Study On Preparation And Antihypertensive Activity Of Almond Oligopeptides

China is one of the main producer countries of apricot and apricot almond, the resource of apricot almond is abundant. Almond is nutritional and contains above 25% protein, but lack of the research and products development of almond protein. Now researchers are focus on the development of functional oligopeptides by the materials of food protein, which can release activity peptides by specific protease hydrolysis. So the almond meal cold pressing was used as material to build preparation process of the almond oligopeptides with high yield, high DH, and the functional activity was evaluated.The objective of this work was to provide some instruction and academic information for reasonable utilization of almond.The The hydrolysis effect of Alcalase, Protamex, Falvorzyme, N120P, Neutrase and Papain was evaluated, and Neutrase and N120P were used to hydrolyze almond meal. The factors affected the preparation of almond oligopeptides were studied such as protease dosage, substrate concentration, pH, temperature and hydrolysis time etc. The regression models of yield rate (TCA-NSI) and degree of hydrolysis (DH) were established by orthogonal rotation design. As the practical matter and experiments, The factors were optimized: Neutrase:N120P=2:1 (U/U), pH values 6.5, temperature 52.5℃, substrate concentration 4% , Neutrase and N120P dosage 7200 U/g, reaction time 1073min. While TCA-NIS was 65.49±1.22%, DH was 26.20±0.35%.The molecular weight of almond oligopeptides was low and percentage of below 1 kDa oligopeptides was 74.39%. In order to acquire oligopeptides of different molecular weight distribution, almond oligopeptides were separated by ultra-filtration technology. The effects of pressure, concentrations and temperature during ultra-filtration were studied. The results show that pressure 20 psi, peptides concentration 4%, temperature 35℃as the optimum conditions of the first-degree ultra-filtratio and pressure 25 psi, peptides concentration 6%, temperature 40℃as optimum conditions of the second-degree. After separation with ultra-filtration, the yield rate of almond oligopeptides below 5 kDa and1 kDa was 84.67% and 62.86% respectively.Antioxidative activity and ACE inhibitory activity of almond oligopeptides were determined using different tests in vitro. The results showed that almond oligopeptides possessed higher antioxidative activity. The free radical scavenging activities below 1 kDa were strongest and IC50 value to superoxide radical, hydroxyl radical, DPPH radical was 99.31 mg/mL, 4.70 mg/mL, 0.48 mg/mL respective. Moreover, this peptide exhibited strong reducing capacity and inhibition capacity of peroxidation in linoleic acid system. The ACE inhibitory activity of different molecular components stand out very much, and below 1 kDa possessed the strongeat activity, the IC50 values of raw peptides, above 5 kDa, 1-5 kDa and below 1 kDa were 0.37 mg/mL, 0.82 mg/mL, 0.43 mg/mL and 0.14 mg/mL respectively.The ACE inhibitory mechanism of oligopeptides below 1 kDa was studied, and oligopeptides below 1 kDa were found to belong to non-competitive inhibition to ACE by Lineweaver-Burk model, and stability tests of oligopeptides below 1 kDa showed that they were of better heat and acid resistant, and they could resist hydrolysis of digestive enzyme. The results of animals in vivo test showed that the almond oligopeptide below 1 kDa with above 100 mg/kg dosages administration could reduced the SBP of SHRs markedly in 6 h and the heart rate of SHRs were not influenced, and the SBP of SHRs were steadily reduced markedly after 28 days administration by above 400 mg/kg bodyweight dosages.One novel ACE inhibitory peptide f1 with strong activities was isolated from the almond peptides by sequential chromatographic methods (Sephadex G-15 gel filtration chromatography, semi-preparative RP-HPLC and RP-HPLC).And its amino acid sequences was identified by MALDI- TOF-TOF/MS and 2D-NMR as ILILLP with molecular weight 679.6 Da and IC50 values of 0.067 mg/mL(98.58μM). According to the results above, an oligopeptide ILILLP was synthesis by solid-phase synthesis, and the ACE inhibitory activity was confirmed, its IC50 values was 0.077 mg/mL(114μM).The parameters of manufacturing technology of almond oligopeptides were amplified. The results showed that the recovery rate of almond oligopeptides manufactured by amplified experiment was 58.40%, DH was 23.90%, the purity was above 50%, the oligopeptides of moleculer weight below 10 kDa was 61.40%. The ACE inhibition IC50 value of raw almond oligopeptides was 0.46 mg/mL. So the manufacturing technology of almond oligopeptides can be applied to almond oligopeptides industrialization.