Chemical Profile and Antioxidant Activity of Various Cysteine-Proteases' Impact on Spirulina Protein Hydrolysate

https://doi.org/10.22146/ijc.103465

Heder Djamaludin(1*), Inayatussakinah Inayatussakinah(2), Andhika Alfanda Kusdiyarlistio(3), Zidan Armanda(4), Dinia Rizqi Dwijayanti(5), Inggit Kresna Maharsih(6), Kartika Dyah Palupi(7), Pamungkas Rizki Ferdian(8), Rizki Rabeca Elfirta(9), Hartoyo Notonegoro(10)

(1) Fish Product Technology Study Program, Faculty of Fisheries and Marine Science, Brawijaya University, Jl. Veteran, Malang 65145, Indonesia
(2) Fish Product Technology Study Program, Faculty of Fisheries and Marine Science, Brawijaya University, Jl. Veteran, Malang 65145, Indonesia
(3) Fish Product Technology Study Program, Faculty of Fisheries and Marine Science, Brawijaya University, Jl. Veteran, Malang 65145, Indonesia
(4) Fish Product Technology Study Program, Faculty of Fisheries and Marine Science, Brawijaya University, Jl. Veteran, Malang 65145, Indonesia
(5) Department of Biology, Faculty of Mathematics and Natural Sciences, Brawijaya University, Jl. Veteran, Malang 65145, Indonesia
(6) Department of Bioprocess Engineering, Faculty of Agriculture Technology, Brawijaya University, Jl. Veteran, Malang 65145, Indonesia
(7) Research Center for Pharmaceutical Ingredients and Traditional Medicine, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km. 46, Cibinong 16911, Indonesia
(8) Research Center for Applied Zoology, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km. 46, Cibinong 16911, Indonesia
(9) Research Center for Applied Microbiology, National Research and Innovation Agency (BRIN), Jl. Raya Bogor Km. 46, Cibinong 16911, Indonesia
(10) Capture Fisheries Study Program, Faculty of Agriculture, Fisheries, and Marine Science, Universitas Bangka Belitung, Jl. Merdeka, Bangka 33172, Indonesia
(*) Corresponding Author

Abstract


Spirulina is a type of microalgae that contains many useful compounds having antioxidant properties. It has low biological activity and limited protein content when used in dry form. Proteins can be broken down through a hydrolysis reaction to increase their bioactivity, producing smaller peptides and free amino acids. This study aims to evaluate the effects of two cysteine-protease enzymes, bromelain and papain, on the hydrolysis of Spirulina protein. The research examined how these enzymes affect the degree of hydrolysis, protein content, molecular weight, and antioxidant activity of the resulting protein hydrolysate. A non-factorial, completely randomized design was used with three replicates per treatment. The results showed that the type of enzyme used significantly influenced all measured parameters. Bromelain was found to be more effective than papain. Spirulina protein hydrolyzed with bromelain had 32.15 ± 0.74% protein, 48.51 ± 0.94% hydrolysis, a density of 0.786 mg/mL, and 29.64 ± 0.82 ppm antioxidant activity. It also contained 18 types of amino acids, totaling 14.41 g/kg. The most efficient of physical extraction methods—particularly the combination of freeze-thaw and ultrasonication—for obtaining high-yield, high-quality protein from Spirulina. Further purification is needed to obtain the smallest peptide.

Keywords


amino acids; bromelain; enzyme; microalgae; papain

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References

[1] Alshuniaber, M.A., Krishnamoorthy, R., and AlQhtani, W.H., 2021, Antimicrobial activity of polyphenolic compounds from Spirulina against food-borne bacterial pathogens, Saudi J. Biol. Sci., 28 (1), 459–464.

[2] Fortuin, J., Hellebois, T., Iken, M., Shaplov, A.S., Fogliano, V., and Soukoulis, C., 2024, Stabilising and functional effects of Spirulina (Arthrospira platensis) protein isolate on encapsulated Lacticaseibacillus rhamnosus GG during processing, storage and gastrointestinal digestion, Food Hydrocolloids, 149, 109519.

