Identification of Alkaline Protease Producing Bacilli from Sludge of Bactofuge and Separator Using Culture–Based and Molecular Techniques

Authors
M. Toloue Naddaf Abkouhi 1
F. Tabatabaei Yazdi 1
S. A. Mortazavi 2
M. R. Edalatian 1
1 Department of Food Science and Technology, College of Agriculture, Ferdowsi University of Mashhad, Mashhad, Islamic Republic of Iran.
2 Department of Food Science and Technology, Faculty of Agriculture, Ferdowsi University of Mashhad, Mashhad, Iran
Abstract
Dairy factories produce high volume of sludge from bactofuge and separator. Meantime, global demand for the proteases is increasing. Recently, utilization and conversion of the waste materials into value added product is a sustainable process. The objective of this study was to investigate the potential of bactofuge and separator sludge to produce alkaline protease enzymes. Total viable aerobic and anaerobic counts were determined on Plate Count Agar at 37 and 50ºC for both types of sludge. Lactobacillus count in MRS Agar plates corresponded to 3.12±0.25 log CFU mL-1 for sludge of bactofuge and 3.085±0.2 log CFU mL-1 for sludge of separator. Mold and yeast had population levels of 2.3±0.1 log CFU mL-1 for bactofuge and 2.08±0.1 log CFU mL-1 for separator. Proteolytic bacteria were isolated from dairy sludge using Skim Milk Agar media. A clear zone of Skim Milk hydrolysis indicated protease-producing organisms. Different cultural parameters (temperature, pH, thermal shock, and kind of sludge) were optimized for maximal enzyme production. Maximum proteolytic activity was observed at 37◦C (P< 0.05). Isolated alkaline protease producing Bacilli were identified by Polymerase Chain Reaction (PCR). The species were identified as Bacillus cereus strain zk2, Bacillus sp. cp-h71, Bacillus thuringiensis strain ILBB224, and Bacillus sp. Bac6D2.

