Comparative evaluation of casein hydrolysates obtained by chymotrypsin and starter-culture hydrolysis under optimized conditions
DOI:
https://doi.org/10.5219/scifood.117Keywords:
casein, degree of hydrolysis, chymotrypsin, starter cultures, Lactobacillus acidophilusAbstract
Hydrolysis parameters are critical in the production of protein hydrolysates, as they directly determine the degree of hydrolysis (DH), which in turn governs the resulting technological properties. The aim of the research is to conduct comparative studies of the properties of casein hydrolysates obtained using optimized hydrolysis parameters with chymotrypsin and acidophilic bacteria. The resulting hydrolysates were characterized for their chemical composition using standard methods, chemical structure by Fourier-transform infrared (FTIR) spectroscopy, particle size distribution using a particle size analyzer, and technological indicators, including oil-holding capacity, water-holding capacity, oil-emulsifying capacity, and solubility. The results indicated that the casein hydrolysate samples exhibited no significant differences in moisture content and only slight variations in protein content. FTIR spectral analysis revealed similar profiles across all samples, indicating the presence of the same key functional groups, albeit at different relative concentrations. These spectroscopic findings corroborated the degree of hydrolysis: the hydrolysate produced with chymotrypsin, which exhibited the highest DH, also demonstrated the greatest absorbance intensity. This suggests a more extensive accumulation of hydrolysis products, including peptides. Particle size analysis confirmed that this sample contained a larger proportion of smaller particles, with 93.67% of the particles measuring between 43 and 85.9 nm. The technological characterization revealed a clear dependence on the degree of hydrolysis. The sample with the highest DH (sample 6) displayed a lower oil-binding capacity (21.08%). Water-holding capacity did not vary substantially across samples, remaining relatively low (26.13% to 30.20%). Hydrolysates with a higher DH exhibited a reduced emulsifying capacity. Conversely, solubility increased progressively with increasing DH, ranging from 89.09% to 94.16%. In conclusion, these findings underscore that to obtain a protein ingredient with tailored techno-functional properties, it is essential to conduct controlled hydrolysis to a specific degree.
References
1. Secchi, G., Amalfitano, N., Cecchinato, A., Lante, A., Pegolo, S., Dettori, M. L., Pazzola, M., Vacca, G. M., & Bittante, G. (2025). Detection and quantification of true proteins, casein fractions, and their genetic variants, and whey proteins in goat milk by Reverse-Phase High-Performance Liquid Chromatography. Journal of Dairy Science. Advance online publication. https://doi.org/10.3168/jds.2024-25514
2. Zhou, S., Xu, T., Zhang, X., Luo, J., An, P., & Luo, Y. (2022). Effect of casein hydrolysate on cardiovascular risk factors: A systematic review and meta-analysis of randomized controlled trials. Nutrients, 14(19), Article 4207. https://doi.org/10.3390/nu14194207
3. Huppertz, T., Fox, P. F., & Kelly, A. L. (2018). The caseins: Structure, stability, and functionality. In Proteins in Food Processing, 2nd ed., 49–92. Woodhead Publishing. https://doi.org/10.1016/B978-0-08-100722-8.00004-8
4. Runthala, A., Mbye, M., Ayyash, M. M., Xu, Y., & Kamal-Eldin, A. (2023). Caseins: Versatility of their micellar organization in relation to the functional and nutritional properties of milk. Molecules, 28(5), Article 2023. https://doi.org/10.3390/molecules28052023
5. Du, Z., Xu, N., Yang, Y.-C., Li, G.-H., Tai, Z., Li, N., & Sun, Y.-Y. (2022). Study on internal structure of casein micelles in reconstituted skim milk powder. International Journal of Biological Macromolecules, 224, 437–452. https://doi.org/10.1016/j.ijbiomac.2022.10.135
6. Tenenbaum, M., Deracinois, B., Dugardin, C., Matéos, A., Romelard, A., Auger, J., Boulier, A., Ravallec, R., Flahaut, C., & Cudennec, B. (2022). Identification, production and bioactivity of casein phosphopeptides - A review. Food Research International, 157, Article 111360. https://doi.org/10.1016/j.foodres.2022.111360
