Isolation, identification, and enzyme optimization of proteolytic lactic acid bacteria from tuna viscera by-products
Article Sidebar
Abstract Views: 192
File Views: 24
Main Article Content
Abstract
Abstract. Jatmiko YD, Muarif AMR. 2025. Isolation, identification, and enzyme optimization of proteolytic lactic acid bacteria from tuna viscera by-products. Biodiversitas 26: 2762-2772. Fish processing by-products, particularly protein-rich viscera, are underutilized despite their potential to be converted into bioactive peptides using protease enzymes produced by Lactic Acid Bacteria (LAB). This study aimed to isolate proteolytic LAB from tuna viscera, identify the strain with the highest proteolytic activity, and optimize the growth conditions for enhanced protease enzyme production. Proteolytic LAB were isolated from tuna viscera and screened for enzyme activity using skim milk agar and tyrosine-based assays. The isolate with the highest proteolytic activity was identified through 16S rDNA sequencing. Protease production was then optimized using Response Surface Methodology (RSM) based on a Central Composite Design (CCD) with Design-Expert software, evaluating the effects of pH, tuna viscera concentration, and incubation time. The proteolytic potential of 12 LAB isolates from tuna viscera was obtained. The isolate of VT11 showed the highest enzyme activity of 0.55 U mL-1 among the proteolytic LAB strains. After conducting 16S rDNA sequencing, VT11 was identified as Pediococcus pentosaceus, exhibiting 99.72% similarity. Furthermore, RSM analysis identified 24 hours of incubation at pH 5 with 15% tuna viscera as the optimal conditions for protease production by P. pentosaceus VT11.
Article Details
Issue
Section
This work is licensed under a Creative Commons Attribution-NonCommercial-ShareAlike 4.0 International License.
References
A'yunin Q, Sulistyono DA, Syawli A, Rahmawati A, Intyas CA, Alviyanti D, Wiratno EN, Setyawan FO, Supriatin FE, Djamaludin H, Tambunan JE, Rihmi MK, Wardani NH, Rijal SS, Anitasari S, Ma'rifat TN, Sari WK. 2021. Sustainable Fisheries. UB Press, Malang. [Indonesian]
Amin M. 2018. Marine protease-producing bacterium and its potential use as an abalone probiont. Aquac Rep 12: 30-35. DOI: 10.1016/j.aqrep.2018.09.004.
Adetunji AI, Olaniran AO. 2020. Statistical modeling and optimization of protease production by an autochthonous Bacillus aryabhattai Ab15-ES: A response surface methodology approach. Biocatal Agric Biotechnol 24: 101528. DOI: 10.1016/j.bcab.2020.101528.
Birahy DC, Sunarti TC, Meryandini A. 2025. Selection of proteolytic lactic acid bacteria with probiotic properties for fish protein hydrolyzate production. Jurnal Ilmu Pertanian Indonesia 30 (1): 31-39. DOI: 10.18343/jipi.30.1.31.
Breig SJM, Luti KJK. 2021. Response surface methodology: A review on its applications and challenges in microbial cultures. Materials Today: Proceedings 42: 2277-2284. DOI: 10.1016/j.matpr.2020.12.316.
Rosales Cavaglieri LA, Isgro MC, Aminahuel C, Parada J, Poloni VL, Montenegro MA, Alonso V, Falcone RD, Cavaglieri LR. 2025. Exploring the potential of lactic acid bacteria to produce postbiotics with antimicrobial and antioxidant properties: Focus on the probiotic strain Pediococcus pentosaceus RC007 for industrial-scale production. Intl J Food Sci Technol 60 (1): vvae003. DOI: 10.1093/ijfood/vvae003.
Cupp-Enyard C. 2008. Sigma's non-specific protease activity assay-casein as a substrate. J Vis Exp 19: 899. DOI: 10.3791/899.
Djimtoingar SS, Derkyi NSA, Kuranchie FA, Yankyera JK. 2022. A review of response surface methodology for biogas process optimization. Cogent Eng 9 (1): 2115283. DOI: 10.1080/23311916.2022.2115283.
