Malaysian Journal of Analytical Sciences, Vol 27 No 4 (2023): 754 - 765

PURIFICATION OF POTENT ANGIOTENSIN CONVERTING ENZYME (ACE)-INHIBITORY PEPTIDES DERIVED FROM RED TILAPIA (Oreochromis Sp.) BY-PRODUCTS

(Penulenan Peptida Perencat Enzim Penukaran Angiotensin (ACE) daripada Produk Sampingan Ikan Tilapia Merah (Oreochromis Sp.))

Nur Suraya Abdul Wahab¹, Emmy Liza Anak Yaji¹, Norfahana Abd-Talib^1,, Mohammad Zulkeflee Sabri^2,, Kelly Yong Tau Len³, Fadzlie Wong Faizal Wong⁴, and Khairul Faizal Pa’ee^1*

¹Food Engineering Technology, Universiti Kuala Lumpur, Branch Campus Malaysian Institute of Chemical and Engineering Technology, 78000 Alor Gajah, Melaka, Malaysia

²Bioengineering Technology, Universiti Kuala Lumpur, Branch Campus Malaysian Institute of Chemical and Engineering Technology, 78000 Alor Gajah, Melaka, Malaysia.

³Process Engineering Technology, Universiti Kuala Lumpur, Branch Campus Malaysian Institute of Chemical and Engineering Technology, 78000 Alor Gajah, Melaka, Malaysia.

⁴Faculty of Biotechnology and Biomolecular Sciences, Universiti Putra Malaysia, 43400, UPM Serdang, Selangor, Malaysia.

^*Corresponding author: khairulfaizal@unikl.edu.my

Received: 7 October 2022; Accepted: 17 January 2023; Published: 22 August 2023

Abstract

Diet and lifestyle changes are essential alternative treatments for hypertension. Consumers are increasingly more interested in health-promoting ingredients. The use of red tilapia (Oreochromis sp.) by-products (RTBP) has been established as a precursor for protein hydrolysate with angiotensin I-converting (ACE)-inhibitory activity. However, the complexity of the protein hydrolysate reduces its potency. Thus, this work aimed to purify and characterise the ACE-inhibitory hydrolysate derived from red tilapia (Oreochromis sp.). Thermoase PC10F (EC 3.4.24.27) was used in this study to hydrolyse red tilapia by-products (Oreochromis sp.). Meanwhile, ultrafiltration (UF), anion-exchange principle (AEX) and hydrophobic interaction chromatogram (HIC) were applied to purify the hydrolysate. Two molecular weight cut-offs (MWCO) were used for ultrafiltration: 3 kDa and 1 kDa. The 1 kDa hydrolysate showed the highest bioactivity of 85.42% (IC₅₀=0.41 mg mL^-). Subsequently, the 1 kDa RTBP hydrolysate was purified based on charge using AEX. Positively charged hydrolysate demonstrated significant bioactivity of 72.32%. In the final purification steps, the hydrophobic fractions showed the highest ACE-inhibitory activity obtained through the hydrophobic interaction chromatogram. The chromatogram yielded two fractions of peaks of high ACE-inhibitory activity on the hydrophobic fraction, which were 90.44% and 95.28%, respectively. Thus, ACE-inhibitory hydrolysate of small molecular size, positive charged and hydrophobic contributed significantly to its potency.

Keywords: ACE-inhibitory activity, hydrophobicity, red tilapia by-product. protein hydrolysate, purification

