Ion Exchange and Gel Filtration Chromatography Used in Isolation of Rennet Substitute

Enzymes that curdle milk are a key ingredient in cheesemaking. However, since traditional rennet comes from the stomachs of slaughtered calves, both its limited supply and the ethical issues surrounding its production have pushed researchers to look for bacteria-based alternatives. A paper published in Food Science & Nutrition¹ reports on a study where the purification (done using diethylaminoethyl [DEAE]-cellulose ion exchange chromatography followed by gel filtration chromatography) and characterization of a milk-clotting protease from Bacillus stercoris NCCP-3139, a soil isolate from Dera Ismail Khan, Pakistan.

Why Are Researchers Looking for Bacterial Alternatives to Traditional Animal-Derived Rennet in Cheesemaking?

Protein-breaking enzymes are key ingredients used across many industries, including dairy, food, and pharmaceuticals. A specific group of these enzymes plays a central role in cheesemaking — they work by breaking down a protein in milk that triggers it to thicken and form curds. Traditionally, this has been done using rennet sourced from animal stomachs, but growing cheese consumption worldwide, along with animal welfare concerns, has pushed researchers to look for non-animal alternatives. Enzymes derived from bacteria, especially from the Bacillus family, have emerged as strong candidates, since they're easy to produce in large quantities, hold up well under various conditions, and can be tweaked at the genetic level to improve.^2-4 performance.

B. stercoris is a soil-based bacterium known for producing enzymes that remain effective at high temperatures and in alkaline conditions, making it useful in food processing. These protein-breaking enzymes are valued in industry because they can function across a wide range of temperatures and acidity levels. When it comes to cheesemaking, the ideal bacterial enzyme should be good at curdling milk without being too aggressive at breaking down other proteins, since excessive protein breakdown can ruin the texture of cheese and affect the amount produced. This is why scientists are actively searching for, refining, and studying new bacterial enzymes, with the goal of finding ones that are both effective and affordable for use in dairy production.^5,6

Can the Enzyme Produced by B. stercoris NCCP-3139 Replace Animal Rennet in Cheesemaking?

For this study, milk-clotting activity was assessed in goat, sheep, cow, buffalo, and camel milk. The researchers found that milk from sheep and buffalo curdled better than milk from other animals. After purifying the enzyme, it became much more potent and was significantly better at clotting milk without breaking down other proteins unnecessarily. The enzyme worked best at 55 °C and a slightly alkaline pH, was given a boost by certain minerals (manganese and strontium), but was effectively shut down by EDTA, mercury, and zinc. Laboratory analysis confirmed it's a simple, single-unit enzyme. Overall, the bacteria B. stercoris NCCP-3139 produces an enzyme that is precise and effective enough to potentially replace animal-derived rennet in cheesemaking.

“This study,” write the authors of the paper,¹ “adds to the growing body of research on microbial milk-clotting enzymes by identifying Bacillus stercoris NCCP-3139 as a source of a protease with relatively favorable clotting selectivity.”

The researchers state, however, that before this can be confidently used on an industrial scale, more testing is needed, such as studying reaction speeds and running real cheese-making trials.¹

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References

1. Sibtain, M.; Qurtam, A. A.; Javaria, S. et al. Milk-Clotting Protease From Bacillus stercoris NCCP-3139: A Potential Microbial Rennet for Cheese Production. Food Sci Nutr. 2026, 14, e71864. DOI: 10.1002/fsn3.71864
2. Bilal, M.; Iqbal, H. M. N. State-of-the-Art Strategies and Applied Perspectives of Enzyme Biocatalysis in Food Sector - Current Status and Future Trends. Crit Rev Food Sci Nutr. 2020, 60 (12), 2052-2066. DOI: 10.1080/10408398.2019.1627284
3. Zhang, X.; Tao, L.; Wei, G. et al. Plant-Derived Rennet: Research Progress, Novel Strategies for their Isolation, Identification, Mechanism, Bioactive Peptide Generation, and Application in Cheese Manufacturing. Crit Rev Food Sci Nutr. 2025, 65 (3), 444-456. DOI: 10.1080/10408398.2023.2275295
4. Zhang, T.; Yang, Z.; Zhang, Y. et al. Proteomics-Guided Isolation of a Novel Serine Protease with Milk-Clotting Activity from Tamarillo (Solanum betaceum Cav.). Food Chem. 2025, 465 (Pt 1), 141956. DOI: 10.1016/j.foodchem.2024.141956
5. Banerjee, G.; Ray, A. K. Impact of Microbial Proteases on Biotechnological Industries. Biotechnol Genet Eng Rev. 2017, 33 (2), 119-143. DOI: 10.1080/02648725.2017.1408256
6. Ali, T. H., Mohamed, L. A., Abdellah, E. M. et al. Biochemical and Functional Characterization of a Penicillium purpurescens Milk-Clotting Enzyme as an Animal Rennet Alternative. World Acad. Sci. J. 2025, 7 (6), 111. DOI: 10.3892/wasj.2025.399