Dubey CK, Mishra J, Nagar A, Gupta MK, Sharma A, Kumar S, Mishra V, Pandey HP. 2024. Microbial Protease: An Update on Sources, Production Methods, and Applications. In: Mishra V, Mishra J, Arora NK (eds.). Bioactive Microbial Metabolites. Academic Press, Massachusetts. DOI: 10.1016/C2022-0-00301-9.
Ellouz Y, Bayoudh A, Kammoun S, Gharsallah N, Nasri M. 2001. Production of protease by Bacillus subtilis grown on sardinelle heads and visceral flour. Bioresour Technol 80 (1): 49-51. DOI: 10.1016/s0960-8524(01)00057-8.
Emon TH, Hakim A, Chakraborthy D, Azad AK. 2024. Enhanced production of dehairing alkaline protease from Bacillus subtilis mutant E29 by consolidated bioprocessing using response surface modeling. Biomass Convers Bioref 14: 19501-19517. DOI: 10.1007/s13399-023-04244-3.
Escobar-Sánchez M, Carrasco-Navarro U, Juárez-Castelán C, Lozano-Aguirre Beltrán L, Pérez-Chabela ML, Ponce-Alquicira E. 2022. Probiotic properties and proteomic analysis of Pediococcus pentosaceus 1101. Foods 12 (1): 46. DOI: 10.3390/foods12010046.
Fahmy NM, El-Deeb B. 2023. Optimization, partial purification, and characterization of a novel high molecular weight alkaline protease produced by Halobacillus sp. HAL1 using fish wastes as a substrate. J Genet Eng Biotechnol 21 (1): 48. DOI: 10.1186/s43141-023-00509-6.
Fitriana N, Asri MT. 2022. Proteolytic activity of protease enzymes from rhizosphere bacteria of soybean plants (Glycine max L.) in Trenggalek. LenteraBio: Berkala Ilmiah Biologi 11 (1): 144-152. DOI: 10.26740/lenterabio.v11n1.p144-152. [Indonesian]
Garofalo SF, Cavallini N, Demichelis F, Savorani F, Mancini G, Fino D, Tommasi T. 2023. From tuna viscera to added-value products: A circular approach for fish-waste recovery by green enzymatic hydrolysis. Food Bioprod Process 137: 155-167. DOI: 10.1016/j.fbp.2022.11.006.
Govindaraj K, Samayanpaulraj V, Narayanadoss V, Uthandakalaipandian R. 2021. Isolation of lactic acid bacteria from intestine of freshwater fishes and elucidation of probiotic potential for aquaculture application. Probiotics Antimicrob Proteins 13 (6): 1598-1610. DOI: 10.1007/s12602-021-09811-6.
Hadinoto S, Idrus S. 2018. Proportion and proximate analysis parts of body yellowfin tuna (Thunnus albacares) from Maluku. Majalah BIAM 14 (2): 51-57. DOI: 10.29360/mb.v14i2.4212. [Indonesian]
Ismail YS, Yulvizar C, Mazhitov B. 2018. Characterization of lactic acid bacteria from local cow's milk kefir. IOP Conf Ser: Earth Environ Sci 130: 012019. DOI: 10.1088/1755-1315/130/1/012019.
Dilini Jayaweera D. 2023. Biorefinery for yellowfin tuna (Thunnus albacares) byproducts for characterization and identification of value-added materials. GRÓ Fisheries Training Programme under the auspices of UNESCO, Iceland. Final project. https://www. grocentre.is/static/gro/publication/1730/document/DiliniJayaweera22prf.pdf.
Jiang S, Cai L, Lv L, Li L. 2021. Pediococcus pentosaceus, a future additive or probiotic candidate. Microb Cell Fact 20 (1): 45. DOI: 10.1186/s12934-021-01537-y.
