Sesame-sourced low-bitterness ACE inhibitory peptide as well as preparation method and application thereof

[0033] Example 1

[0034] A method of preparing a low bitter ACE inhibiting peptide from skim sesame powder, which includes the following steps:

[0035] 1) Exase fat sesame powder preparation:

[0036] The sesatist cake was pulverized, and degreasing was carried out by a rope extract, and the solvent was selected as petroleum ether (boiling point 40-60 ° C), and the extraction temperature was 60 ° C, and the degreasing 7 h was separated. The protoxed sesame melamine raw material was pulverized, and the mesh is collected, which is the degreasing sesame powder, and the protein content is 51.23% by the detection of sesame nitrogen (GB5009.5-2003);

[0037] 2) Protein enzymatic solution:

[0038] Step 1) The resulting degreasing sesame powder is mixed with 10 g: 100 mL of the distilled water. The pH was adjusted with 6 mol / lnaOh, then 9000 U / g was added under 50-55 ° C. 原料蛋白 Alkaline protease Alcalase, after 50-55 ° C, add 4000 U / g 原料蛋白 Flavor protease, continuing the enzymatic solution 2 h. The enzymatic hydrolysis was rapidly placed in a boiling water bath for 10 min, cooled to room temperature, then centrifuged at 5000 r / min for 20 min, taking the supernatant, 0.2-6 ° C low temperature placement, spare;

[0039] 3) Press the bitterness:

[0040] The D201 molar resin is added to step 2), in the supernatant obtained, the large pore resin concentration is 0.9 g / ml, the pH is 7, stirred with stirring under 35 ° C, stir adsorption, over 200 mesh screen, collect the filtrate ;

[0041] 4) Separation and purification

[0042]Step 3) The filtered solution was ultrafiltrated with the ultrafiltration membrane obtained by the intercepting molecular weight 3 kDa, and then further separated purification was carried out using a high pressure chromatographic chromatographic system. The chromatographic conditions were as follows: the flow phase A is an aqueous solution containing 0.1% trifluoroacetic acid, flow phase B is a acetonitrile solution containing 0.1% trifluoroacetate; elution gradient: 0 ~ 30% mobile phase B elution time 3 min, 30% ~ 65% mobile phase B elution time 5 min, 65% ~ 60% flow phase B elution time 25 min, 60% ~ 0% mobile phase B elution time 5 min; flow rate 6ml / min, detection wavelength is 220 nm, chromatography column is Shim-Pack GIS C 18 Columns (250 mm inner diameter, column length 20 cm, particle size 10 μm). The components corresponding to the eluting peaks corresponding to the high ACE suppression activity are collected and filtered using the collector, i.e., the collection of retained time is 10.95-11.10 min (see figure 1 There are mainly 6 elution peaks, and the fifth peak P5 is collected, and the water bath is allowed to allow for 2 h.

[0043] The collected polypeptide sample is used for mass spectrometry identification using nano-lc-eSI-ms / ms (see figure 2 ), Identified the amino acid sequence of the low bitter ACE inhibited peptide as: trp-leu-tyr-arg (WLYR).

[0044] Example 2

[0045] A method of artificial synthesis of low bitter sesame ACE inhibits peptide, which is composed of solid phase synthesis. The basic process is as follows: First, an amino acid is attached to the immunogenic acid from the FMoc group in the insoluble solid support WANG resin, then remove the protective group of the amino group, the first amino acid is attached to the solid support; secondly, the amino group is fmoc The second amino acid carboxyl group protected by the group is activated, and the activated amino acid is formed to form a peptide bond at an amino acid having a first amino acid that has been connected to the solid phase. Toppeptide with protective group. The above peptide bonds were repeated, allowing the peptide chain from the C-terminal n-end until the desired peptide chain length was reached, and finally the peptide was obtained. The method synthesized by the low bitter sesame ACE inhibits peptide Trp-Leu-Tyr-Arg (WLYR), which is shown in liquid phase and mass spectrometry. image 3 and Figure 4. This high-purity synthetic peptide is as long as the ion-peak quality is 636.74, which meets the molecular weight of the synthesis sequence, indicating that the solid phase synthesis is successful.

[0046] The specific procedures of the above-mentioned solid phase synthesis method to inhibit the peptide inhibition of the peptide: 1 Huang Wei Die, Chen Changqing, peptide synthesis, scientific publishing house, 1985. 2.N. Jukukkki, Liu Keliang et al, Peptide: Chemistry and Biology, Science Press, 2005. The low bitter sesame ACE inhibitory peptide can also entrust the corresponding polypeptide biocide to synthesize, because the chemical synthesis process is not the focus of the present application, so the present application will not repeatedly described the chemical synthesis process.