Comparative characteristics of enzymatic hydrolysates of cow milk proteins for the use in enteral nutrition

Introduction . Enteral nutrition is a critical component of treatment for seriously ill patients. It is prescribed to patients with a functioning gastrointestinal tract in situations where regular oral nutrition fails to meet their physiological needs. Due to its high biological value and optimal amino acid composition, cow milk proteins are most often used to produce protein hydrolysates for enteral nutrition products. The goal of the research was to comparatively evaluate the physicochemical properties of enzymatic whey protein hydrolysates (EWPHs) with varying degrees of hydrolysis, as well as to assess their biological value and in vivo efficacy for their introduction into specialized food products, including enteral nutrition. The objectives and methods . Hydrolysate obtained according to a previously developed protocol (FHWPC 1 “Protozyme N”) and three samples of industrial hydrolysates (FHWPC 2 “Uglich”, FHWPC 3 “Yaroslavl”, and FHWPC 4 “Voronezh”) were used in the research. In the first stage, the molecular weight distribution of peptide fractions, the degree of hydrolysis, the antigenicity reduction factor, and the amino acid composition of the samples were assessed. In the second stage, the biological value of the hydrolysate samples in an experiment on male Wistar rats was assessed. Feed intake and body weight gain was evaluated, a threeday exchange experiment in metabolic cells was conducted, and the protein efficiency ratio (PER), true digestibility, and the biological value adjusted for the amino acid score (PDCAAS) were calculated. The results and discussion. FHWPC 1 “Protozyme N” was characterized by a moderate degree of hydrolysis, without a clear reduction in antigenicity, with a high FER value, high intrinsic digestibility, and a PDCAAS coefficient . Conclusion . The enzymatic hydrolysate obtained using our developed scheme has a number of technological advantages over commercial samples and possesses a biological value indistinguishable from the original whey protein concentrate.

1. Improving enteral nutrition in patients requiring neurological intensive care unit care: A retrospective study on a novel bedside nasointestinal tube placement technique / Niuchenglin [et al.] // Nutrition in Clinical Practice. 2026. Vol. 2026. DOI: 10.1002/ncp.70106.

2. ESPEN guideline on clinical nutrition in the intensive care unit / Singer P. [et al.] // Clin Nutr. 2019. Vol. 38, No. 1. P. 48-79. DOI:10.1016/j.clnu.2018.08.037.

3. Semi-Elemental and Elemental Formulas for Enteral Nutrition in Infants and Children with Medical Complexity-Thinking about Cow's Milk Allergy and Beyond / Verduci E. [et al.] // Nutrients. 2021. Vol. 13, No. 12. P. 4230. DOI: 10.3390/nu1312423.

4. Milk Whey Hydrolysates as High Value-Added Natural Polymers: Functional Properties and Applications / León-López A. [et al.] // Polymers (Basel). 2022. Vol. 14, No. 6. P. 1258. DOI: 10.3390/polym14061258.

5. Enzymes for production of whey protein hydrolysates and other value-added products / Irazoqui J.M. [et al.] // Appl Microbiol Biotechnol. 2024. Vol. 108, No. 1. P. 354. DOI: 10.1007/s00253-024-13117-2.

6. A new high protein-to-energy enteral formula with a whey protein hydrolysate to achieve protein targets in critically ill patients: a prospective observational tolerability study // Tedeschi-Jockers F. [et al.] // Eur J Clin Nutr. 2022. Vol. 76, No. 3. P. 419-427. DOI: 10.1038/s41430-021-00956-9.

7. Peptide profiles and allergy-reactivity of extensive hydrolysates of milk protein / Cui Q [et al.] // Food Chem. 2023. Vol. 411. P. 135544. DOI: 10.1016/j.foodchem.2023.135544.

8. Whey Protein Enzymatic Breakdown: Synthesis, Analysis, and Discovery of New Biologically Active Peptides in Papain-Derived Hydrolysates / Czelej M. [et al.] // Molecules. 2025. Vol. 30, No. 7. P. 1451. DOI: 10.3390/molecules30071451.

9. Glucose Management, and Muscle Protein Synthesis Properties of Fish Protein Hydrolysates and Peptides / Shekoohi N. [et al.] // J Agric Food Chem. 2024. Vol. 72, No. 39. P. 21301-21317. DOI: 10.1021/acs.jafc.4c02920.

