Statistical optimization of culture conditions for milk-clotting enzyme production by Bacillus amyloliquefaciens using wheat bran-an agro-industry waste
Open accessAbstract
In order to improve the production of the milk-clotting enzyme under submerged fermentation, two statistical methods were applied to optimize the culture conditions of Bacillus amyloliquefaciens D4 using wheat bran as nutrient source. First, initial pH, agitation speed, and fermentation time were shown to have significant effects on D4 enzyme production using the Plackett-Burman experimental design. Subsequently, optimal conditions were obtained using the Box-Behnken method, which were as follows: initial pH 7.57, agitation speed 241 rpm, fermentation time 53.3 h. Under these conditions, the milk-clotting enzyme production was remarkably enhanced. The milk-clotting enzyme activity reached 1996.9 SU/mL, which was 2.92-fold higher than that of the initial culture conditions, showing that the Plackett-Burman design and Box-Behnken response surface method are effective to optimize culture conditions. The research can provide a reference for full utilization of wheat bran and the production of milk-clotting enzyme by B. amyloliquefaciens D4 under submerged fermentation.
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Wheat bran provides an excellent medium for microbial growth and enzyme biosynthesis because it is a nutrient-rich material containing about 15% protein, along with cellulose, starch, and trace minerals.
“In contrast, MCE production is inhibited by inorganic nitrogen sources as a nutrientrich material containing ~15% protein; along with cellulose, starch, and trace minerals, wheat bran provides an excellent medium for microbial growth and enzyme biosynthesis (Zhang et al., 2013).”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhang et al., 2013
The passage on wheat bran (section labeled Abstract; page unavailable) says it contains cellulose, starch, crude protein, and trace elements that can promote microbial growth and enzyme production. It does not give the claimed protein content of about 15%. The cited paper also does not establish the citing sentence’s separate assertion that inorganic nitrogen sources inhibit MCE production. (LLM, conf=0.88)
Experimental — accuracy not yet measuredThe highest concentrations of milk-clotting enzyme from Bacillus licheniformis and Amylomyces rouxii were obtained when the initial pH of the medium was set at 7.0, and from Bacillus amyloliquefaciens when the initial pH was 7.6.
“Furthermore, in the study by Yu and Chou (2005; for B. licheniformis and Amylomyces rouxii) and Zhang et al. (2013;for B. amyloliquefaciens), the highest concentrations of MCE were obtained when the initial pH of the medium was set at 7.0 and 7.6, respectively.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhang et al., 2013
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Cites
Deng, A., Wu, J., Zhang, Y., Zhang, G., & Wen, T. (2010) · Bioresource Technology · doi:10.1016/j.biortech.2010.03.130 · Cited by 201 · see citing works
Milk-clotting enzymes produced by culture of Bacillus subtilis natto.
Shieh, C.-J., Phan Thi, L.-A., & Shih, I.-L. (2009) · Biochemical Engineering Journal · doi:10.1016/j.bej.2008.09.003 · Cited by 99 · see citing works
Yang, H., Liu, L., Shin, H., Chen, R. R., Li, J., Du, G., & Chen, J. (2012) · Journal of Biotechnology · doi:10.1016/j.jbiotec.2012.12.007 · Cited by 76 · see citing works
Haddar, A., Fakhfakh, N., Hmidet, N., Frikha, F., Nasri, M., & Sellami-Kamoun, A. (2010) · Journal of Bioscience and Bioengineering · doi:10.1016/j.jbiosc.2010.03.015 · Cited by 74 · see citing works
Das, K., Doley, R., & Mukherjee, A. K. (2004) · Biotechnology and Applied Biochemistry · doi:10.1042/ba20040034 · Cited by 71 · see citing works
Application of Thermostable Xylanase of Bacillus pumilus in Textile Processing
Battan, B., Dhiman, S. S., Ahlawat, S., Mahajan, R., & Sharma, J. (2011) · Indian Journal of Microbiology · doi:10.1007/s12088-011-0118-1 · Cited by 61 · see citing works
Annamalai, N., Thavasi, R., Vijayalakshmi, S. P., & Balasubramanian, T. (2011) · Indian Journal of Microbiology · doi:10.1007/s12088-011-0160-z · Cited by 57 · see citing works
Ageitos, J. M., Vallejo, J. A., Sestelo, A. B. F., Poza, M., & Villa, T. G. (2007) · Journal of Applied Microbiology · doi:10.1111/j.1365-2672.2007.03460.x · Cited by 56 · see citing works
Christiansen, T., Christensen, B. B., & NIELSEN, J. K. (2002) · Metabolic Engineering · doi:10.1006/mben.2001.0219 · Cited by 46 · see citing works
Purification and properties of a milk-clotting enzyme produced by Bacillus amyloliquefaciens D4
He, X., Ren, F., Guo, H., Zhang, W., Song, X., & Gan, B. (2010) · Korean Journal of Chemical Engineering · doi:10.1007/s11814-010-0347-8 · Cited by 42 · see citing works
Self-citationProduction of milk-clotting protease from Bacillus subtilis.
