Optimization of Protein Hydrolysate from Earthworms ( Perionyx excavatus ) of Angiotensin-I-Converting Enzyme Inhibitory Activity by One Variable at a Time

Phong Thanh Bui; Truc Anh Hoang; My Thi Thuy Pham; Linh Le Phuong Tran; Oanh Kim Nguyen

doi:10.22146/ijc.99059

Optimization of Protein Hydrolysate from Earthworms (Perionyx excavatus) of Angiotensin-I-Converting Enzyme Inhibitory Activity by One Variable at a Time

https://doi.org/10.22146/ijc.99059

Phong Thanh Bui^(1*), Truc Anh Hoang⁽²⁾, My Thi Thuy Pham⁽³⁾, Linh Le Phuong Tran⁽⁴⁾, Oanh Kim Nguyen⁽⁵⁾

(1) Department of Food Technology, Faculty of Chemical Engineering, Ho Chi Minh City University of Technology (HCMUT), 268 Ly Thuong Kiet Street, District 10, Ho Chi Minh City 731000, Vietnam; Vietnam National University, Linh Trung Ward, Thu Duc City, Ho Chi Minh City 711000, Vietnam; Hong Bang International University, 215 Dien Bien Phu, Ward 15, Binh Thanh District, Ho Chi Minh City 723000, Vietnam
(2) Hong Bang International University, 215 Dien Bien Phu, Ward 15, Binh Thanh District, Ho Chi Minh City 723000, Vietnam
(3) Hong Bang International University, 215 Dien Bien Phu, Ward 15, Binh Thanh District, Ho Chi Minh City 723000, Vietnam
(4) Hong Bang International University, 215 Dien Bien Phu, Ward 15, Binh Thanh District, Ho Chi Minh City 723000, Vietnam
(5) Hong Bang International University, 215 Dien Bien Phu, Ward 15, Binh Thanh District, Ho Chi Minh City 723000, Vietnam
(*) Corresponding Author

Abstract

Hypertension, characterized by elevated blood pressure, is commonly treated with angiotensin-I-converting enzyme (ACE) inhibitors. This study evaluated the ACE inhibitory activity (ACEIA) of protein hydrolysate from earthworms (Perionyx excavatus) by commercial proteases. The results showed that the protein hydrolysate of earthworms gave the highest ACEIA of 85.38 ± 2.31% (at the protein concentration of 2 mg/mL) and IC₅₀ value of 844.64 μg/mL when the earthworms were hydrolyzed with Flavourzyme® 500 MG under optimized conditions such as earthworm:phosphate buffer ratio of 1:6 (w/v), hydrolysis temperature of 50 °C, pH 7, enzyme:substrate ratio of 600 U/g protein and hydrolysis time of 2 h. The membrane further fractionated the hydrolysate, and the < 1 kDa fraction had the highest ACEIA with an IC₅₀ value of 261.94 μg/mL. The stability of ACEIA was assessed under various conditions, including in vitro digestion, heat treatment at 100 °C for 180 min, and pH adjustments from pH 1 to 11. The < 1 kDa fraction maintained ACEIA activity at 133.34, 76.25, and 84.95%, respectively, after these treatments. These results suggest that earthworm protein hydrolysates, particularly the <1 kDa fraction, exhibit strong stability and could be promising candidates for the development of functional foods or pharmaceuticals targeting hypertension.

Keywords

ACE; angiotensin-I-converting enzyme; hypertensive; Perionyx excavatus

Full Text:

Full Text PDF

References

[1] Dzau, V.J., and Balatbat, C.A., 2019, Future of hypertension: The need for transformation, Hypertension, 74 (3), 450–457.

[2] Li, G.H., Le, G.W., Shi, Y.H., and Shrestha, S., 2004, Angiotensin I–converting enzyme inhibitory peptides derived from food proteins and their physiological and pharmacological effects, Nutr. Res., 24 (7), 469–486.

