Screening of a Bacillus subtilis strain producing both nattokinase and milk-clotting enzyme and its application in fermented milk with thrombolytic activity
Open accessAbstract
Bacillus subtilis is a generally recognized as safe probiotic, which is used as a starter for natto fermentation. Natto is a functional food with antithrombus function due to nattokinase. Compared with natto, fermented milk is a more popular fermented food, which is commonly fermented by Lactobacillus bulgaricus and Streptococcus. However, there is no report on B. subtilis-fermented milk. In this study, to produce a functional fermented milk with antithrombus function, a B. subtilis strain (B. subtilis JNFE0126) that produced both nattokinase and milk-clotting enzyme was isolated from traditionally fermented natto and used as the starter for the functional fermented milk. In liquid fermentation culture, the peak values of thrombolytic activity and milk-clotting activity were 3,511 U/mL at 96 h and 874.5 Soxhlet unit/mL at 60 h, respectively. The optimal pH and temperature were pH 7.0 at 40°C for nattokinase and pH 6.5 and 55°C for milk-clotting enzyme, respectively. The thrombolytic activity in the fermented milk reached 215.1 U/mL after 8 h of fermentation. Sensory evaluation showed that the acceptance of the milk fermented by B. subtilis JNFE0126 was similar to the traditional milk fermented by L. bulgaricus and S. thermophilus. More importantly, oral intake of the fermented milk by the thrombosis-model mice prevented the development of thrombosis. Our results suggest that B. subtilis JNFE0126-fermented milk has potential as a novel, functional food in the prevention of thrombosis-related cardiovascular diseases.
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B. subtilis JNFE0126 possesses milk coagulation properties, forming a firm curd after 8 h of fermentation with no noticeable whey separation.
“However, a study by Zhang et al. (2021) showed that B. subtilis JNFE0126 possesses milk coagulation properties, forming a firm curd after 8 h of fermentation with no noticeable whey separation observed.”
From Invited review: Milk-clotting enzymes of microbial origin and their role in cheesemaking-A review. (2025) · cites this paper as Zhang et al., 2021
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- Authors
- Zhang, X., Tong, Y., Wang, J., Lyu, X., & Yang, R.
- Affiliations
- Jiangnan University (CN)
- Corresponding author
- Yang, Ruijin
- Venue
- Journal of Dairy Science
- Publisher
- American Dairy Science Association
- Published
- 2021-09
- Volume
- 104
- Issue
- 9
- Pages
- 9437-9449
- ISSN
- 0022-0302 (ISSN-L)
- OpenAlex
- W3182911774
- Cited by
- 33
- References
- 30
- Citations by year
- 2026: 5 · 2025: 7 · 2024: 10 · 2023: 5 · 2022: 6
- FWCI
- 3.14
- Type
- journal article
- Language
- en
- Open access
- bronze
- doi.org
- Locations
- Journal of Dairy Science (publishedVersion)
- PubMed (publishedVersion)
- Topics
- Life Sciences › Agricultural and Biological Sciences › Food Science › Probiotics and Fermented Foods
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Biotechnology › Enzyme Production and Characterization
- Life Sciences › Biochemistry, Genetics and Molecular Biology › Molecular Biology › Protein Hydrolysis and Bioactive Peptides
- Keywords
- Bacillus subtilis, nattokinase, milk-clotting enzyme, fermented milk, thrombolytic activity, probiotic starter cultures, natto, thromboprophylaxis, cardiovascular disease, sensory evaluation
- MeSH terms
- Animals, Bacillus subtilis, Fermentation, Milk, Subtilisins, Aspartic Acid Endopeptidases, Mice
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