foodsystFood systemsПищевые системы2618-97712618-7272Федеральный научный центр пищевых систем им. В.М. Горбатова РАН10.21323/2618-9771-2025-8-4-616-623foodsyst-927Research ArticleСтатьиOptimization of technological parameters of casein hydrolysisОптимизация технологических параметров гидролиза казеинаhttps://orcid.org/0000-0003-4817-1645ЗининаО. В.ZininaO. V.

Зинина Оксана Владимировна — доктор технических наук, профессор, кафедра «Пищевые и биотехнологии»

454080, Челябинск, пр. Ленина, 76 

Oksana V. Zinina, Doctor of Technical Sciences, Professor, Department of Food and Biotechnology

76, Lenin av., 454080, Chelyabinsk

zininaov@susu.ruhttps://orcid.org/0000-0001-7484-3252ЧановИ. М.ChanovI. M.

Чанов Илья Михайлович — аспирант, кафедра «Пищевые и биотехнологии»

454080, Челябинск, пр. Ленина, 76 

Ilya M. Chanov, Postgraduate Student, Department of Food and Biotechnology

76, Lenin av., 454080, Chelyabinsk

chanovi2000@mail.ruhttps://orcid.org/0000-0003-0857-5143РебезовМ. Б.RebezovM. B.

Ребезов Максим Борисович — доктор сельскохозяйственных наук, профессор, главный научный сотрудник

109316, Москва, ул. Талалихина, 26 

Maksim B. Rebezov, Doctor of Agricultural Sciences, Professor, Chief Researcher

26, Talalikhin str., Moscow, 109316

rebezov@ya.ruhttps://orcid.org/0000-0001-8758-5512ЛиЯ.LiY.

Ли Ян — профессор, доктор технических наук, Колледж пищевых наук, Школа питания

150036, Харбин, Дорога Чанцзян, 600 

Yang Li, Professor, Doctor of Engineering, College of Food Science, School of Food

150036, Harbin, Changjiang Road, 600 

liyanghuangyu@163.comhttps://orcid.org/0000-0003-1603-7222ЛиЧ.LiCh.

Ли Чунь — доктор продовольственных наук, профессор, колледж продовольственных наук

150036, Харбин, Дорога Чанцзян, 600 

Chun Li, Doctor of food Science, Professor, College of Food Science

150036, Harbin, Changjiang Road, 600 

lchun@neau.edu.cnhttps://orcid.org/0000-0002-7061-779XЧжаоИ.ZhaoY.

Чжао Инин — кандидат в докторантуру, колледж продовольственных наук

150036, Харбин, Дорога Чанцзян, 600 

Yingying Zhao, Doctoral Candidate, College of Food Science

150036, Harbin, Changjiang Road, 600 

zyy210125@126.comЮжно-Уральский государственный университет (национальный исследовательский университет)РоссияSouth Ural State UniversityRussian FederationФедеральный научный центр пищевых систем им. В. М. ГорбатоваРоссияV. M. Gorbatov Federal Research Center for Food SystemsRussian FederationСеверо-Восточный сельскохозяйственный университетКитайNortheasts Agricultural UniversityChina20252801202684616623Copyright © Zinina O.V., Chanov I.M., Rebezov M.B., Li Y., Li C., Zhao Y., 20262026Зинина О.В., Чанов И.М., Ребезов М.Б., Ли Я., Ли Ч., Чжао И.Zinina O.V., Chanov I.M., Rebezov M.B., Li Y., Li C., Zhao Y.Данная работа распространяется под лицензией Creative Commons Attribution 4.0.This work is licensed under a Creative Commons Attribution 4.0 License.https://www.fsjour.com/jour/article/view/927

