Novel Bacillus Milk-Clotting Enzyme Produces Diverse Functional Peptides in Semihard Cheese
Abstract
Although rennet is one of the best choices for cheese manufacturing, its production cannot meet the growing demands of the cheese industry. Thus, new milk-clotting enzymes (MCEs) with similar or better properties as/than those of calf chymosin are needed urgently. Here, three MCEs, BY-2, BY-3, and BY-4, were mined by bioinformatic analysis and then expressed in and isolated from Escherichia coli . BY-4 had the highest milk-clotting activity/proteolytic activity (238.76) with enzyme properties similar to those of calf chymosin. BY-4 cheese had a composition, appearance, consistency/texture, and overall acceptability proximate to calf chymosin cheese. The EC 50 values of peptides extracted from BY-4 cheese for 2,2-diphenyl-1-picrylhydrazyl inhibition (antioxidant property), angiotensin-converting enzyme inhibition (antihypertensivity), and growth inhibition of liver cancer cells (antitumor property) were found to be 81, 49, and 238 μg/mL, respectively, which were 2.35, 2.59, and 2.12 folds higher than those of calf chymosin cheese. These results indicated the potential of BY-4 as a supplement to calf chymosin in cheese manufacturing, especially for functional and health care purposes.
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Sensory analysis revealed no differences between the tested cheese with E. coli MCE and the control cheese with chymosin in terms of bitterness, tartness, mustiness, sourness, and ammonia.
“Sensory analysis revealed no differences between the tested cheese with E. coli MCE and the control cheese with chymosin in terms of bitterness, tartness, mustiness, sourness, and ammonia (Meng et al., 2021).”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Meng et al., 2021
MCE isolated from Escherichia coli exhibited high PA (238.76 g/mL) and properties comparable to those of calf chymosin.
“The MCE isolated from Escherichia coli exhibited high PA (238.76 g/mL) and properties comparable to those of calf chymosin.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Meng et al., 2021
The taste of cheese produced with E. coli MCE, particularly bitterness, is linked to casein breakdown and the presence of hydrophobic peptides.
“The taste of cheese produced with E. coli MCE, particularly bitterness, is linked to casein breakdown and the presence of hydrophobic peptides.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Meng et al., 2021
The composition, appearance, texture, and overall appeal of Cheddar cheese produced with the tested MCE were similar to the control cheese.
“The composition, appearance, texture, and overall appeal of Cheddar cheese produced with the tested MCE were similar to the control cheese.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Meng et al., 2021
Cites
Molecular cloning: A laboratory manual
Deininger, P. L. (1990) · Analytical Biochemistry · doi:10.1016/0003-2697(90)90595-z · Cited by 84,684 · see citing works
Bioactive milk peptides: a prospectus.
Clare, D. A., & Swaisgood, H. E. (2000) · Journal of Dairy Science · doi:10.3168/jds.s0022-0302(00)74983-6 · Cited by 718 · see citing works
Phelan, M., Aherne, A., Fitzgerald, R. J., & O’Brien, N. M. (2009) · International Dairy Journal · doi:10.1016/j.idairyj.2009.06.001 · Cited by 354 · see citing works
Recent Advances in Milk Clotting Enzymes
JACOB, M., JAROS, D., & ROHM, H. (2011) · International Journal of Dairy Technology · doi:10.1111/j.1471-0307.2010.00633.x · Cited by 295 · see citing works
Megías, C., Yust, M. D. M., Pedroche, J., Lquari, H., Girón‐Calle, J., Alaiz, M., Millán, F., & Vioque, J. (2004) · Journal of Agricultural and Food Chemistry · doi:10.1021/jf034707r · Cited by 220 · see citing works
