Aqueous Enzymatic Extraction Of Rapeseed Oil And Bioactive Peptides

Rapeseed is one of the most important oilseeds and the production of rapeseed in China ranks first now in the world.Rapeseed contains rich oil and protein.The composition of amino acids in rapeseed protein is well-balanced.Conventional industrial processing of rapeseed involves pressing and hexane extraction,which yields two products-the oil and a low-valued meal that is mainly used as animal feed or fertilizer.It is necessary to effectively utilize the rapeseed protein for edible use when the protein sources for human are increasingly scarce.Aqueous enzymatic extraction (AEE) has emerged as a novel oil extraction technique by which oil and protein can be obtained simultaneously without use of organic solvent.In this dissertation,AEE was applied to "double-low" rapeseeds(Brassica napus) for simultaneous production of free oil and bioactive peptides.The results are as follows:The wet-heating inactivation of endo-myrosinases in intact rapeseeds and the stability of glucosinolates during the heat treatment were investigated.Results indicate that boiling intact rapeseed for 5min can inactivate myrosinases effectively and 11.28% of glucosinolates is degraded due to heating.Through screening the enzymes,the formula of pectinase,cellulase andβ-glucanase(4:1:1,v/v/v) was used to treat the rapeseed slurries.The experimental results of SEM threw light on the mechanisms of the AEE.The optimal conditions for the complex enzyme hydrolysis are as follows:the ratio of solid to liquid 1:5,enzymes/rapeseeds 3%(v/w) and hydrolysis time 4 h.The properties of emulsions from the AEE during different pH conditions was studied,including the zeta potential,the amount of bound protein and the microstructures of the surface membrane of oil droplet.The mechanisms of alkaline extraction were proposed:the pH value of the slurries is raised which would lead to an increase in the charges of the proteins on the surface of the oil droplet.As a result,the electrostatic repulsive power among the protein molecules is enhanced and then they are easily desorbed from the surface.Thus,the membrane would become thin which is in favor of the protein hydrolysis and the aggregation of oil droplets.Further,the relationship between the degree of protein hydrolysis(DH) and its emulsifying capacity was investigated.A conclusion was drawn that notable amounts of free oil could be obtained due to the decrease of the protein emulsifying capacity as the DH rose above 10%.Following the carbohydrase treatment,sequential treatments were carried out consisting of alkaline extraction and an alkaline protease(Alcalase 2.4L) hydrolysis to simultaneously produce free oil and protein hydrolysates.Response surface methodology(RSM) was used to study and optimize the effects of alkaline extraction pH,Alcalase 2.4L concentration and hydrolysis time.Increasing Alcalase 2.4L concentration and hydrolysis time significantly(p＜0.05) increased free oil,protein hydrolysates yields and the DH while the alkaline extraction pH had a significant (p＜0.05) effect on the protein hydrolysates yield.The optimal conditions for the alkaline extraction and Alcalase 2.4L hydrolysis are as follows:alkaline extraction pH 10, temperature 60℃,extraction time 45 min;Alcalase 2.4L concentration 1.3%(v/w), hydrolysis time 160min.Under these conditions,the yields of free oil and protein hydrolysates were 74.2-75.1%and 80.9～82.5%,respectively,and the protein DH was 18.9～21.0%.Through washing the wet precipitate and a stepwise demulsification procedure consisting of storage-centrifugation and freezing-thawing followed by centrifugation,notable amounts of free oil and protein hydrolysates could be obtained. The total yields of free oil and protein hydrolysates were 88～90%and 94～97%, respectively.Compared with Soxlet-extracted oil,the content of free fatty acid of enzyme-extracted oil is higher while the peroxide value is lower.The color of enzyme-extracted oil is slightly darker than that of Soxlet-extracted oil.The iodine value,sapo(?)ificati(?)n value and fatty acid composition between them are similar.The total content ot the unsaturated fatty acids in the enzyme-extracted oil is about 90%. Therefore,the quality of the oil is high.The macroporous adsorption resins(type:DA201-C) were used to treat the aqueous phase.Aqueous phases were pooled and adsorbed onto macroporous adsorption resins to remove salts and sugars.Following extensive rinsing with deionized water (pH4),desorption was achieved by washing with 85%ethanol(v/v) to obtain crude rapeseed peptides(CRPs).The protein recovery was 66.7%and the protein content was enriched from 47.04 to 73.51%in the CRPs.No glucosinolates and phytic acid were detected in the CRPs.In a separate experiment,stepwise desorption was carried out with 25,55 and 85%ethanol to separate the bitter peptides from the other peptides. From the stepwise desorption,a non-bitter fraction RP25(containing 64～66%of total desorbed protein) had bland color and significantly higher protein content(81.04%) and hence was the more desirable product.The in vitro antioxidant and antithrombotic activities of crude rapeseed peptides (CRPs) and peptide fractions(RP25 and RP55) were determined.The results indicate that rapeseed peptides possess potent antioxidant activities and they are dose-dependent. The median effective dose(ED₅₀) values of CRPs,RP25 and RP55 forα,α-diphenyl-β-picrylhydrazyl(DPPH) radical scavenging were 72,499 and 41μg/mL, respectively.The ED₅₀ values for RP55 and CRPs for O₂^-·scavenging were 0.91 and 1.70mg/mL,respectively.The ED₅₀ values for RP25 and RP55 for hydroxyl radicals scavenging were 2.53 and 6.79mg/mL,respectively while the ED₅₀ values of RP55 and CRPs for inhibition of lipid peroxidation in a liposome model system were 4.06 and 4.69mg/mL,respectively.The difference between RP55 and ascorbic acid with respect to inhibition of lipid peroxidation was insignificant(p＞0.05) at a concentration of 5mg/mL.With addition of RP55 in the cookies,the lipid peroxidation was significantly (p＜0.05) inhibited during the storage.RP55 generally showed more potent antioxidant activities except for hydroxyl radicals scavenging ability than RP25 and CRPs at the same concentrations,which was thought to relate to the significantly higher contents of hydrophobic amino acid,tannin,and the brown color substances in RP55.Rapeseed peptides possess marked inhibitory activities on the thrombin-catalyzed coagulation of fibrinogen at certain concentrations.It can be established that both tannin and peptides contribute much to the potent antioxidant activities of RP55 according to the fractionation and reconstitution experimental results.A peptide(RP55-E2-G5-RS-F1) showing strong antioxidant activity was isolated from RP55 using consecutive chromatographic methods including ton-exchange chromatography,gel-filtration chromatography and RP-HPLC.Its ED₅₀ value for DPPH radical scavenging was 0.063mg/mL.The molecular mass(487.2) and the amino acid sequence of the purified peptide(Pro-Ala-Gly-Pro-Phe) were determined using electrospray ionization-quadrupole-time of flight(ESI-Q-TOF) mass spectrometry. Another two peptides(the molecular mass is 661.4 and 683.3,respectively) were also analyzed for their amino acid sequences using tandem mass spectrometry.The respective sequences were Arg-Asn-Leu(Ile)-Pro-Tyr and Tyr-Pro-Leu(Ile)-Tyr-Glu. The amino acid compositions and sequences of these peptides agreed with the reported characteristics of antioxidant peptides.