Milk proteins are valuable raw materials for obtaining protein hydrolysates and active peptides formed during hydrolysis. Various proteolytic enzymes and bacteria are most often used for hydrolysis. Important parameters for regulating the degree of protein hydrolysis are duration, temperature of hydrolysis and concentration of a biological agent. The aim of the study was to establish optimum parameters for casein hydrolysis with chymotrypsin and bacterial starter of acidophilus bacillus. A three-factor experiment was used to determine optimum conditions for the hydrolysis process. Three factors were varied during the experiment: hydrolysis time (from 6 to 26 h) with a step of 10 h; temperature (from 32 to 42 °C) with a step of 5 °C; starter amount (from 0.5 to 1.5 %) with a step of 0.5 %. The dependent variable was the degree of hydrolysis. The content of total nitrogen in casein was determined by the Kjeldahl method, and that of amine nitrogen was determined by formol titration. The influence of independent variables on the dependent variable was analyzed using MathCAD, where response surfaces were also constructed. For the obtained multiple regression equations, the authors determined the reliability of the equation by the determination coefficient (R2) and Fisher’s criterion (Fkr), and the statistical significance of the parameters of the multiple regression equation by the Student’s criterion (t). Two regression equations were obtained for the process of casein hydrolysis with chymotrypsin and bacterial starter. Determination coefficients were established, confirming the high significance of the models. Response surfaces were constructed showing the dependence of the hydrolysis degree on the hydrolysis parameters. The optimum parameters of hydrolysis with the enzyme chymotrypsin and acidophilus starter were established. For example, when using chymotrypsin with a hydrolysis duration of 21.99 h, a temperature of 43.3 °C, and an enzyme amount of 0.02 %, the hydrolysis degree will be 28.6 % and when using acidophilus starter with a hydrolysis duration of 31.1 h, a temperature of 35.8 °C, and an amount of starter of 1.5 %, the hydrolysis degree will be 17.33 %.) Thus, the results of the multifactorial experiment made it possible to establish the optimum hydrolysis parameters for obtaining casein hydrolysates with chymotrypsin and acidophilus starter.

Молочные белки являются ценным сырьем для получения белковых гидролизатов и активных пептидов, образуемых при гидролизе. Наиболее часто для проведения гидролиза используют различные протеолитические ферменты и бактерии. Важными параметрами регулирования степени гидролиза белков являются продолжительность, температура гидролиза и концентрация биологического агента. Целью исследований является установление оптимальных параметров гидролиза казеина химотрипсином и бактериальной закваской ацидофильной палочки. Для определения оптимальных условий процесса гидролиза использовали трехфакторный эксперимент. В ходе эксперимента варьировались три фактора: время гидролиза (от 6 до 26 ч) с шагом 10 ч; температура (от 32 до 42 °C) с шагом 5 °C; количество закваски (от 0,5 до 1,5 %) с шагом 0,5 %. Зависимой переменной являлась степень гидролиза. Содержание общего азота в казеине определяли методом Кьельдаля, аминного азота — формольным титрованием. Влияние независимых переменных на зависимую переменную анализировали с помощью MathCAD, где также были построены поверхности отклика. Для полученных уравнений множественной регрессии определяли надежность уравнения по коэффициенту детерминации (R2) и критерию Фишера (Fkr), статистическую значимость параметров уравнения множественной регрессии по критерию Стьюдента (t). Получено два уравнения регрессии процесса гидролиза казеина химотрипсином и бактериальной закваской. Установлены коэффициенты детерминации, подтверждающие высокую значимость моделей. Построены поверхности отклика, показывающие зависимость степени гидролиза от параметров гидролиза, установлены оптимальные параметры гидролиза ферментом химотрипсином (например, при продолжительности гидролиза 21,99 ч, температуре 43,3 °C, количестве фермента 0,02 % степень гидролиза составит 28,6 %) и закваской ацидофильной палочки (например, при продолжительности гидролиза 31,1 ч, температуре 35,8 °C, количестве закваски 1,5 % степень гидролиза составит 17,33 %). Таким образом, результаты проведенного многофакторного эксперимента позволили установить оптимальные параметры гидролиза для получения гидролизатов казеина химотрипсином и закваской ацидофильной палочки.

