Preparation, Purification And Characterization Of Angiotensin Ⅰ-converting Enzyme Inhibitory Peptides Derived From Channel Catfish Skin Gelatin

In this research, ACE inhibitory peptide was isolated from channel catfish skin gelatin.Firstly, hydrolysis conditions for extracting ACE inhibitory peptides were investigated, theactivity of ACE inhibitory peptide was estimated by ACE inhibitory activities in vitro;secondly, a nonapeptide and octadecapeptide were purified by purification system; finally,basic physicochemical properties of these two peptides were estimated.（1）The channel catfish skin was dealed with n-batanol, sulpuric acid and sodiumhydroxide solutions, then dealed with hot water. Five proteases, including pepsin, neutrualprotease, alkaline protease, trypsin and papain were chosen to prepare ACE inhibitorypeptides from channel catfish （Ictalurus punctatus） skin gelatin. The activity of ACEinhibitory peptide was estimated by ACE inhibitory activities in vitro. The results showedthat the hydrolysates by alkaline exhibited the highest ACE inhibitory activities and thehydrolysates by pepsin took the second place. Box-Behnken experimental design combinedwith response surface methodology was taken to optimize the hydrolysis conditions ofalkaline protease for production of ACE inhibitory peptides from channel catfish skingelatin. The results showed that the optimal condition for extracting ACE inhibitorypeptides by alkaline protease were temperature50℃, pH value8.0, concentration ofsubstrate4.5%, enzyme-to-substrate （E/S） ratio2.2%, extract time3h, with the ACEinhibitory activities of84.57%. The hydrolysate was then hydrolyzed by pepsin under itsoptimal conditions for3h. The results showed that the hydrolysate hydrolyzed by Alkalineand pepsin showed the highest ACE inhibitory activities of97.83%.（2）Ultra filtration, DEAE iron exchange, G-15gel filtration, reverse-phase HPLC andultraflex TOF/TOF were used to isolate the ACE inhibitory peptide. Three fractions weregot after ultra filtration with the cut-off molecular weight membrances of3000Da and1000Da, fraction of Da<1000and1000<Da<3000showed the higher ACE inhibitoryactivity with82.36%and80.29%respectively. Merging these two fractions into one, thenew fraction was further purified by DEAE iron exchange. Three fractions including F₁, F₂and F₃were obtained and F₂showed the highest ACE inhibitory activity of88.36%. F2wasfurther separated into two fractions after G-15gel filtration and F₃₂showed the highestACE inhibitory activity of89.65%. RP-HPLC was used to purify F32and R5showed the highest ACE inhibitory activity of90.00%.The amino acid sequences of these purifiedsubstrates determined by ultraflex TOF/TOF were FTHNGYLNA （IC50=0.3041mg/mL）and SNTRVSAHKHCGSYLIIN (IC₅₀=0.8064mg/mL).（3）Effects of temperature, pH and common metal ion on ACE inhibitory peptidestability were investigated.The result showed that: the ACE inhibitory peptides had strongthermo tolerance, its ACE inhibitory activity could maintain90%when heating at100℃for2h; ACEIP1remain stable ACE inhibitory activity under alkalinity condition andACEIP2remain stable ACE inhibitory activity under acidity condition; K+、Zn²⁺almosthad no impact on the stability of ACE inhibitory peptide, Ca²⁺had little impact on thestability of ACEIP1and ACEIP2, Mg²⁺had no impact on ACEIP1and little impact onACEIP2, Cu²⁺had no impact on ACEIP1and little impact on ACEIP2.