[3] Sotiroudis, S.T., and Sotiroudis, G.T., 2013, Health aspects of Spirulina (Arthrospira) microalga food supplement, J. Serb. Chem. Soc., 78 (3), 395–405.

[4] Priyanka, S., Varsha, R., Verma, R., and Ayenampudi, S.B., 2023, Spirulina: A spotlight on its nutraceutical properties and food processing applications, J. Microbiol., Biotechnol. Food Sci., 12 (6), e4785.

[5] Asmaz, E.D., and Seyidoglu, A., 2022, The prevention role of Spirulina platensis (Arthrospira platensis) on intestinal health, Food Sci. Hum. Wellness, 11 (5), 1342–1346.

[6] Carrizzo, A., Conte, G.M., Sommella, E., Damato, A., Ambrosio, M., Sala, M., Scala, M.C., Aquino, R.P., De Lucia, M., Madonna, M., Sansone, F., Ostacolo, C., Capunzo, M., Migliarino, S., Sciarretta, S., Frati, G., Campiglia, P., and Vecchione, C., 2019, Novel potent decameric peptide of Spirulina platensis reduces blood pressure levels through a PI3K/AKT/eNOS-dependent mechanism, Hypertension, 73 (2), 449–457.

[7] Zhang, J., Zou, Y., Yan, B., Zhang, N., Zhao, J., Zhang, H., Chen, W., and Fan, D., 2023, Microwave treatment on structure and digestibility characteristics of Spirulina platensis protein, Curr. Res. Food Sci., 7, 100581.

[8] Saorin Puton, B.M., Demaman Oro, C.E., Lisboa Bernardi, J., Exenberger Finkler, D., Venquiaruto, L.D., Dallago, R.M., and Tres, M.V., 2025, Sustainable valorization of plant residues through enzymatic hydrolysis for the extraction of bioactive compounds: Applications as functional ingredients in cosmetics, Processes, 13 (5), 1314.

[9] Solanki, P., Putatunda, C., Kumar, A., Bhatia, R., and Walia, A., 2021, Microbial proteases: Ubiquitous enzymes with innumerable uses, 3 Biotech, 11 (10), 428.

[10] Hendarto, T., and Budiyanto, D., 2022, Anticholesterol bioactive peptides from bromelain hydrolysates of mangrove Sonneratia alba protein, Int. J. Multidiscip. Res. Anal., 05 (12), 3589–3597.

[11] Wang, Z., and Zhang, X., 2017, Isolation and identification of anti-proliferative peptides from Spirulina platensis using three-step hydrolysis, J. Sci. Food Agric., 97 (3), 918–922.

[12] Safi, C., Ursu, A.V., Laroche, C., Zebib, B., Merah, O., Pontalier, P.V., and Vaca-Garcia, C., 2014, Aqueous extraction of proteins from microalgae: Effect of different cell disruption methods, Algal Res., 3, 61–65.

[13] Sun, Y., Chang, R., Li, Q., and Li, B., 2016, Isolation and characterization of an antibacterial peptide from protein hydrolysates of Spirulina platensis, Eur. Food Res. Technol., 242 (5), 685–692.

[14] Yathisha, U.G., Karunasagar, I., and Mamatha, B.S., 2021, Bioactivity and functional properties of protein hydrolysate from muscle and visceral waste of ribbon fish (Lepturacanthus savala) extracted by three different proteolytic enzymes, J. Biol. Act. Prod. Nat., 11 (4), 363–379.

[15] Mohammadi, M., Soltanzadeh, M., Ebrahimi, A.R., and Hamishehkar, H., 2022, Spirulina platensis protein hydrolysates: Techno-functional, nutritional and antioxidant properties, Algal Res., 65, 102739.

[16] Djamaludin, H., Hardoko, H., Dailami, M., Nurhadianty, V., Uluwwi, M.S., Muhammad, N.Y., and Tristany, B.J., 2023, The compounds of tuna-shredded (Thunnus sp.) fortified banana blossom extracts’ antioxidant activity and xanthine oxidase enzyme inhibition capacity: An in vitro-in silico study, Indones. J. Chem., 23 (3), 782–795.

