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Title:	Characterization and antioxidant activities of bioactive peptides from catfish (Pangasius sutchi) protein hydrolysates
Authors:	Leila Najafian (P55918)
Supervisor:	Abdul Salam Babji, Prof. Dr.
Keywords:	Antioxidant activities Bioactive peptides Catfish Proteins (Analysis)
Issue Date:	5-Dec-2014
Description:	Patin (Pangasius sutchi) merupakan ikan air tawar yang mempunyai kepentingan ekonomi dan hidup dalam sungai, tasik atau diternak dalam kolam. Hidrolisat protein sarkoplasmik (SPHs) dan hidrolisat protein miofibrilar (MPHs) telah dihasilkan menggunakan tiga jenis protease: papain, alkalase dan flavourzyme. Asai aktiviti perencatan radikal 2,2-difenil-1-pikrilhidrazil (DPPH), 2-azino-bis(3-etilbenzotiazolin-6-sulfonik) garam diammonium (ABTS), kuasa penurunan dan pengkelat logam untuk kajian aktiviti antioksida telah dijalankan ke atas SPHs dan MPHs. Hidrolisat ditulenkan menggunakan ultraturasan, penurusan gel dan kromatografi cecair prestasi tinggi fasa terbalik (RP-HPLC), manakala kromatografi cecair dengan pengesan spektrometri jisim tandem (LC-MS/MS) digunakan untuk menentukan jujukan peptida. Hasil kajian menunjukkan jenis protease mempengaruhi darjah hidrolisis (DH), di mana kesemua enzim menunjukkan kadar hidrolisis yang tinggi pada jam pertama, dan menurun secara perlahan. DH paling tinggi (82.40%) untuk SPHs diperolehi selepas 60 min eraman dengan papain. Untuk protein miofibrilar, semua enzim meningkatkan nilai DH apabila masa hidrolisis meningkat. DH (89.17%) paling tinggi untuk protein miofibrilar telah dihasilkan menggunakan papain selepas rawatan selama 120 min. Kesan DH ke atas aktiviti antioksida SPHs dan MPHs telah dikaji. Pada nilai DH 65.83%, papain-MPH menunjukkan aktiviti pemerangkapan radikal DPPH (71.14%) maksimum dengan kuasa penuruan 0.310. Pada kepekatan 1 mg/ml, papian-MPH menunjukkan kapasiti antioksida setara Trolox (TEAC) 70.50 ± 1.22 μmol/g peptida, walaupun tidak menunjukkan perbezaan ketara antara sampel papain-MPH dan alkalase-MPH dengan nilai DH yang sama. Alcalase-MPH mempunyai aktiviti pengkelat logam paling tinggi pada DH 83.60%. Pada DH 51.60%, flavourzyme-SPH menunjukkan aktiviti pemerangkapan radikal DPPH paling tinggi. Nilai TEAC dan pengkelat logam masing-masing mencatatkan 42.58 ± 1.70 μmol TE/ g protein and 83% untuk hidrolisat disediakan menggunakan alkalase pada DH 79.80%. Papain-SPH menunjukkan kuasa penurunan yang paling tinggi. Antara hidrolisat, papain-MPH dan alkalase-SPH yang mempunyai aktiviti antioksida yang paling tinggi telah ditulenkan menggunakan ultraturasan, kromatografi penurusan gel dan RP-HPLC. Alkalase-SPH-III dan papain-MPH-III dengan MWCO < 3 kDa mempunyai aktiviti pemerangkapan radikal DPPH paling tinggi dengan IC50 1.26 dan 1.76 kali ganda lebih tinggi berbandingan SPH dan MPH. Pecahan penurasan gel dengan aktiviti antioksida paling tinggi diperoleh daripada sarkoplasmik alkalase (SI) dan hidrolisat miofibrilar papain (MI) telah dikaji untuk menentukan komposisi asid amino. Keputusan juga menunjukan kedua-dua fraksi mempunyai kandungan asid amino hidrofobik dan aromatik yang tinggi berbanding hidrolisat. Fraksi hidrolisat sarkoplasmik (SI 3) and miofibrilar (MI 4) diperolehi daparida RP-HPLC menunjukkan aktiviti pemerangkapan radikal DPPH 1.83 dan 2.97 kali ganda lebih tinggi berbanding SPH dan MPH. Jujukan peptida bagi peptida antioksida daripada SPH dan MPH telah ditentukan menggunakan kromatografi cecair-spektrometer jisim LC-MS. Jujukan peptida SPH telah di kenal pasti sebagai DPQHPVMPR, LVVDIPAALQHA dan GVDNPGHP dengan berat molikul 539, 312 dan 397 Da. VPKNYFHDIV, LVMFLDNQHRVIRH dan FVNQPYLLYSVHMK dengan berat molikul 616, 593 dan 580 Da masing-masing dikenalpasti dalam peptida MPH yang mempunyai aktiviti antioksida. Peptida berdaya antipengoksida daripada SPH dan MPH mangandungi asid amino hidrofobik, iaitu leusina (L), Valina (V) dan fenilalanina (F). Kehadiran asid amino bercas negatif, asid aspartik (D) dalam peptide SPH dan asid amino neutral, asparagina (N) dalam peptida MPH turut dikenalpasti. Dengan ini, kehadiran asid amino hidrofobik, asid amino asid aspartic dan asparabina, asid amino hidrofilik, histidin (H) dan prolina (P), daripada peptida SPH dan MPH dipercayai menyumbangkan kepada aktiviti antioksida yang tinggi. Kesimpulan, SPH dan MPH ikan patin mempunyai potensi sebagai bahan berfungsi semulajadi dalam industri makanan dan farmasi.,Patin (Pangasius sutchi) is a freshwater catfish of great economic importance found in rivers, lakes or cultured in ponds. The fish sarcoplasmic protein hydrolysates (SPHs) and myofibrillar protein hydrolysates (MPHs) were produced using three types of proteases: papain, alcalase and flavourzyme. 