Hydrolysis of food proteins with isolated enzymes has been subject to extensive research carried out with a view to produce bioactive peptides. In this study, 0.5% (w/v) of papain and bromelain, and 1.25% (v/v) of Flavourzyme™ were used to hydrolyse soluble protein fractions leatherjacket (Meuchenia sp.) and trevally (Pseudocaranx sp.) and 1% (w/v) of papain and bromelain and 2.5% (v/v) of Flavourzyme™ for insoluble protein fraction. The resulting hydrolysates were analysed for the degree of hydrolysis (DH), anti-microbial activity, and angiotensin I converting enzyme (ACE) inhibition. It was found that the DH of the hydrolysates ranged from 22- 25% for soluble protein hydrolysates, and from 30-33% for the insoluble protein hydrolysates after 10-hour hydrolysis. Results from anti-microbial assays showed that 8 h bromelain hydrolysate of leatherjacket insoluble protein fraction (labeled as LBI8H) showed strongest activity. Two active fractions, fraction 9 (labeled as LBI9) and 12 (labeled as LBI12), showed activities against Staphylococcus aureus and Bacillus cereus. Fraction LBI9 exhibited some activity against B. cereus without an MIC being reached at 5.35 mg/ml peptide concentration. Peptide fraction LBI12, on the other hand, showed activity against both B. Cereus and S. aureus with an MIC value of 4.3 mg/ml. Edman N-terminal sequence analysis revealed active fraction LBI12 consisting mainly heptapeptide EQIDNLQ (MW = 858.9 Da, net charge = -2), an anionic peptide. Anti-microbial activity assays using synthetic EQIDNLQ revealed that the heptapeptide is active against both B. Cereus and S. aureus bacteria having MIC values of 5.3 mg/ml and 7.96 mg/ml, respectively. Results from ACE inhibition studies, expressed as IC50 values, showed that leatherjacket soluble protein hydrolysates have IC50 values ranged from 1.35 mg/ml to 1.89 mg/ml, while the IC50 values of the leatherjacket insoluble protein fractions ranged from 0.77 mg/ml to 6.78 mg/ml. The IC50 values of trevally soluble protein fraction ranged from 1.99 mg/ml to 3.34 mg/ml, while the IC50 values of the trevally insoluble protein fractions ranged between 2.45 mg/ml to 4.74 mg/ml. Purification with C18 RP-HPLC column produced 5 purified fractions from leatherjacket hydrolysates and 7 purified fractions from trevally hydrolysates. The 5 leatherjacket fractions were labelled as LPI5, LPI6, LBI2, LBI5, and LFI5, while the 7 trevally fractions were labelled as TPI3, TPI4, TBS1, TBS2, TBS6, TBI2, and TBI4. The primary structures of the four leatherjacket peptide fractions LPI5, LPI6, LBI5, and LFI5 were determined as EPLYV, DPHI, AER, and WDDME, having molecular weights of 619.71, 480.52, 374.40, and 694.71 Da, respectively. The primary structure of three trevally peptide fractions TBS1, TBS2, and TBI2 are AR, AV, and APER, having molecular weight of 245.28, 188.23, and 471.51 Da, respectivelly. In addition, leatherjacket fraction LBI12 with sequence of EQIDNLQ, an anionic antimicrobial peptide also showed ACE inhibitory activity with an IC50 value of 0.24 mg/ml. Kinetic study showed that heptapeptide Glu-Gln-Ile-Asp-Asn-Leu-Gln (EQIDNLQ) is a competitive anti-hypertensive peptide that will bind to the active site of ACE. Chemical shift index (CSI) nuclear Overhauser effect (NOEs) from NMR experiments in aqueous and SDS solutions showed that the peptide has an extended structure with a tendency to take a turn between isoleucine (Ile-3, I) and aspartic acid (Asp-4, D). With such an open structure, this small peptide can attach into the active site of ACE though partially negative charge of glutamic acid (Glu-1, E) as well as glutamine (Gln-2 and Gln-7, Q) to bind to the zinc ion that is bound at the active site of ACE and to bind to microbial membrane through hydrogen bonds or van der Waals interactions. Results from this study suggest that papain, bromelain, Flavourzyme can be used in production of ACE inhibitory peptides. In addition, bromelain is also able to release anionic anti-microbial peptide from leatherjacket proteins. Further study is needed to investigate the possibility to utilise these active ACE inhibitory peptides in preparation of anti-hypertensive functional foods. Further characterisation of the anionic anti-microbial peptide may be beneficial to understand and to improve its activity.
| Date of Award | 2010 |
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| Original language | English |
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