Purification And Structure Identification Of Antihypertensive Peptide,preparation By Mixed Solid Fermentation Of Corylus Mandshurica Maxim Hazelnut Meal

The Corylus mandshurica Maxim.,which produces a large number of byproducts during processing.Hazelnut meal,is a good source of vegetable protein.However,its low utilization rate which is often sold at a low price as feed or fertilizer,and its development can improve the economic value of Corylus mandshurica Maxim.In this paper,defatted hazelnut pods were used as raw materials to prepare antihypertensive peptides by mixed bacteria solid state fermentation method,and the best combination of fermentation bacteria was selected and the fermentation process was optimized.The prepared antihypertensive peptide was isolated and purified,and the amino acid sequence of the best blood pressure lowering component was identified.Finally,the function of the blood-pressure-lowering peptides of Hazelnut meal was verified by cell experiments.1.Through the determination of the basic components of the hazelnut pod,the protein content in the hazelnut pod was as high as 51.19 g/100 g,which is a potential raw material for biological peptide preparation.Its high reducing sugar content meets the carbon source and nitrogen source required for microbial fermentation,so it can be used as raw material for solid state fermentation.Through solid-state fermentation methods,strains with higher protease yields were screened for protease activity,degree of hydrolysis,and crude peptide yield.The results showed that Bacillus subtilis,Bacillus licheniformis,and Aspergillus oryzae are more suitable for solid state fermentation of hazelnut meal.In order to obtain the polypeptide sample with the highest ACE inhibition rate,the equal inoculum amounts of the three strains were mixed in pairs to ferment the hairy hazelnut pods under certain conditions,and the ACE inhibition of the fermentation product of the combined strain of Bacillus subtilis and Aspergillus oryzae was found.The rate,degree of hydrolysis and crude polypeptide yield were higher,which were 22.11%,21.74%and 19.66%,respectively.It was determined that the combination strains of Bacillus subtilis and Aspergillus oryzae were the best combination of strains for solid-state fermentation.2.On the basis of determining the best combinations of bacteria and solid-state fermentation,the single-factor test and orthogonal test were used to optimize the fermentation process for preparation of antihypertensive peptides from solid-state fermented hazelnut quail,and the optimized process conditions were optimized.The ACE inhibitory rate,degree of hydrolysis,and crude peptide of the fermented samples under the conditions of Bacillus subtilis and Aspergillus oryzae strain ratio 2:3,inoculation amount 15%,moisture content 60%,fermentation temperature 40?,and fermentation time 72 hours Got it.The rates were 45.24%,30.13%and 25.53%respectively,of which the ACE inhibition rate increased by 104.60%.3.The blood pressure peptides prepared by the microbiological method were separated and purified.The polypeptide extracts of fermentation products were separated into three components by ultrafiltration.The ACE inhibition of component II(<3KD)was found after determining the ACE inhibition rate.The highest activity.The Sephadex G-25 was used to further purify the peptides in Fraction II.In the best separation process for Sephadex chromatography,deionized water was used as the eluent and the sample concentration was 50 mg.With a flow rate of 0.4 mL/min at a flow rate of 0.4 mL/min,the Hazeleen antihypertensive peptide was divided into 3 different fractions.The inhibitory rate of ACE inhibition of G-2 component was up to 75.46 ± 0.72%,and the ACE inhibition rate of component ii after ultrafiltration was increased by 20.06%.4.The G-2 components were detected by Nano-UPLC-MS and their amino acid sequences were identified.Four polypeptides were obtained by comparison with the BIOPEP database,Ala-Ser-Phe-Leu-Leu-Arg-Asn-Val-Leu(ASFLLRNVL),Ile-Asp-Tyr-Pro-Asn-Val-Leu-Arg(IDYPNVLR),Ala-Pro-Leu-Lys-Ala-Phe-Gly-Arg-Thr(APLKAFGRT),Lys The molecular weights of-Lys-Lys-Pro-Glu(KKKPE)were 1031.6128 Da,988.5342 Da,959.5521 Da,628.3907 Da,respectively.Unmatched peptides were detected by De Novo protein sequencing analysis.There were 61 short-chain peptides with 6-14 amino acids in the G-2 fraction.Their molecular weights were all less than 1.5 KD.After amino acid composition analysis,they were found to have A large number of hydrophobic amino acids,basic amino acids and aromatic amino acids account for 59.97%of the total number of amino acids.This proves that the Hazeleen antihypertensive peptide is a polypeptide having the characteristics of an ACE inhibitory peptide.5.Used purified G-2 fraction for cell experiments,observe the culture of human umbilical vein endothelial cells(HUVEC),and determine the release of NO,ET in the culture fluid.The results showed that the Hazeleen antihypertensive peptide can promote the release of NO from human umbilical vein endothelial cells and reduce the release of ET.It was proved that Hazeleen antihypertensive peptide has the similar blood pressure lowering effect with captopril at certain concentration range.