Preparation And ACE Inhibitory Activity Of Casein Non-Phosphopeptides

There are many bioactive peptides in casein, among which casein phosphopeptides (CPPs) was researched and developed earlier. In the course of the industrializing production of CPPs, nearly 80% casein byproduct - casein non-phosphopeptides (CNPPs) can not be utilized with reason, only serving as feedstuff and waste. In this dissertation, CNPPs was recovered in the same time of CPPs preparation. Meanwhile, the effect of desalting CNPPs using macroporous adsorption resin, the angiotensin-converting enzyme (ACE) inhibitory activity of CNPPs were researched, which would give the theoretical foundations for utilization of CNPPs.Initially, the conditions of CPPs and CNPPs preparation using Alcalase 2.4 L were researched. Results showed that the optimum conditions for Alcalase 2.4 L hydrolysis were substrate concentration of 5% (w/v), the ratio of enzyme of 0.03 mL·g-1 protein, temperature of 60℃and pH 9.0. The recovery of CPPs and CNPPs and N/P (mol/mol) of CPPs were all influenced by alcohol concentration and the DH of casein. CPPs was completely released and precipitated entirely with DH 18% and alcohol concentration of 70% (v/v) respectively. The recovery of CPPs was 23.94%; N/P (mol/mol) of CPPs was 9.61; the recovery of CNPPs was 76.06%. The relative molecular weight of CPPs and CNPPs were in the range of 200~5000, and the peptides with relative molecular weight under 700 represented 61.52% and 67.26% respectively. CNPPs contained more hydrophobic amino acid than CPPs.Because a lot of salts were introduced into CNPPs in the process of casein hydrolysis and CPPs precipitation, the content of ash was as high as 20.88%. This could impact the research of CNPPs activities and its quality. It was then necessary to remove the salts from CNPPs. The effect of desalination of CNPPs was researched. It was simple and available to desalt CNPPs using DA201-C macroporous adsorption resin. 60 mg·mL-1 CNPPs solution was carried out to desalt using a DA201-C column, the outflow was detected by ultraviolet detector in the wavelength of 220 nm, regarded A220=0.05 as the through point. Then the column was eluted using water, and CNPPs were desorbed using 70% (v/v) ethanol. The desalting ratio of CNPPs reached 95.40% and the recovery of peptides was more than 85%. Moreover, ACE inhibitory activity was enhanced as seen from its IC50 value decreased from 0.35 mg·mL-1 to 0.21 mg·mL-1. From digestive experiment in vitro, it could be found that ACE inhibitory activity of CNPPs increased after being digested by digestive enzymes. Because of the effect of digestive enzymes, CNPPs were hydrolyzed to smaller peptides, which were more effective towards ACE inhibitory activity. Peptides map showed that the components of CNPPs were complicated, so separation and identification a pure ACE inhibitory peptide became a difficult but useful work.In order to realize the potential effect of CNPPs to decrease blood pressure, this dissertation managed to isolate a pure component marked as CNPPs-8-2 with highest ACE inhibiting activity from CNPPs by gel filtration chromatography and semi-preparative reversed phase high performance liquid chromatography (RP-HPLC). The identification by amino acid composition analysis and liquid chromatography / electrospray ionization mass spectrometry (LC/ESI-MS) showed that the relative molecular weights of CNPPs-8-2 was 291.1, its amino acid sequence was Ser-Trp probablely, and its IC50 (ACE inhibiting activity) was 92.8μmol·L-1.