Jini R, Swapna HC, Rai AK, Vrinda R, Halami PM, Sachindra NM, Bhaskar N. 2011. Isolation and characterization of potential Lactic Acid Bacteria (LAB) from freshwater fish processing wastes for application in fermentative utilization of fish processing waste. Braz J Microbiol 42: 1516-1525. DOI: 10.1590/S1517-838220110004000039.
Kabense R, Ginting EL, Wullur S, Kawung NJ, Losung F, Tombokan JL. 2019. Screening of the proteolytic bacteria symbiont with algae Gracillaria sp. Jurnal Ilmiah PLATAX 7 (2): 421-426. DOI: 10.35800/jip.7.2.2019.24487.
Kieliszek M, Pobiega K, Piwowarek K, Kot AM. 2021. Characteristics of the proteolytic enzymes produced by lactic acid bacteria. Molecules 26 (7): 1858. DOI: 10.3390/molecules26071858.
Lim ES, Lee NG. 2016. Control of histamine-forming bacteria by probiotic lactic acid bacteria isolated from fish intestine. Korean J Microbiol 52 (3): 352-364. DOI: 10.7845/kjm.2016.6041.
Lim YH, Foo HL, Loh TC, Mohamad R, Abdullah N. 2019. Comparative studies of versatile extracellular proteolytic activities of lactic acid bacteria and their potential for extracellular amino acid productions as feed supplements. J Anim Sci Biotechnol 10: 15. DOI: 10.1186/s40104-019-0323-z.
Linh NTH, Sakai K, Taoka Y. 2018. Screening of lactic acid bacteria isolated from fermented food as potential probiotics for aquacultured carp and amberjack. Fish Sci 84: 101-111. DOI: 10.1007/s12562-017-1150-9.
Rohmatussolihat, Lisdiyanti P, Sari MN, Sukara E. 2021. Response surface methodology for optimization of medium components for extracellular protease production by Enterococcus faecalis InaCC B745. IOP Conf Ser: Earth Environ Sci 762: 012078. DOI: 10.1088/1755-1315/762/1/012078.
Luong HQ, Le TN, Lee PH, Hsieh PC. 2023. Optimization of nonspecific protease activity fabrication by Bacillus subtilis N30 isolated from Taiwan using different models of response surface methodology. Biocatal Agric Biotechnol 50: 102686. DOI: 10.1016/j.bcab.2023.102686.
Matti A, Utami T, Hidayat C, Rahayu ES. 2019. Isolation, screening, and identification of proteolytic lactic acid bacteria from indigenous chao product. J Aquat Food Prod Technol 28 (7): 781-793. DOI: 10.1080/10498850.2019.1639872.
Migaw S, Ghrairi T, Belguesmia Y, Choiset Y, Berjeaud JM, Chobert JM, Hani K, Haertlé T. 2014. Diversity of bacteriocinogenic lactic acid bacteria isolated from Mediterranean fish viscera. World J Microbiol Biotechnol 30 (4): 1207-1217. DOI: 10.1007/s11274-013-1535-6.
Moranda DP, Handayani L, Nazlia S. 2018. Utilization of yellowfin tuna (Thunnus albacares) skin waste as gelatin: Hydrolysis using HCl solvent with different concentrations. Acta Aquatica: Aquatic Sci J 5 (2): 81-87. DOI: 10.29103/aa.v5i2.850. [Indonesian]
Mugendiran V, Gnanavelbabu A, Ramadoss R. 2014. Parameter optimization for surface roughness and wall thickness on AA5052 aluminum alloy by incremental forming using response surface methodology. Procedia Eng 97: 1991-2000. DOI: 10.1016/j.proeng.2014.12.442.
Nugroho G, Ekawati AW, Kartikaningsih H. 2020. Characteristics of tuna viscera (Thunnus sp.) protein hydrolysate fermented by Bacillus licheniformis. Res J Life Sci 7 (2): 101-107. DOI: 10.21776/ub.rjls.2020.007.02.4.
