In the present study, to establish the optimum gelatin extraction conditions from pangasius catfish (Pangasius sutchi) bone, Response Surface Methodology (RSM) with a 4-factor, 5-level Central Composite Design (CCD) was conducted. The model equation was proposed with regard to the effects of HCl concentration (%, X 1 ), treatment time (h, X 2 ), extraction temperature (°C, X 3 ) and extraction time (h, X 4 ) as independent variables on the hydroxyproline recovery (%, Y) as dependent variable. X 1 02.74 %, X 2 021.15 h, X 3 074.73°C and X 4 05.26 h were found to be the optimum conditions to obtain the highest hydroxyproline recovery (68.75 %). The properties of optimized catfish bone gelatin were characterized by amino acid analysis, SDS-PAGE, gel strength, TPA and viscosity in comparison to bovine skin gelatin. The result of SDS-PAGE revealed that pangasius catfish bone gelatin consisted of at least 2 different polypeptides (α 1 and α 2 chains) and their cross-linked chains. Moreover, the pangasius catfish bone gelatin was found to contain 17.37 (g/100 g) imino acids (proline and hydroxyproline). Pangasius catfish bone gelatin also indicated physical properties comparable with that of bovine and higher than those from cold water fish gelatin. Based on the results of the present study, there is a potential for exploitation of pangasius catfish bone for gelatin production. Furthermore, RSM provided the best method for optimizing the gelatin extraction parameters.

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.

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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.

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In a successful fortification program, the stability of micronutrients added to the food is one of the most important factors. The added vitamin D3 is known to sometimes decline during storage of fortified milks, and oxidation through fatty acid lipoxidation could be suspected as the likely cause. Identification of vitamin D3 oxidation products (VDOPs) in natural foods is a challenge due to the low amount of their contents and their possible transformation to other compounds during analysis. The main objective of this study was to find a method to extract VDOPs in simulated whole milk powder and to identify these products using LTQ-ion trap, Q-Exactive Orbitrap and triple quadrupole mass spectrometry. The multistage mass spectrometry (MS) spectra can help to propose plausible schemes for unknown compounds and their fragmentations. With the growth of combinatorial libraries, mass spectrometry (MS) has become an important analytical technique because of its speed of analysis, sensitivity, and accuracy. This study was focused on identifying the fragmentation rules for some VDOPs by incorporating MS data with in silico calculated MS fragmentation pathways. Diels-Alder derivatization was used to enhance the sensitivity and selectivity for the VDOPs' identification. Finally, the confirmed PTAD-derivatized target compounds were separated and analyzed using ESI(+)-UHPLC-MS/MS in multiple reaction monitoring (MRM) mode. Graphical Abstract ᅟ.