Separation and Purification,Structural Analysis and Stabilization of ACE Inhibitory Peptides Derived from Sunflower Seeds

Author:Luo Peng

Supervisor:pan si zuo he dong ping


Degree Year:2018





Sunflower seeds are the fifth largest oil crops in China,with the annual output of about 2.5 million tons.The sunflower seed meal still contains 29%~43% protein,and is an important protein resource.In recent years,the preparation of ACE inhibitory peptides derived from different protein sources has gradually become a hot research topic,also provides a new way for developing of antihypertensive drugs.Using the low temperature defatted sunflower meal as raw material,this paper aimed at the preparation,separation and purification,identification and stabilization of ACE inhibitory peptides(ACEIPs)derived from sunflower seeds,in order to provide a scientific basis for the development and utilization of sunflower protein resource.The research contents and results are listed as follows:1 Optimization of the preparation of ACE inhibitory peptides(ACEIPs)derived from sunflower seedsThe single protease hydrolysis,the fractional hydrolysis of alcalase and flavourzyme,and the combined hydrolysis of alcalase and flavourzyme for ACE inhibitory peptides from sunflower seeds were researched respectively in this study.Under the condition of single protease hydrolysis,alkaline protease enzymolysis effect is best,the sunflower protein hydrolysis degree(DH)was 21.14±0.76%,ACE inhibition rate was 62.34±0.57%.In the fractional hydrolysis of alcalase and flavourzyme,the sunflower protein hydrolysis degree was 32.32%,ACE inhibition rate was up to 67.80%.In the alcalase and flavourzyme synergetic hydrolysis processing,the sunflower protein hydrolysis degree was 31.25±1.24%,ACE inhibition rate of hydrolysate was 65.23±0.58%.ACE inhibitory IC50 concentration of ACEIPs was 1.39 mg/m L.Most of molecular weight of ACEIPs distribution of under 3000 Da(accounted for 97.34%),Most of molecular weight(81.52%)was less than 1000 Da.Compared with sunflower seeds protein,the hydrophobic amino acids,neutral amino acids,aromatic amino acid and alkaline amino acids of ACEIPs had a certain degree of reduced,while the content of acidic amino acids was significantly increased,growth of 50%.2 Purification and identification of ACEIPs derived from sunflower seedsThe separation and purification of ACEIPs derived from sunflower seeds was carried by macroporous resin adsorption,ultrafiltration,gel chromatography and reversed-phase liquid chromatography,and the amino acid sequence was analyzed by mass spectrometry.Comparison of five kinds of macroporous resins of the adsorption and desorption performance,respectively S-8,X-5,D-101,DA201-C and HPD-100,the adsorption and desorption effect of DA201-C resin is the best.The desalination rate of ACEIPs can reach more than 85% and ACE inhibitory IC50 value fall to 1.09 mg/m L by DA201-C resin adsorption.There are three fractions,respectively Mw<1 kDa,1 kDa<Mw<3 kDa,Mw>3 kDa by ultrafiltration,the amino acid composition and content of them have certain differences,but mainly by glutamate(Glu),arginine(Arg),leucine(Leu)and phenylalanine(Phe).Including Mw<1k Da ultrafiltration fraction of IC50 alue concentration of the lowest point of 0.41 mg/m L.The ultrafiltration fraction(Mw<1 k Da)by Sephadex G-10 and Sephadex G-25 gel chromatography included four peaks and three peaks,respectively.ACE inhibitory IC50 concentration of B Ⅲ peak can reduce to 83±5 μg/m L in the Sephadex G-10 gel chromatography.By using reversed phase liquid chromatography,B Ⅲ peak can be isolated from the six peaks,including 4# peak of ACE inhibitory IC50 concentration was5.6±0.5 μg/m L.Based on the molecular weight identification of spectrometry,the amino acid sequence of 4# peak was supposedly G–L/I-R-P.The above two tetrapeptides were acquired by solid-phase peptide synthesis,the ACE inhibitory IC50 concentration of them were 4.5±0.5 μg/m L and 5.0±0.4 μg/m L respectively,and there was no significant difference.Comparison of two tetrapeptides(G-L-R-P and G-I-R-P)by solid-phase synthesis and sunflower seed peptides using high performance liquid chromatography can be concluded that the amino acid sequence of the ACE inhibitory peptides derived from sunflower seeds should be G-L-R-P.3 Preliminary study on ACE inhibitory activity in vivoA single administration