7. Broyard, C., & Gaucheron, F. (2015). Modifications of structures and functions of caseins: A scientific and technological challenge. Dairy Science & Technology, 95(6), 831–862. https://doi.org/10.1007/S13594-015-0220-Y
8. Chourasia, R., Phukon, L. C., Singh, S. P., Rai, A. K., & Sahoo, D. (2020). Role of enzymatic bioprocesses for the production of functional food and nutraceuticals. In Biomass, Biofuels, Biochemicals (pp. 315–342). Elsevier. https://doi.org/10.1016/B978-0-12-819820-9.00015-6
9. Moita, T., Pedroso, L., Santos, I., & Lima, A. (2025). Casein and casein-derived peptides: Antibacterial activities and applications in health and food systems. Nutrients, 17(10), Article 1615. https://doi.org/10.3390/nu17101615
10. Ren, R., Liu, L., Li, X., Zhang, X., Xu, M., Zhang, Z., Wang, X., Mu, B., Zhang, X., Zhou, A. (2026). Identification and in-silico screening of ACE-inhibitory peptides from casein hydrolysate via fermentation with different probiotics. International Dairy Journal, 172, Article 106425. https://doi.org/10.1016/j.idairyj.2025.106425
11. Coscueta, E. R., Batista, P., Gomes, J. E. G., da Silva, R., & Pintado, M. M. (2022). Screening of novel bioactive peptides from goat casein: In silico to in vitro validation. International Journal of Molecular Sciences, 23(5), Article 2439. https://doi.org/10.3390/ijms23052439
12. Meleti, E., Koureas, M., Manouras, A., Giannouli, P., & Malissiova, E. (2025). Bioactive peptides from dairy products: A systematic review of advances, mechanisms, benefits, and functional potential. Dairy, 6(6), 65. https://doi.org/10.3390/dairy6060065
13. Saubenova, M., Oleinikova, Y., Rapoport, A., Maksimovich, S., Yermekbay, Z., & Khamedova, E. (2024). Bioactive peptides derived from whey proteins for health and functional beverages. Fermentation, 10(7), 359. https://doi.org/10.3390/fermentation10070359
14. Basavaprabhu Haranahalli Nataraj, & Behare, P. V. (2026). In vivo studies on isoleucine-proline-proline-rich peptides derived from milk protein hydrolysates. In S. Hati & S. Mandal (Eds.), Non-Bovine Milk Derived Bioactive Peptides (pp. 153–166). Academic Press. https://doi.org/10.1016/B978-0-443-23561-0.00007-4
15. Jiang, Y., Li, S., Yuan, R., Tuo, Y., Mu, G., & Jiang, S. (2024). Preparation of casein hydrolysate by two-step enzymatic hydrolysis and its immunomodulatory activities in vitro and in vivo. Food Bioscience, 59, Article 104007. https://doi.org/10.1016/j.fbio.2024.104007
16. Ryan, G., Nongonierma, A. B., O’Regan, J., & FitzGerald, R. J. (2018). Functional properties of bovine milk protein isolate and associated enzymatic hydrolysates. International Dairy Journal, 81, 113–121. https://doi.org/10.1016/j.idairyj.2018.01.013
17. Kaur, S., Vasiljevic, T., & Huppertz, T. (2023). Influence of actinidin-induced hydrolysis on the functional properties of milk protein and whey protein concentrates. Foods, 12(20), 3806. https://doi.org/10.3390/foods12203806
18. Zinina, O. V., Chanov, I. M., Rebezov, M. B., Li, Y., Li, C., & Zhao, Y. (2025). Optimization of technological parameters of casein hydrolysis. Food Systems, 8(4), 616–623. https://doi.org/10.21323/2618-9771-2025-8-4-616-623
19. Assaad, H., Zhou, L., Carroll, R. J., & Wu, G. (2014). Rapid publication-ready MS-Word tables for one-way ANOVA. SpringerPlus, 3, Article 474. https://doi.org/10.1186/2193-1801-3-474
20. Santos, B. A. S., Azambuja, S. P. H., Ávila, P. F., Pacheco, M. T. B., & Goldbeck, R. (2020). n-Butanol production by Saccharomyces cerevisiae from protein-rich agro-industrial by-products. Brazilian Journal of Microbiology, 51, 1655–1664. https://doi.org/10.1007/s42770-020-00370-6
21. Mirzaei Teshnizi, Z., Robatjazi, S. M., & Mosaabadi, J. M. (2020). Optimization of the enzymatic hydrolysis of poultry slaughterhouse wastes using alcalase enzyme for the preparation of protein hydrolysates. Applied Food Biotechnology, 7(3), 153–160. https://doi.org/10.22037/afb.v7i3.28417
