Microbiology, 14, 1180463. https://doi.org/10.3389/fmicb.2023.1180463
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Husain, Q. (2010). β Galactosidases and their potential applications: a review. Critical Reviews in Biotechnology, 30(1), 41-62. https://doi.org/10.3109/07388550903330497
Jaturapiree, P., Phuengjayaeam, S., Seangsawang, P., Srila, W., & Muangnapoh, C. (2012). Isolation and production of novel [beta]-galactosidase from a newly isolated, moderate thermophile, Bacillus sp. strain B1. 1. Journal of Food Science and Engineering, 2(7), 395-402. https://doi.org/10.17265/2159-5828/2012.07.006
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Lyngwi, N. A., and Joshi, S. R. (2014). Economically important Bacillus and related genera: A mini review. In A. Sen (editor) Biology of Useful Plants and Microbes (33-43). New Delhi: Narosa Publishing House.
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Nguyễn, T. T., Trần, T. N., Nguyễn, T. L. Đ., Nguyễn, H. A. (2021). –galactosidase của chủng Lactobacillus fermentum FV4: Từ tuyển chọn chủng đến xác định đặc tính tạo galacto-oligosaccharide của enzyme. Tạp chí Khoa học Nông nghiệp Việt Nam, 19(6), 745-755.
Nguyen, T. H., Splechtna, B., Steinböck, M., Kneifel, W., Lettner, H. P., Kulbe, K. D., & Haltrich, D. (2006). Purification and characterization of two novel β-galactosidases from Lactobacillus reuteri. Journal of Agricultural and Food Chemistry, 54(14), 4989-4998. 10.1021/jf053126u
Princely, S., Basha, N. S., Kirubakaran, J. J., & Dhanaraju, M. D. (2013). Biochemical characterization, partial purification, and production of an intracellular beta-galactosidase from Streptococcus thermophilus grown in whey. European Journal of Experimental Biology, 3(2), 242-251.
Raveendran, S., Parameswaran, B., Ummalyma, S. B., Abraham, A., Mathew, A. K., Madhavan, A., & Pandey, A. (2018). Applications of microbial enzymes in food industry. Food Technology and Biotechnology, 56(1), 16-30. https://doi.org/10.17113/ftb.56.01.18.5491
Rehamnia, B., Lee, N. M., Kuktaite, R., & Kacem Chaouche, N. (2022). Screening of spore-forming bacteria with probiotic potential in Pristine Algerian Caves. Microbiology Spectrum, 10(5), e00248-22. https://doi.org/10.1128/spectrum.00248-22
Saqib, S., Akram, A., Halim, S. A., & Tassaduq, R. (2017). Sources of β-galactosidase and its applications in food industry. 3 Biotech, 7, 1-7. https://doi.org/10.1007/s13205-017-0645-5
Shelef, L.A. (2003). Bacillus: Detection. In Benjamin Caballero (Editor-in-Chief), Encyclopedia of Food Sciences and Nutrition (2nd ed.), 358-365. Cambridge: Academic Press. https://doi.org/10.1016/B0-12-227055-X/00075-4
Sewalt, V., Shanahan, D., Gregg, L., La Marta, J., & Carrillo, R. (2016). The generally recognized as safe (GRAS) process for industrial microbial enzymes. Industrial Biotechnology, 12(5), 295-302. https://doi.org/10.1089/ind.2016.0011
Sonune, N. & Garode, A. (2018). isolation, characterization and identification of extracellular enzyme producer Bacillus licheniformis from municipal wastewater and evaluation of their biodegradability. Biotechnology Research and Innovation, 2(1), 37-44. https://doi.org/10.1016/j.biori.2018.03.001
Su, Y., Liu, C., Fang, H., & Zhang, D. (2020). Bacillus subtilis: A universal cell factory for industry, agriculture, biomaterials and medicine. Microbial Cell Factories, 19, 1-12.
Volford, B., Varga, M., Szekeres, A., Kotogán, A., Nagy, G., Vágvölgyi, C., & Takó, M. (2021). β-galactosidase-producing isolates in mucoromycota: Screening, enzyme production, and applications for functional oligosaccharide synthesis. Journal of Fungi, 7(3), 229. https://doi.org/10.3390/jof7030229
Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173(2), 697-703. https://doi.org/10.1128/jb.173.2.697-703.1991
Al-Jazairi, M., Abou-Ghorrah, S., & Bakri, Y. (2014). Isolation and identification of a new yeast isolate with high beta-galactosidase activity from Syrian dairy products. International Food Research Journal, 21(2), 541-546.