Proteolysis in Cheese during Ripening
Upadhyay, V. K., McSweeney, P. L. H., Magboul, A. A. A., & Fox, P. F. (2004) · Cheese · doi:10.1016/s1874-558x(04)80076-9 · Cited by 206 · see citing works
Dairy protein hydrolysates: Peptides for health benefits
Hernández‐Ledesma, B., García‐Nebot, M. J., Fernández‐Tomé, S., Amigo, L., & Recio, I. (2013) · International Dairy Journal · doi:10.1016/j.idairyj.2013.11.004 · Cited by 205 · see citing works
Chymosin and Other Milk Coagulants: Sources and Biotechnological Interventions
Kumar, A., Grover, S., Sharma, J., & Batish, V. K. (2010) · Critical Reviews in Biotechnology · doi:10.3109/07388551.2010.483459 · Cited by 160 · see citing works
The Thermolysin Family (M4) of Enzymes: Therapeutic and Biotechnological Potential
Adekoya, O. A., & Sylte, I. (2008) · Chemical Biology & Drug Design · doi:10.1111/j.1747-0285.2008.00757.x · Cited by 160 · see citing works
Amira, A. B., Besbes, S., Attia, H., & Blecker, C. S. (2017) · International Journal of Food Properties · doi:10.1080/10942912.2017.1289959 · Cited by 129 · see citing works
Comparison of the Milk-Clotting Properties of Three Plant Extracts
Mazorra-Manzano, M. A., Perea-Gutiérrez, T. C., Lugo-Sánchez, M. E., Ramirez-Suarez, J. C., Torres-Llanez, M. J., González-Córdova, A. F., & Vallejo-Cordoba, B. (2013) · Food Chemistry · doi:10.1016/j.foodchem.2013.05.042 · Cited by 117 · see citing works
Sah, B. N. P., Vasiljevic, T., McKechnie, S., & Donkor, O. N. (2015) · Comprehensive Reviews in Food Science and Food Safety · doi:10.1111/1541-4337.12126 · Cited by 112 · see citing works
Baum, F., Fedorova, M., Ebner, J., Hoffmann, R., & Pischetsrieder, M. (2013) · Journal of Proteome Research · doi:10.1021/pr4003273 · Cited by 97 · see citing works
Burbank, H. M., & Qian, M. C. (2005) · Journal of Chromatography A · doi:10.1016/j.chroma.2005.01.027 · Cited by 92 · see citing works
A mini-review on health and nutritional aspects of cheese with a focus on bioactive peptides
López‐Expósito, I., Amigo, L., & Recio, I. (2012) · Dairy Science and Technology · doi:10.1007/s13594-012-0066-5 · Cited by 78 · see citing works
Rennet-induced Coagulation of Milk
Horne, D. S., & Banks, J. M. (2004) · Cheese · doi:10.1016/s1874-558x(04)80062-9 · Cited by 69 · see citing works
Mohanty, A. K., Mukhopadhyay, U. K., Grover, S., & Batish, V. K. (1999) · Biotechnology Advances · doi:10.1016/s0734-9750(99)00010-5 · Cited by 64 · see citing works
Raposo, S., & Domingos, A. (2008) · Process Biochemistry · doi:10.1016/j.procbio.2007.11.003 · Cited by 64 · 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
Merheb‐Dini, C., Garcia, G. A. C., Penna, A. L. B., Gomes, E., & da Silva, R. (2011) · Food Chemistry · doi:10.1016/j.foodchem.2011.07.105 · Cited by 54 · see citing works
Kumari Narwal, R., Bhushan, B., Pal, A., Panwar, A., & Malhotra, S. (2016) · LWT · doi:10.1016/j.lwt.2015.08.065 · Cited by 42 · see citing works
Production of volatile sulfur compounds in Cheddar cheese slurries
Dias, B., & Weimer, B. C. (1999) · International Dairy Journal · doi:10.1016/s0958-6946(99)00130-2 · Cited by 41 · see citing works
Fan, F., Tu, M., Liu, M., Shi, P., Wang, Y., Wu, D., & Du, M. (2017) · Journal of Agricultural and Food Chemistry · doi:10.1021/acs.jafc.7b02067 · Cited by 41 · see citing works
Fed-batch process for the psychrotolerant marine bacterium Pseudoalteromonas haloplanktis
Wilmes, B., Hartung, A., Lalk, M., Liebeke, M., Schweder, T., & Neubauer, P. B. (2010) · Microbial Cell Factories · doi:10.1186/1475-2859-9-72 · Cited by 39 · see citing works
Meng, F., Chen, R., Zhu, X., Lu, Y., Nie, T., Lu, F., & Lu, Z. (2018) · Journal of Agricultural and Food Chemistry · doi:10.1021/acs.jafc.8b01697 · Cited by 36 · see citing works