казеинстепень гидролизамикробная ферментацияацидофильная палочкауравнение регрессииcaseindegree of hydrolysismicrobial fermentationacidophilus bacillusregression equationReferencesZaky, A. A., Simal-Gandara, J., Eun, J. B., Shim, J. H., Abd El-Aty, A. M. (2022). Bioactivities, applications, safety, and health benefits of bioactive peptides from food and by-Products: A review. Frontiers in Nutrition, 8, Article 815640. https://doi.org/10.3389/fnut.2021.815640Zaky, A. A., Simal-Gandara, J., Eun, J. B., Shim, J. H., Abd El-Aty, A. M. (2022). Bioactivities, applications, safety, and health benefits of bioactive peptides from food and by-Products: A review. Frontiers in Nutrition, 8, Article 815640. https://doi.org/10.3389/fnut.2021.815640Lorenzo, J. M., Munekata, P. E. S., Gómez, B., Barba, F. J., Mora, L., Pérez-Santaescolástica, C. et al. (2018). Bioactive peptides as natural antioxidants in food products — A review. Trends in Food Science and Technology, 79, 136–147. https://doi.org/10.1016/j.tifs.2018.07.003Lorenzo, J. M., Munekata, P. E. S., Gómez, B., Barba, F. J., Mora, L., Pérez-Santaescolástica, C. et al. (2018). Bioactive peptides as natural antioxidants in food products — A review. Trends in Food Science and Technology, 79, 136–147. https://doi.org/10.1016/j.tifs.2018.07.003Nielsen, S.D., Beverly, R.L., Qu, Y., Dallas, D.C. (2017). Milk bioactive peptide database: A comprehensive database of milk protein-derived bioactive peptides and novel visualization. Food Chemistry, 232, 673–682. https://doi.org/10.1016/j.foodchem.2017.04.056Nielsen, S.D., Beverly, R.L., Qu, Y., Dallas, D.C. (2017). Milk bioactive peptide database: A comprehensive database of milk protein-derived bioactive peptides and novel visualization. Food Chemistry, 232, 673–682. https://doi.org/10.1016/j.foodchem.2017.04.056Najafian, L. (2023). A review of bioactive peptides as functional food ingredients: Mechanisms of action and their applications in active packaging and food quality improvement. Food and Function, 14, 5835–5857. https://doi.org/10.1039/D3FO00362KNajafian, L. (2023). A review of bioactive peptides as functional food ingredients: Mechanisms of action and their applications in active packaging and food quality improvement. Food and Function, 14, 5835–5857. https://doi.org/10.1039/D3FO00362KLemes, A. C., de Oliveira Filho, J. G., Fernandes, S. S., Gautério, G. V., Egea, M. B. (2023). Bioactive Peptides from Protein-Rich Waste. Chapter in a book: Agricultural Waste: Environmental Impact, Useful Metabolites and Energy Production. Sustainable Development and Biodiversity. Springer, Singapore, 2023. https://doi.org/10.1007/978-981-19-8774-8_6Lemes, A. C., de Oliveira Filho, J. G., Fernandes, S. S., Gautério, G. V., Egea, M. B. (2023). Bioactive Peptides from Protein-Rich Waste. Chapter in a book: Agricultural Waste: Environmental Impact, Useful Metabolites and Energy Production. Sustainable Development and Biodiversity. Springer, Singapore, 2023. https://doi.org/10.1007/978-981-19-8774-8_6Chourasia, 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. Chapter in a book: Biomass, Biofuels, Biochemicals, Elsevier, 2020. https://doi.org/10.1016/B978-0-12-819820-9.00015-6Chourasia, 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. Chapter in a book: Biomass, Biofuels, Biochemicals, Elsevier, 2020. https://doi.org/10.1016/B978-0-12-819820-9.00015-6Castañeda-Valbuena, D., Berenguer-Murcia, Á., Fernandez-Lafuente, R., Morellon-Sterling, R., Tacias-Pascacio, V. G. (2022). Biological activities of peptides obtained by pepsin hydrolysis of fishery products. Process Biochemistry, 120, 