Study On Extraction And Purification,Nanoparticles Preparation And Stability Of Proanthocyanidins From Chokeberry

Chokeberry is a fruit with rich nutritional value and economic value,rich in phenolic compounds,in which the content of proanthocyanidins can be more than 66%of the total phenol.Proanthocyanidins have many functions like antioxidant,nerve protection,antiviral,heart protection and antibacterial.But its stability is poor.Therefore,it is needy to study the extraction and purification of proanthocyanidins from chokeberry and improve its stability.In this study,the effects of different methods on extracting proanthocyanidins from chokeberry were compared and the extraction methods were optimized.The proanthocyanidin extract was enriched and purified.And finally,zein nanoparticles were used to load proanthocyanidins to improve their stability.The followings are specific research results:(1)Extraction of proanthocyanidins from chokeberries:The extraction process of proanthocyanidins from chokeberries by ultrasonic microwave assisted extraction(UMAE)was optimized by single factor and Box-Behnken response surface design.The best extraction process parameters were as below:the liquid material ratio was 62:1m L/g,the ethanol concentration was 62%(v/v),the microwave power was 434 W,the extraction time was 4.5 min,the yield of proanthocyanidins was 31.52±0.8 mg/g,and the purity was16.34±0.59%.By comparing UMAE,ultrasonic-assisted extraction(UAE),microwave-assisted extraction(MAE),and traditional solvent extraction(TSE),it was found that yield and purity of the UMAE extraction were the highest.The antioxidant activity indexes such as ABTS~+,DPPH,superoxide anion free radical and total reducing power were measured.The results showed that the antioxidant activity of chokeberry proanthocyanidins extracted by UMAE was the highest.The microscopic morphology of chokeberry residue extracted by UMAE was observed by Field scanning electron microscope(FE-SEM).It was found that the residue was more thoroughly damaged and the cell wall was more damaged,which was conducive to the dissolution of proanthocyanidins.And the extraction time is shortest(4 min),which is far less than the other three extraction methods(UAE-60 min;MAE-40 min;TSE-360min),which improves the efficiency and reduces the energy consumption.Hence,UMAE is the best extraction method.(2)Purification of proanthocyanidins from chokeberry:Through static and dynamic adsorption,HP-20 resin with the best separation effect of proanthocyanidins was selected from ten resins.The purification process of chokeberry proanthocyanidins was optimized,and the best purification process was acquired:the rate of loading flow was 1 BV per h,the loading concentration was 3 mg per m L,and the rate of elution flow was 1 BV per h.Firstly,the proanthocyanidin extract was enriched by collecting 35%(v/v)ethanol eluted components.The purity of the enrichment was 65.37±3.52%.Then the enrichment was purified.The purity of the 30%ethanol gradient elution component(30%EC-PPA)was the highest,93.59±2.73%,followed by 40%ethanol gradient elution,and its purity was 84.93±4.18%.The antioxidant activities of extracts,concentrates and purified gradients were compared.It was found that 30%EC-PPA had the highest antioxidant activity.Based on the good physiological activity of high purity 30%EC-PPA,it was analyzed by UPLC-Q-TOF-MS and average degree of polymerization.UPLC-Q-TOF-MS results showed that most of the purified products were B-type proanthocyanidins with epicatechin as the basic unit,and there were many isomers of B-type proanthocyanidins dimer,trimer and tetramer.The average degree of polymerization of the purified products was 5.(3)Study on embedding proanthocyanidin nanoparticles:Proanthocyanidins are sensitive to light and temperature.In this study,the self-assembly characteristics of zein were used to form nanoparticles to embed proanthocyanidins and improve their stability.In this study,zein nanoparticles loaded proanthocyanidins were prepared by anti-solvent precipitation method(ASP)and citric acid crosslinking method(CAC).The optimum preparation conditions of the two methods were determined by single factor test.The results showed that the particle size of ASP nanoparticles was 522.0±9.46 nm and the embedding rate was 91.56±0.98%;The nanoparticles prepared by CAC have smaller particle size(284.1±7.34 nm)and higher embedding rate(94.13±1.04%).By comparing the FE-SEM,fluorescence spectrum,Fourier infrared transform,circular dichroism,X-ray diffraction and interaction force analysis of the nanoparticles prepared by the two methods,it is found that hydrophobic interaction is the main force in the nanoparticles prepared by ASP.After the crosslinking of zein and citric acid,in addition to hydrophobic interaction,hydrogen bond and electrostatic interaction are also involved in the formation of zein and citric acid.The secondary structure and crystallization are changed,making the nanoparticles form a more compact and smooth shell.(4)Stability test of proanthocyanidin nanoparticlesA series of stability analysis and gastrointestinal simulation digestion of zein nanoparticles loaded with proanthocyanidins prepared by ASP and CAC were carried out.The nanoparticles prepared by CAC were significantly better than those prepared by ASP in terms of thermal stability,redispersibility,freeze-thaw,p H stability,Na Cl stability and temperature storage stability.It can significantly improve the environmental stability of proanthocyanidins.The simulated gastrointestinal digestion test showed that citric acid crosslinked zein nanoparticles had stronger resistance to gastrointestinal and other harsh environments,and the biological accessibility of procyanidins also increased significantly.