[17] Parimi, N.S., Singh, M., Kastner, J.R., Das, K.C., Forsberg, L.S., and Azadi, P., 2015, Optimization of protein extraction from Spirulina platensis to generate a potential co-product and a biofuel feedstock with reduced nitrogen content, Front. Energy Res., 3, 30.

[18] Lee, S.Y., Show, P.L., Ling, T.C., and Chang, J.S., 2017, Single-step disruption and protein recovery from Chlorella vulgaris using ultrasonication and ionic liquid buffer aqueous solutions as extractive solvents, Biochem. Eng. J., 124, 26–35.

[19] Htoo, N.Y.M., Kraseasintra, O., Buncharoen, W., Kaewkod, T., Pekkoh, J., Tragoolpua, Y., Khoo, K.S., Chaipoot, S., Srinuanpan, S., and Pumas, C., 2024, In vitro immunomodulation activity of protein hydrolysate from Spirulina (Arthrospira platensis): The ingredient of future foods, Front. Mar. Sci., 11, 1303025.

[20] Zhu, X., Das, R.S., Bhavya, M.L., Garcia-Vaquero, M., and Tiwari, B.K., 2024, Acoustic cavitation for agri-food applications: Mechanism of action, design of new systems, challenges and strategies for scale-up, Ultrason. Sonochem., 105, 106850.

[21] Suchintita Das, R., Tiwari, B.K., Chemat, F., and Garcia-Vaquero, M., 2022, Impact of ultrasound processing on alternative protein systems: Protein extraction, nutritional effects and associated challenges, Ultrason. Sonochem., 91, 106234.

[22] Alahmad, K., Xia, W., Jiang, Q., and Xu, Y., 2022, Effect of the degree of hydrolysis on nutritional, functional, and morphological characteristics of protein hydrolysate produced from bighead carp (Hypophthalmichthys nobilis) using ficin enzyme, Foods, 11 (9), 1320.

[23] Noman, A., Xu, Y., AL-Bukhaiti, W.Q., Abed, S.M., Ali, A.H., Ramadhan, A.H., and Xia, W., 2018, Influence of enzymatic hydrolysis conditions on the degree of hydrolysis and functional properties of protein hydrolysate obtained from Chinese sturgeon (Acipenser sinensis) by using papain enzyme, Process Biochem., 67, 19–28.

[24] El-Moataaz, S., Ismael, H., and Aborhyem, S., 2019, Assessment of chemical composition of Spirulina Platensis and its effect on fasting blood glucose and lipid profile in diabetic rats, J. High Inst. Public Health, 49 (3), 199–211.

[25] Feng, X., Hang, S., Zhou, Y., Liu, Q., and Yang, H., 2018, Bromelain kinetics and mechanism on myofibril from golden pomfret (Trachinotus blochii), J. Food Sci., 83 (8), 2148–2158.

[26] Limsukon, W., Rubino, M., Rabnawaz, M., Lim, L.T., and Auras, R., 2023, Hydrolytic degradation of poly (lactic acid): Unraveling correlations between temperature and the three phase structures, Polym. Degrad. Stab., 217, 110537.

[27] Zhu, Y., Zhao, X., Zhang, X., Liu, H., and Ao, Q., 2021, Amino acid, structure and antioxidant properties of Haematococcus pluvialis protein hydrolysates produced by different proteases, Int. J. Food Sci. Technol., 56 (1), 185–195.

[28] Ketemepi, H.K., Awang, M.A., Seelan, J.S.S., and Mohd Noor, N.Q.I., 2024, Extraction process and applications of mushroom-derived protein hydrolysate: A comprehensive review, Future Foods, 9, 100359.

[29] Yang, N., Matthew, M.A., and Yao, C., 2023, Roles of cysteine proteases in biology and pathogenesis of parasites, Microorganisms, 11 (6), 1397.

[30] Tejano, L.A., Peralta, J.P., Yap, E.E.S., and Chang, Y.W., 2019, Bioactivities of enzymatic protein hydrolysates derived from Chlorella sorokiniana, Food Sci. Nutr., 7 (7), 2381–2390.

[31] Mulyasari, M., Subaryono, S., Utomo, B.S.B., Yosmaniar, Y., Taufik, I., Yamin, M., Kusmini, I.I., and Marwati, T., 2023, Protein hydrolysate from waste of catfish fillet processing for snakehead fish feed formulation, Scientifica, 2023 (1), 815122.