10. Adler-Nissen J. Determination of the degree of hydrolysis of food protein hydrolysates by trinitrobenzenesulfonic acid // J Agric Food Chem. 1979. Vol. 27, No. 6. P. 1256-1262. DOI: 10.1021/jf60226a042.

11. Hydrolysates of mare milk proteins. Immunochemical and physicochemical characteristics / Semenova E.S. [et al.] // Food systems. 2024. Vol. 7, No. 3. P. 466–472. DOI: 10.21323/2618-9771-2024-7-3-466-472. [In Russ.]

12. Modified method for obtaining phycocyanin concentrate of Arthrospira platensis biomass / Biryulina N.A. [et al.] // Nutrition issues. 2023. Vol. 92, No. 5. P. 110-116. DOI: 10.33029/0042-8833-2023-92-5-110-116. [In Russ.]

13. In vitro study of antifungal effects of earthworm coelomic fluid obtained from Eisenia fetida on three opportunistic fungal pathogens / Sanaiirad S. [et al.]. // Curr Med Mycol. 2025. Vol. 11. P. 1637. DOI: 10.22034/cmm.2025.345248.1637.

14. Reeves P.G. Components of the AIN-93 diets as improvements in the AIN-76A diet // J Nutr. 1997. Vol. 127, No. 5. P. 838S-841S. DOI: 10.1093/jn/127.5.838S.

15. Quantification of total endogenous nitrogen losses in broilers using a minimally invasive 15N-isotope dilution method / Habibi M.F. [et al.] // Animal. 2026. Vol. 20, No. 3. P. 101768. DOI: 10.1016/j.animal.2026.101768.

16. Genotypic influence on soybean protein quality: Linking molecular structure, functional properties, and nutritional quality through principal component and correlation analyses / Poddar S. [et al.] // Food Res Int. 2026. Vol. 232. P. 118869. DOI: 10.1016/j.foodres.2026.118869.

17. Cow's Milk Protein Allergy, a Systematic Review of Clinical Characteristics, Diagnosis, Management, and Economic Impact / Menco Contreras F. [et al.] // Diseases. 2026. Vol. 14, No. 4. P. 146. DOI: 10.3390/diseases14040146.

18. Myricetin suppresses whey protein allergenicity: In vitro and in vivo study / Yang W. [et al.] // Food Chem (Oxf). 2026. Vol. 12. P. 100380. DOI: 10.1016/j.fochms.2026.100380.

19. The use of extensively hydrolysed and amino acid feeds beyond cow's milk allergy: a national survey / Meyer R. [et al.] // J Hum Nutr Diet. 2021. Vol. 34, No. 1. P. 13-23. DOI: 10.1111/jhn.12794.

20. Characteristics and Absorption Rate of Whey Protein Hydrolysates Prepared Using Flavourzyme after Treatment with Alcalase and Protamex / Chang Y.B. [et al.] // Molecules. 2023. Vol. 28, No. 24. P. 7969. DOI: 10.3390/molecules28247969.

21. Nutritional, biological, and structural properties of bioactive peptides from bellflower, Persian-willow, and bitter-orange pollens. / Sarabandi K. [et al.] // J Food Sci. 2023. Vol. 88, No. 7. P. 3119-3133. DOI: 10.1111/1750-3841.16658.

22. Egg White Hydrolysate Retains the Nutritional Value of Proteins and Is Quickly Absorbed in Rats / Matsuoka R. [et al.] // Scientific World Journal. 2019. Vol. 2019. P. 5475302. DOI: 10.1155/2019/5475302.

23. Serum amino acid concentrations in growing rats fed intact protein versus enzymatic protein hydrolysate-based diets / Baró L. [et al.] // Biol Neonate. 1995. Vol. 68, No. 1. P. 55-61. DOI: 10.1159/000244218.

24. Standardized amino acid digestibility and protein quality in extruded canine diets containing hydrolyzed protein using a precision-fed rooster assay / Hsu C. [et al.] // J Anim Sci. 2023. Vol. 101. P. skad289. DOI: 10.1093/jas/skad289.

25. Comparison of digestibility and quality of intact proteins with their respective hydrolysates / Potier M. [et al.] // J AOAC Int. 2008. Vol. 91, No. 4. P. 1002-1005. PMID: 18727562.