Enhancement of Antibacterial Activity from Chicken Head Protein Hydrolysate Using Dual-Enzyme Hydrolysis


  • Pramudya Andiana Animal Product Technology Department, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java, 65145, Indonesia
  • Moch. Geerhan Miraja Syahdan Animal Product Technology Department, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java, 65145, Indonesia
  • Arif Hendra Utama Animal Product Technology Department, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java, 65145, Indonesia
  • Kasri Kasri Animal Nutrition and Feed Department, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java, 65145, Indonesia
  • Khothibul Umam Al Awwaly Animal Product Technology Department, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java, 65145, Indonesia
  • Abdul Manab Animal Product Technology Department, Faculty of Animal Science, Universitas Brawijaya, Jl. Veteran, Malang, East Java, 65145, Indonesia




By-product, chicken head, antibacterial activity, bioactive peptide


The chicken head is one of the by-products with a high protein content. Therefore, chicken heads can be used as raw materials to produce protein hydrolysates containing bioactive peptides that have biological activities, such as antibacterial, anti-inflammatory, and antioxidant activities. This research aimed to evaluate the use of the combined ratio of papain and bromelain enzymes to produce chicken head protein hydrolysate that has antibacterial activity. The research method used in this study was a laboratory experiment using a completely randomized design (CRD) with four treatments and five replications. Statistical significance was done using one-way analysis of variance (ANOVA) followed by Duncan’s multiple range test (DMRT). The inhibition zones of chicken head protein hydrolysate using a combination of papain enzymes against Lactobacillus casei, Escherichia coli, Staphylococcus aureus, Pseudomonas aeruginosa, and Salmonella typhimurium were 1.72-2.68, 1.19-4.47, 0.93-1.45, 1.64-2.46, and 1.01-3.62 mm, respectively. The result showed that the highest antibacterial activities against Lactobacillus casei, Escherichia coli, and Staphylococcus aureus were in A1 (hydrolysis using papain 75% and bromelain 25%), the highest antibacterial activities against Pseudomonas aeruginosa was in A3 (hydrolysis using papain 25% and bromelain 75%), and the highest antibacterial activity against Salmonella typhimurium was in A2 (hydrolysis using papain 50% and bromelain 50%). However, all the hydrolysate didn’t exhibit antibacterial activity against Bacillus subtilis. Chicken head protein hydrolysate had the potential to be an antibacterial agent against pathogenic bacteria.


Abd Rashid, N. Y., Manan, M. A., Pa’ee, K. F., Saari, N., & Faizal Wong, F. W. (2022). Evaluation of antioxidant and antibacterial activities of fish protein hydrolysate produced from Malaysian fish sausage (Keropok Lekor) by-products by indigenous Lactobacillus casei fermentation. Journal of Cleaner Production. 347. https://doi.org/10.1016/j.jclepro.2022.131303

Aidat, O., Belkacemi, L., Belalia, M., Zainol, M. khairi, & Barhoum, H. S. (2023). Physicochemical, rheological, and textural properties of gelatin extracted from chicken by-products (feet-heads) blend and application. International Journal of Gastronomy and Food Science, 32, 1-8. https://doi.org/10.1016/j.ijgfs.2023.100708

Akimova, D., Suychinov, A., Kakimov, A., Kabdylzhar, B., Zharykbasov, Y., & Yessimbekov, Z. (2023). Effect of chicken by-products on the physicochemical properties of forcemeat formulations. Future Foods, 7, 1-8. https://doi.org/10.1016/j.fufo.2023.100238

Al Awwaly, K. U., Thohari, I., Apriliyani, M. W., & Amertaningtyas, D. (2020). Extraction of Chicken Head Proteins and Evaluation of Their Functional Properties. Paper presented at The International Conference of Environmentally Sustainable Animal Industry (ICESAI), (pp. 97-102). Malang, Indonesia.

AOAC. Official Methods of Analysis. Method 2007.04 Fat, Moisture, and Protein in Meat and Meat Products. FOSS Foodscan Near-Infrared (NIR) Spectrophotometer with FOSS Artificial Neural Network (ANN) Calibration Model and Associated Database. AOAC International Gaithersburg, MD, USA, 2015.