22. López-Medina, F. A., Dublán-García, O., Morachis-Valdez, A. G., Saucedo-Vence, K., López-García, G., Díaz-Bandera, D., & Gómez-Espinoza, R. M. (2025). Biopolymeric hydrolysates from Dosidicus gigas: Functional applications and shelf-life extension in squid sausages. Polymers, 17(7), 839. https://doi.org/10.3390/polym17070839
23. Djellouli, M., López-Caballero, M. E., Arancibia, M. Y., Karam, N., & Martínez-Alvarez, O. (2019). Antioxidant and antimicrobial enhancement by reaction of protein hydrolysates derived from shrimp by-products with glucosamine. Waste and Biomass Valorization, 11, 2491–2505. https://doi.org/10.1007/s12649-019-00607-y
24. Cui, Q., Sun, Y., Zhou, Z., Cheng, J., & Guo, M. (2021). Effects of enzymatic hydrolysis on physicochemical properties and solubility and bitterness of milk protein hydrolysates. Foods, 10(10), 2462. https://doi.org/10.3390/foods10102462
25. Wilkins, D. K., Grimshaw, S. B., Receveur, V., Dobson, C. M., Jones, J. A., & Smith, L. J. (1999). Hydrodynamic radii of native and denatured proteins measured by pulse field gradient NMR techniques. Biochemistry, 38(50), 16424–16431. https://doi.org/10.1021/bi991765q
26. Vreeke, G. J. C., Vincken, J.-P., & Wierenga, P. A. (2023). The path of proteolysis by bovine chymotrypsin. Food Research International, 165, 112485. https://doi.org/10.1016/j.foodres.2023.112485
27. Rubio-Reyes, A. Y., Delgado-Enciso, I., Casas, E., Garza-Brenner, E., & Sifuentes-Rincón, A. M. (2025). Influence of the Casein Genotype on Goat Milk Bioactivity: An In Silico Analysis of the Casein Peptidome. Molecules, 30(12), 2601. https://doi.org/10.3390/molecules30122601
28. Pescuma, M., Espeche Turbay, M.B., Mozzi, F., Font de Valdez, G., Savoy de Giori, G., & Hebert E. M. (2013). Diversity in proteinase specificity of thermophilic lactobacilli as revealed by hydrolysis of dairy and vegetable proteins. Applied Microbiology and Biotechnology, 97(17), 7831–7844. https://doi.org/10.1007/s00253-013-5037-0
29. Fira, D., Kojic, M., Banina, A., Spasojevic, I., Strahinic, I., & Topisirovic, L. (2001). Characterization of cell envelope-associated proteinases of thermophilic lactobacilli. Journal of Applied Microbiology, 90(1), 123–130. https://doi.org/10.1046/j.1365-2672.2001.01226.x
30. Murtaza, M. A., Irfan, S., Hafiz, I., Ranjha, M. M. A. N., Rahaman, A., Murtaza, M. S., Ibrahim, S. A., & Siddiqui, S. A. (2022). Conventional and Novel Technologies in the Production of Dairy Bioactive Peptides. Frontiers in Nutrition, 9, 780151. https://doi.org/10.3389/fnut.2022.780151
31. Liu, Y., Nawazish, H., Farid, M.S., Abdul Qadoos, K., Habiba, U.E., Muzamil, M., Tanveer, M., Sienkiewicz, M., Lichota, A., & Łopusiewicz, Ł. (2024). Health-Promoting Effects of Lactobacillus acidophilus and Its Technological Applications in Fermented Food Products and Beverages. Fermentation, 10, 380. https://doi.org/10.3390/fermentation10080380
32. Gruppi, A., Dermiki, M., Spigno, G., & FitzGerald, R.J. (2022). Impact of Enzymatic Hydrolysis and Heat Inactivation on the Physicochemical Properties of Milk Protein Hydrolysates. Foods, 11, 516. https://doi.org/10.3390/foods11040516
33. Kubyshkin, V., & Budisa, N. (2017). Hydrolysis, polarity, and conformational impact of C-terminal partially fluorinated ethyl esters in peptide models. Beilstein Journal of Organic Chemistry, 13, 2442–2457. https://doi.org/10.3762/bjoc.13.241
34. Roman, J.A., & Sgarbieri, V.C. (2005). Efeito da hidrólise enzimática sobre propriedades funcionais de caseína bovina coagulada pela ação da quimosina. Ciência e Tecnologia de Alimentos, 25(3), 468–474. https://www.redalyc.org/articulo.oa?id=395940075013
35. Sindayikengera, S., & Wen-Shui, X. (2006). Enzymatic Hydrolysis of Caseins by Two Different Proteases and Their Effect on Functional Properties of Resulting Protein Hydrolysates[J]. Food Science, 27(2), 64-71. https://www.spkx.net.cn/EN/
36. Ewert, J., Glück, C., Zeeb, B., Weiss, J., Stressler, T., & Fischer, L. (2018). Modification of the interfacial properties of sodium caseinate using a commercial peptidase preparation from Geobacillus stearothermophilus. Food Hydrocolloids, 81, 60-70. https://doi.org/10.1016/j.foodhyd.2018.02.036.
