Study On Preparation Of Bitter Almond Protein And Antioxidative Oligopeptides

In this research, slightly denatured bitter almond dreg flour that was obtained from oil making process by low temperature pressing methods was used as raw material to prepare protein isolates. The flour was pretreated by extracting with petroleum ether and ethanol respectively to remove residual oil and amygdalin. First, the bitter almond protein was extracted and prepared by diluted salt solution combined with isoelectric point-salt precipitation methods. The optimum extraction conditions were analyzed and determined through quadratic orthogonal regressive rotational combination test. Then, factors effecting functional properties of bitter almond protein were investigated. Moreover, the suitable conditions of dual-enzyme hydrolysis in preparation of bitter almond oligopeptides were analysed and optimized. At last, the antioxidant properties of oligopeptides were examined. The main results were as follows:1. The optimized conditions of diluted salt solution extraction were:the extraction solvent:0.05mol/L phosphate buffered saline (pH7.0) containing0.19M NaCl; the liquid to solid ratio:37:1(mL/g); extraction temperature:56℃; extracting time:50min; extraction times:3; and the centrifugal speed:4500r/min (15min). Under such conditions, the extraction rate of bitter almond protein was up to70.95%.2. The optimum preparation conditions of bitter almond protein were:adjusting the pH of the protein solution close to its isoelectric point (pI4.1), then, isolating and purifying the protein by two-step ammonium sulfate precipitation method (with the concentrations of45%and60%respectively). Under the optimum conditions, the protein content in the final product was reached to90.23%.3. The protein concentration, pH, temperature, and NaCl concentration had significant effects on functional properties of bitter almond protein. The result showed that solubility, water-holding capacity, emulsifying capacity and emulsion stability, foaming capacity of bitter almond protein were lowest around isoelectric point (pI4.1), while they were improved by increasing NaCl concentrations within the range of0～0.8mol/L. Higher NaCl concentration however, would inhibit those properties. Its emulsifying properties and foaming properties were improved by increased protein concentration, but when the protein concentration reached to3%or4%, there were no obvious changes in emulsifying capacity, emulsion stability, foaming capacity and foam stability. Functional properties were significantly improved by properly heating (25～45℃). However, those properties would remarkably decline when the temperature was over55℃.4. The optimal enzymolysis conditions for combinated application of Flavourzyme and Alealase protease were:enzyme/substrate ratio:13000U/g; protein concentration2%(w/v); activity ratio between Flavourzyme and Alcalase:6:1; reaction temperature:55℃; pH7.3; adding two enzymes simultaneously and then hydrolyzing for3h. After actual hydrolysis experiment, the hydrolysis rate could reach to41.23%.5. Anion and cation mixed bed was used to improve the purity of hydrolysate, the oligopoptide recovery rate was88.9%and desalting rate was91.2%. Antioxidative activity of bitter almond oligopeptides were determined using different tests in vitro. The results showed that DPPH radical scavenging activity of oligopeptides reached to79.2%, hydroxyl radical scavenging activity was85.7%and superoxide radical scavenging activity was72.7%. With the increase of oligopoptide concentraction, the radical scavenging activity was improved.