[3] Gu, X., Hou, Y.K., Li, D., Wang, J.Z., and Wang, F.J., 2015, Separation, purification, and identification of angiotensin I–converting enzyme inhibitory peptides from walnut (Juglans regia L.) hydrolyzate, Int. J. Food Prop., 18 (2), 266–276.

[4] Ma, F.F., Wang, H., Wei, C.K., Thakur, K., Wei, Z.J., and Jiang, L., 2019, Three novel ACE inhibitory peptides isolated from Ginkgo biloba seeds: Purification, inhibitory kinetic and mechanism, Front. Pharmacol., 9, 1579.

[5] Nuchprapha, A., Paisansak, S., Sangtanoo, P., Srimongkol, P., Saisavoey, T., Reamtong, O., Choowongkomon, K., and Karnchanatat, A., 2020, Two novel ACE inhibitory peptides isolated from longan seeds: Purification, inhibitory kinetics and mechanisms, RSC Adv., 10 (22), 12711–12720.

[6] Sarbon, N.M., Howell, N.K., and Wan Ahmad, W.A.N., 2019, Angiotensin-I converting enzyme (ACE) inhibitory peptides from chicken skin gelatin hydrolysate and its antihypertensive effect in spontaneously hypertensive rats, Int. Food Res. J., 26 (3), 903–911.

[7] Jahangiri, R., Soltani, S., and Barzegar, A., 2014, A review QSAR studies to predict activity of ACE peptide inhibitors, Pharm. Sci., 20 (3), 122–129.

[8] Daskaya-Dikmen, C., Yucetepe, A., Karbancioglu-Guler, F., Daskaya, H., and Ozcelik, B., 2017, Angiotensin-I-converting enzyme (ACE)-inhibitory peptides from plants, Nutrients, 9 (4), 316.

[9] Iwaniak, A., Minkiewicz, P., and Darewicz, M., 2014, Food‐originating ACE inhibitors, including antihypertensive peptides, as preventive food components in blood pressure reduction, Compr. Rev. Food Sci. Food Saf., 13 (2), 114–134.

[10] Prakash, M., Gunasekaran, G., and Elumalai, K., 2008, Effect of earthworm powder on antioxidant enzymes in alcohol induced hepatotoxic rats, Eur. Rev. Med. Pharmacol. Sci., 12, 237–243.

[11] Chen, J., Liu, Y., Wang, G., Sun, S., Liu, R., Hong, B., Gao, R., and Bai, K., 2018, Processing optimization and characterization of angiotensin-Ι-converting enzyme inhibitory peptides from lizardfish (Synodus macrops) scale gelatin, Mar. Drugs, 16 (7), 228.

[12] Deng, Z., Gao, S., Xiao, X., Yin, N., Ma, S., Li, W., and Li, Y., 2019, The effect of earthworm extract on mice S180 tumor growth and apoptosis, Biomed. Pharmacother., 115, 108979.

[13] Ansari, A.A., and Sitaram, K., 2010, An investigation on the anti-microbial and antifungal properties of earthworm powder obtained from Eisenia foetida, Am. J. Food Technol., 6 (4), 329–335.

[14] Istiqomah, L., Sofyan, A., Damayanti, E., and Julendra, H., 2009, Amino acid profile of earthworm and earthworm meal (Lumbricus rubellus) for animal feedstuff JITAA, 34 (4), 253-257.

[15] Vo, T.D.L., Pham, K.T., Le, L.T., and Nguyen, T.T.H., 2018, Identification of a new calcium‐binding peptide from enzymatic proteolysate of Acetes japonicus, J. Food Process. Preserv., 42 (12), e13837.

[16] Ngo, D.H., Ryu, B., and Kim, S.K., 2014, Active peptides from skate (Okamejei kenojei) skin gelatin diminish angiotensin-I converting enzyme activity and intracellular free radical-mediated oxidation, Food Chem., 143, 246–255.

