Purification, characterization and molecular docking study of angiotensin-I converting enzyme (ACE) inhibitory peptide from shortfin scad (Decapterus macrosoma) protein hydrolysate.

Hypertension is a threatening chronic disease, which become a global killer among the adult population. The mortality rate increasing day by day even several Angiotensin I-converting enzyme (ACE) inhibitor drugs were introduced. Bioactive peptides derived from aquatic resources exhibits potential ACE inhibitory activity. The objective of this work is to report the purification and molecular docking studies of angiotensin-I converting enzyme (ACE) inhibitory peptide isolated from shortfin scad (Decapterus macrosoma) waste protein hydrolysate (SWH), enzymatically prepared by using alcalase. The purification process included ultrafiltration, gel filtration and reverse phase high performance liquid chromatography (RP-HPLC). Results showed that ultra-filtered peptide fraction (< 3 kDa) possessed the highest ACE inhibitory activity, followed by the fraction 14 by gel filtration. Fraction P obtained by RP-HPLC, with the amino acid sequence of RGVGPVPAA (IC50 = 0.20 mg/ml) was identified. In terms of ACE inhibition, the Lineweaver-Burk plot showed that the SWH peptide obtained acted as a competitive ACE inhibitor. The molecular docking studies showed that the SWH peptide exhibit hydrogen bonds and Pi-interactions with ACE by Z-dock scores. These results showed that the purified peptide isolated from shortfin scad waste hydrolysate has potential antihypertensive properties which could potentially be used as functional food ingredients.

Determining the Cytotoxicity of Oxidized Lipids in Cultured Caco-2 Cells Using Bioimaging Techniques.

Fish lipids are comprised of considerable quantities of polyunsaturated acids and are prone to oxidation, producing reactive oxygen species and hydroperoxides. This study aimed to evaluate the biochemical and structural alterations in Caco-2 cells following exposure to 100 µg/mL methyl linoleate or fish oil, and then radiated for 24, 48 or 72 h. Electron spin resonance spectroscopy detected free radicals in the lipid membrane, Raman microscopy observed biochemical alterations and atomic force microscopy identified changes in morphology, such as the breakdown of DNA bonds. The study showed that bioimaging and biochemical techniques can be effective at detecting and diagnosing cellular injuries incurred by lipid peroxidation.

Proximate and biochemical characterization of burrito (Bachydeuterus auritus) and flying gurnard (Dactylopterus volitans).

With limited protein resources and depleting commercial fish species there is the need to improve utilization of some of the lesser known species which are underutilized, for example, big eye grunt (burrito), Bachydeuterus auritus, and the flying gurnard (Dactylopterus volitans), (other names Cephalocanthus volitans (local) Pampansre). This study was to characterize some of the proximate and biochemical properties of burrito and the flying gurnard so as to evaluate their potential for use in human nutrition and other value-added products. Proximate and chemical analysis were determined by the methods of AOAC. Fatty acid profiles were determined following the method of Saaed and Howell (1999). Amino acid profiles for the species were determined according to Bidlingmeyer et al. (1987). The protein content of both the water soluble and salt soluble protein extracts of the fish species were determined by the Bradford Protein Assay method (Bradford 1976). Rancidity of the fish species was assessed by thiobarbituric acid reactive substances (TBARS) and Peroxide value (PV) as described by Saeed and Howell (1999). Burrito contained 18% protein, whereas the flying gurnard contained 22.3%. Calcium content was 296 mg/100 g for burrito and 185 mg/100 g for flying gurnard, whereas iron content was 4.1 mg/100 g and 1.0 mg/100 g for burrito and the flying gurnard, respectively. Palmitic acid (C16) was 27% and 14.3% for the flying gurnard and burrito, respectively. C17: 1ω8 was 3% in the flying gurnard and 0.2% in burrito. Oleic (C18:1ω9) was 17% in the flying gurnard and 6% in burrito. C20:4ω6 was 1.6% in the flying gurnard and 3% in burrito. Docosahexaenoic acid (C22:6ω3) was 4.9% in the flying gurnard and 4.0% in burrito. Both burrito and the flying gurnard are of high nutritional quality as they had a high protein content, good general amino acid profile and abundance of polyunsaturated fatty acids.

