Gorakh Mal*, Birbal Singh, Gauri Jairath, Rinku Sharma, Ajayta Rialch, Devi Gopinath and Suma N

ICAR-Indian Veterinary Research Institute, Regional Station, Palampur (HP), India

*Corresponding Author: Gorakh Mal, ICAR-Indian Veterinary Research Institute, Regional Station, Palampur (HP), India.

Received: November 13, 2025; Published: December 31, 2025

Abstract

Gaddi goat milk casein protein fractions were hydrolysed with proteolytic enzyme alcalase, and further subjected to in vitro digestion using gastric (pepsin) and intestinal (trypsin & pancreatin) enzymes. Bioactive peptides obtained after enzymatic hydrolysis and in vitro digestion of hydrolysates were centrifuged. Supernatants were used for protein profiling by SDS-PAGE and estimation of various antioxidant, antihypertensive and antimicrobial activities. SDS-PAGE revealed that major of the Gaddi goat casein proteins were degraded after enzymatic hydrolysis and, degraded completely after in vitro digestion. OPA activity was noted to be increased with enzymatic hydrolysis and further enhanced after in vitro digestion. OPA activity was observed highest (37.46 ± 0.26 mg/ml) in overnight in vitro digested casein hydrolysates generated with enzyme alacalase A1 (1:100). FRAP antioxidant activities were also noted highest (22.59 ± 0.18 mg/100ml) in overnight in vitro digested Gaddi goat casein protein hydrolysates generated with enzyme alcalase A1 (1:100) as compare to Gaddi goat casein in vitro digested hydrolysates prepared with alcalase A2 (0.01:100) and A3 (0.005:100). DPPH activity was observed higher in pepsin digested hydrolysates than the corresponding undigested hydrolysates and overnight in vitro digested hydrolysates. DPPH activity was noted maximum (36.76 ± 0.14 %) in pepsin digested Gaddi goat casein protein hydrolysates generated with enzyme alcalase (A1). Dry dot- TLC showed that DPPH antioxidant activity remains stable subsequently overnight in Gaddi goat casein hydrolysates and in vitro digested hydrolysates prepared with various concentrations of alcalase. Antihypertensive activity was noted highest (48.24 ± 0.35 %) in overnight in vitro digested Gaddi goat casein protein hydrolysates prepared with enzyme alcalase (A1). Antimicrobial activity was noted maximum (14 mm) in pepsin digested hydrolysates of Gaddi goat casein protein hydrolysates generated with enzyme alcalase (A1) against Rhodococcus equi. Among the three concentrations of alcalase (A1, A2 and A3) used in the present study, antioxidant, antihypertensive and antimicrobial activities were found to be highest in Gaddi goat casein protein hydrolysates prepared with enzyme alcalase (A1), indicating likely application as bioactive and purposeful components in various food preparations.

Keywords: Gaddi Goat; Casein Proteins; Enzymatic Hydrolysis; In Vitro Digestion; OPA Activity; Antioxidant Activity; Antihypertensive Activity; Antimicrobial Activity

