Separation and partial characterization of agiotensin Ⅰ-converting enzyme inhibitory peptides from enzymic hydrolysates of red tilapia skin collagen

Mingyong ZENG; Shengjun CHEN; Laihao LI; Bafang LI

ABSTRACT

BACKGROUND:

Agiotensin Ⅰ -converting enzyme (ACE) inhibitory peptides which are separated from red tilapia skin collagen should be researched further.

OBJECTIVE:

To obtain a peptide of high ACE inhibitory activity through enzymic hydrolysates of red tilapia skin collagen.

DESIGN:

Enzymic hydrolysates were done with orthogonal experimental method; decompression peptide was separated with ultrafiltration, gel chromatography and hypertensive liquid chromatography.

SETTING:

Laboratory of Aquatic Products, Chinese Marine University.MATERIALS The red tilapia weighing (500+50)g was donated by Branch Factory of Jimo Redian Factory. ACE was isolated from hog lung.

METHODS:

The experiment was carried out in the Laboratory of Aquatic Products, Chinese Marine University from May Bergman.①The collagen from red tilapia skin was extracted using the method described by Grossman and Bergman.The collagen extraction was hydrolyzed with compound enzymes in the order of bromelain and Alcalase 2.4 L.The red tilapia collagen hydrolysates (RTCH) were subsequently boiled to inactivate the enzyme. The resultant RTCH was fractionated through three different UF membrane bioreactor system having a range of molecular weight cut-offs (MWCO) of Mr 6 000, 4 000 and 1 000. Three portions were obtained RTCH-I (M, 6000-4000), RTCH- Ⅱ (Mr 4000-1000)and RTCH-Ⅲ(Mr＜1000).②The ACE inhibitory 50%of ACE activity was defined as the IC50 value.③RTCH-Ⅲ(1.5 Ml) with the highest activity among RTCHs was loaded onto a Sephadex G-25 column (1.6×100 cm), and the absorbance of theeluent was monitored at 220 nm. Four samples of primary hydrolysates, RTCH- Ⅰ, RTCH- Ⅱ and RTCHⅢ were separated to collect active fractions which were pooled and lyophilized, immediately. The lyophilized fraction was separated with HPLC ODS-C18 analysis column to obtain component of high activity. Meanwhile, the same method was used to measure ACE inhibitory rate.④Each sample was hydrolyzed with 6 mol/L hydrochloric acid containing 1g/L thioglycolic acid at 110 ℃ for 24 hours in vacuum. The amino acid compositions of the resultant hydrolysates were determined on an amino acid auto analyzer, and molecular weight was measured with HPLC technique.MAIN OUTCOME MEASURES：①ACE inhibitory activity of fractionated RTCHS;②Purification of ACE inhibitory peptide;③Amino acid analysis of ACE inhibitory peptides.

RESULTS:

①ACE inhibitory activityof fractionated RTCHsIC50values of RTCH-Ⅰ,RTCH-Ⅱand RTCH-Ⅲ were 3.30,2.23 and 0.31 g/L,and inhibitory of RTCH-Ⅲ was the highest.②Purification of ACE inhibitory peptide The IC50 value of the four peak were 3.5, 2.12, 1.56 and 0.65 g/L, respectively. Results in Figures 2, 3, 4 and 5 showed that the high active fractions were 6K4, 4K2, 1K2 and ACF2, and the inhibitory ratio were 11.1%, 89.9%, 65.0% and 49.7%, respectively.Among of these fractions,the 4K2 exhibited the highest inhibitory rate.③Amino acid analysis of ACE inhibitory peptides Separated peptide products had more aromatic and hydrophobic amino acids.CONCLUSTON Enzymic hydrolysates of red tilapia skin collagen have high ACE inhibitory peptide which is separated with ultrafiltration, gel chromatography and hypertensive liquid chromatography.