Antiproliferative Activity of Egg Yolk Peptides in Human Colon Cancer Cells.

Egg yolk peptides were successfully prepared from egg yolk protein by-products after lecithin extraction. Defatted egg yolk protein was hydrolyzed with pepsin and pancreatin and purified by gel filtration to produce egg yolk gel filtration fraction (EYGF-33) with antiproliferative activity. The highlight of this study was that the peptide EYGF-33 (1.0 mg/ml) significantly inhibits cell viability of colon cancer cells (Caco-2) with no inhibitory effects on the viability of human colon epithelial normal cells (HCEC) after 48 h. Reduced cell viability can be explained by cell cycle arrest in the S-phase in which DNA replication normally takes place. EYGF-33 significantly enhanced the production of superoxide anions in the mitochondria of Caco-2 cells; this could activate a mitochondrial apoptotic pathway leading to typical Poly Adenosine diphosphate-ribose polymerase (PARP) cleavage as observed in the Western blot result. The induction of apoptotic cell death by EYGF-33 was supported by the externalization of phosphatidylserine (PS). However, further elucidation of the mechanism of EYGF-33-mediated apoptosis would provide further support for its use as a potential therapeutic and chemopreventive agent.

Cytotoxicity of oxidised lipids in cultured colonal human intestinal cancer cells (caco-2 cells).

In this study, we investigated the extent of the cytotoxicity effect of oxidised lipids and whether tea catechins namely (-)epigallocatechin-3 gallate (EGCG) decreased lipid peroxidation in caco-2 cells. Cells treated with 0-100 microg/ml fish oil or methyl linoleate (ML) oxidised by UV irradiation for 24 h, 48 h and 72 h, indicated a substantial decrease in cell viability especially in samples treated with 100 microg/ml oxidised lipid. Addition of malondialdehyde (MDA) and hydroxynonenal (50 microM) also reduced cell viability. Using EGCG (50 microM) increased the viability of cells treated with 24 h oxidised mackerel oil (72% live and 28% dead) compared with 48 h oxidised mackerel oil (89% live and 11% dead) and 72 h oxidised mackerel oil (71% live and 29% dead) as monitored by the MTT assay. Apoptosis of caco-2 cells by oxidised fish oil and ML and protection by EGCG was confirmed using fluorescence microscopy and caspase-3 presence by Western blotting.

Aldehyde formation in frozen mackerel (Scomber scombrus) in the presence and absence of instant green tea.

The effect of frozen storage on lipid peroxidation in Atlantic mackerel (Scomber scombrus) stored for up to 26 weeks at -10 or -80°C (control), with and without green tea antioxidants, was investigated. Hydroperoxides (PV) and aldehydes (TBARS) were measured by HPLC and LC-MS and hexanal by GC. There was an increase in peroxide value which was associated with an increase in aldehydes, followed by hexanal increase with storage time and at a higher temperature of -10°C compared with samples stored at -80°C. Although TBARS is a common assay used to follow malondialdehyde formation, other aldehyde products can also react with thiobarbituric acid to give the red chromogen. Analysis of aldehyde-TBA adducts by LC-MS confirmed the presence of malondialdehyde and, in particular, we report the production of gluteraldehyde for the first time in stored frozen fish. Green tea (at 250ppm) substantially slowed down the oxidation process, whereas at 500ppm it was less effective.

Fourier transform raman spectroscopy study of heat-induced gelation of plasma proteins as influenced by pH.

Fourier transform (FT) Raman spectroscopy was used to elucidate heat-induced structural changes of albumin, globulins, serum, and plasma protein solutions (15% w/w) as affected by pH (4.5, 6.0, and 7.5). Reduction of alpha-helix and formation of beta-sheet, disulfide bond reactions, and exposure and buriedness of hydrophobic groups and amino acid residues were observed. All of these features contributed to the formation of strong, irreversible heat-induced gels. The application of a dimensionality reducing technique such as principal component analysis proved to be useful to determine the most influential qualities of protein samples, as well as the pH-dependent behavior of some of the attributes of both unheated and heated solutions. Analysis of Raman spectra in terms of differences demonstrated the interactions of albumin and globulins in co-occurrence and the significant role of fibrinogen on the gel's attributes.

