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In this study, the angiotensin I-converting enzyme (ACE) inhibitory peptides were isolated from snakehead fish sarcoplasmic protein hydrolysates. Enzymatic hydrolysis of sarcoplasmic protein was performed using various commercial enzymes. The alcalase hydrolysate with the highest ACE inhibition activity was purified with gel chromatography and reversed phased high-performance liquid chromatography. The purified fractions were then subjected to electrospray ionization quadrupole-micro-time-of-flight mass spectrometry for amino acid characterization. Two novel ACE inhibitory peptides LYPPP and YSMYPP with IC50 values of 1.3 and 2.8 mM were identified, respectively. The pattern of ACE inhibition, resistance to hydrolysis by gastrointestinal proteases and cytotoxic potencies of isolated peptides were described. The results showed no cytotoxicity of peptides on human embryonic fibroblast cell line (MRC-5) and human hepatocarcinoma cell line (HepG2). PRACTICAL APPLICATIONSFreshwater fish muscle proteins and their hydrolysates offer huge potential as novel sources of natural bioactive peptides with angiotensin I-converting enzyme (ACE) inhibitory activity. The present study revealed the identification of two strong ACE inhibitory peptides obtained from alcalase hydrolysis of snakehead fish sarcoplasmic protein. However, further studies are required to determine the in vivo antihypertensive activity of the purified potent ACE inhibitory peptides. bs_bs_banner Journal of Food Biochemistry ISSN 1745-4514 ACE INHIBITORS OF SNAKEHEAD SARCOPLASMIC PROTEIN HYDROLYSATE M. GHASSEM ET AL.

Edible bird's nest (EBN) is widely consumed as a delicacy and traditional medicine amongst the Chinese. In the present study, for the first time, the antioxidant properties of an EBN pepsin-trypsin hydrolysate of the swiftlet species Aerodramus fuciphagus and its ultrafiltration fractions were investigated. Thirteen peptides with molecular weights between 514.29 and 954.52 Da were identified in the EBN fraction with the use of mass spectrometry. Two novel pentapeptides Pro-Phe-His-Pro-Tyr and Leu-Leu-Gly-Asp-Pro, corresponding to f134-138 and f164-168 of cytochrome b of A. fuciphagus, indicated the highest ORAC values of 14.95 and 14.32 μM of TE μM peptide, respectively. Both purified peptides showed resistance against simulated gastrointestinal proteases. In addition, both peptides had no in vitro cytotoxicity on human lung MRC-5 cells and prevented human liver carcinoma HepG2 cellular damage caused by hydroxyl radicals. Therefore, it is suggested that EBN protein hydrolysates are a good source of natural antioxidants and could be applied as nutraceutical compounds.

This study was conducted to evaluate the kinetic characteristics of proteolytic activity of proteases on Channa striatus protein fractions. Degree of hydrolysis (DH), amino acid composition and kinetic parameters of sarcoplasmic and myofibrillar proteins were investigated when incubated with proteinase K and thermolysin, separately. After 30 min incubation with proteases, a decrease in DH of sarcoplasmic protein was observed whereas, hydrolysis of myofibrillar protein with proteases took 2 h with an increase in DH. The major amino acids were glutamic acid (16.6%) in thermolysin-myofibrillar hydrolysate followed by aspartic acid (11.1%) in sarcoplasmic protein fraction with no enzyme treatment and lysine (10%) in thermolysinmyofibrillar hydrolysate. The apparent Michaelis constant of proteinase K was lower than thermolysin for both sarcoplasmic and myofibrillar proteins. However, rate of turnover and enzyme efficiency suggested that sarcoplasmic and myofibrillar proteins are suitable substrates for proteinase K and thermolysin hydrolytic reaction, respectively.

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Skin and bone gelatins of pangasius catfish (Pangasius sutchi) were hydrolyzed with alcalase to isolate Angiotensin Converting Enzyme (ACE) inhibitory peptides. Samples with the highest degree of hydrolysis (DH) were separated into different fractions with molecular weight cutoff (MWCO) sizes of 10, 3 and 1 kDa, respectively and assayed for ACE inhibitory activity. Skin and bone gelatins had highest DH of 64.87 and 68.48 % after 2 and 1 h incubation, respectively. Results from this study indicated that by decreasing the molecular weight of fractions, ACE inhibitory activity was increased. Therefore, F 3 permeates (MWCO< 1 kDa) of skin (IC 50 03.2 μg/ml) and bone (IC 50 01.3 μg/ml) gelatins possessed higher ACE inhibitory activity compared to their untreated gelatins and corresponding hydrolyzed fractions. In this study, the major amino acids were Glycine followed by Proline with an increased amount of hydrophobic amino acid content in F 3 permeates of skin (4.01 %) and bone (5.79 %) gelatin. Digestion stability against gastrointestinal proteases did not show any remarkable change on ACE inhibition potency of these permeates. It was concluded that alcalase hydrolysis of P. sutchi by-products could be utilized as a part of functional food or ingredients of a formulated drug in order to control high blood pressure.

Summary The angiotensin I‐converting enzyme (ACE)‐inhibitory activities of catfish (Clarias batrachus) muscle protein hydrolysates were investigated. Thermolytic digests of C. batrachus sarcoplasmic and myofibrillar proteins exhibited inhibitory activity towards ACE and were purified with the aim of ultrafiltration, gel filtration and reversed‐phase high‐performance liquid chromatography (RP‐HPLC). The amino acid sequences of hydrolysates with the highest ACE‐inhibitory activities were determined using electrospray quadrupole time‐of‐flight tandem mass spectrometry (ESI‐TOFQ MS/MS). The sequences of GPPP (IC50 = 0.86 μm) and IEKPP (IC50 = 1.2 μm) corresponding to the fragments 986–989 and 441–445 of myosin‐I heavy chain were identified for the sarcoplasmic and myofibrillar protein hydrolysates, respectively. Peptide GPPP exhibited a mixed‐type inhibition whereas peptide IEKPP could only bind to the active sites of ACE. The results demonstrate that hydrolysates of C. batrachus muscle proteins obtained by thermolysin may contain bioactive peptides.

Pretreatments with different types of alkali and acid were compared to determine their effects on gelatin extraction from African catfish (Clarias gariepinus) skin. The study was divided into three parts. In the first part, the skins were only treated with alkaline (Ca(OH)2 or NaOH) solution or pretreated with acetic acid solution. For second part, combination of alkali and acid pretreatment was carried out. For the third part, the skins were first treated with NaOH solution, followed by the treatment with acetic acid, citric acid or sulfuric acid solution. Functional properties including the yield of protein recovery, gel strength, viscosity, pH and viscoelastic properties were determined on gelatins obtained with different pretreatment conditions. Pretreatment with alkali removed noncollagenous proteins effectively, whilst acid pretreatment induced some loss of collagenous proteins. Combination of alkali and acid pretreatment not only removed the noncollagenous proteins and caused a significant amount of swelling, but also provided the proper pH condition for extraction, during which some cross-linkages could be further destroyed but with less breakage of intramolecular peptide chains. Pretreatment of catfish skins with 0.2 N NaOH followed by 0.05 M acetic acid improved yield of protein recovery, gel strength, viscosity, melting temperature and gelling temperature of gelatin extract.