Borrajo, P., Pateiro, M., Gagaoua, M., Franco, D., Zhang, W., & Lorenzo, J. M. (2020). Evaluation of the antioxidant and antimicrobial activities of porcine liver protein hydrolysates obtained using Alcalase, Bromelain, and Papain. Applied Sciences (Switzerland), 10 (7), 1-16. https://doi.org/10.3390/app10072290

Colletti, A., Li, S., Marengo, M., Adinolfi, S., & Cravotto, G. (2021). Recent advances and insights into bromelain processing, pharmacokinetics and therapeutic uses. Applied Sciences (Switzerland), 11 (18), 1-21. https://doi.org/10.3390/app11188428

Cruz-Casas, D. E., Aguilar, C. N., Ascacio-Valdés, J. A., Rodríguez-Herrera, R., Chávez-González, M. L., & Flores-Gallegos, A. C. (2021). Enzymatic hydrolysis and microbial fermentation: The most favorable biotechnological methods for the release of bioactive peptides. Food Chemistry: Molecular Sciences, 3, 1-12. https://doi.org/10.1016/j.fochms.2021.100047

Gal, R., Mokrejs, P., Mrazek, P., Pavlackova, J., Janacova, D., & Orsavova, J. (2020). Chicken heads as a promising by-product for preparation of food gelatins. Molecules, 25 (3), 1-12. https://doi.org/10.3390/molecules25030494

Gomez, H. L. R., Peralta, J. P., Tejano, L. A., and Chang, Y. W. (2019). In Silico and In Vitro Assessment of Portuguese Oyster (Crassostrea angulata) Proteins as Precursor of Bioactive Peptides. International Journal of Molecular Sciences. 20, 1-12. https://doi.org/10.3390%2Fijms20205191

Hakim, A. R. H., Hartoyo, B., Rahayu, S., Tugiyanti, E., & Munasik, M. (2023). Supplementation of Biopeptide from Chicken Feet to the Immune System and Growth of Broiler Chicken. Buletin Peternakan, 47 (2), 70-75. https://doi.org/10.21059/buletinpeternak.v47i2.82452

Henriques, G., McGovern, S., Neef, J., Antelo-Varela, M., Götz, F., Otto, A., Becher, D., van Dijl, J. M., Jules, M., & Delumeau, O. (2020). SppI Forms a Membrane Protein Complex with SppA and Inhibits Its Protease Activity in Bacillus subtilis. MSphere, 5 (5), 1-6. https://doi.org/10.1128/msphere.00724-20

Hou, Y., Wu, Z., Dai, Z., Wang, G., & Wu, G. (2017). Protein hydrolysates in animal nutrition: Industrial production, bioactive peptides, and functional significance. Journal of Animal Science and Biotechnology, 8 (24), 1-13. https://doi.org/10.1186/s40104-017-0153-9

Jung, S. H., Hong, D. K., Bang, S. J., Heo, K., Sim, J. J., & Lee, J. L. (2021). The functional properties of lactobacillus casei hy2782 are affected by the fermentation time. Applied Sciences (Switzerland), 11 (6), 1-11. https://doi.org/10.3390/app11062481

Li, D., Li, P., Yu, X., Zhang, X., Guo, Q., Xu, X., Wang, M., & Wang, M. (2021). Molecular characteristics of Escherichia coli causing bloodstream infections during 2010–2015 in a tertiary hospital, Shanghai, China. Infection and Drug Resistance, 3 (14), 2079-2086. https://doi.org/10.2147/IDR.S305281

Lima, C. A., Campos, J. F., Filho, J. L. L., Converti, A., da Cunha, M. G. C., & Porto, A. L. F. (2015). Antimicrobial and radical scavenging properties of bovine collagen hydrolysates produced by Penicillium aurantiogriseum URM 4622 collagenase. Journal of Food Science and Technology, 52(7), 4459–4466. https://doi.org/10.1007/s13197-014-1463-y

Malinggas, F., Pangemanan, D. H. C., & Mariati, N. W. (2015). Uji daya hambat ekstrak buah mengkudu (M. citrifolia , L) terhadap pertumbuhan Streptococcus mutans secara in vitro. Pharmacon Jurnal Ilmiah Farmasi – Unsrat. 4 (4), 22-26. https://doi.org/10.35799/pha.4.2015.10187

Moghaddam, M. M., Aghamollaei, H., Kooshki, H., Barjini, K. A., Mirnejad, R., & Choopani, A. (2015). The development of antimicrobial peptides as an approach to prevention of antibiotic resistance. Reviews and Research in Medical Microbiology, 26 (3), 98-110. https://doi.org/10.1097/MRM.0000000000000032

Qin, S., Xiao, W., Zhou, C., Pu, Q., Deng, X., Lan, L., Liang, H., Song, X., & Wu, M. (2022). Pseudomonas aeruginosa: pathogenesis, virulence factors, antibiotic resistance, interaction with host, technology advances and emerging therapeutics. Signal Transduction and Targeted Therapy, 7 (199), 1-27. https://doi.org/10.1038/s41392-022-01056-1