Dutt, K., Gupta, P., Saran, S., Misra, S., & Saxena, R. K. (2009) · Applied Biochemistry and Biotechnology · doi:10.1007/s12010-008-8504-9 · Cited by 31 · see citing works
Production of milk-clotting enzyme by Bacillus subtilis B1 from wheat bran
Zhongyang, D., Shuangping, L., Zhenghua, G., Liang, Z., Kechang, Z., & Guiyang, S. (2011) · AFRICAN JOURNAL OF BIOTECHNOLOGY · doi:10.5897/ajb10.1647 · Cited by 28 · see citing works
Optimization of extraction of β-endoglucanase from the fermented bran of Aspergillus niger
Chandra, M. S., Viswanath, B., & Reddy, B. R. (2010) · Indian Journal of Microbiology · doi:10.1007/s12088-010-0020-2 · Cited by 20 · see citing works
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Research works printed but not linked: 1
- Gan BZ, Song X, He XL, Zhang WB, Li F (2009) Separation and identification of a chymosin producing bacterium from soil of yak grazing district in Tianzhu country of Gansu province. Food Sci 30(11):158-162 (in chinese)
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Patel, S. V., & Shukla, S. (2016) · Elsevier eBooks · doi:10.1016/b978-0-12-802309-9.00030-3 · Cited by 20 · see citing works
Guleria, S., Walia, A., Chauhan, A., & Shirkot, C. K. (2016) · Bioresources and Bioprocessing · doi:10.1186/s40643-016-0108-6 · Cited by 16 · see citing works
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Zhang, Y., Hu, J., Liu, X., Jiang, C., Sun, J., Song, X., & Wu, Y. (2022) · AMB Express · doi:10.1186/s13568-022-01493-9 · Cited by 8 · see citing works
Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review.
Wiśniewski, P., Małkowska-Kowalczyk, M., Łobacz, A., Zadernowska, A., & Żulewska, J. (2025) · Journal of dairy science · doi:10.3168/jds.2025-26973 · Cited by 10 · see citing works
Huang, J., Zhang, D., Leng, B. S., Lin, Z., & Pan, Y.-T. (2019) · The Journal of General and Applied Microbiology · doi:10.2323/jgam.2018.06.002 · Cited by 4 · see citing works
Potential applications of food industrial by-products in the dairy industry
Nazir, A., Maan, A. A., & Shaukat, M. N. (2021) · Elsevier eBooks · doi:10.1016/b978-0-12-820563-1.00009-3 · Cited by 2 · see citing works
Ho, H., Soh, L., & Ong, S. (2015) · British Microbiology Research Journal · doi:10.9734/bmrj/2016/20766
Zhang, Y., Hu, J., Liu, X., Jiang, C., Sun, J., Song, X., & Wu, Y. (2022) · Figshare · doi:10.6084/m9.figshare.21629238
Yang, W., Gan, L., Zhang, Y., Wang, Y., Zhang, W., Wang, T., & Qiao, H. (2026) · Journal of Agricultural and Food Chemistry · doi:10.1021/acs.jafc.6c07588
Self-citation
- Authors
- Zhang, W., He, X., Liu, H., Guo, H., Ren, F., & Wen, P.
- Affiliations
- Gansu Agricultural University (CN)
- China Agricultural University (CN)
- Corresponding author
- Ren, Fazheng
- Venue
- Indian Journal of Microbiology
- Published
- 2013-03-21
- Volume
- 53
- Issue
- 4
- Pages
- 492-495
- ISSN
- 0046-8991 (ISSN-L)
- 0973-7715
- PMC
- PMC3779303
- OpenAlex
- W2032330374
- Cited by
- 13
- References
- 13
- Citations by year
- 2026: 1 · 2025: 1 · 2022: 2 · 2021: 1 · 2020: 1 · 2019: 1 · 2018: 1 · 2017: 1 · 2016: 3 · 2015: 1
- FWCI
- 1.17
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Licence
- www.springer.com/tdm (tdm)
- Locations
- Indian Journal of Microbiology (publishedVersion)
- PubMed Central (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
- Life Sciences › Agricultural and Biological Sciences › Food Science › Advanced Scientific Research Methods
- Social Sciences › Decision Sciences › Management Science and Operations Research › Optimal Experimental Design Methods
- Keywords
- milk-clotting enzyme, Bacillus amyloliquefaciens, wheat bran, agro-industrial waste utilization, submerged fermentation, culture condition optimization, Plackett-Burman design, Box-Behnken design, initial pH, stirring speed, fermentation time
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