53–63. https://doi.org/10.1016/j.procbio.2022.05.029Castañeda-Valbuena, D., Berenguer-Murcia, Á., Fernandez-Lafuente, R., Morellon-Sterling, R., Tacias-Pascacio, V. G. (2022). Biological activities of peptides obtained by pepsin hydrolysis of fishery products. Process Biochemistry, 120, 53–63. https://doi.org/10.1016/j.procbio.2022.05.029Vaishnav, A., Mehta, N. K., Hussain, S. A., Acharya, P. Ch., Biswal, S., Nath, H. et al. (2025). Bromelain excised hydrolysates with potent bioactivity from Bellamya bengalensis soft tissues: Process optimization and characterization. Journal of Agriculture and Food Research, 19, Article 101595. https://doi.org/10.1016/j.jafr.2024.101595Vaishnav, A., Mehta, N. K., Hussain, S. A., Acharya, P. Ch., Biswal, S., Nath, H. et al. (2025). Bromelain excised hydrolysates with potent bioactivity from Bellamya bengalensis soft tissues: Process optimization and characterization. Journal of Agriculture and Food Research, 19, Article 101595. https://doi.org/10.1016/j.jafr.2024.101595Coscueta, 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/ijms23052439Coscueta, 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/ijms23052439Korhonen, H. (2009). Milk-derived bioactive peptides: From science to applications. Journal of Functional Foods, 1(2), 177–187. https://doi.org/10.1016/j.jff.2009.01.007Korhonen, H. (2009). Milk-derived bioactive peptides: From science to applications. Journal of Functional Foods, 1(2), 177–187. https://doi.org/10.1016/j.jff.2009.01.007Ugwu, C. P., Abarshi, M. M., Mada, S. B., Sanusi, B., Nzelibe, H. Ch. (2019). Camel and horse milk casein hydrolysates exhibit angiotensin converting enzyme inhibitory and antioxidative effects in vitro and in silico. International Journal of Peptide Research and Therapeutics 25, 1595–1604. https://doi.org/10.1007/s10989-018-09802-2Ugwu, C. P., Abarshi, M. M., Mada, S. B., Sanusi, B., Nzelibe, H. Ch. (2019). Camel and horse milk casein hydrolysates exhibit angiotensin converting enzyme inhibitory and antioxidative effects in vitro and in silico. International Journal of Peptide Research and Therapeutics 25, 1595–1604. https://doi.org/10.1007/s10989-018-09802-2Bamdad, F., Shin, S. H., Suh, J. -W., Nimalaratne, C., Sunwoo, H. (2017). Antiinflammatory and antioxidant properties of casein hydrolysate produced using high hydrostatic pressure combined with proteolytic enzymes. Molecules, 22(4), Article 609. https://doi.org/10.3390/molecules22040609Bamdad, F., Shin, S. H., Suh, J. -W., Nimalaratne, C., Sunwoo, H. (2017). Antiinflammatory and antioxidant properties of casein hydrolysate produced using high hydrostatic pressure combined with proteolytic enzymes. Molecules, 22(4), Article 609. https://doi.org/10.3390/molecules22040609Moita, T., Pedroso, L., Santos, I., Lima, A. (2025). Casein and casein-derived peptides: Antibacterial activities and applications in health and food systems. Nutrients, 17, Article 1615. https://doi.org/10.3390/nu17101615Moita, T., Pedroso, L., Santos, I., Lima, A. (2025). Casein and casein-derived peptides: Antibacterial activities and applications in health and food systems. Nutrients, 17, Article 1615. https://doi.org/10.3390/nu17101615Ren, R., Liu, L., Li, X., Zhang, X., Xu, M., Zhang, Z. et al. (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.2025.106425Ren, R., Liu, L., Li, X., Zhang, X., Xu, M., Zhang, Z. et al. (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.2025.106425Mada, S. B., Abaya, P. C., James, D. B., Abarshi, M. M., Tanko, M. S. (2020). Milkderived bioactive peptides with antiosteoporotic effect: A mini review. FUDMA Journal of Sciences, 4(3), 351–357. https://doi.org/10.33003/fjs-2020-0403-277Mada, S. B., Abaya, P. C., James, D. B., Abarshi, M. M., Tanko, M. S. (2020). Milkderived