1,1-diphenyl-2-picrylhydrazyl (DPPH), 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulphonic acid) diammonium salt (ABTS) radical scavenging activities, reducing power and metal chelating activity tests for antioxidant activities of SPHs and MPHs were assayed. The hydrolysates were purified by ultrafiltration, gel filtration and reverse phase high performance liquid chromatography (RP-HPLC) and liquid chromatography with tandem mass spectrometry detection (LC-MS/MS) and used in identifying their peptide sequences. Results of this study indicated that type of protease affected the degree of hydrolysis (DH), where all of the enzymes showed high rate of hydrolysis during the first hour, and then gradually decreased. The highest DH (82.40%) among the SPHs resulted from 60 min of incubation with papain. For the myofibrillar protein all enzymes increased the DH values with increasing hydrolysis time. The highest DH (89.17%) for myofibrillar protein was produced with a papain treatment for 120 min. The effect of DH on antioxidant activities of SPHs and MPHs was determined. With a DH of 65.83%, the papain-MPH exhibited the maximum of DPPH radical-scavenging activity (71.14%) and a reducing power with absorbance of 0.310. At a concentration of 1 mg/ml, the papain-MPH exhibited a Trolox equivalent antioxidant capacity (TEAC) of 70.50 ± 1.22 μmol/g protein, although significant differences were not observed (p < 0.05) between samples of papain-MPH and alcalase-MPH with similar DH values. Alcalase-MPH had the highest metal chelating activity at 83.60% DH. When the DH was 51.60%, flavourzyme-SPH exhibited the strongest DPPH radical scavenging activity. The maximum values of the TEAC and metal chelating activity were 42.58 ± 1.70 μmol TE/ g protein and 83%, respectively for the hydrolysates prepared by alcalase at DH 79.80%. Papain-SPH showed the highest reducing power. Among the hydrolysates, papain-MPH and alcalase-SPH, which had the highest antioxidant activities, were further purified using ultrafiltration, gel filtration chromatography and RP-HPLC. Alcalase-SPH-III and papain-MPH-III with MWCO < 3 kDa had the highest DPPH radical scavenging activity with IC50 1.26 and 1.76 times respectively higher than SPH and MPH. Gel filtrated fractions with the highest antioxidant activity separated from alcalase sarcoplasmic (SI) and papain myofibrillar hydrolysates (MI), were tested for amino acid composition. The results revealed that both fractions had higher amount hydrophobic and aromatic amino acids than their hydrolysates. The potent fractions obtained from RP-HPLC of sarcoplasmic (SI 3 fraction) and myofibrillar (MI 4 fraction) hydrolysates showed DPPH radical scavenging activity of 1.83 and 2.97 times higher than the SPH and MPH. Peptide sequences of antioxidative peptide from SPH and MPH were identified using liquid chromatography mass spectrometer, LC-MS. The peptide sequences for SPH were identified as DPQHPVMPR, LVVDIPAALQHA and GVDNPGHP with molecular weight of 539, 312 and 397 Da. VPKNYFHDIV, LVMFLDNQHRVIRH and FVNQPYLLYSVHMK with molecular weight of 616, 593 and 580 Da, respectively were identified in antioxidative peptides of MPH. Antioxidant peptides from SPH and MPH contained hydrophobic amino acids, namely leucine (L), valine (V) and phenylalanine (F). In addition, the presence of negatively charged amino acids, aspartic acid (D) in the peptide of SPH and neutral amino acid, asparagine (N) in the peptide of MPH was also observed. With this, the presence of hydrophobic amino acid, aspartic acid and asparagine amino acids and hydrophilic amino acids, histidine (H) and proline (P), in the peptide sequences of SPH and MPH is believed to contribute to high antioxidant activity. In conclusion, SPH and MPH from patin have the potential as a natural functional ingredient in food and pharmaceutical industries.,PhD
Pages:	160
Call Number:	QP551.N345 2014 tesis
Publisher:	UKM, Bangi
Appears in Collections:	Faculty of Science and Technology / Fakulti Sains dan Teknologi

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