Self-citationProduction 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
Hang, F., Wang, Q., Hong, Q., Liu, P., Wu, Z., Liu, Z., Zhang, H., & Chen, W. (2016) · International Journal of Biological Macromolecules · doi:10.1016/j.ijbiomac.2016.01.028 · Cited by 27 · see citing works
Zhao, X., Cai, M., Yang, Z.-J., Luo, T.-Q., Sarwar, A., Megrous, S., Aziz, T., & Yang, Z.-N. (2019) · European Food Research and Technology · doi:10.1007/s00217-019-03361-6 · Cited by 24 · see citing works
Bonomi, F., Brandt, R., Favalli, S., et al. (2011) · International Dairy Journal · doi:10.1016/j.idairyj.2011.04.012 · Cited by 22 · see citing works
Li, Y., Liang, S., Zhi, D.-J., Chen, P., Su, F., & Li, H.-Y. (2012) · European Food Research and Technology · doi:10.1007/s00217-012-1663-5 · Cited by 22 · see citing works
Wehaidy, H. R., Abdel Wahab, W. A., Kholif, A. M. M., Elaaser, M., Bahgaat, W. K., & Abdel-Naby, M. A. (2020) · Biocatalysis and Agricultural Biotechnology · doi:10.1016/j.bcab.2020.101589 · Cited by 21 · see citing works
Manuelian, C. L., Boselli, C., Vigolo, V., Giangolini, G., & MARCHI, M. D. (2020) · Journal of Dairy Science · doi:10.3168/jds.2019-17208 · Cited by 18 · see citing works
Taha, S., Abd, M. E., Gobba, C. D., Abdel-Hamid, M., Khalil, E. M., Hassan, F., & Fathy, D. (2020) · Journal of Food Processing and Preservation · doi:10.1111/jfpp.14434 · Cited by 12 · see citing works
Phengnuam, T., Goroncy, A. K., Rutherfurd, S. M., et al. (2013) · Journal of Agricultural and Food Chemistry · doi:10.1021/jf4035964 · Cited by 9 · see citing works
Fileto-Pérez, H. A., Ayón, L. G. M., Soto-García, E., Páez‐Lerma, J. B., Soto-Cruz, N. O., García-Caballero, B. E., & Rutiaga-Quiñones, J. G. (2020) · Journal of Dairy Research · doi:10.1017/s0022029920000655 · Cited by 4 · see citing works
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Plant Milk-Clotting Enzymes for Cheesemaking
Nicosia, F. D., Puglisi, I., Pino, A., Caggia, C., & Randazzo, C. L. (2022) · Foods · doi:10.3390/foods11060871 · Cited by 95 · see citing works
Zhang, Y., Hu, J., Wang, J., Liu, C., Liu, X., Sun, J., Song, X., & Wu, Y. (2023) · Journal of Dairy Science · doi:10.3168/jds.2023-23450 · Cited by 10 · 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
- Authors
- Meng, F., Zhao, H., Lu, F., Bie, X., Lu, Z., & Lu, Y.
- Affiliations
- Nanjing Agricultural University (CN)
- College of Food Science and Technology, Nanjing Agriculture University, 1 Weigang, Nanjing 210095, China
- Nanjing University of Finance and Economics (CN)
- Corresponding author
- Lu, Zhaoxin
- Lu, Yingjian
- Venue
- Journal of Agricultural and Food Chemistry
- Publisher
- American Chemical Society (ACS)
- Published
- 2021-02-25
- Volume
- 69
- Issue
- 9
- Pages
- 2784-2792
- ISSN
- 0021-8561 (ISSN-L)
- 1520-5118
- OpenAlex
- W3131219400
- Cited by
- 11
- References
- 35
- Citations by year
- 2026: 3 · 2025: 3 · 2023: 3 · 2022: 2
- FWCI
- 0.39
- Type
- journal article
- Language
- en
- Open access
- closed
- Licence
- Locations
- Journal of Agricultural and Food Chemistry (publishedVersion)
- PubMed (publishedVersion)
- Funders
- National Natural Science Foundation of China (31771948, 32072182)
- Topics
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Hydrolysis and Bioactive Peptides
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
- Life Sciences › Agricultural and Biological Sciences › Food Science › Proteins in Food Systems
- Keywords
- semi-hard cheese, functional peptides, antioxidant peptides, angiotensin-converting enzyme inhibition, antihypertensive peptides, anticancer peptides, proteolytic activity
- MeSH terms
- Animals, Bacillus, Cheese, Milk, Peptides, Chymosin, Aspartic Acid Endopeptidases
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