bioactive peptides with antiosteoporotic effect: A mini review. FUDMA Journal of Sciences, 4(3), 351–357. https://doi.org/10.33003/fjs-2020-0403-277Wang, X., Yuan, X., Yan, R., Song, J., Ren, C., Li, H. et al. (2025). Purification, characterization, and functional validation of a novel casein complex enzyme hydrolysate-binding calcium. Food Chemistry, 476, Article 143438. https://doi.org/10.1016/j.foodchem.2025.143438Wang, X., Yuan, X., Yan, R., Song, J., Ren, C., Li, H. et al. (2025). Purification, characterization, and functional validation of a novel casein complex enzyme hydrolysate-binding calcium. Food Chemistry, 476, Article 143438. https://doi.org/10.1016/j.foodchem.2025.143438Duan, C., Zhang, L., Wu, C., Zhang, Y., Ma, F., Li, X. et al. (2025). An exploration on the structural characteristics and antioxidant activities of casein hydrolysates fermented by Lactiplantibacillus plantarum A56 and A157. Food Bioscience, 65, Article 106069. https://doi.org/10.1016/j.fbio.2025.106069Duan, C., Zhang, L., Wu, C., Zhang, Y., Ma, F., Li, X. et al. (2025). An exploration on the structural characteristics and antioxidant activities of casein hydrolysates fermented by Lactiplantibacillus plantarum A56 and A157. Food Bioscience, 65, Article 106069. https://doi.org/10.1016/j.fbio.2025.106069Jauhiainen, T., Rönnback, M., Vapaatalo, H., Wuolle, K., Kautiainen, H., Korpela, R. (2007). Lactobacillus helveticus fermented milk reduces arterial stiffness in hypertensive subjects. International Dairy Journal, 17(10), 1209–1211. https://doi.org/10.1016/j.idairyj.2007.03.002Jauhiainen, T., Rönnback, M., Vapaatalo, H., Wuolle, K., Kautiainen, H., Korpela, R. (2007). Lactobacillus helveticus fermented milk reduces arterial stiffness in hypertensive subjects. International Dairy Journal, 17(10), 1209–1211. https://doi.org/10.1016/j.idairyj.2007.03.002Quirós, A., Ramos, M., Muguerza, B., Delgado, M. A., Miguel, M., Aleixandre, A. et al. (2007). Identification of novel antihypertensive peptides in milk fermented with Enterococcus faecalis. International Dairy Journal, 17(1), 33–41. https://doi.org/10.1016/j.idairyj.2005.12.011Quirós, A., Ramos, M., Muguerza, B., Delgado, M. A., Miguel, M., Aleixandre, A. et al. (2007). Identification of novel antihypertensive peptides in milk fermented with Enterococcus faecalis. International Dairy Journal, 17(1), 33–41. https://doi.org/10.1016/j.idairyj.2005.12.011Baars, T., van Esch, B., Diks, M., van Ooijen, L., Zhang, Z., Dekker, P. et al. (2025). Bacterial diversity, bioactive peptides, and enhanced immunomodulatory effects in raw milk kefir made with defined starter cultures versus backslopping. International Dairy Journal, 164, Article 106202. https://doi.org/10.1016/j.idairyj.2025.106202Baars, T., van Esch, B., Diks, M., van Ooijen, L., Zhang, Z., Dekker, P. et al. (2025). Bacterial diversity, bioactive peptides, and enhanced immunomodulatory effects in raw milk kefir made with defined starter cultures versus backslopping. International Dairy Journal, 164, Article 106202. https://doi.org/10.1016/j.idairyj.2025.106202Zhou, 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/nu14194207Zhou, 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/nu14194207Thomsen, J. P. S., Poulsen, N. A., Larsen, L. B. (2026). Effect of enzymatic dephosphorylation on in vitro digestion of purified bovine caseins. International Dairy Journal, 172, Article 106426. https://doi.org/10.1016/j.idairyj.2025.106426Thomsen, J. P. S., Poulsen, N. A., Larsen, L. B. (2026). Effect of enzymatic dephosphorylation on in vitro digestion of purified