References

1. Mal G., et al. “Bioactivities of Gaddi Goat Milk Whey Protein Hydrolysates Processed with Papain". Acta Scientific Veterinary Sciences2 (2024): 84-92.
2. Jung TH., et al. “Hydrolysis by alcalase improves hypoallergenic properties of goat milk protein”. Korean Journal for Food Science of Animal Resources4 (2016): 516-522.
3. “1996 Production Yearbook”. Food and Agriculture Organisation, UN. Rome, Italy (1997).
4. Amrein-Boyes D. “200 Easy Homemade Cheese Recipes”. Robert Rose Inc. Toronto (2009).
5. Lordan R., et al. “Phospholipids of Animal and Marine Origin: Structure, Function, and Anti-Inflammatory Properties”. Molecules11 (2017).
6. Nayik GA., et al. “Recent insights into processing approaches and potential health benefits of goat milk and its products: A Review”. Frontiers in Nutrition8 (2021): 789117.
7. Miller BA., et al. “Current status of global dairy goat production: an overview”. Asian-Australasian Journal of Animal Sciences8 (2019): 1219-1232.
8. Haenlein G F W., et al. “Goat milk in human nutrition”. Small Ruminant Research2 (2004): 155-163.
9. Kaminarides S E., et al. “Comparative study of the separation of casein from bovine, ovine and caprine milks using HPLC”. Journal of Dairy Research 60 (1993): 495-504.
10. Urbiene S., et al. “Physical and chemical properties and the biological value of goat's, cow's and human milk”. Milchwissenschaft 52 (1997): 427-430.
11. Im YS., et al. “Characteristics of goat milk and current utilizing trends in Korea”. Journal of Korean Dairy Technology Science 24 (2006): 1-9.
12. Ceballos LS., et al. “Evaluation of the allergenicity of goat milk, cow milk, and their lactosera in a guinea pig model”. Journal of Dairy Science 92 (2009): 837-846.
13. Bernacka H. “Health-promoting properties of goat milk”. Medycyna Weterynaryjna 67 (2011): 507-511.
14. Hinz K., et al. “Comparison of the principal proteins in bovine, caprine, buffalo, equine and camel milk”. Journal of Dairy Research 79 (2012): 185-191.
15. Yangilar F., et al. “As a potentially functional food: Goat milk and products”. Journal of Food and Nutrition Research 4 (2013): 68-81.
16. Park YW., et al. “Bioactive components in goat milk”. Bioactive Components in Milk and Dairy Products4381 (2009): 43-81.
17. Korhonen H., et al. “Bioactive peptides: production and functionality”. International Dairy Journal9 (2006): 945-960.
18. Muro Urista C., et al. “Production and functionality of active peptides from milk”. Food Science and Technology International4 (2011): 293-317.
19. Singh H. “Interactions of milk proteins during the manufacture of milk powders”. Le Lait4-5 (2007): 413-423.
20. De Gobba C., et al. “Antioxidant peptides from goat milk protein fractions hydrolysed by two commercial proteases”. International Dairy Journal 39 (2014): 28-40.
21. Hernandez-Ledesma B., et al. “Antihypertensive peptides: Production, bioavailability and incorporation into foods”. Advances in Colloid and Interface Science1 (2011): 23-35.
22. Power O., et al. “Antioxidative peptides: enzymatic production, in vitro and in vivo antioxidant activity and potential applications of milk-derived antioxidative peptides”. Amino Acids3 (2013): 797-820.
23. Brandelli A., et al. “Whey as a source of peptides with remarkable biological activities”. Food Research International 73 (2015): 149-161.
24. Nongonierma AB., et al. “Strategies for the discovery, identification and validation of milk protein-derived bioactive peptides”. Trends in Food Science and Technology 50 (2016): 26-43.
25. Pintado ME., et al. “Characterization of whey cheese packed under vacuum”. Journal of Food Protection63 (2000): 216-221.
26. Jeewanthi RKC., et al. “Comparative analysis of improved soy-mozzarella cheeses made of ultrafiltrated and partly skimmed soy blends with other mozzarella types”. Journal of Food Sciences and Technology 52 (2015): 5172-5179.
27. Lopez-Garcia G., et al. “Antioxidant and antimicrobial peptides derived from food proteins”. Molecules 27 (2022): 1343.
