Preparation and Characterization of Cholesterol Esterase Inhibition Protein Hydrolysate from Whiteleg Shrimp ( Litopenaeus vannamei ) Head

Bao Chi Vo; Ha Thu Nguyen; Linh Thi Truc Vo; Khang Tran Gia Cao; Tam Dinh Le Vo

doi:10.22146/ijc.109424

Preparation and Characterization of Cholesterol Esterase Inhibition Protein Hydrolysate from Whiteleg Shrimp (Litopenaeus vannamei) Head

https://doi.org/10.22146/ijc.109424

Bao Chi Vo⁽¹⁾, Ha Thu Nguyen⁽²⁾, Linh Thi Truc Vo⁽³⁾, Khang Tran Gia Cao⁽⁴⁾, Tam Dinh Le Vo^(5*)

(1) Department of Food Technology, Faculty of Chemical Engineering, Ho Chi Minh City University of Technology (HCMUT), VNU-HCM, Ho Chi Minh City 700000, Vietnam
(2) Department of Food Technology, Faculty of Chemical Engineering, Ho Chi Minh City University of Technology (HCMUT), VNU-HCM, Ho Chi Minh City 700000, Vietnam
(3) Department of Food Technology, Faculty of Chemical Engineering, Ho Chi Minh City University of Technology (HCMUT), VNU-HCM, Ho Chi Minh City 700000, Vietnam
(4) Department of Food Technology, Faculty of Chemical Engineering, Ho Chi Minh City University of Technology (HCMUT), VNU-HCM, Ho Chi Minh City 700000, Vietnam
(5) Department of Food Technology, Faculty of Chemical Engineering, Ho Chi Minh City University of Technology (HCMUT), VNU-HCM, Ho Chi Minh City 700000, Vietnam
(*) Corresponding Author

Abstract

Protein hydrolysates with cholesterol esterase inhibition activity (CEIA) can reduce the de-esterification and absorption of dietary cholesterol, helping manage hypercholesterolemia. With a high protein content, whiteleg shrimp (Litopenaeus vannamei) heads (WLSH) are a promising source for producing such hydrolysates. Using a single-factor test by sequentially varying WLSH powder-to-water ratio (1:3–1:8 w/v), enzyme-to-substrate (E:S) ratio (10–50 U/g protein), and hydrolysis time (2–7 h), the best alcalase-catalyzed hydrolysis condition was determined as a 1:6 (w/v) WLSH powder-to-water ratio, a 30 U/g protein E:S ratio, and a 4-h hydrolysis time. The resulting hydrolysate displayed a CEIA of 58.00 ± 0.64% and retained over 70% of its activity after treatment with pH (1.0–6.0) or heat (100 °C for 2 h). Arg, the predominant amino acid in the hydrolysate, together with abundant hydrophobic amino acids, contributed to the hydrolysate’s activity. Furthermore, the notable foaming and emulsifying properties of the hydrolysate indicate its promise as a surface-active agent in food applications. Besides, the 1–3 kDa fraction from the hydrolysate exhibited the greatest CEIA, probably due to its easier access to the enzyme’s active site. With higher CEIA, compared to other hydrolysates, the WLSH hydrolysate and its 1–3 kDa fraction could serve as a new natural cholesterol-lowering agent.

Keywords

protein hydrolysate; cholesterol esterase inhibition activity; whiteleg shrimp head; functional property

Full Text:

Full Text PDF

References

[1] Mudgil, P., Baby, B., Ngoh, Y.Y., Vijayan, R., Gan, C.Y., and Maqsood, S., 2019, Identification and molecular docking study of novel cholesterol esterase inhibitory peptides from camel milk proteins, J. Dairy Sci., 102 (12), 10748–10759.

[2] Fawale, O.S., Mudgil, P., Abdelrahman, R., Baba, W.N., Nirmal, N.P., and Maqsood, S., 2023, Anti-hypercholesteraemic and antioxidative activities of camel skin gelatin hydrolysate: Effect of enzyme type, enzyme: substrate ratio and time of hydrolysis, Int. J. Food Sci. Technol., 58 (4), 2151–2160.

[3] Alnuaimi, A., Fisayo Ajayi, F., Hamdi, M., Mudgil, P., Kamal, H., Yuen Gan, C., and Maqsood, S., 2023, A comparative analysis of anti-lipidemic potential of soybean (Glycine max) protein hydrolysates obtained from different ripening stages: Identification, and molecular interaction mechanisms of novel bioactive peptides, Food Chem., 402, 134192.

[4] Prados, I.M., Marina, M.L., and García, M.C., 2018, Isolation and identification by high resolution liquid chromatography tandem mass spectrometry of novel peptides with multifunctional lipid-lowering capacity, Food Res. Int., 111, 77–86.

[5] Ashraf, Z.U., Shah, A., Gani, A., and Gani, A., 2024, Effect of enzymatic hydrolysis of pulse protein macromolecules to tailor structure for enhanced nutraceutical properties, LWT - Food Sci. Technol., 205, 116502.