bovine caseins. International Dairy Journal, 172, Article 106426. https://doi.org/10.1016/j.idairyj.2025.106426Stokes, T., Hector, A. J., Morton, R. W., McGlory, C., Phillips, S. M. (2018). Recent perspectives regarding the role of dietary protein for the promotion of muscle hypertrophy with resistance exercise training. Nutrients, 10(2), Article 180. https://doi.org/10.3390/nu10020180Stokes, T., Hector, A. J., Morton, R. W., McGlory, C., Phillips, S. M. (2018). Recent perspectives regarding the role of dietary protein for the promotion of muscle hypertrophy with resistance exercise training. Nutrients, 10(2), Article 180. https://doi.org/10.3390/nu10020180Jiang, 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.2024.104007Jiang, 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.2024.104007Bueno-Gavilá E., Abellán A., Girón-Rodríguez F., Cayuela J. M., Salazar E., Gómez R. et al. (2019). Bioactivity of hydrolysates obtained from bovine casein using artichoke (Cynara scolymus L.) proteases. Journal of Dairy Science, 102(12), 10711–10723. https://doi.org/10.3168/jds.2019-16596Bueno-Gavilá E., Abellán A., Girón-Rodríguez F., Cayuela J. M., Salazar E., Gómez R. et al. (2019). Bioactivity of hydrolysates obtained from bovine casein using artichoke (Cynara scolymus L.) proteases. Journal of Dairy Science, 102(12), 10711–10723. https://doi.org/10.3168/jds.2019-16596Китаевская, С. В., Пономарев, В. Я., Решетник, О. А. (2022). Оценка протеолитической активности новых штаммов лактобацилл с криорезистентными свойствами. Известия вузов. Прикладная химия и биотехнология, 12(1), 76–86. https://doi.org/10.21285/2227-2925-2022-12-1-76-86Kitaevskaya, S. V., Ponomarev, V. Y., Reshetnik, O. A. (2022). Evaluation of the proteolytic activity of new cryoresistant lactobacillus strains. Proceedings of Universities. Applied Chemistry and Biotechnology, 12(1), 76–86. (In Russian) https://doi.org/10.21285/2227-2925-2022-12-1-76-86Zhao, L., Gao, P., Zhang, Y., Wang, X., lu, S., Yue, C. et al. (2024). Measurement of degree of hydrolysis and molecular weight distribution of protein hydrolysates by liquid chromatography-mass spectrometry. Talanta, 268(Part 1), Article 125347. https://doi.org/10.1016/j.talanta.2023.125347Zhao, L., Gao, P., Zhang, Y., Wang, X., lu, S., Yue, C. et al. (2024). Measurement of degree of hydrolysis and molecular weight distribution of protein hydrolysates by liquid chromatography-mass spectrometry. Talanta, 268(Part 1), Article 125347. https://doi.org/10.1016/j.talanta.2023.125347Cruz-Casas, D. E., Chávez-García, S. N., García-Flores, L. A., Martínez-Medina, G. A., Ramos-González, R., Prado-Barragán, L. A. et al. (2023). Bioactive peptides from fermented milk products. Chapter in a book: Foundations and Frontiers in Enzymology, Enzymes Beyond Traditional Applications in Dairy Science and Technology, Academic Press, 2023. https://doi.org/10.1016/B978-0-323-96010-6.00010-2Cruz-Casas, D. E., Chávez-García, S. N., García-Flores, L. A., Martínez-Medina, G. A., Ramos-González, R., Prado-Barragán, L. A. et al. (2023). Bioactive peptides from fermented milk products. Chapter in a book: Foundations and Frontiers in Enzymology, Enzymes Beyond Traditional Applications in Dairy Science and Technology, Academic Press, 2023. https://doi.org/10.1016/B978-0-323-96010-6.00010-2Doneva, M., Dyankova, S., Terziyska, M., Metodieva, P., Nacheva, I. (2024). Antioxidant protein hydrolysates from hemp seed oil cake — optimization of the process using response surface methodology. Applied Sciences, 14(19), Article 8602. https://doi.org/10.3390/app14198602Doneva, M., Dyankova, S., Terziyska, M., Metodieva, P., Nacheva, I. (2024). Antioxidant protein hydrolysates from hemp seed oil cake — optimization of the process using response surface methodology. Applied Sciences, 14(19), Article 8602. https://doi.org/10.3390/app14198602Santos, 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/s42770-020-00370-6Santos, 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/s42770-020-00370-6Mirzaei 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.v7i3.28417Mirzaei 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.v7i3.28417Fang, K.