Nurjanah N, Baharuddin TI, Nurhayati T. 2021. Collagen extraction of yellowfin tuna (Thunnus albacares) skin using pepsin and papain enzymes. Jurnal Pengolahan Hasil Perikanan Indonesia 24 (2): 174-187. DOI: 10.17844/jphpi.v24i2.35410. [Indonesian]
Nurmiah S, Syarief R, Sukarno S, Peranginangin R, Nurmata B. 2013. Application of response surface methodology on optimization of Alkali Treated Cottonii (ATC) processing conditions. Jurnal Pascapanen dan Bioteknologi Kelautan dan Perikanan 8 (1): 9-22. DOI: 10.15578/jpbkp.v8i1.49. [Indonesian]
Ojo-Okunola A, Claassen-Weitz S, Mwaikono KS, Gardner-Lubbe S, Zar HJ, Nicol MP, du Toit E. 2020. The influence of DNA extraction and lipid removal on human milk bacterial profiles. Methods Protoc 3 (2): 39. DOI: 10.3390/mps3020039.
Phupaboon S, Hashim FJ, Phumkhachorn P, Rattanachaikunsopon P. 2023. Molecular and biotechnological characteristics of proteolytic activity from Streptococcus thermophilus as a proteolytic lactic acid bacteria to enhance protein-derived bioactive peptides. AIMS Microbiol 9 (4): 591-611. DOI: 10.3934/microbiol.2023031.
Prastujati AU, Hilmi M, Khusna A, Arief II, Makmur S, Maulida Q. 2022. Isolation and identification of lactic acid bacteria of bekamal (Banyuwangi traditional fermented meat). IOP Conf Ser: Earth Environ Sci 1020: 012026. DOI: 10.1088/1755-1315/1020/1/012026.
Raccach M. 2014. Encyclopedia of Food Microbiology (Second Edition). Elsevier, Amsterdam.
Rahayu HM. 2021. Isolation and identification of lactic acid bacteria from tempoyak for making yogurt by morphological and biochemical characterization. EPiC Ser Biol Sci 1: 118-124. DOI: 10.29007/vvbj.
Ramadhan AR, Bachruddin Z, Erwanto Y, Hanim C. 2021. Isolation and selection of proteolytic lactic acid bacteria from colostrum of dairy cattle. IOP Conf Ser: Earth Environ Sci 788: 012077. DOI: 10.1088/1755-1315/788/1/012077.
Ramkumar A, Sivakumar N, Victor R. 2016. Fish waste-potential low cost substrate for bacterial protease production: A brief review. Open Biotechnol J 10: 335-341. DOI: 10.2174/1874070701610010335.
Rebah BF, Miled N. 2013. Fish processing wastes for microbial enzyme production: A review. 3 Biotech 3 (4): 255-265. DOI: 10.1007/s13205-012-0099-8.
Rosnawita M, Agustien A, Nasir A. 2015. Effect of abiotic factors on protease production from bacterial isolate ML-23. Jurnal Biologi Universitas Andalas 4: 45-49. DOI: 10.25077/jbioua.4.1.%25p.2015. [Indonesian]
Gómez-Sala B, Muñoz-Atienza E, Sánchez J, Basanta A, Herranz C, Hernández PE, Cintas LM. 2015. Bacteriocin production by lactic acid bacteria isolated from fish, seafood and fish products. Eur Food Res Technol 241: 341-356. DOI: 10.1007/s00217-015-2465-3.
Saravanan A, Yuvaraj D, Kumar PS, Karishma S, Rangasamy G. 2023. Fish processing discards: A plausible resource for valorization to renewable fuels production, optimization, byproducts and challenges. Fuel 335: 127081. DOI: 10.1016/j.fuel.2022.127081.
Sodagar N, Jalal R, Najafi MF, Bahrami AR. 2024. A novel alkali and thermotolerant protease from Aeromonas spp. retrieved from wastewater. Sci Rep 14 (1): 26000. DOI: 10.1038/s41598-024-76004-w.