Abstrak

Penukaran enzim yang dikenali sebagai Angiotensin-1 kepada Angiotensin-2 bertanggungjawab ke atas kenaikan tekanan darah yang menyebabkan hipertensi. Perubahan diet dan gaya hidup perlu diterapkan sebagai pelan rawatan alternatif. Pasaran untuk kompoun penggalak kesihatan didalam makanan semakin dicari-cari pengguna. Sejak sedekad lalu, pelbagai kajian dijalankan ke atas hasil sampingan ikan tilapia (Oreochromis sp.) kerana sifat biologinya terutamanya sebagai agen antihipeternsi. Kenaikkan permintaan bekalan terhadap filet ikan turut meningkatkan kadar penangkapan ikan. Maka, terdapat lebihan model pengeluaran yang dapat dieksploitasi dengan cara yang menguntungkan. Di dalam kajian ini,Thermoase PC10F menghasilkan hidrolisat penghambat ACE daripada hasil sampingan ikan tilapia merah (Oreochromis sp.). Manakala hidrolisat ini ditulenkan melalui ultrafiltrasi (UF), prinsip pertukaran anion (AEX) dan kromatogram interaksi hidrofobik (HIC). Dua berat molekul 3 kDa dan 1 kDa digunakan didalam proses ultrafiltrasi. Penghambatan aktiviti ACE untuk berat molekul 1 kDa lebih tinggi berbanding 3 kDa dengan jumlah 85.42%. Seterusnya, hidrolisat 1 kDa ditulenkan melalui prinsip pertukaran ion dan hidrolisat bercas positif mempunyai penghambatan aktiviti ACE yang tertinggi (72.32%) berbandingkan dengan hidrolisat bercas negatif. Akhir sekali, proses penulenan dilakukan melalui kromatogram interaksi hidrofobik dan pecahan hidrofobik mempunyai penghambatan aktiviti ACE yang tertinggi. Kromatogram menghasilkan dua bagian puncak hidrofobik yang mempunya peringkat aktiviti penghambatan ACE tertinggi sebanyak 90.44% dan 95.28%. Maka, sifat molekul yang kecil, bercas positif dan hidrofobik merupakan ciri-ciri hidrolisat penghambatan aktiviti ACE yang manjur.

Kata kunci: penghambatan aktiviti ACE, kehidrofobikan, produk sampingan ikan tilapia merah, protein hidrosilat, penulenan

References

1. Gao, R., Yu, Q., Shen, Y., Chu, Q., Chen, G., Fen, S., Yang, M., Yuan, L., McClements, D. J. and Sun, Q. (2021). Production, bioactive properties, and potential applications of fish protein hydrolysates: Developments and challenges. Trends in Food Science & Technology, 110: 687-699.

2. Memarpoor-Yazdi, M., Zare-Zardini, H., Mogharrab, N. and Navapour, L. (2020). Purification, characterization and mechanistic evaluation of angiotensin converting enzyme inhibitory peptides derived from Zizyphus jujuba fruit. Scientific Reports, 10(1): 1-10.

3. Caballero, J. (2020). Considerations for docking of selective angiotensin-converting enzyme inhibitors. Molecules, 25(2): 295.

4. Hua, X., Sun, L., Zhong, C., Wu, Q., Xiao, P., Yoshida, A., Liu, G. and Cao, M. (2020). Successive digestion of tilapia collagen by serine proteinase and proline specific endopeptidase to produce novel angiotensin I-converting enzyme inhibitory peptides. Marine Life Science & Technology, 2: 268-278.

5. Ling, Y., Liping, S. and Yongliang, Z. (2018). Preparation and identification of novel inhibitory angiotensin-I-converting enzyme peptides from tilapia skin gelatin hydrolysates: Inhibition kinetics and molecular docking. Food & Function, 9(10): 5251-5259.

6. Ghassem, M., Arihara, K., Babji, A. S., Said, M. and Ibrahim, S. (2011). Purification and identification of ACE inhibitory peptides from Haruan (Channa striatus) myofibrillar protein hydrolysate using HPLC–ESI-TOF MS/MS. Food chemistry, 129(4): 1770-1777.

7. Ghassem, M., Babji, A. S., Said, M., Mahmoodani, F. and Arihara, K. (2014). Angiotensin I–converting enzyme inhibitory peptides from snakehead fish sarcoplasmic protein hydrolysate. Journal of Food Biochemistry, 38(2): 140-149.

8. Ishak, N. H. and Sarbon, N. M. (2018). A review of protein hydrolysates and bioactive peptides deriving from wastes generated by fish processing. Food and Bioprocess Technology, 11: 2-16.

9. Hoa, H. Q. and Duy, N. X. (2016). Ace-Inhibitory Activity Of Protein Hydrolysate From The Skin Of Striped Catfish (Pangasius hypophthalmus). Journal Fish. Science Technology, 3: 1-10.

10. Rincón, C. T. S. and Montoya, J. E. Z. Effects of enzymatic hydrolysis conditions on the antioxidant activity of red tilapia (Oreochromis spp.) viscera hydrolysates. Current Pharmaceutical Biotechnology, 21:1-13.

11. Pędziwiatr, P., Zawadzki, D. and Michalska, K. (2017). Aquaculture waste management. Acta Innovation, 22(22): 20-29.

12. Chen, J., Wang, Y., Zhong, Q., Wu, Y. and Xia, W. (2012). Purification and characterization of a novel angiotensin-I converting enzyme (ACE) inhibitory peptide derived from enzymatic hydrolysate of grass carp protein. Peptides, 33(1): 52-58.