-X., Sun, X., Chen, L.-K., Wang, K., Yang, C.-J., Mei, S.-S. et al. (2025). A novel integrated strategy for discovering absorbable anticoagulant bioactive peptides: A case study on leech protein hydrolysates. Molecules, 30(15), Article 3184. https://doi.org/10.3390/molecules30153184Fang, K.-X., Sun, X., Chen, L.-K., Wang, K., Yang, C.-J., Mei, S.-S. et al. (2025). A novel integrated strategy for discovering absorbable anticoagulant bioactive peptides: A case study on leech protein hydrolysates. Molecules, 30(15), Article 3184. https://doi.org/10.3390/molecules30153184Остроумов, Л. А., Бабич, О. О., Милентьева, И. С. (2013). Оценка состава и физико-химических свойств ферментативных гидролизатов казеина. Вестник ВСГУТУ, 1(40), 82–85.Ostroumov, L. A., Babich, O. O., Milentyeva, I. S. (2013). Evaluation of composition and physicochemical properties of the enzyme casein hydrolysates. ESSUTM Bulletin,1(40), 82–85. (In Russian)Knuf, F., Caspers-Weiffenbach, R., Schieber, A., Fontana, A. (2025). Peptidomics profiling and biological activities of grape pomace protein hydrolysates. Food Chemistry, 463(Part 1), Article 141032. https://doi.org/10.1016/j.foodchem.2024.141032Knuf, F., Caspers-Weiffenbach, R., Schieber, A., Fontana, A. (2025). Peptidomics profiling and biological activities of grape pomace protein hydrolysates. Food Chemistry, 463(Part 1), Article 141032. https://doi.org/10.1016/j.foodchem.2024.141032Damen, D., Thibodeau, J., Gaaloul, S., Fliss, I., Labrie, S., Hamoudi, S. et al. (2024). Influence of enzymatic hydrolysis conditions on antimicrobial activities and peptide profiles of milk protein-derived hydrolysates from white wastewater. Cleaner Waste Systems, 9, Article 100172. https://doi.org/10.1016/j.clwas.2024.100172Damen, D., Thibodeau, J., Gaaloul, S., Fliss, I., Labrie, S., Hamoudi, S. et al. (2024). Influence of enzymatic hydrolysis conditions on antimicrobial activities and peptide profiles of milk protein-derived hydrolysates from white wastewater. Cleaner Waste Systems, 9, Article 100172. https://doi.org/10.1016/j.clwas.2024.100172Habib, M., Singh, S., Hanan, E., Jan, K., Bashir, K. (2025). Optimization of enzymatic hydrolysis for obtaining antioxidant hydrolysates from pumpkin seed protein: Improvement of the physicochemical, structural and functional properties. Applied Food Research, 5(2), Article 101272. https://doi.org/10.1016/j.afres.2025.101272Habib, M., Singh, S., Hanan, E., Jan, K., Bashir, K. (2025). Optimization of enzymatic hydrolysis for obtaining antioxidant hydrolysates from pumpkin seed protein: Improvement of the physicochemical, structural and functional properties. Applied Food Research, 5(2), Article 101272. https://doi.org/10.1016/j.afres.2025.101272Лепилкина, О. В., Григорьева, А. И. (2023). Ферментативный протеолиз при преобразовании молока в сыр. Пищевые системы, 6(1), 36–45. https://doi.org/10.21323/2618-9771-2023-6-1-36-45Lepilkina, O. V., Grigorieva, A. I. (2023). Enzymatic proteolysis during the conversion of milk into cheese. Food Systems, 6(1), 36–45. (In Russian) https://doi.org/10.21323/2618-9771-2023-6-1-36-45Lotfi, M., Keshvari, T., Taghizadeh, M. S., Afsharifar, A., Moghadam, A., Aram, F. et