Keywords

Subjects

1. Adav, S. S., Lee, D. J. and Lai, J.Y.2009. Proteolytic Activity in Stored Aerobic Granular Sludge and Structural Integrity. Bioresour. Technol., 100(1): 68–73.
2. Akcan, N. and Uyar, F. 2011. Production of Extracellular Alkaline Protease from Bacillus Subtilis RSKK96 with Solid State Fermentation. Eurasia. J. Biosci., 5: 64-72
3. Allaf, M. 2011. Isolation of Bacillus spp. from Some Sources and Study of Its Proteolytic Activity. Tikrit J. Pure Sci., 16(4): 59-63
4. Asker, M. M. S., Mahmoud, M. G., El Shebwy, K. H. and Abd El Aziz, M. S. 2013. Purification and Characterization of Two Thermo Stable Protease Fractions from Bacillus megaterium. J. Genet. Eng. Biotechnol., 11: 103–109.
5. Bah, C. S. F., Bekhit, A. E. A., McConnell, M. A. and Carne, A. 2016. Generation of Bioactive Peptide Hydrolysates from Cattle Plasma Using Plant and Fungal Proteases. Food Chem., 213: 98-107.
6. Baur, C., Krewinkel, M., Kranz, B., Neubeck, M., Wenning, M. Scherer, S., Hinrichs, M. S. J., Stressler, T., Fischera L., 2015. Quantification of the Proteolytic and Lipolytic Activity of Microorganisms Isolated from Raw Milk. Int. Dairy J., 49: 23-29.
7. Davati, N., Tabatabaei Yazdi, F., Zibaee, S., Shahidi, F. and Edalatian, M. R. 2015. Study of Lactic Acid Bacteria Community from Raw Milk of Iranian One-Humped Camel and Evaluation of Their Probiotic Properties. Jundishapur J. Microbiol., 8(5): 1-6.
8. Edalatian, M. R., Habibi Najafi, M. B. and Mortazavi, S. A. 2012. Microbial Diversity of the Traditional Iranian Cheeses Lighvan and Koozeh, as Revealed by Polyphasic Culturing and Culture-Independent Approaches. Dairy Sci. Technol., 92: 75–90.
9. Ellaiah, P., Srinivasulu, B. and Adinarayana, K. 2002. A Review on Microbial Alkaline Proteases. J. Sci. Ind. Res., 61: 690-704.
10. Ercolini, D., Russo, F., Ferrocino, I. and Villani, F. 2009. Molecular Identification of Mesophilic and Psychrotrophic Bacteria from Raw Cow’s Milk. Food Microbiol., 26: 228–231.
11. Furhan, J. and Sharma, S. 2014. Microbial Alkaline Proteases: Findings and Applications. Int. J. Inv. Pharm. Sci., 2(4): 823-834.
12. Gaur, R., Tiwari, S. and Sharma, A. 2014. Isolation and Characterization of Thermo Tolerant Alkaline Serine Protease of Bacillus sp. P-02. Am. J. Food Technol., 9(5):1-11.
13. Gésan Guiziou, G. 2010. Removal of Bacteria, Spores and Somatic Cells from Milk by Centrifugation and Microfiltration Techniques. Wood head publishing in Food Science, Technology and Nutrition, 1, CRC Press, Boca-Raton, USA, PP. 349-371.
14.
15. Ghorbel, B., Sellami-Kamoun, A. and Nasri, M. 2003. Stability studies of protease from Bacillus cereus BG1. Enzymee. Microb. Technol., 32: 513–518.
16. Gohel, S. and Singh, S. 2013. Characteristics and Thermodynamics of a Thermo Stable Protease from a Salt-Tolerant Alkaliphilicactinomycete. Int. J. Biol. Macromol., 56: 20–27.
17. Harrigan, W. F. 1998. Laboratory Methods in Food Microbiology. 3rd Edition, Academic Press, San Diego, London, Boston, New York, Sydney, Tokyo, Toronto, 282 PP.
18. Hermet, A., Mounier, J., Keravec, M., Vasseur, V., Barbier, G. and Jany, J. L. 2014. Application of Capillary Electrophoresis Single-Stranded Conformation Polymorphism (CE-SSCP) Analysis for Identification of Fungal Communities in Cheese. Food Microbiol., 41: 82-90.
19. Hosseini, S. V., Saffari, Z., Farhanghi, A., Atyabi, S. M. and Norouzian, D. 2016. Kinetics of Alkaline Protease Production by Streptomyces griseoflavus PTCC1130. Iran. J. Microbiol., 8(1): 8-13.
20. McCarthy, O. J. 2011. Plant and Equipment. “Centrifuges and Separators: Applications in the Dairy Industry”. Encyclopedia Dairy Sci., 175-183. DOI:10.1016/b978-0-12-374407-4.00463-5
21. Mehrban Sang Atash, M., Saraby Jamab, M., Karajian, R., Norbakhsh, R., Ghalasy, F., Vosogh, A. S. and Mohsenzadeh, M. 2011. Evaluation of Microbiological Contamination Sources on Swelling of Iranian Yoghurt Drink during Production Processes. Food Indu. Res., 21(1): 45-55.
22. Mhone, T. A., Matope, G. and Saidi, P. T. 2011. Aerobic Bacterial, Coliform, Escherichia coli and Staphylococcus aureus Counts of Raw and Processed Milk from Selected Smallholder Dairy Farms of Zimbabwe. Int. J. Food Microbiol., 151(2): 223–228
23. Mir Mohammad Sadeghi, H., Rabbani, M. and Naghitorabi, M. 2009. Cloning of Alkaline Protease Gene from Bacillus subtilis 168. Res. Pharm. Sci., 4(1): 43-46.
24. Molva, C., Sudagidan, M. and Okuklu, B. 2009. Extracellular Enzymee Production and Enterotoxigenic Gene Profiles of Bacillus cereus and Bacillus thuringiensis strains Isolated from Cheese in Turkey. Food Control, 20: 829–834.
25. Mukesh Kumar, D. J., Venkatachalam, P., Govindarajan, N., Balakumaran, M. D. and Kalaichelvan, P. T. 2012. Production and Purification of Alkaline Protease from Bacillus sp. MPTK 712 Isolated from Dairy Sludge. Global Vet., 8(5): 433-439.
26. Ozer, B. and Yaman, H. 2014. Milk and Milk Product, Microbiology of Liquid Milk. Encyclopedia Food Microbiol. (Second Edition), 2: 721-727.
27. Patil, P., Sabale, S. and Devale, A. 2015. Isolation and Characterization of Protease Producing Bacteria from Rhizosphere Soil and Optimization of Protease Production Parameters. Int. J. Curr. Microbiol. Sci., 2(Special Issue): 58-64.
28. Rathod, M. G. and Pathak, A. P. 2016. Optimized Production, Characterization and Application of Alkaline Proteases from Taxonomically Assessed Microbial Isolates from Lonar Soda Lake, India. Biocatal. Agric. Biotechnol., 7: 164-173.
29. Saran, S., Jasmine, I. and Rajendra, K. 2007. A Modified Method for the Detection of Microbial Proteases on Agar Plates Using Tannic Acid. J. Biochem. Biophys. Methods, 70: 697–699.
30. Saxena, S., Verma, J. and Shikha Modi, D. R. 2014. RAPD-PCR and 16S rDNA Phylogenetic Analysis of Alkaline Protease Producing Bacteria Isolated from Soil of India: Identification and Detection of Genetic Variability. Genet. Eng. Biotechnol. J., 12: 27–35.
31. Settanni, L., Gaglio, R., Guarcello, R., Francesca, N., Carpino, S., Sannino, C. and Todaro, M. 2013. Selected Lactic Acid Bacteria as a Hurdle to the Microbial Spoilage of Cheese: Application on a Traditional Raw Ewes’ Milk Cheese. Int. Dairy J., 32(2): 126-132.
32. Standard Iran 2008. Milk and Its Products, Sampling. No. 326.
http://standard.isiri.gov.ir/StandardView.aspx?Id=5117.
33. Yavarmanesh, M., Mortazavi, S. A. and Habibi Najafi, M. B. 2007. Predicting the Microbiological Quality of Raw Milk Based on Mathematical Models. Iran. J. Food Sci. Technol., 4(15): 53-63.
Journal of Agricultural Science and Technology
Volume 21, Issue 7
November and December 2019
Pages 1819-1830