Effects Of Different Wall-broken Methods On The Extraction And Activity Of Astaxanthin From Haematococcus Pluvialis

Astaxanthin,as a fat-soluble ketocarotenoid,which chemical structure is connected by conjugated double bonds of four isoprene units,each end of side connected by a six-membered ring.The special chemical structure makes astaxanthin have high antioxidant activity,which has been widely used in medicine,health products,cosmetics and aquaculture.Astaxanthin is widely existed in a various algae,fungi and crustacean aquatic crusts.Among them,Haematococcus pluvialis(H.pluvialis)(thick-walled spores)is the richest sources of natural astaxanthin,and the astaxanthin accumulation in a single algae can reach up to 4%.Although H.pluvialis has the characteristics of high astaxanthin accumulation and easy to scale production.The tough and three-layers cell wall,making the cell wall remarkably resistant to organic solvent penetration and complicating the astaxanthin extraction process.Therefore,the extraction of astaxanthin from H.pluvialis as a key link in downstream production processed,which has attracted great attention from researchers.In this paper,H.pluvialis was used as raw material.First,the extraction effects of different wall breaking(ionic liquid,enzyme,high pressure homogenization,acid treatment)methods were compared.The mechanism of wall breaking of different treatment methods was studied in depth.Finally,the effects of different disruption methods on the astaxanthin antioxidant activity and anti-proliferative capacity were compared,which providing a theoretical guidance for the efficient extraction and high value application of H.pluvialis.The main research contents and results are as follows:(1)Study on the extraction effect of astaxanthin by different disruption methods.In this study,the extraction of astaxanthin from H.pluvialis by ionic liquids,enzyme,high-pressure homogenization and hydrochloric acid were investigated.The response surface methodology was employed to optimize the disruption conditions of ionic liquid and enzyme.The results of ionic liquid indicated that take [Bmim] Cl ionic liquid as an example,the optimal disruption conditions was as follow: ionic liquid concentration 45%,ionic liquid pretreatment temperature 63 ?,extraction temperature 54 ?,the theoretical and actual crude yield of astaxanthin were reached 89.27% and 90.76±0.38%,with a relative error of1.6%.The results of enzymatic method showed that when pectinase and cellulase mixed with the ratio of 1:1(U/U),enzyme dosage 7000 U/mL,the optimal hydrolysis condition was as follow: p H 4.9,temperature 49 ?,and time 6 h,Under this condition,the theoretical and actual crude yield of astaxanthin were reached 70.21% and 71.08±0.26%,with a relative error of 1.2%.The results of high-pressure homogenization showed that when the homogenization pressure was 100 Mpa,the extraction rate of astaxanthin was 92.05±1.12%.The results of acid hydrolysis showed that when the concentration of hydrochloric acid was4 mol/L,the extraction rate of astaxanthin was 80.52±0.14%.Therefore,the effect of the four extraction methods followed this: pressure microfluidization > ionic liquid >hydrochloric acid > enzyme.(2)Study on the mechanism of different disruption techniques on H.pluvialis.In this study,the mechanism of cell wall breakdown by ILs,ME and HCl was conducted,which was reflected from the results of light microscope,scanning electron microscopy(SEM),transmission electron microscopy(TEM),fourier transform infrared spectroscopy(FT-IR),X-Ray diffraction(XRD),nuclear magnetic resonance(NMR)and gas chromatograph mass spectrometer(GC-MS).The light microscope,SEM and TEM shown that the three chemical techniques have significant differences in the destruction of the three-layer cell wall of H.pluvialis.After treated by ILs,the cell wall appeared a wide range of depressions,and the entire cell surface generating honeycomb-like morphology.After treated by EM,the cell wall appeared multiple discontinuous pores.After treated by HCl,the cell wall has been completely decomposed,and the integrity of the cell has been destruction which showed the strongest disruption effect of cell walls.Results of FTIR demonstrated that after treated by ILs,EM and HCl,the characteristic absorption peaks of cellulose,hemicellulose and lignin has been weaken significantly.Among then ME treatment has less effect on lignin than other two methods.X-ray diffraction results showed that after treated by ILs,EM and HCl,the crystallinity of cellulose of cell wall from 12.93% increased to 21.56%,24.28% and 37.52%respectively.NMR implied that the amorphous region of cellulose of cell wall was susceptible to be hydrolyzed/breakdown by three techniques.The GC-MS results showed that the monosaccharide composition of the supernatants were significant difference.Glucose and mannose were detected in the supernatant after HCl treatment.Glucose,mannose,arabinose and xylose were detected in the supernatant after enzyme treatment.In the ILs treatment supernatant,only detected the glucose.(3)Study on the antioxidant activity of astaxanthin based on different extraction methods and its ability to inhibit the proliferative of liver cancer cells.In this study the methods of DPPH,ABTS,Oxygen radical absorbance capacity(ORAC)and peroxyl radical scavenging capacity(PSC)were used to analysis the antioxidant of astaxanthin based on different extraction methods.Meanwhile,Hep G2 cells were carried to manifest the antiproliferative effects of astaxanthin based on different extraction methods.There are significant differences in the results of different antioxidant systems.The antioxidant strength obtained by DPPH and ABTS follows this: HCl> ILs> HPMF >ME.The oxygen radical absorption capacity is as follow: ILs > ME > HPMF > HCl,the ORAC values are110.4±2.28 ?mol TE / g,98.87±1.91 ?mol TE / g,84.6±2.45 ?mol,46.3±1.21 ?mol TE / g respectively.Peroxide free radical scavenging ability(PSC)is as follow:ME>ILs>HCl>HPMF,the PSC value are 30.37±1.49 ?mol VCE / g DW,23.55±0.57 ?mol VCE / g DW,12.78±1.15 ?mol VCE / g DW and 10.68±0.82 ?mol VCE / g DW respectively.The antiproliferative ability of astaxanthin by the four methods is as follows:ILs>ME>HCl>HPMF,IC50 value were 6.98 ?g/mL,11.17 ?g/mL,17.21 ?g/mL and 19.46?g/mL respectively.