al. (2025). Optimizing Camelina sativa oil extraction and its cytotoxicity using RSM by emphasis on antioxidant properties, physical characteristics, and molecular docking insights. Industrial Crops and Products, 224, Article 120334. https://doi.org/10.1016/j.indcrop.2024.120334Lotfi, M., Keshvari, T., Taghizadeh, M. S., Afsharifar, A., Moghadam, A., Aram, F. et al. (2025). Optimizing Camelina sativa oil extraction and its cytotoxicity using RSM by emphasis on antioxidant properties, physical characteristics, and molecular docking insights. Industrial Crops and Products, 224, Article 120334. https://doi.org/10.1016/j.indcrop.2024.120334Ren, J., Yu, D., Li, N., Liu, S., Xu, H., Li, J. et al. (2023). Biological characterization and whole-genome analysis of Bacillus subtilis MG 1 isolated from mink fecal samples. Microorganisms, 11(12), Article 2821. https://doi.org/10.3390/microorganisms11122821Ren, J., Yu, D., Li, N., Liu, S., Xu, H., Li, J. et al. (2023). Biological characterization and whole-genome analysis of Bacillus subtilis MG 1 isolated from mink fecal samples. Microorganisms, 11(12), Article 2821. https://doi.org/10.3390/microorganisms11122821Reale, A., Di Stasio, L., Di Renzo, T., De Caro, S., Ferranti, P., Picariello, G. et al. (2021). Bacteria do it better! Proteomics suggests the molecular basis for improved digestibility of sourdough products. Food Chemistry, 359, Article 129955. https://doi.org/10.1016/j.foodchem.2021.129955Reale, A., Di Stasio, L., Di Renzo, T., De Caro, S., Ferranti, P., Picariello, G. et al. (2021). Bacteria do it better! Proteomics suggests the molecular basis for improved digestibility of sourdough products. Food Chemistry, 359, Article 129955. https://doi.org/10.1016/j.foodchem.2021.129955Zhao, Q., Shi, Y., Wang, X., Huang, A. (2020). Characterization of a novel antimicrobial peptide from buffalo casein hydrolysate based on live bacteria adsorption. Journal of Dairy Science, 103(12), 11116–11128. https://doi.org/10.3168/jds.2020-18577Zhao, Q., Shi, Y., Wang, X., Huang, A. (2020). Characterization of a novel antimicrobial peptide from buffalo casein hydrolysate based on live bacteria adsorption. Journal of Dairy Science, 103(12), 11116–11128. https://doi.org/10.3168/jds.2020-18577Курбанова, М. Г., Бондарчук, О. Н., Масленникова, С. М. (2013). Практические аспекты гидролиза казеина молока эндопептидазами. Техника и технология пищевых производств, 2(29), 34–39.Kurbanova, M. G., Bondarchuk, O. N., Maslennikova, S. M. (2013). Practical aspects of hydrolysis of milk casein by endopeptidases. Food Processing: Techniques and Technology, 2(29), 34–39. (In Russian)Милентьева, И. С., Давыденко, Н. И., Расщепкин, А. Н. (2020). Подбор рабочих параметров для проведения направленного протеолиза казеина с целью получения биопептидов. Техника и технология пищевых производств, 50(4), 726–735. https://doi.org/10.21603/2074-9414-2020-4-726-735Milentyeva, I. S., Davydenko, N. I., Rasshchepkin, A. N. (2020). Casein Proteolysis in Bioactive Peptide Production: Optimal Operating Parameters. Food Processing: Techniques and Technology, 50(4), 726–735. (In Russian) https://doi.org/10.21603/2074-9414-2020-4-726-735Агаркова, Е. Ю., Кручинин, А. Г. (2018). Ферментативная конверсия как способ получения биологически активных пептидов. Вестник МГТУ. Труды Мурманского государственного технического университета, 21(3), 412– 419.Agarkova, E. Yu., Kruchinin, A. G. (2018). Enzymatic conversion as a method of producing biologically active peptides. Vestnik of MSTU, 21(3), 412–419. (In Russian)

The authors declare that there are no conflicts of interest present.