Studies On Preparation And Bioactivities Of Chlorogenic Acid And Enzymatically Hydrolyzed Peptides From Seed Sunflower (Helianthus Annuus L.)

Sunflower seed produces high-quality edible vegetable oil, and sunflower mealriches in protein and chlorogenic acid. Chlorogenic acid is a good antioxidant; andsunflower is important sources of vegetable protein and bioactive peptides, becausesunflower has rich and balanced amino acids which are of high bioavailability noantinutritional factors. However, intensive studies on bioactivies of chlorogenic acid,protein and peptides from sunflower are still rare. In this study, we adopted reponsesurface design, carried out antioxidant and enzymatically hydrolyzed experiments,introduced gel chromatography, high-performance liquid chromatography and massspectrometry into the systematic studies, including the extraction process andphysicochemical properties of chlorogenic acid and protein, technological parameters inenzymatical hydrolysis of protein, antioxidant activity in vitro of enzymaticallyhydrolyzed substance and its inhibition of angiotensin I converting enzyme (ACE),separation and structural identification of antioxidant peptides and antihypertensivepeptides. This study provided theoretical basis for the development of functional productsfrom sunflower, such as antioxidant peptide and antihypertensive peptide. The mainresults are as follows:1. Optimized extraction conditions for chlorogenic acid:99-minute extraction in75%ethanol (ratio of sample to solution was1:14, pH=4.9) at40℃. The extraction yield ofchlorogenic acid was64.73%. Extracted chlorogenic acid was of high oxidationresistance. Optimal extraction conditions for protein:1.5h extraction in a liquid of1mol/L(ratio of sample to solution was1:12) at50℃. Globulin from sunflower meal, comparedwith that from soybean, is a complete protein of high oil absorption, good foamingproperty and stability. Emulsifying activity and emulsification stability was also higherthan that in isolated soybean protein, only water absorptivity is a little lower.2. When the substrate was globulin extracted from sunflower seed, the enzymesproducing active antioxidant peptide were Alcalase and papain, the enzyme producinghypertensive active peptides was a neutral protease. In each single factor experiment,response surface design was used to optimize the experiments. In combination with factorlevel, the effects of factor level on response values and integrated cost, optimal conditionsfor enzymatic hydrolysis were determined. Alcalase: substrate concentration,2%; pH, 9.64;[E]/[S],3%; temperature,51.31℃and reaction time,115.4minutes. Percentage ofDPPH removed by globulin hydrolysate prepared from sunflower meal was50.85%. Theoptimal enzymatic hydrolysis conditions for papain: substrate concentration,3%; pH,6.5;[E]/[S],5.54%; temperature,61.02℃and reaction time,116.39min. Percentage of DPPHremoved by globulin hydrolysate prepared from sunflower meal was73.11%. Neutralprotease: the substrate concentration,4%;[E]/[S],2.72%; temperature,52℃; pH,7.0andhydrolysis time,90min. Rate of ACE inhibited by hydrolysate prepared from sunflowermeal under such conditions was61.55%, and the IC50were0.45mg/mL3. Sunflower protein hydrolysates of Alcalase and papain showed strong antioxidantactivity in five antioxidant systems in vitro. In the dose-effect relationships betweenhydrolysate and·OH, O2-·, DPPH· clearance and reduction, the IC50were5.7mg/mL,2.5mg/mL, and1.5mg/mL, respectively. Protein hydrolysates significantly inhibited lipidperoxidation of soybean lecithin and oxidation of soybean oil; antioxidant effectincreased with the addition of hydrolysates.4. Gel chromatography and liquid chromatography are good techniques for theseparation and purification of hydrolysates, mass spectrometry provides methodsdetermining the molecular structure. The molecular weight (hydrolysate peak II)produced by neutral protease is1114.5, and that of peak III is1114.6. The relevantsequences are DLWLRLDPS (Asp-Leu-Trp-Leu-Arg-Leu-Asp-Pro-Ser) andDLPWPFRSP (Asp-Leu-Pro-Trp-Pro-Phe-Arg-Ser-Pro), respectively. The molecularweights of hydrolysate at peak II produced by papain is1261.6, the relevant sequenceis FDIVKADNVGPS (Phe-Asp-Ile-Val-Lys-Ala-Asp-Asn-Val-Gly-Pro-Ser). Themolecular weight of Alcalase is1038.5, the relevant sequence is RHAKTPSNK(Arg-His-Ala-Lys-Thr-Pro-Ser-Asn-Lys).