ESR and NMR spectroscopy studies on protein oxidation and formation of dityrosine in emulsions containing oxidised methyl linoleate.

Oxidised lipids are reported to interact with proteins causing undesirable changes in nutritional and functional properties including a loss of amino acids, cross-linking and damage to proteins and DNA. ESR spectroscopy with spin trapping was used to study the type of radical species generated in methyl linoleate and the transfer of the radical to protein beta-lactoglobulin. Antioxidants vitamins C and E reduced lipid oxidation and subsequent transfer of the radical to the protein as shown by the shape and size of the radical adduct. Changes to protein molecular structure due to oxidation were investigated by multidimensional NMR spectroscopy and liquid chromatography. NMR spectra indicated that as a result of oxidation and protein denaturation, there was an increase in structural flexibility and some initially protected backbone amide groups were exposed as they become sharper and easily identifiable. Dityrosine was detected in all samples tested which is indicative of oxidative damage to proteins. Monitoring tyrosyl radicals and formation of dityrosine is of practical value in order to enhance the acceptability, nutritional and safety aspects of proteins.

Elucidation of the effect of formaldehyde and lipids on frozen stored cod collagen by FT-Raman spectroscopy and differential scanning calorimetry.

The effect of frozen storage (-10 and -30 degrees C), formaldehyde, and fish oil on collagen, isolated from cod muscle, was investigated. Salt- and acid-soluble collagen fractions as well as insoluble collagen indicated changes in solubility on frozen storage. Differential scanning calorimetry (DSC) showed a highly cooperative transition at 28.2 degrees C for isolated collagen. Changes in the thermodynamic properties of collagen were observed on frozen storage at -10 degrees C compared with the control at -30 degrees C because of changes in structure. In the presence of formaldehyde, there were no changes in the DSC collagen transition; however, in the presence of fish oil there was an increase in enthalpy and an extra peak was observed at 44.6 degrees C, indicating collagen-fish oil interaction. Structural changes resulted in a decrease in the solubility of collagen in salt and acid solution. FT-Raman spectra obtained for collagen at -10 degrees C and -30 degrees C confirmed the alteration of the conformation of collagen not only at -10 degrees C but also in the presence of formaldehyde and fish oil.

Effect of antioxidants, citrate, and cryoprotectants on protein denaturation and texture of frozen cod (Gadus morhua).

To investigate the role of antioxidants and cryoprotectants in minimizing protein denaturation in frozen lean fish, cod fillets were treated with either antioxidants (vitamin C (500 mg kg(-1)) or vitamin C (250 mg kg(-1)) + vitamin E (250 mg kg(-1))), antioxidants (vitamins C + E 250 mg kg(-1)each) with citrate (100 mg kg(-1)), cryoprotectants (4% (w/w) sucrose + 4% (w/w) sorbitol), or a mixture of antioxidants (vitamins C + E 250 mg kg(1)), citrate (100 mg kg(-1)), and cryoprotectants (sucrose 40 g kg(-1) + sorbitol 40 g kg(-1)). Untreated and treated fish samples were stored at -10 degrees C; cod fillets stored at -30 degrees C were used as a control. Stored frozen samples were analyzed at intervals for up to 210 days for changes in protein extractability, thermodynamic parameters (transition temperature T(m) and enthalpy DeltaH), structure by FT-Raman spectroscopy, and rheological properties by large and small deformation tests. Results indicated that protein denaturation and texture changes were minimized in the presence of cryoprotectants, as well as in the presence of antioxidants with citrate, antioxidants alone, or the mixture of antioxidants, citrate, and cryoprotectants. In the presence of increased formaldehyde levels in fish treated with vitamin C, toughening was still lower compared to that of the -10 degrees C control due to the antioxidant property of vitamin C. Thus, ice crystal formation and lipid oxidation products are the major factors that cause protein denaturation in lean frozen fish, and antioxidants in addition to cryoprotectants can be used to minimize toughness.