[6] Zhang, Z., Jiang, S., Li, Y., Xie, D., and Li, J.X., 2024, Residue profiles of peptides with cholesterol esterase and pancreatic lipase inhibitory activities through virtual screening and sequence analysis, Food Chem., 460 (Pt. 2), 140708.

[7] Huang, F., Dai, Q., Zheng, K., Ma, Q., Liu, Y., Jiang, W., and Yan, X., 2023, Unmasking the efficacy of Skipjack Tuna (Katsuwonus Pelamis) dark muscle hydrolyzate in lipid regulation: A promising component for functional food, Int. J. Food Prop., 26 (2), 3014–3029.

[8] Wang, Y.M., Ge, M.X., Ran, S.Z., Pan, X., Chi, C.F., and Wang, B., 2025, Antioxidant peptides from miiuy croaker swim bladders: Ameliorating effect and mechanism in NAFLD cell model through regulation of hypolipidemic and antioxidant capacity, Mar. Drugs, 23 (2), 63.

[9] Dou, B., Wu, X., Xia, Z., Wu, G., Guo, Q., Lyu, M., and Wang, S., 2023, Multiple bioactivities of peptides from hydrolyzed Misgurnus anguillicaudatus, Molecules, 28 (6), 2589.

[10] Lu, J., Shi, P., Cao, Y., Shi, B., Shen, H., Zhao, S., Gao, Y., Chi, H., Wang, L., and Shi, Y., 2025, Isolation and purification of novel antioxidant peptides from mussel (Mytilus edulis) prepared by marine Bacillus velezensis Z-1 protease, Mar. Drugs, 23 (8), 294.

[11] VASEP, 2025, Report on Vietnam Seafood Exports in 2024, Vietnam Association of Seafood Exporters and Producers (VASEP), Hanoi, Vietnam, 39–57.

[12] Liu, Z., Liu, Q., Zhang, D., Wei, S., Sun, Q., Xia, Q., Shi, W., Ji, H., and Liu, S., 2021, Comparison of the proximate composition and nutritional profile of byproducts and edible parts of five species of shrimp, Foods, 10 (11), 2603.

[13] Yin, H., Zhu, J., Zhong, Y., Wang, D., and Deng, Y., 2024, Kinetic and thermodynamic-based studies on the interaction mechanism of novel R. roxburghii seed peptides against pancreatic lipase and cholesterol esterase, Food Chem., 447, 139006.

[14] AOAC, 2023, Official Methods of Analysis, 22^nd Ed., Oxford University Press, UK.

[15] Vo, T.D.L., Huynh, T., Le, T.T., Tran, A.T.T., and Vo, B.C., 2025, Iron-binding capacity and antidiabetic activity of baby clam (Corbiculidae sp.) meat protein hydrolysate, Indones. J. Chem., 25 (1), 121–132.

[16] Vo, T.D.L., Huynh, T., Le, T.T., Tran, A.T.T., and Vo, B.C., 2025, Recovery of a protein hydrolysate from baby clam (Corbiculidae sp.) meat with angiotensin I‑converting enzyme inhibition activity and functionalities, ACS Food Sci. Technol., 5 (4), 1482–1491.

[17] Vo, T.D.L., Tran, T.N., Vo, B.C., Ma, H.T., Tran, H.G., Nguyen, S.M., Hoang, Q.P., Nguyen, V.T.T., Nguyen, M.T.N., Nguyen, T.H.N., Pham, V.T., Cao, K.T.G., and Pham, C.V., 2025, Study on zinc-binding capacity of featherback (Chitala ornata) skin hydrolysate, Indones. J. Chem., 25 (2), 590–598.

[18] Mudgil, P., Alblooshi, M., Singh, B.P., Devarajan, A.R., and Maqsood, S., 2023, Pearl millet protein hydrolysates exhibiting effective in-vitro antioxidant, antidiabetic and anti-lipidemic properties as potential functional food ingredient, Int. J. Food Sci. Technol., 58 (6), 3264–3272.

[19] Yin, H., Zhu, J., Zhong, Y., Wang, D., Deng, Y., Jiang, Y., Yang, L., and Gao, L., 2025, Insights into rapid screening and molecular mechanism of novel Rosa roxburghii seeds pancreatic lipase/cholesterol esterase inhibitory peptides: A combined in silico and in vitro perspective, Food Sci. Hum. Wellness, 14 (7), 9250163.

[20] Mostafa, H., Al-Ahbabi, N., Adiamo, O.Q., Mudgil, P., and Maqsood, S., 2022, Phoenix dactylifera L. seed protein hydrolysates as a potential source of peptides with antidiabetic and anti-hypercholesterolemic properties: An in vitro study, Food Biosci., 49, 101916.

[21] Vo, T.D.L., Tran, T.N., Vo, B.C., Tran, M.C., Nguyen, Q.V.N., Nguyen, B.N., Le, T.M.X., Bui, N.H.Y., and Nguyen, H.T.N., 2023, Preparation, amino acid composition, peptide fractionation, thermal and pH activity stability of featherback (Chitala Ornata) skin gelatin hydrolysate with zinc-binding capacity, Chem. Eng. Trans., 106, 871–876.