[17] Kang, B., Skonberg, D.I., and Myracle, A.D., 2020, Anti-hyperglycemic effects of green crab hydrolysates derived by commercially available enzymes, Foods, 9 (3), 258.

[18] Sripokar, P., Benjakul, S., and Klomklao, S., 2019, Antioxidant and functional properties of protein hydrolysates obtained from starry triggerfish muscle using trypsin from albacore tuna liver, Biocatal. Agric. Biotechnol., 17, 447–454.

[19] Tacias-Pascacio, V.G., Morellon-Sterling, R., Siar, E.H., Tavano, O., Berenguer-Murcia, Á., and Fernandez-Lafuente, R., 2020, Use of Alcalase in the production of bioactive peptides: A review, Int. J. Biol. Macromol., 165, 2143–2196.

[20] Kamran, F., Phillips, M., and Reddy, N., 2021, Functional properties of Australian blue lupin (Lupinus angustifolius) protein and biological activities of protein hydrolysates, Legume Sci., 3 (1), e65.

[21] Torruco-Uco, J., Chel-Guerrero, L., Martínez-Ayala, A., Dávila-Ortíz, G., and Betancur-Ancona, D., 2009, Angiotensin-I converting enzyme inhibitory and antioxidant activities of protein hydrolysates from Phaseolus lunatus and Phaseolus vulgaris seeds, LWT-Food Sci. Technol., 42 (10), 1597–1604.

[22] Fu, Y., Liu, J., Hansen, E.T., Bredie, W.L.P., and Lametsch, R., 2018, Structural characteristics of low bitter and high umami protein hydrolysates prepared from bovine muscle and porcine plasma, Food Chem., 257, 163–171.

[23] Deng, Y., Butré, C.I., and Wierenga, P.A., 2018, Influence of substrate concentration on the extent of protein enzymatic hydrolysis, Int. Dairy J., 86, 39–48.

[24] Shu, G., Zhang, B., Zhang, Q., Wan, H., and Li, H., 2016, Effect of temperature, pH, enzyme to substrate ratio, substrate concentration and time on the antioxidative activity of hydrolysates from goat milk casein by alcalase, Acta Univ. Cibiniensis, Ser. E: Food Technol., 20 (2), 29–38.

[25] Le, 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.

[26] Nikhita, R., and Sachindra, N.M., 2021, Optimization of chemical and enzymatic hydrolysis for production of chicken blood protein hydrolysate rich in angiotensin-I converting enzyme inhibitory and antioxidant activity, Poult. Sci., 100 (5), 101047.

[27] Vo, T.D.L., Pham, K.T., and Doan, K.T., 2021, Identification of copper-binding peptides and investigation of functional properties of Acetes japonicus proteolysate, Waste Biomass Valorization, 12, 1565–1579.

[28] Gao, W.W., Zhang, F.X., Zhang, G.X., and Zhou, C.H., 2015, Key factors affecting the activity and stability of enzymes in ionic liquids and novel applications in biocatalysis, Biochem. Eng. J., 99, 67–84.

[29] Zhang, H., Yu, L., Yang, Q., Sun, J., Bi, J., Liu, S., Zhang, C., and Tang, L., 2012, Optimization of a microwave-coupled enzymatic digestion process to prepare peanut peptides, Molecules, 17 (5), 5661–5674.

[30] Mongkonkamthorn, N., Malila, Y., Yarnpakdee, S., Makkhun, S., Regenstein, J.M., and Wangtueai, S., 2020, Production of protein hydrolysate containing antioxidant and angiotensin-I-converting enzyme (ACE) inhibitory activities from tuna (Katsuwonus pelamis) blood, Processes, 8 (11), 1518.

[31] Bao, C., Chen, H., Chen, L., Cao, J., and Meng, J., 2016, Comparison of ACE inhibitory activity in skimmed goat and cow milk hydrolyzed by alcalase, flavourzyme, neutral protease and proteinase K, Acta Univ. Cibiniensis, Ser. E: Food Technol., 20 (1), 77–84.