Amin, A. A., & Ali, S. M. (2023). Characterization of an isolated lactase enzyme produced by Bacillus licheniformis ALSZ2 as a potential pharmaceutical supplement for lactose intolerance. Frontiers in Microbiology, 14, 1180463. https://doi.org/10.3389/fmicb.2023.1180463
Bahaddad, S. A., Almalki, M. H., Alghamdi, O. A., Sohrab, S. S., Yasir, M., Azhar, E. I., & Chouayekh, H. (2023). Bacillus species as direct-fed microbial antibiotic alternatives for monogastric production. Probiotics and Antimicrobial Proteins, 15(1), 1-16. https://doi.org/10.1007/s12602-022-09909-5
Batra, N., Singh, J., Banerjee, U. C., Patnaik, P. R., & Sobti, R. C. (2002). Production and characterization of a thermostable β‐galactosidase from Bacillus coagulans RCS3. Biotechnology and Applied Biochemistry, 36(1), 1-6. https://doi.org/10.1042/ba20010091
Dominici, S., Marescotti, F., Sanmartin, C., Macaluso, M., Taglieri, I., Venturi, F., Zinnai, A. & Facioni, M. S. (2022). Lactose: Characteristics, food and drug-related applications, and its possible substitutions in meeting the needs of people with lactose intolerance. Foods, 11(10), 1486. https://doi.org/10.3390/foods11101486
Elsayed, E. A., Danial, E. N., Wadaan, M. A., & El-Enshasy, H. A. (2019). Production of β-galactosidase in shake-flask and stirred tank bioreactor cultivations by a newly isolated Bacillus licheniformis strain. Biocatalysis and Agricultural Biotechnology, 20, 101231. https://doi.org/10.1016/j.bcab.2019.101231
Ghani, M., Ansari, A., Aman, A., Zohra, R. R., Siddiqui, N. N., Qader, S. A. (2013). Isolation and characterization of different strains of Bacillus licheniformis for the production of commercially significant enzymes. Pakistan Journal of Pharmaceutical Sciences, 26(4): 691-697.
Husain, Q. (2010). β Galactosidases and their potential applications: a review. Critical Reviews in Biotechnology, 30(1), 41-62. https://doi.org/10.3109/07388550903330497
Jaturapiree, P., Phuengjayaeam, S., Seangsawang, P., Srila, W., & Muangnapoh, C. (2012). Isolation and production of novel [beta]-galactosidase from a newly isolated, moderate thermophile, Bacillus sp. strain B1. 1. Journal of Food Science and Engineering, 2(7), 395-402. https://doi.org/10.17265/2159-5828/2012.07.006
Juajun, O., Nguyen, T. H., Maischberger, T., Iqbal, S., Haltrich, D., & Yamabhai, M. (2011). Cloning, purification, and characterization of β-galactosidase from Bacillus licheniformis DSM 13. Applied Microbiology and Biotechnology, 89, 645-654. https://doi.org/10.1007/s00253-010-2862-2
Kamran, A., Bibi, Z., Aman, A., & Qader, S. A. U. (2016). Lactose hydrolysis approach: isolation and production of β-galactosidase from newly isolated Bacillus strain B-2. Biocatalysis and Agricultural Biotechnology, 5, 99-103. https://doi.org/10.1016/j.bcab.2015.12.010
Kuribayashi, L. M., do Rio Ribeiro, V. P., De Santana, R. C., Ribeiro, E. J., Dos Santos, M. G., Falleiros, L. N. S. S., & Guidini, C. Z. (2021). Immobilization of β-galactosidase from Bacillus licheniformis for application in the dairy industry. Applied Microbiology and Biotechnology, 105, 3601-3610. https://doi.org/10.1007/s00253-021-11325-8
Logan, N. A., Hoffmaster, A. R., Shadomy, S. V. & Stauffer, K. E. (2011). Bacillus and other aerobic endospore-forming bacteria. In J. Versalovic, K. C. Carroll, G. Funke, J. H. Jorgensen, M. L. Landry & D. W. Warnock (editors), Manual of Clinical Microbiology, 10th ed., vol. 1 (381-402). Washington, DC: American Society for Microbiology.