13. Chen, J., Chen, Y., Xia, W., Xiong, Y. L., Ye, R. and Wang, H. (2016). Grass carp peptides hydrolysed by the combination of Alcalase and Neutrase: Angiotensin‐I converting enzyme (ACE) inhibitory activity, antioxidant activities and physicochemical profiles. International Journal of Food Science & Technology, 51(2): 499-508.

14. Roslan, J., Yunos, K. F. M., Abdullah, N. and Kamal, S. M. M. (2014). Characterization of fish protein hydrolysate from tilapia (Oreochromis niloticus) by-product. Agriculture and Agricultural Science Procedia, 2: 312-319.

15. Sierra-Lopera, L. M. and Zapata-Montoya, J. E. (2021). Optimization of enzymatic hydrolysis of red tilapia scales (Oreochromis sp.) to obtain bioactive peptides. Biotechnology Reports 30:1-10.

16. Zahiri, J., Emamjomeh, A., Bagheri, S., Ivazeh, A., Mahdevar, G., Tehrani, H. S., Mirzaie, M., Fakgheri, B. A. and Mohammad-Noori, M. (2020). Protein complex prediction: a survey. Genomics, 112(1): 174-183.

17. Dycka, F., Bobal, P., Mazanec, K. and Bobalova, J. (2012). Rapid and efficient protein enzymatic digestion: an experimental comparison. Electrophoresis, 33(2): 288-295.

18. Adekoya, O. A. and Sylte, I. (2009). The thermolysin family (M4) of enzymes: therapeutic and biotechnological potential. Chemical Biology & Drug Design, 73(1): 7-16.

19. MacLeod-Carey, D., Solis-Céspedes, E., Lamazares, E. and Mena-Ulecia, K. (2020). Evaluation of new antihypertensive drugs designed in silico using Thermolysin as a target. Saudi Pharmaceutical Journal, 28(5): 582-592.

20. Mahmoodani, F., Ghassem, M., Babji, A. S., Yusop, S. M. and Khosrokhavar, R. (2014). ACE inhibitory activity of pangasius catfish (Pangasius sutchi) skin and bone gelatin hydrolysate. Journal of Food Science and Technology, 51(9): 1847-1856.

21. Ke, M., Shen, H., Wang, L., Luo, S., Lin, L. and Yang, J. (2016). Modern proteomics-sample preparation, analysis and practical applications. Springer International. Switzerland: pp. 345-382.

22. Baynes, J. W. and Dominiczak, M. H. (2019). Medical Biochemistry. Elsevier. China.

23. Acquah, C., Wei, Y. Sharadwata, C. and Dominic P. (2018). Structure ‐ informed separation of bioactive peptides. Journal Food Biochemistry, 43(1): 1-10.

24. Nielsen, P. M., Petersen, D. and Dambmann, C. J. (2001). Improved method for determining food protein degree of hydrolysis. Journal of Food Science, 66(5): 642-646.

25. Pa’ee, K. F., Razali, N., Sarbini, S. R., Ramonaran Nair, S. N., Yong Tau Len, K. and Abd-Talib, N. (2021). The production of collagen type I hydrolyzate derived from tilapia (Oreochromis sp.) skin using thermoase PC10F and its in silico analysis. Food Biotechnology, 35(1): 1-21.

26. Roslan, J., Kamal, S. M. M., Yunos, K. F. M. and Abdullah, N. (2017). Assessment on multilayer ultrafiltration membrane for fractionation of tilapia by-product protein hydrolysate with angiotensin I-converting enzyme (ACE) inhibitory activity. Separation and Purification Technology, 173: 250-257.

27. Ghassem, M., Arihara, K. and Babji, A.S. (2012). Isolation, purification and characterisation of angiotensin I-converting enzyme-inhibitory peptides derived from catfish (Clarias batrachus) muscle protein thermolysin hydrolysates. International Journal of Food Science Technology, 47(11): 2444–2451.

28. Pa’ee, K. F., Gibson, T., Marakilova, B. and Jauregi, P. (2015). Production of acid whey hydrolysates applying an integrative process: Effect of calcium on process performance. Process Biochemistry, 50(2): 302-310.

29. Alonso, G., del Valle, E. and Ramirez, J. R. (2020). Desalination in nuclear power plants. Woodhead Publishing, Elsevier, pp: 31–42.

