Effect of Acid Type and Buffer-assisted Stimulation on Pepsin Recovery from Skipjack Tuna (Katsuwonus pelamis) Stomach
Article Sidebar
Main Article Content
Abstract
The global tuna processing industry generates substantial by-products, including stomach tissues that remain underutilized despite their potential as a source of industrial enzymes. This study evaluated the effects of acid type, acid concentration, and buffer-assisted acid stimulation on crude pepsin recovery from skipjack tuna (Katsuwonus pelamis) stomach. Acetic acid (CH₃COOH) provided better enzymatic performance than hydrochloric and sulfuric acids. Among the concentrations tested, 1 M CH₃COOH produced the highest pepsin activity (1.03±0.05 U·mL⁻¹) and specific activity (0.115±0.005 U·mg⁻¹). Buffer-assisted acid stimulation yielded higher pepsin activities than direct acid extraction. Direct extraction produced substantially higher total protein concentration, suggesting greater co-extraction of non-pepsin proteins. The recovered crude pepsin exhibited both optimal activity and stability at pH 2, while the highest pepsin activity was observed at 50 °C, with stability maintained at 30 °C or below. More than 80% of the activity was retained within moderately acidic and mid-temperature ranges. These findings demonstrate that acid selection, concentration, and extraction strategy strongly influence functional pepsin recovery. Buffer-assisted acid stimulation with acetic acid provides a simple yet promising approach for obtaining catalytically active crude pepsin from tuna stomach by-products, supporting their valorization as a sustainable source of acid proteases for potential food and biotechnological applications.
Article Details
This work is licensed under a Creative Commons Attribution-NonCommercial-NoDerivatives 4.0 International License.
References
Amnuaikit, T., R. Shankar and S. Benjakul. 2022. Hydrolyzed fish collagen serum from by-product of food industry: cosmetic product formulation and facial skin evaluation. Sustainability 14(24): 16553. DOI: 10.3390/su142416553.
Anggarkasih, M.G. and P. Resma. 2022. The importance of halal certification for the processed food by SMEs to increase export opportunities. E3S Web of Conferences 348: 00039. DOI: 10.1051/e3sconf/202234800039.
Anson, M.L. 1938. The estimation of pepsin, trypsin, papain, and cathepsin with hemoglobin. Journal of General Physiology 22(1): 79–89. DOI: 10.1085/jgp.22.1.79.
Asaduzzaman, F. and S. Salmon. 2023. Protease immobilization in solution-blown poly (ethylene oxide) nanofibrous nonwoven webs. ACS Applied Engineering Materials 1(1): 447–457. DOI: 10.1021/acsaenm.2c00111.
Binh, N.C. and N.M.X. Hong. (2025). Zero-waste approach in tuna fish processing industry. HUIT Journal of Science 25: 58–70. DOI: 10.62985/j.huit_ojs.vol25.nos1_ica.246.
Borrajo, P., M. Pateiro, M. Gagaoua, D. Franco, W. Zhang and J.M. Lorenzo. 2020. Evaluation of the antioxidant and antimicrobial activities of porcine liver protein hydrolysates obtained using alcalase, bromelain, and papain. Applied Sciences 10(7): 2290. DOI: 10.3390/app10072290.
Brier, S., G. Maria, V. Carginale, A. Capasso, Y. Wu, R.M. Taylor, N.B. Borotto, C. Capasso and J.R. Engen. 2007. Purification and characterization of pepsins A1 and A2 from the Antarctic rock cod (Trematomus bernacchii). FEBS Journal 274(23): 6152–6166. DOI: 10.1111/j.1742-4658.2007.06136.x.
Cadar, E., A. Pesterau, I. Prasacu, A.M. Ionescu, C. Pascale, A.M.L. Dragan, R. Sirbu and C.L. Tomescu. 2024. Marine antioxidants from marine collagen and collagen peptides with nutraceuticals applications: A review. Antioxidants 13(8): 919. DOI: 10.3390/antiox13080919.