Raharjo, T. J., Utami, W. M., Fajr, A., Haryadi, W., & Swasono, R. T. (2021). Antibacterial peptides from tryptic hydrolysate of Ricinus communis seed protein fractionated using cation exchange chromatography. Indonesian Journal of Pharmacy. 32(1), 74-85. https://doi.org/10.22146/ijp.1260

Roy, M., Sarker, A., Azad, M. A. K., Shaheb, M. R., & Hoque, M. M. (2020). Evaluation of antioxidant and antimicrobial properties of dark red kidney bean (Phaseolus vulgaris) protein hydrolysates. Journal of Food Measurement and Characterization, 14, 303–313. https://doi.org/10.1007/s11694-019-00292-4

Santos, J. C. P., Sousa, R. C. S., Otoni, C. G., Moraes, A. R. F., Souza, V. G. L., Medeiros, E. A. A., Espitia, P. J. P., Pires, A. C. S., Coimbra, J. S. R., & Soares, N. F. F. (2018). Nisin and other antimicrobial peptides: Production, mechanisms of action, and application in active food packaging. Innovative Food Science and Emerging Technologies, 48, 179-194. https://doi.org/10.1016/j.ifset.2018.06.008

Sukarno, A. S., Nurliyani, N., Erwanto, Y., Rakhmatulloh, S., & Rifqi, R. (2023). Antibacterial and Antioxidant Activity of Protein Hydrolysate Extracted from different Indonesian Avian Egg White. Jurnal Sain Peternakan Indonesia, 18 (1), 27-33. https://doi.org/10.31186/jspi.id.18.1.27-33

Sultana, A., Luo, H., & Ramakrishna, S. (2021). Harvesting of antimicrobial peptides from insect (Hermetia illucens) and its applications in the food packaging. Applied Sciences (Switzerland), 11 (5), 1-16. https://doi.org/10.3390/app11156991

Susanto, E., Rosyidi, D., Radiati, L. E., & Subandi, S. (2018). Optimasi Aktivitas Antioksidan Peptida Aktif dari Ceker Ayam Melalui Hidrolisis Enzim Papain. Jurnal Ilmu Dan Teknologi Hasil Ternak, 13 (1), 14-26. https://doi.org/10.21776/ub.jitek.2018.013.01.2

Sutrisno, A. (2019). Teknologi Enzim. (pp. 14-15). Malang, Indonesia: Universitas Brawijaya Press.

Thammasena, R., & Liu, D. C. (2020). Antioxidant and antimicrobial activities of different enzymatic hydrolysates from desalted duck egg white. Asian-Australasian Journal of Animal Sciences, 33 (9), 1487-1496. https://doi.org/10.5713/ajas.19.0361

Tian, F., Rodtong, S., Thumanu, K., Hua, Y., Roytrakul, S., & Yongsawatdigul, J. (2022). Molecular Insights into the Mode of Action of Antibacterial Peptides Derived from Chicken Plasma Hydrolysates. Foods, 11 (22): 1-17. https://doi.org/10.3390/foods11223564

Ulagesan, S., Kuppusamy, A., & Kim, H. J. (2018). Antimicrobial and antioxidant activities of protein hydrolysate from terrestrial snail Cryptozona bistrialis. Journal of Applied Pharmaceutical Science, 8 (12), 12-19. https://doi.org/10.7324/JAPS.2018.81202

Vimalraj, E., Ramani, R., Rao, V. A., Parthiban, M., Narendrababu, R., & Arulkumar, S. (2022). Antidiabetic Activity of Peptides Extracted from Chicken Intestine Hydrolysate. The Pharma Innovation Journal, 11 (2), 1718-1721.

Wickramasinghe, H. S., Abeyrathne, E. D. N. S., Nam, K.-C., & Ahn, D. U. (2022). Antioxidant and Metal-Chelating Activities of Bioactive Peptides from Ovotransferrin Produced by Enzyme Combinations. Poultry, 1 (4), 220-228. https://doi.org/10.3390/poultry1040019

Yuan, J., Zheng, Y., Wu, Y., Chen, H., Tong, P., & Gao, J. (2020). Double enzyme hydrolysis for producing antioxidant peptide from egg white: Optimization, evaluation, and potential allergenicity. Journal of Food Biochemistry, 44 (10), 1-12. https://doi.org/10.1111/jfbc.13113