28. Najafian L. “A review of bioactive peptides as functional food ingredients: mechanisms of action and their applications in active packaging and food quality improvement”. Journal of Food and Function 14 (2023): 5835-5857.
29. Xu Q., et al. “Bioavailability of bioactive peptides derived from food proteins across the intestinal epithelial membrane: a review”. Trends in Food Science and Technology86 (2019): 399-411.
30. Nath A., et al. “Bioactive peptides from liquid milk protein concentrate by sequential tryptic and microbial hydrolysis”. Processes9 (2021): 1688.
31. Chalamaiah M., et al. “Regulatory requirements of bioactive peptides (protein hydrolysates) from food proteins”. Journal of Functional Foods 58 (2019): 123-129.
32. Ulug SK., et al. “Novel technologies for the production of bioactive peptides”. Trends in Food Science and Technology108 (2021): 27-39.  
33. Tacias-Pascacio VG., et al. “Use of alcalase in the production of bioactive peptides: A review”. International Journal of Biological Macromolecules 165 (B) (2020): 2143-2196.
34. Sharma M., et al. “A review on microbial alkaline protease: an essential tool for various industrial approaches”. Industrial Biotechnology2 (2019): 69-78.
35. Ibrahim HR., et al. “Novel angiotensin-converting enzyme inhibitory peptides from caseins and whey proteins of goat milk”. Journal of Advanced Research1 (2017): 63-71.
36. Singh A., et al. “Milk-Derived Antimicrobial Peptides: Overview, Applications, and Future Perspectives”. National Library of Medicine1 (2022): 44-62.
37. Al-Shamsi KA., et al. “Camel milk protein hydrolysates with improved technofunctional properties and enhanced antioxidant potential in in vitro and in food model systems”. Journal of Dairy Science 1 (2018): 47-60.
38. Abdel Hamid M., et al. “Hepatoprotective action of papain-hydrolyzed buffalo milk protein on carbon tetrachloride oxidative stressed albino rats”. Journal of Dairy Science2 (2020): 1884-1893.
39. Kumar D., et al. “Enzymatic hydrolysis of camel milk casein and its antioxidant properties”. Dairy Science and Technology3 (2016): 391-404.
40. Holt C., et al. “Invited review: Modeling milk stability”. Journal of Dairy Science8 (2024): 5259-5279.
41. Rahmawati IS., et al. “Effects of fermentation and storage on bioactive activities in milks and yoghurts”. Procedia Chemistry 18 (2016): 53-62.
42. Kanik et al. “Antioxidant Potential of Fermented Milk Supplemented with Various Aqueous Herbal Proteins”. Elsevier (2019): 519-547.
43. Penta-Ramos EA., et al. “Antioxidant activity of soy protein hydrolysates in a liposomal system”. Journal of Food Science 67 (2002): 2952-2956.
44. Mal G., et al. “Milk composition, antioxidant activities and protein profile of Gaddigoat milk”. Journal of Food Biochemistry6 (2018).
45. Osman A., et al. “Antibacterial peptides generated by alcalase hydrolysis of goat whey”. Food Science and Technology 65 (2016): 480-486.
46. Espejo-Carpio FJ., et al. “Angiotensin I-converting enzyme inhibitory activity of enzymatic hydrolysates of goat milk protein fractions”. International Dairy Journal 32 (2013): 175-183.
47. Kamali A., et al. “Antimicrobial peptides derived from milk. A review”. Journal of Food Biosciences and Technology 7 (2017): 49-56.
48. Bielecka M., et al. “Antioxidant, antimicrobial and anticarcinogenic activities of bovine milk proteins and their hydrolysates. A review”. International Dairy Journal 127 (2022): 105208.
49. McCann KB., et al. “Isolation and characterization of a novel antibacterial peptide from bovine aS1-casein”. International Dairy Journal 16 (2006): 316-323.

Citation

Citation: Gorakh Mal.,et al. “Bioactive Properties of Gaddi Goat Milk Casein Protein Hydrolysates Treated with Alcalase".Acta Scientific Veterinary Sciences 7.12 (2025): 18-29.

Copyright

Copyright: © 2025 Gorakh Mal.,et al. This is an open-access article distributed under the terms of the Creative Commons Attribution License, which permits unrestricted use, distribution, and reproduction in any medium, provided the original author and source are credited.