[22] Jafar, S., Kamal, H., Mudgil, P., Hassan, H.M., and Maqsood, S., 2018, Camel whey protein hydrolysates displayed enhanced cholesteryl esterase and lipase inhibitory, anti-hypertensive and anti-haemolytic properties, LWT - Food Sci. Technol., 98, 212–218.

[23] Zhang, Z.Q., Ren, X.R., Geng, J., Chen, S.C., Wang, Q.L., Liu, C.Q., Xiao, J., and Huang, D.W., 2024, Identification, characterization and hypolipidemic effect of novel peptides in protein hydrolysate from Protaetia brevitarsis larvae, Food Res. Int., 176, 113813.

[24] Mudgil, P., Ajayi, F.F., Alkaabi, A., Alsubousi, M., Singh, B.P., and Maqsood, S., 2023, Flaxseed- and chia seed-derived protein hydrolysates exhibiting enhanced in vitro antidiabetic, anti-obesity, and antioxidant properties, Front. Sustainable Food Syst., 7, 1223884.

[25] Wang, P., Song, X., and Liang, Q., 2024, Study on the inhibitory effect of bioactive peptides derived from yak milk cheese on cholesterol esterase, Foods, 13 (18), 2970.

[26] Vo, T.D.L., Pham, K.T., Le, V.M.V., Lam, H.H., Huynh, O.N., and Vo, B.C., 2020, Evaluation of iron-binding capacity, amino acid composition, functional properties of Acetes japonicus proteolysate and identification of iron-binding peptides, Process Biochem., 91, 374–386.

[27] Fuentes, C., Verdú, S., Grau, R., Barat, J.M., and Fuentes, A., 2024, Impact of raw material and enzyme type on the physico-chemical and functional properties of fish by-products hydrolysates, LWT - Food Sci. Technol., 201, 116247.

[28] Roy, T., Singh, A., Sari, T.P., and Gupta, R., 2024, Microwave-assisted enzymatic hydrolysis: A sustainable approach for enhanced structural and functional properties of broken rice protein, Process Biochem., 136, 301–310.

[29] Akcay, F.A., and Avci, A., 2024, Direct hydrolysis of einkorn whole grain flour proteins for the generation of bioactive peptides using various proteases, Int. J. Biol. Macromol., 275, 133565.

[30] Islam, M.S., Hongxin, W., Admassu, H., Noman, A., Ma, C., and An wei, F., 2021, Degree of hydrolysis, functional and antioxidant properties of protein hydrolysates from grass turtle (Chinemys reevesii) as influenced by enzymatic hydrolysis conditions, Food Sci. Nutr., 9 (8), 4031–4047.

[31] Luo, X., Wu, S., Xue, J., Hu, H., He, Z., Liu, X., and Wu, F., 2021, The bioactive peptide screening from Torreya grandis meal protein hydrolysates, Food Biosci., 44, 101419.

[32] Li, G., Zuo, X., Luo, X., Chen, Z., Cao, W., Lin, H., Qin, X., Wu, L., and Zheng, H., 2023, Functional, physicochemical, and structural properties of the hydrolysates derived from the abalone (Haliotis discus subsp hannai Ino) foot muscle proteins, Food Chem.: X, 19, 100841.

[33] Adav, S.S., 2025, Advances in the study of protein deamidation: Unveiling its influence on aging, disease progression, forensics and therapeutic efficacy, Proteomes, 13 (2), 24.

[34] De Sciscio, M.L., Nardi, A.N., Centola, F., Rossi, M., Guarnera, E., and D’Abramo, M., 2023, Molecular modeling of the deamidation reaction in solution: A theoretical–computational study, J. Phys. Chem. B, 127 (44), 9550–9559.

[35] Pastrana, B., Culyba, E., Nieves, S., Sazinsky, S.L., Canto, E.I., and Noda, I., 2024, Streamlined multi-attribute assessment of an array of clinical-stage antibodies: Relationship between degradation and stability, Appl. Spectrosc., 79 (1), 87–103.

[36] Damodaran, S., and Parkin, K.L., 2017, Fennema’s Food Chemistry, CRC Press, Boca Raton, FL, US.

[37] Pu, L., Kong, X., Xing, R., Wang, Y., Zhang, C., Hua, Y., Chen, Y., and Li, X., 2024, Identification, rapid screening, docking mechanism and in vitro digestion stability of novel DPP-4 inhibitory peptides from wheat gluten with ginger protease, Food Funct., 15 (7), 3848–3863.

[38] Cheng, Y., Chen, X., Lin, Q., Wang, J., Chen, Y., Mo, H., Chen, M., Xu, Z., Wen, L., and Jiao, Y., 2024, Improvement effect of different polysaccharides on the functional properties of curcumin-loaded rice protein hydrolysate nanoparticles, LWT - Food Sci. Technol., 204, 116471.

DOI: https://doi.org/10.22146/ijc.109424