Studies On Preparation Of Cottonseed Protein, Antioxidative Activity And Agiotensin Converting Enzyme Inhibitory Activity Of The Hydrolysates Derived From Cottonseed Protein

Cottonseed meal,which is the by-product of cottonseed after oil extraction,is rich in protein and cheap,yet with low utilization rate.This paper investigated the preparation (degossypol method),enzymatic hydrolysis of cottonseed protein,and the purification antioxidant activity and ACE-inhibitory activity of the hydrolysates,so as to obtain cottonseed protein isolate(CPI) with good properties.This can provide theoretical bases and technical support for the industrially producing of cottonseed protein(CP) and ACE-inhibitory peptides.The main research content and results are following:1.Extraction technology and degossypol of cottonseed protein from cottonseed meal. Several different methods of extraction and degossypol of cottonseed protein were compared with cottonseed meal as the raw material.The optimal extraction conditions were determined using orthogonal rotation combination design.The results showed that hot aqueous alkali method had high protein yield and content,and good degossypol effect. The optimal conditions were pH 10.7,temperature 60.2℃,time 1.07h and the ratio of liquid to solid 16.7:1.The protein yield was 67.48%and the percentage of protein was 90.78%,free gossypol content was 16.8mg/kg under the above optimal extraction conditions.2.Preparation of low bitter peptide by multienzyme-induced continuous hydrolysis. Cottonseed bioactive peptides were prepared through enzymatic hydrolysis using DH as the hydrolysis index.The two enzyme hydrolysis technique was investigated on the basis of single enzyme hydrolysis.Alcalase and trypsin were the best combination and the optimum conditions were as follows:temperature 50℃,pH 8.2,substrate concentration 5%,time 3.5h,and total enzyme activity 6000u/g,the ratio of enzyme activity(Alcalase to Trypsin) 3:1.DH and TCA-SN%can reach 26.67%,87.35%respectively.Further flavorzyme hydrolysis effectively decreased the bitterness,TCA-SN%can reach 85.26%.3.Desalination method for ACE-inhibitory peptide from cottonseed protein. Cottonseed hydrolysates were desalted by 001×7,201×7 iron exchange resin,MWCO100 dialytic bags and DA201-C macroporous adsorption resin,their desalination effects were compared comprehensively.The results showed that DA201-C macroporous adsorption resin had the best desalination effect,desalination ratio reached to 94.78%and nitrogen recovery ratio was 86.32%when the velocity of flow was controlled at 3mL/min. Hydrophobic amino acid was enriched and ACE inhibitory activity was improved effectively after desalination.4.Amino acid(AA) composition and molecular weight distribution of the hydrolysates from cottonseed protein.The AA composition analysis of cottonseed protein and hydrolysates were compared,the hydrolysates were separated using Sephadex G-25, the molecular weight distribution was analysed with mass spectrometry(MS),and the ACE-inhibitory activity of different compositions was compared.AA composition analysis showed that AA composition of cottonseed proteins which were rich in Glu and Arg yet with low content of Met wasn't significantly influenced by proteolysis.Gel chromatography graph demonstrated that the molecular weight was distributed continuously and most of the peptides were lower than 13000Da.The compositionⅢhad the best ACE-inhibitory activity.HPLC-MS analysis proved that the main short peptides which had ACE-inhibitory activity were between 400 and 1000Da.5.Comparison with antioxidant activities of different compositions.The antioxidant activities of CPI,CH,CH(3-8kD) and CH(＜3kD) were studied.Their total antioxidant activities,DPPH and OH radical scavenging activity within a certain concentration range were measured;the antioxidant activity of different compositions which separated from gel chromatography was compared.The results showed that all cottonseed protein fractions had antioxidant activity and the dose-effect relationship existed between the antioxidant activity and sample concentration.CPI had the lowest antioxidant activity but was improved after hydrolysis.After ultrafiltration,antioxidant activity of CH(3-8kD) and CH(＜3kD) was the strongest and weakest respectively.The compositionⅡwhich separated from gel chromatography had the highest antioxidant activity.6.Effect of cottonseed hydrolysates on hypertension of SHR.The ACE inhibition abilities of different fractions of cottonseed protein were compared.Then the SBP of tail and ACE inhibition abilities in serum were determined after single and continuous oral gavage.The results showed ACE-inhibitory abilities of cottonseed protein were increased after proteolysis,and further improved by desalination.The fraction(＜3kD) obtained by ultrafiltration had the best ACE inhibitiory effect.Hydrolysates with high dosage (3000mg/kg·bw) could reduce the blood pressure of SHR during 2～8h after gavage,then restore to the initial level,however they had no influence on normal Wistar rats. Continuous gavage with high dose decreased the blood pressure significantly;however they had no influence on the ACE activity of SHR in serum.The hydrolysates from cottonseed protein had hypertension reducing effect in SHR,and there were correlations between the effect and dosage.