of ACEIPs derived from sunflower seeds was adopted in this part.ACEIPs were administrated to SHRs rats,with low dose group,middle dose group and high dose group.Results indicated that ACEIPs could lower the systolic blood pressure of SHR,and there is an obvious concentration-effect relationship.In addition,the lowest of the systolic blood pressure of SHR in 3 h after a single administration could be observed,and then systolic blood pressure began to slowly recover.The results showed that the hypotensive activity Oof ACEIPs has a certain timeliness.Each group of SHR weight had increased on long-term administration of ACEIPs,including the blank group rats was one of the biggest gain,positive control group gain minimum,ACEIPs group center,and there was no significant difference among low dose group,middle dose group and high dose group.The experimental group and control group in SHR systolic blood pressure in the feeding of 4 weeks,except the systolic blood pressure of low dose group and blank group had no significant difference,compared with the control group other groups had significant difference.Heart rate of the experimental SHR had no significant difference,the results showed that ACEIPs derived from sunflower seeds had no significant adverse effect on the heart rate.4 Stabilization of ACEIPs derived from sunflower seedsThe change of inhibitory activity and color which ACEIPs derived from sunflower seeds by heat treatment and the stabilization in simulated gastric intestinal fluids were studied.The results showed that the ACEIPs was very sensitive to heat and p H value,the stability of ACEIPs was good only when p H was less than 4.0,heat temperature was not higher than 40 ℃,heat time was less than 2.0 h.However,the inhibitory activity of ACEIPs in simulated gastric and intestinal fluids was not losses,on the contrary slightly increased.The optimum preparation parameters of microencapsulation were the ratio of core to wall material(ACEIPs: beta-cyclodextrin)of 1:18,embedding temperature of 30 ℃,intensity of ultrasonic of 150 W,ultrasonic time of 70 min,under the condition loading quantity of the microcapsule can be up to 4.9±0.5%.In vitro release test in simulated intestinal fluid and simulated gastric fluid showed that the microencapsulation of ACEIPs derived from sunflower seeds with beta cyclodextrin as the wall material play a certain controlled-release effect,and ACE inhibitory activity of microcapsules keep well in opening storage,ACE inhibition rate can reach 24.6% after 9 days.Liposomal ACEIPs were prepared by the thin-film ultrasonic method.Response surface methodology(RSM),in combination with central composite design methods were utilized to optimize entrapment efficiency.We found that the ratio of phospholipids to cholesterol,ultrasound time and the ratio of phospholipids to ACEIPs were significant factors affecting entrapment efficiency(p<0.001).Optimal preparation conditions of liposomal-ACEIPs were the ratio of soybean phospholipids to cholesterol of 4.1:1,PEG-2000 dosage(%)of 4,Na Cl concentration in PBS of 50 mmol/L,hydration temperature of 45℃,ultrasound time of 8.05 min and the ratio of soybean phospholipids to ACEIPs of 15:1 The experimental entrapment efficiency of liposomal-ACEIPs was91.25±0.48%.The percentage of ACEIPs released in phosphate buffer was 95.74 % after 12 h,while liposomal ACEIPs was 77.83 after 12 h.The release effect of liposome entrapped ACEIPs had been certain degree delayed.Moreover,the Logistic model and One-order dynamic model were well-fitted with the release percentage of ACEIPs(R-Square of0.9883 and 0.9841,respectively).The entrapment rate of liposomal ACEIPs were88.40%,86.74%,84.52% and 88.40% respectively,ACE inhibition rate of liposomal ACEIPs were 75.65%,74.32%,70.79% and 68.83% respectively in the preservation 15,30,45 and 60 days respectively,the results showed that liposomal ACEIPs has good stability,and play a certain protective effect for the ACE inhibitory activity of sunflower seed peptide.In conclusion,we got a higher new ACEIPs from sunflower seed polypeptides,the amino acid sequence(G-L-R-P)of ACEIPs was identified,and the ACE inhibitory activity was verified by using solid phase peptide synthesis.The microencapsulation and liposome preparation technology of ACEIPs derived from sunflower seeds was original systematic and valuable researched.