30. Roslan, J., Kamal, S. M. M., Yunos, K. F. M. and Abdullah, N. (2019). Assessment on flux reduction and protein rejection behavior in fractionating tilapia by-product protein hydrolysate by ultrafiltration membrane. Pertanika Journal of Science and Technology, 27(S1): 67-80.

31. Ali, N. A., Hassan, F. and Hamzah, S. (2012). Preparation and characterization of asymmetric ultrafiltration membrane for effective recovery of proteases from surimi wash water. Frontiers of Chemical Science and Engineering, 6(2): 184-191.

32. Singh, R. (2005). Introduction to membrane technology. Hybrid Membrane System of Water Purification: pp. 1-56.

33. Sofiah, H., Nora’aini, A., Asmadi, A. and Abdul Wahab, M. (2014). Preparation and characterization of asymmetric ultrafiltration membrane for lysozyme separation: Effect of polymer concentration. ARPN Journal of Engineering and Applied Science, 9(12): 2543-2550.

34. Zain, M. M., Mohammad, A. W. and Hairom, N. H. H. (2017). Flux and permeation behaviour of ultrafiltration in sugaring out cellulose hydrolysate solution: A membrane screening. Journal of Physical Science, 28(1): 25-38.

35. Ishak, N. H., Shaik, M. I., Yellapu, N. K., Howell, N. K. and Sarbon, N. M. (2021). Purification, characterization and molecular docking study of angiotensin-I converting enzyme (ACE) inhibitory peptide from shortfin scad (Decapterus macrosoma) protein hydrolysate. Journal of Food Science Technology, 58(12): 4567-4577.

36. Lin, H. C., Alashi, A. M., Aluko, R. E., Sun Pan, B. and Chang, Y. W. (2017). Antihypertensive properties of tilapia (Oreochromis spp.) frame and skin enzymatic protein hydrolysates. Food & Nutrition Research, 61(1): 1-12.

37. Aykul, S. and Martinez-Hackert, E. (2016). Determination of half-maximal inhibitory concentration using biosensor-based protein interaction analysis. Analytical biochemistry, 508: 97-103.

38. Liu, C., Fang, L., Min, W., Liu, J. and Li, H. (2018). Exploration of the molecular interactions between angiotensin-I-converting enzyme (ACE) and the inhibitory peptides derived from hazelnut (Corylus heterophylla Fisch.). Food Chemistry, 245(2888): 471-480.

39. de Vos, W. M. and Lindhoud, S. (2019). Overcharging and charge inversion: Finding the correct explanation(s). Advance Colloid Interface Science, 274(3): 1-8.

40. Fekete, S., Beck, A., Veuthey, J. L. and Guillarme, D. (2015). Ion-exchange chromatography for the characterization of biopharmaceuticals. Journal of Pharmaceutical and Biomedical Analysis, 113: 43-55.

41. Je, J. Y., Park, P. J., Kwon, J. Y. and Kim, S. K. (2004). A novel angiotensin I converting enzyme inhibitory peptide from Alaska pollack (Theragra chalcogramma) frame protein hydrolysate. Journal of Agricultural and Food Chemistry, 52(26): 7842-7845.

42. Sungperm, P., Khongla, C. and Yongsawatdigul, J. (2020). Physicochemical properties and angiotensin I converting enzyme inhibitory peptides of freshwater fish skin collagens. Journal of Aquatic Food Product Technology, 29(7): 650-660.

43. Manoharan, S., Shuib, A. S. and Abdullah, N. (2020). Structural characteristics and antihypertensive effects of angiotensin-I-converting enzyme inhibitory peptides in the renin-angiotensin and kallikrein kinin systems. African Journal of Traditional Complement Alternative Medicines, 14(2): 383-406.

44. 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): 1-19.

45. De Leo, F., Panarese, S., Gallerani, R. and Ceci, L. R. (2009). Angiotensin converting enzyme (ACE) inhibitory peptides: Production and implementation of functional food. Current Pharmaceutical Design, 15(31): 3622-3643.

46. Ko, A. J., Kang, N., Kim, J. L. J., Park, W. K. S. and Jeona, Y. K. Y. (2016). Angiotensin I-converting enzyme inhibitory peptides from an enzymatic hydrolysate of flounder fish (Paralichthys olivaceus ) muscle as a potent anti-hypertensive agent. Process Biochemistry, 51(4): 535-541.

47. Acquah, C., Di Stefano, E. and Udenigwe, C. C. (2018). Role of hydrophobicity in food peptide functionality and bioactivity. Journal of Food Bioactives, 4: 88-98.