Preparation Of Porcine Haemoglobin Biopeptides By Ultrafiltration Methodology

In order to develop efficient industrialization process of prepareing hemoglobin activity peptide, the condition optimization of hydrolysis and the membrane processes were analyzed, then we could obtain angiotensin I-converting enzyme(ACE) inhibition peptide. The main contents and results were as follows:1. Optimize the hydrolysis condition. Firstly, the hemoglobin powder was hydrolysised by pepsin for 12 hours, the enzymatic hydrolysis conditions: the substrate concentration was 3.0 %, the enzyme concentration [E]/[S]% was 1.0 %, the temperature was 37 ℃,p H value was 3. The degree of hydrolysis was 18.1 %. The ultrafiltration membranes of Mw cut-off 30 k Da, 10 k Da and 6 k Da were used to separate inhibitory peptides from pepsin hydrolysate. We can get four parts, then made the hydrolysate freeze-dried, the ACE inhibitory rate of molecular weight beyond 30 k Da was 16.5 %, molecular weight between10-30 k Da was 21.7%, molecular weight between6-10 k Da was 24.3%, molecular weight lower than 6 k Da was 55.6% and the color was white. The content of inhibition peptides in lower than 6 k Da was measured by high performance liquid chromatography, the content of VVPWT was 0.094 % and the content of LGFPTTKTYFPHF was 0.891 %.2. Optimization of membrane separation conditions. The pressure, temperature and p H value which was the main factors that affect the permeation flux were studied. The optimized conditions were as follows: operating pressure is 0.17 MPa, liquid temperature is 40 ℃, p H value is 3, the optimal activation process was determined. It was cleaned by 2.0 % hydrochloric acid for 15 min and then 0.5 % Na OH solution for 15 min, the recovery rate reached above 95 %.3. The determination of multi-step hydrolysis conditions. The parts which had lower ACE inhibitory rate were hydrolysised again by enzymes and the optimal enzymatic hydrolysis conditions were as follows: P-I(≥30 k Da) and P-II(10-30 k Da) were hydrolysised by alkaline for 1 hour; P-III(6-10 k Da) was hydrolysised by neutaral for 5 hours. P-I-I(≥30 k Da) was hydrolysised by alkaline for 1 hour; P-II-I(10-30 k Da) was hydrolysised by alkaline for 3 hours( the substrate concentration was 2.0 %, the enzyme concentration [E]/[S]% was 1.0%,the temperature and p H were the optimal conditions for enzyme).