Suberu Y, Akande I, Samuel T, Lawal A, Olaniran A. 2019. Optimization of protease production in indigenous Bacillus species isolated from soil samples in Lagos, Nigeria using response surface methodology. Biocat Agric Biotechnol 18: 101011. DOI: 10.1016/j.bcab.2019.01.049.
Sulistyantini C, Mustafa I, Jatmiko YD. 2023. Isolation and Identification of Chitinolytic Bacteria as Biocontrol Agents of Fungal Pathogens in Cocoons of Cricula trifenestrata (Lepidoptera: Saturnidae). [Thesis]. Institut Pertanian Bogor, Bogor 13 (1): 29-34. DOI: 10.21776/ub.jels.2023.013.01.05. [Indonesian]
Sulthoniyah STM, Handoko, Nursyam HH. 2015. Characterization of extracellular protease lactic acid bacteria from shrimp paste. J Life Sci Biomed 5 (1): 1-5.
Sun F, Hu Y, Chen Q, Kong B, Liu Q. 2019. Purification and biochemical characteristics of the extracellular protease from Pediococcus pentosaceus isolated from Harbin dry sausages. Meat Sci 156: 156-165. DOI: 10.1016/j.meatsci.2019.05.030.
Ter ZY, Chang LS, Babji AS, Zaini NAM, Fazry S, Sarbini SR, Peterbauer CK, Lim SJ. 2024. A review on proteolytic fermentation of dietary protein using lactic acid bacteria for the development of novel proteolytically fermented foods. Intl J Food Sci Technol 59 (3): 1213-1236. DOI: 10.1111/ijfs.16888.
Thirukumaran R, Anu Priya VK, Krishnamoorthy S, Ramakrishnan P, Moses JA, Anandharamakrishnan C. 2022. Resource recovery from fish waste: Prospects and the usage of intensified extraction technologies. Chemosphere 299: 134361. DOI: 10.1016/j.chemosphere.2022.134361.
Toe CJ, Foo HL, Loh TC, Mohamad R, Abdul Rahim R, Idrus Z. 2019. Extracellular proteolytic activity and amino acid production by lactic acid bacteria isolated from Malaysian foods. Intl J Mol Sci 20 (7): 1777. DOI: 10.3390/ijms20071777.
Vinoth J, Murugan S, Stalin C. 2014. Optimization of alkaline protease production and its fibrinolytic activity from the bacterium Pseudomonas fluorescens isolated from fish waste discharged soil. Afr J Biotechnol 13 (30): 3052. DOI: 10.5897/AJB2014.13863.
Ruby-Figueroa R. 2016. Design of Experiments. In: Drioli E, Giorno L. (eds) Encyclopedia of Membranes. Springer, Berlin, Heidelberg. DOI: 10.1007/978-3-662-44324-8_1997.
Xu Y, Dai M, Zang J, Jiang Q, Xia W. 2015. Purification and characterization of an extracellular acidic protease of Pediococcus pentosaceus isolated from fermented fish. Food Sci Technol Res 21 (5): 739-744. DOI: 10.3136/fstr.21.739.
Xing CF, Hu HH, Huang JB, Fang HC, Kai YH, Wu YC, Chi SC. 2013. Diet supplementation of Pediococcus pentosaceus in cobia (Rachycentron canadum) enhances growth rate, respiratory burst and resistance against photobacteriosis. Fish Shellfish Immunol 35 (4): 1122-1128. DOI: 10.1016/j.fsi.2013.07.021.
Yin LJ, Wu CW, Jiang ST. 2003. Bacteriocins from Pediococcus pentosaceus L and S from pork meat. J Agric Food Chem 51 (4): 1071-1076. DOI: 10.1021/jf025838f.
Zamora-Sillero J, Gharsallaoui A, Prentice C. 2018. Peptides from fish byproduct protein hydrolysates and its functional properties: An overview. Mar Biotechnol (NY) 20 (2): 118-130. DOI: 10.1007/s10126-018-9799-3.