[32] Ding, Y., Ko, S.C., Moon, S.H., and Lee, S.H., 2019, Protective effects of novel antioxidant peptide purified from alcalase hydrolysate of velvet antler against oxidative stress in chang liver cells in vitro and in a zebrafish model in vivo, Int. J. Mol. Sci., 20 (20), 5187.

[33] Kasemsuk, T., Vivithanaporn, P., and Unchern, S., 2018, Anti-inflammatory effects of bromelain in LPS-induced human U937 macrophages, Chiang Mai J. Sci., 45 (1), 299–307.

[34] Qing, R., Hao, S., Smorodina, E., Jin, D., Zalevsky, A., and Zhang, S., 2022, Protein design: From the aspect of water solubility and stability, Chem. Rev., 122 (18), 14085–14179.

[35] Lisboa, C.R., Pereira, A.M., and Costa, J.A.V., 2016, Biopeptides with antioxidant activity extracted from the biomass of Spirulina sp. LEB 18, Afr. J. Microbiol. Res., 10 (3), 79–86.

[36] Ma, J., Zeng, X., Zhou, M., Cheng, L., and Ren, D., 2021, Inhibitory effect of low-molecular-weight peptides (0–3 kDa) from Spirulina platensis on H2O2-induced oxidative damage in L02 human liver cells, Bioresour. Bioprocess., 8 (1), 36.

[37] Xu, B., Dong, Q., Yu, C., Chen, H., Zhao, Y., Zhang, B., Yu, P., and Chen, M., 2024, Advances in research on the activity evaluation, mechanism and structure-activity relationships of natural antioxidant peptides, Antioxidants, 13 (4), 479.

[38] Duan, X., Ocen, D., Wu, F., Li, M., Yang, N., Xu, J., Chen, H., Huang, L., Jin, Z., and Xu, X., 2014, Purification and characterization of a natural antioxidant peptide from fertilized eggs, Food Res. Int., 56, 18–24.

[39] Senadheera, T.R.L., Dave, D., and Shahidi, F., 2021, Antioxidant potential and physicochemical properties of protein hydrolysates from body parts of North Atlantic Sea cucumber (Cucumaria frondosa), Food Prod., Process. Nutr., 3 (1), 3.

[40] Rajakumar, M.S., and Muthukumar, K., 2018, Influence of pre-soaking conditions on ultrasonic extraction of Spirulina platensis proteins and its recovery using aqueous biphasic system, Sep. Sci. Technol., 53 (13), 2034–2043.

[41] Naghdi, S., Rezaei, M., Tabarsa, M., and Abdollahi, M., 2013, Fish protein hydrolysate from sulfated polysaccharides extraction residue of tuna processing by-products with bioactive and functional properties, Global Challenges, 7 (4), 2200214.

[42] Tavakoli, Z., Kavoosi, G., Siahbalaei, R., and Karimi, J., 2025, Spirulina maxima as a valuable ingredient: Determination of broad fatty acid and amino acid profiles and nutritional quality and anti-amylase capacity, Appl. Food Res., 5 (1), 100741.

[43] Paoletti, A., Courtney-Martin, G., and Elango, R., 2024, Determining amino acid requirements in humans, Front. Nutr., 11, 1400719.

[44] Parthasarathy, A., Cross, P.J., Dobson, R.C.J., Adams, L.E., Savka, M.A., and Hudson, O.A., 2018, A three-ring circus: Metabolism of the three proteogenic aromatic amino acids and their role in the health of plants and animals, Front. Mol. Biosci., 5, 29.

[45] Karami, Z., and Duangmal, K., 2024, Exploring the effect of incubation pH, temperature, and in vitro digestion on antioxidant potential of mung bean protein hydrolysates, Chiang Mai J. Sci., 51 (6), e2024090.

[46] Buglak, A.A., 2024, Antioxidant properties of α-amino acids: A density functional theory viewpoint, Free Radical Res., 58 (6-7), 380–387.



DOI: https://doi.org/10.22146/ijc.103465

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