Cao, M.J., W.Q. Chen, C.H. Du, A. Yoshida, W.G. Lan, G.M. Liu and W.J. Su. 2011. Pepsinogens and pepsins from Japanese seabass (Lateolabrax japonicus). Comparative Biochemistry and Physiology, Part B 158(4): 259–265. DOI: 10.1016/j.cbpb.2010.12.003.
Chen, W.Q., M.J. Cao, A. Yoshida, G.M. Liu, W.Y. Weng, L.C. Sun and W.J. Su. 2009. Study on pepsinogens and pepsins from snakehead (Channa argus). Journal of Agricultural and Food Chemistry 57(22): 10972–10978. DOI: 10.1021/jf902548p.
De Domenico, S., G. De Rinaldis, M. Paulmery, S. Piraino and A. Leone. 2019. Barrel jellyfish (Rhizostoma pulmo) as source of antioxidant peptides. Marine Drugs 17(2): 134. DOI: 10.3390/md17020134.
Delgado, L., N. Shologu, K. Fuller and D.I. Zeugolis. 2017. Acetic acid and pepsin result in high yield, high purity and low macrophage response collagen for biomedical applications. Biomedical Materials 12(6): 065009. DOI: 10.1088/1748-605X/aa838d.
Food and Agriculture Organization (FAO). 2022. The State of World Fisheries and Aquaculture 2022. Towards Blue Transformation. FAO, Rome, Italy. 266 pp.
Food and Agriculture Organization (FAO). 2025. Higher Tuna Trade in 2024. Globefish – Quarterly Tuna Analysis, February 2025. FAO, Rome, Italy. https://openknowledge.fao.org/server/api/core/bitstreams/8b0eac6c-fb9e-47c9-ae84-188d4c016d78/content. Cited 10 Feb 2026.
Fatiranes, E., S. W. Sidauruk and B. Hasan. 2025. Characteristics of crude pepsin enzyme from catfish stomach (Clarias sp.). Aquatic Life Sciences 2(2): 57–63. DOI: 10.58920/aqlis0202418.
Fink, A.L., L.J. Calciano, Y. Goto, T. Kurotsu and D.R. Palleros. 1994. Classification of acid denaturation of proteins: intermediates and unfolded states. Biochemistry 33(41): 12504–12511. DOI: 10.1021/bi00207a018.
Fraterrigo Garofalo, S., N. Cavallini, F. Demichelis, F. Savorani, G. Mancini, D. Fino and T. Tommasi. 2023. From tuna viscera to added-value products: A circular approach for fish-waste recovery by green enzymatic hydrolysis. Food and Bioproducts Processing 137: 155–167. DOI: 10.1016/j.fbp.2022.11.006.
Gaikwad, S. and M.J. Kim. 2024. Fish by-product collagen extraction using different methods and their application. Marine Drugs 22(2): 60. DOI: 10.3390/md22020060.
Gamarro, E.G., W. Orawattanamateekul, J. Sentina and T.K.S. Gopal. 2013. By-products of tuna processing. GLOBEFISH Research Programme, 112. FAO, Rome, Italy. 55 pp.
Hajihama, M. and W. Youravong. 2013. Concentration and desalination of protein derived from tuna cooking juice by nanofiltration. Jurnal Teknologi (Sciences and Engineering) 65(1): 91–96. DOI: 10.11113/jt.v65.2318.
Heidari, M.G. and M. Rezaei. 2022. Extracted pepsin of trout waste and ultrasound-promoted method for green recovery of fish collagen. Sustainable Chemistry and Pharmacy 30: 100854. DOI: 10.1016/j.scp.2022.100854.
Iqbal, A., S.R.A. Shah, I.S. Cetingul and I. Bayram. 2020. A review – halal animal nutrition perspective to the halal meat production. Malaysian Journal of Halal Research 3(1): 17–23. DOI: 10.2478/mjhr-2020-0003.