[32] Arise, A.K., Alashi, A.M., Nwachukwu, I.D., Malomo, S.A., Aluko, R.E., and Amonsou, E.O., 2017, Inhibitory properties of bambara groundnut protein hydrolysate and peptide fractions against angiotensin‐converting enzymes, renin and free radicals, J. Sci. Food Agric., 97 (9), 2834–2841.

[33] Samaranayaka, A.G.P., Kitts, D.D., and Li-Chan, E.C.Y., 2010, Antioxidative and angiotensin-I-converting enzyme inhibitory potential of a Pacific hake (Merluccius productus) fish protein hydrolysate subjected to simulated gastrointestinal digestion and Caco-2 cell permeation, J. Agric. Food. Chem., 58 (3), 1535–1542.

[34] Tejasari, T., Yuwanti, S., Ahmadi, M.B., and Afsari, Y.L., 2020, The anti hypertensive nutraceuticals of Vigna sp bean protein hydrolized by alcalase and flavourzyme, J. Funct. Food Nutraceutical, 2 (1), 63–73.

[35] Esmaeili, S., Ashrafi-Kooshk, M.R., Adibi, H., and Khodarahmi, R., 2017, Captopril/enalapril inhibit promiscuous esterase activity of carbonic anhydrase at micromolar concentrations: An in vitro study, Chem.-Biol. Interact., 265, 24–35.

[36] Ketnawa, S., Benjakul, S., Martínez-Alvarez, O., and Rawdkuen, S., 2017, Fish skin gelatin hydrolysates produced by visceral peptidase and bovine trypsin: Bioactivity and stability, Food Chem., 215, 383–390.

[37] Alemán, A., Gómez-Guillén, M.C., and Montero, P., 2013, Identification of ACE-inhibitory peptides from squid skin collagen after in vitro gastrointestinal digestion, Food Res. Int., 54 (1), 790–795.

[38] Yu, S., Bech Thoegersen, J., and Kragh, K.M., 2020, Comparative study of protease hydrolysis reaction demonstrating normalized peptide bond cleavage frequency and protease substrate broadness index, PLoS One, 15 (9), e0239080.

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

[40] Klomklao, S., and Benjakul, S., 2018, Protein hydrolysates prepared from the viscera of skipjack tuna (Katsuwonus pelmamis): Antioxidative activity and functional properties, Turk. J. Fish. Aquat. Sci., 18 (1), 69–79.

[41] Jang, H.L., Liceaga, A.M., and Yoon, K.Y., 2016, Purification, characterisation and stability of an antioxidant peptide derived from sandfish (Arctoscopus japonicus) protein hydrolysates, J. Funct. Foods, 20, 433–442.

[42] Nourmohammadi, E., SadeghiMahoonak, A., Alami, M., and Ghorbani, M., 2017, Amino acid composition and antioxidative properties of hydrolysed pumpkin (Cucurbita pepo L.) oil cake protein, Int. J. Food Prop., 20 (12), 3244–3255.

[43] Ninomiya, K., Ina, S., Hamada, A., Yamaguchi, Y., Akao, M., Shinmachi, F., Kumagai, H., and Kumagai, H., 2018, Suppressive effect of the α-amylase inhibitor albumin from buckwheat (Fagopyrum esculentum Moench) on postprandial hyperglycaemia, Nutrients, 10 (10), 1503.

[44] López-Sánchez, J., Ponce-Alquicira, E., Pedroza-Islas, R., De la Peña-Díaz, A., and Soriano-Santos, J., 2016, Effects of heat and pH treatments and in vitro digestion on the biological activity of protein hydrolysates of Amaranthus hypochondriacus L. grain, J. Food Sci. Technol., 53 (12), 4298–4307.

DOI: https://doi.org/10.22146/ijc.99059