37. Zheng, Z., Li, J., & Liu, Y. (2020). Effects of partial hydrolysis on the structural, functional and antioxidant properties of oat protein isolate. Food & Function, 11(4), 3144–3155. https://doi.org/10.1039/c9fo01783f
38. Troncoso, R., Torrado, A., Pérez-Guerra, N., & Tovar, C. A. (2024). Viscoelastic properties of biscuit doughs with different lipidic profiles fortified with a casein hydrolysate. Macromol, 4(2), 437-447. https://doi.org/10.3390/macromol4020025
39. Karpukhina, P. A., & Krasnoshtanova, A. A. (2021). Protein fractions poultry egg white hydrolisis and measurement of its functional and technological properties. Success in Chemistry and Chemical Technology, 35(12), 76–78. (in Russian)
40. Padial-Domínguez, M., Espejo-Carpio, F. J., Pérez-Gálvez, R., Guadix, A., & Guadix, E. M. (2020). Optimization of the emulsifying properties of food protein hydrolysates for the production of fish oil-in-water emulsions. Foods, 9(5), 636. https://doi.org/10.3390/foods9050636
41. Klompong, V., Benjakul, S., Yachai, M., Visessanguan, W., Shahidi, F., & Hayes, K. D. (2009). Amino acid composition and antioxidative peptides from protein hydrolysates of yellow stripe trevally (Selaroides leptolepis). Journal of Food Science, 74(2), C126-C133. https://doi.org/10.1111/j.1750-3841.2009.01047.x
42. Badfar, N., Jafarpour, A., Casanova, F., Sales Queiroz, L., Tilahun Getachew, A., Jacobsen, C., Jessen, F., & Gringer, N. (2025). Influence of supercritical fluid extraction process on techno-functionality of enzymatically derived peptides from filter-pressed shrimp waste. Marine Drugs, 23(3), 122. https://doi.org/10.3390/md23030122
43. Schröder, A., Berton-Carabin, C., Venema, P., & Cornacchia, L. (2017). Interfacial properties of whey protein and whey protein hydrolysates and their influence on O/W emulsion stability. Food Hydrocolloids, 73, 129–140. https://doi.org/10.1016/j.foodhyd.2017.06.001
44. Klompong, V., Benjakul, S., Kantachote, D., & Shahidi, F. (2007). Antioxidative activity and functional properties of protein hydrolysate of yellow stripe trevally (Selaroides leptolepis) as influenced by the degree of hydrolysis and enzyme type. Food Chemistry, 102(4), 1317–1327. https://doi.org/10.1016/j.foodchem.2006.07.016
45. Gani, A., Broadway, A.A., Masoodi, F.A., Wani, A.A., Maqsood, S., Ashwar, B.A., Shah, A., Rather, S.A., & Gani, A. (2015). Enzymatic hydrolysis of whey and casein protein- effect on functional, rheological, textural and sensory properties of breads. Journal of Food Science and Technology, 52, 7697–7709. https://doi.org/10.1007/s13197-015-1840-1
46. Peighambardoust, S. H., Karami, Z., Pateiro, M., & Lorenzo, J. M. (2021). A review on health-promoting, biological, and functional aspects of bioactive peptides in food applications. Biomolecules, 11(5), 631. https://doi.org/10.3390/biom11050631
47. Rajarathnam, E., Nongonierma, A. B., O'Sullivan, D., Flynn, C., & FitzGerald, R. J. (2016). Impact of enzyme preparation and degree of hydrolysis on peptide profile and nitrogen solubility of sodium caseinate hydrolysates. International Journal of Food Science and Technology, 51(9), 2123–2131. https://doi.org/10.1111/ijfs.13191
48. Ye, T., Kong, X., Sun, L., Ding, X., & Hua, Y. (2015). Effects of protease and hydrolysis conditions on the properties of casein hydrolysates[J]. Science and Technology of Food Industry, 19, 181–185. https://doi.org/10.13386/j.issn1002-0306.2015.19.030
49. Cingöz, A., & Yildirim, M. (2023). Effects of hydrolysis degree on the functional properties of hydrolysates from sour cherry kernel protein concentrate. Foods and Raw Materials, 11(2), 197–205. https://doi.org/10.21603/2308-4057-2023-2-566
Downloads
Published
Issue
Section
License
Copyright (c) 2026 Oksana Zinina, Ilya Chanov, Maksim Rebezov, Akbota Itbalakova (Author)
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
Articles are published under the Creative Commons Attribution–NonCommercial–NoDerivatives 4.0 International License (CC BY-NC-ND 4.0).
This license permits non-commercial use, sharing, distribution, and reproduction of the work in any medium or format, provided that:
-
appropriate credit is given to the author(s) and the original publication in Scifood,
-
a link to the license is provided,
-
the work is not modified, adapted, or transformed.