Jahromi, R.R.F., P. Morris, R.J. Martinez-Torres and P.A. Dalby. 2011. Structural stability of Escherichia coli transketolase to temperature and pH denaturation. Journal of Biotechnology 155(2): 209–216. DOI: 10.1016/j.jbiotec.2011.06.023.
Jesús-Ramirez, F., C.A. Álvarez-González, H.G. Nolasco-Soria, E. Peña, R. Martínez-García, S. Camarillo-Coop, K. Conway and C. Pohlenz. 2017. Partial characterization of digestive proteases of fat snook (Centropomus parallelus). Hidrobiologica 27(3): 419–427. DOI: 10.24275/uam/izt/dcbs/hidro/2017v27n3/Alvarez. (in Spanish)
Kuepethkaew, S., Y. Zhang, H. Kishimura, Y. Kumagai, B.K. Simpson, S. Benjakul, S. Damodaran and S. Klomklao. 2022. Enzymological characteristics of pepsinogens and pepsins purified from lizardfish (Saurida micropectoralis) stomach. Food Chemistry 366: 130532. DOI: 10.1016/j.foodchem.2021.130532.
Lowry, O.H., N.J. Rosebrough, A.L. Farr and R.J. Randall. 1951. Protein measurement with the Folin phenol reagent. Journal of Biological Chemistry 193(1): 265–275. DOI: 10.1016/S0021-9258(19)52451-6.
Maiz, H., E. Guadix, A. Guadix, M. Gargouri and F. Espejo-Carpio. 2019. Valorisation of tuna viscera by endogenous enzymatic treatment. International Journal of Food Science and Technology 54(4): 1100–1108. DOI: 10.1111/ijfs.14009.
Mamo, J. and F. Assefa. 2018. The role of microbial aspartic protease enzyme in food and beverage industries. Journal of Food Quality 2018: 7957269. DOI: 10.1155/2018/7957269.
Mazumder, S.K., S.K. Das, S.M. Rahim and M. Abd Ghaffar. 2018. Temperature and diet effect on the pepsin enzyme activities, digestive somatic index and relative gut length of Malabar blood snapper (Lutjanus malabaricus Bloch & Schneider, 1801). Aquaculture Reports 9: 1–9. DOI: 10.1016/j.aqrep.2017.11.003.
McCluney, J.K., C.M. Anderson and J.L. Anderson. 2019. The fishery performance indicators for global tuna fisheries. Nature Communication 10(1): 1641. DOI: 10.1038/s41467-019-09466-6
Ministry of Trade of the Republic of Indonesia. 2020. Tuna Export in the Pandemic. Ministry of Trade of the Republic of Indonesia. Jakarta, Indonesia. 32 pp.
Muttaqin, R.A.B., A. Surya, I. Istiarini, M. Duanassurya, W. Samosir, S. Makatita and T. Nurhayati. 2025. Extraction and characterization of pepsin enzyme from the skipjack viscera (Katsuwonus pelamis). BIO Web of Conferences 147: 01018. DOI: 10.1051/bioconf/202414701018.
Nalinanon, S., S. Benjakul, W. Visessanguan and H. Kishimura. 2008. Tuna pepsin: Characteristics and its use for collagen extraction from the skin of threadfin bream (Nemipterus spp.). Journal of Food Science 73(5): C413–C419. DOI: 10.1111/j.1750-3841.2008.00777.x
Nalinanon, S., S Benjakul and H. Kishimura. 2010a. Purification and biochemical properties of pepsins from the stomach of skipjack tuna (Katsuwonus pelamis). European Food Research and Technology 231(2): 259–269. DOI: 10.1007/s00217-010-1275-x.
Nalinanon, S., S. Benjakul, H. Kishimura and F. Shahidi. 2010b. Biochemical properties of pepsinogen and pepsin from the stomach of albacore tuna (Thunnus alalunga). Food Chemistry 121(1): 49–55. DOI: 10.1016/j.foodchem.2009.11.089.