Lyngwi, N. A., and Joshi, S. R. (2014). Economically important Bacillus and related genera: A mini review. In A. Sen (editor) Biology of Useful Plants and Microbes (33-43). New Delhi: Narosa Publishing House.
Mattar, R., de Campos Mazo, D. F., & Carrilho, F. J. (2012). Lactose intolerance: diagnosis, genetic, and clinical factors. Clinical and Experimental Gastroenterology, 113-121. https://doi.org/10.3390/foods11101486
Nguyễn, T. T., Trần, T. N., Nguyễn, T. L. Đ., Nguyễn, H. A. (2021). –galactosidase của chủng Lactobacillus fermentum FV4: Từ tuyển chọn chủng đến xác định đặc tính tạo galacto-oligosaccharide của enzyme. Tạp chí Khoa học Nông nghiệp Việt Nam, 19(6), 745-755.
Nguyen, T. H., Splechtna, B., Steinböck, M., Kneifel, W., Lettner, H. P., Kulbe, K. D., & Haltrich, D. (2006). Purification and characterization of two novel β-galactosidases from Lactobacillus reuteri. Journal of Agricultural and Food Chemistry, 54(14), 4989-4998. 10.1021/jf053126u
Princely, S., Basha, N. S., Kirubakaran, J. J., & Dhanaraju, M. D. (2013). Biochemical characterization, partial purification, and production of an intracellular beta-galactosidase from Streptococcus thermophilus grown in whey. European Journal of Experimental Biology, 3(2), 242-251.
Raveendran, S., Parameswaran, B., Ummalyma, S. B., Abraham, A., Mathew, A. K., Madhavan, A., & Pandey, A. (2018). Applications of microbial enzymes in food industry. Food Technology and Biotechnology, 56(1), 16-30. https://doi.org/10.17113/ftb.56.01.18.5491
Rehamnia, B., Lee, N. M., Kuktaite, R., & Kacem Chaouche, N. (2022). Screening of spore-forming bacteria with probiotic potential in Pristine Algerian Caves. Microbiology Spectrum, 10(5), e00248-22. https://doi.org/10.1128/spectrum.00248-22
Saqib, S., Akram, A., Halim, S. A., & Tassaduq, R. (2017). Sources of β-galactosidase and its applications in food industry. 3 Biotech, 7, 1-7. https://doi.org/10.1007/s13205-017-0645-5
Shelef, L.A. (2003). Bacillus: Detection. In Benjamin Caballero (Editor-in-Chief), Encyclopedia of Food Sciences and Nutrition (2nd ed.), 358-365. Cambridge: Academic Press. https://doi.org/10.1016/B0-12-227055-X/00075-4
Sewalt, V., Shanahan, D., Gregg, L., La Marta, J., & Carrillo, R. (2016). The generally recognized as safe (GRAS) process for industrial microbial enzymes. Industrial Biotechnology, 12(5), 295-302. https://doi.org/10.1089/ind.2016.0011
Sonune, N. & Garode, A. (2018). isolation, characterization and identification of extracellular enzyme producer Bacillus licheniformis from municipal wastewater and evaluation of their biodegradability. Biotechnology Research and Innovation, 2(1), 37-44. https://doi.org/10.1016/j.biori.2018.03.001
Su, Y., Liu, C., Fang, H., & Zhang, D. (2020). Bacillus subtilis: A universal cell factory for industry, agriculture, biomaterials and medicine. Microbial Cell Factories, 19, 1-12.
Volford, B., Varga, M., Szekeres, A., Kotogán, A., Nagy, G., Vágvölgyi, C., & Takó, M. (2021). β-galactosidase-producing isolates in mucoromycota: Screening, enzyme production, and applications for functional oligosaccharide synthesis. Journal of Fungi, 7(3), 229. https://doi.org/10.3390/jof7030229
Weisburg, W. G., Barns, S. M., Pelletier, D. A., & Lane, D. J. (1991). 16S ribosomal DNA amplification for phylogenetic study. Journal of Bacteriology, 173(2), 697-703. https://doi.org/10.1128/jb.173.2.697-703.1991
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