Nolasco-Soria, H., H.R. Nolasco-Alzaga and E. Gisbert. 2020. The importance of pepsin-like acid protease quantification in aquaculture studies: A revision of available procedures and presentation of a new protocol for its assessment. Reviews in Aquaculture 12(3): 1928–1943. DOI: 10.1111/raq.12417.
Oliveira, V.M., R.S. Bezerra and C.R.D. Assis. 2015. Fish pepsin: basic characteristics, extraction, determination and biotechnological applications. Natural Resources 4(1): 6–14. DOI: 10.6008/SPC2237-9290.2014.001.0001.
Pasaribu, E., T. Nurhayati and M. Nurilmala. 2018. Extraction and characterization of pepsin enzyme from tuna (Thunnus albacares). Jurnal Pengolahan Hasil Perikanan Indonesia 21(3): 486–496. DOI: 10.17844/jphpi.v21i3.24727. (in Indonesian)
Pérez-Velázquez, M., C.A. Maldonado-Othón and M.L. González-Félix. 2024. Molecular weights and optimum temperature and pH for pepsin activity of three sciaenid finfish species from the Gulf of California. Archives of Biological Sciences 76(1): 83–90. DOI: 10.2298/ABS240104004P.
Precup, G., E. Marini, P. Zakidou, et al. 2024. Novel foods, food enzymes, and food additives derived from food by-products of plant or animal origin: principles and overview of the EFSA safety assessment. Frontiers in Nutrition 11: 1390734. DOI: 10.3389/fnut.2024.1390734.
Razali, U.H.M., H. Ya'akob, N.M. Sarbon, N.H. Zainan, D.J. Dailin and D.N.A. Zaidel. 2023. Improving collagen processing towards a greener approach: current progress. Journal of Chemical Technology and Biotechnology 98(5): 1063–1082. DOI: 10.1002/jctb.7332.
Sari, F.N., W. Nur, S. Surianti, N. Nurliana and I. Mawar. 2025. Processing of fishery waste as an alternative source of gelatin production. Jurnal Perikanan Unram 15(2): 484–492. DOI: 10.29303/jp.v15i2.963.
Thai Tuna Industry Association. 2025. TTIA/TPFA summary of export products to the world in 2023–2025. Thai Tuna Industry Association. www.moc.go.th. Cited 10 Feb 2026.
Tohmadlae, P., W. Worawattanamateekul and J. Hinsui. 2020. Characterization of acidic tuna protease and its application for extraction of tilapia collagen hydrolysate. Chiang Mai University Journal of Natural Sciences 19(2): 206–221. DOI: 10.12982/CMUJNS.2020.00014.
Wald, M., H. Rehbein, C. Beermann, B. Bußmann and K. Schwarz. 2016. Purification and characterization of pepsinogen and pepsin from the stomach of rainbow trout (Oncorhynchus mykiss). European Food Research and Technology 242(11): 1925–1935. DOI: 10.1007/s00217-016-2692-2.
Weng, W.Y., T. Wu, W.Q. Chen, G.M. Liu, K. Osatomi, W.J. Su and M.J. Cao. 2010. Purification and characterization of pepsinogens and pepsins from the stomach of rice field eel (Monopterus albus Zuiew). Fish Physiology and Biochemistry 37(3): 543–552. DOI: 10.1007/s10695-010-9456-x.
Yu, F., C. Zong, S. Jin, et al. 2018. Optimization of extraction conditions and characterization of pepsin-solubilised collagen from skin of giant croaker (Nibea japonica). Marine Drugs 16(1): 29. DOI: 10.3390/md16010029.
Zhao, W.H., C.F. Chi, Y.Q. Zhao and B. Wang. 2018. Preparation, physicochemical and antioxidant properties of acid- and pepsin-soluble collagens from the swim bladders of miiuy croaker (Miichthys miiuy). Marine Drugs 16(5): 161. DOI: 10.3390/md16050161.
