灰枣多糖大孔径树脂提纯工艺优化.pdf

第 28 卷第 21 期农 业 工 程 学 报Vol.28No.212012年11月Transactions of the Chinese Society of Agricultural EngineeringNov.2012287Optimization of resin based technology for purification ofpolysaccharides from Ziziphus Jujuba cv.HuizaoWang Na1,Fan Huiping1,Sun Xiaorui2,Pan Zhili1,Shi Juling2,Ai Zhilu1(1.College of Food Science and Technology,Henan Agricultural University,Zhengzhou,450002,China;2.Haoxiangni Jujube Industry Co.,Ltd,Zhengzhou,451100,China)Abstract:Ziziphus Jujuba cv.Xinzheng Huizao is rich in kinds of nutrients,especially polysaccharides,which possessmany biological effects on human bodies.However,the purification of polysaccharides from jujube after a primarilyextraction step is still a difficult technology.In present research,enzyme combined with ultrasonic wave extraction,ethanol precipitation and ultrafiltration membrane filtration were employed to extract and preliminarily purify thepolysaccharides from Ziziphus Jujuba cv.Xinzheng Huizao.The obtained raw polysaccharides were employed for furtherpurification.Through the investigation of static adsorption and desorption of resins,the most suitable macroporous resinfor the purification of jujube polysaccharides was selected and its dynamic adsorption and desorption behavior wasstudied.Furthermore,response surface experiments were conducted to investigate the effects of multifactor on theadsorption properties of macroporous resin for purification of jujube polysaccharide.The optimum adsorption conditionswere 1.5 mL/min of in-flux rate,2.2mg/mL of concentration of the feed liquid,5.6 of pH value.Under this condition,themaximum dynamic adsorption rate of 19.52 mg/g could be obtained in theory.The optimized technical condition for dynamicdesorption was obtained with 0.4 mol/L of NaCl,60%of ethanol,0.2 mol/L of HCl and 1.5 mL/min of flow rate.Under thiscondition,maximum dynamic desorption rate of 85.21%was achieved,and the purity of yield polysaccharides was 88.87%.Taken together,the present optimized technology is a highly effective method for the extraction and purification ofpolysaccharides from jujube,and has a great application and market potential in the medical and health care industries.Key words:purification,resins,polysaccharides,Ziziphus Jujuba cv.Xinzheng Huizao,doi：10.3969/j.issn.1002-6819.2012.21.040CLC number:TS201.4Document code:AArticle ID:1002-6819(2012)-21-0287-06Wang Na,Fan Huiping,Sun Xiaorui,et al.Optimization of resin based technology for purification of polysaccharidesfrom Ziziphus Jujuba cv.HuizaoJ.Transactions of the Chinese Society of Agricultural Engineering(Transactions of theCSAE),2012,28(21):287282.(in English with Chinese abstract)王娜，范会平，孙晓瑞，等.灰枣多糖大孔径树脂提纯工艺优化J.农业工程学报，2012，28(21)：287292.0IntroductionJujube(Ziziphus Jujuba cv.Xinzheng Huizao)isfruit of Rhamnaceae tree.Jujube has highly nutritionalvalue as a natural vitamin fruit,which is rich in 18kinds of necessary amino acid for human bodies,proteins,fats,carbohydrate,organic acid,mineralsand vitamine B,C,P,etc1-2.Jujube polysaccharideshave been proved to possess the biological activitiesincludingrelievingcough,enhancingimmunity,anti-aging,anticancer and so on3-8.The most commonly used methods for extractionof polysaccharides from jujube were step-by-stepReceived date：2011-11-01Revised date：2012-08-16Foundation item：Sub-project of“Twelfth Five-Year”National Science andTechnology Support Projects(Project No：2012BAD36B07-12)Biography：Wang Na(1979),Female,Henan Nanyang,lecturer,MasterDegree,Food science.Zhengzhou,450002.Email:Na-Corresponding author：Ai Zhilu(1965),Male,Professor,Ph.D,Industrialization research of traditional food and processing of agriculturalproducts;No.63 Nongye Rd.Zhengzhou,450002.Email:zhila precipitation,quaternary ammonium salt precipitation,DEAE-cellulose column chromatography and differentkinds of gel column chromatography,centrifugalsedimentation chromatography and ion exchange resinmethod9.Macroporous adsorption resin(Model AB-8)is anewly developed organic polymer adsorbent,whichpossesses big hole mesh structure and specific surfacearea,and has been widely applied in the areas ofenvironment,food and biomedicine10-11,as well asthe purification of polysaccharides from jujube12-15.In present research,the performance of AB-8 and thefactors of adsorption and desorption were fullyconsidered to establish the technology of dynamicadsorption and desorption of AB-8 for the purificationof polysaccharides from jujube.1Materials and methods1.1MaterialsJujubefruitcultivarXinzhengHuizaowaspurchased from Haoxiangni Jujube Co.,Ltd.After农业工程学报2012 年288shipping to the lab,the dried jujube fruits were storedunder-20 C.1.2Extraction of crude polysaccharidesClean and de-seed jujube fruits were emulsifiedwith 1:8 of solid-liquid ratio in water by a high shearscatteredemulsifyingmachine,thenpreliminaryextracted by ultrasonic method for 25 min under 70 Cand 70 W.After adding 0.15%of papain and adjustingthe pH value to 6.0,the extracts was heated at 60 Cfor 120 min by water bath,then inactivated for 10 minunder 90 C before refrigeration.The supernatant ofcooling liquid was obtained by centrifugation,andconcentrated to 1/6 volume of original by a rotaryevaporator.Thecrudepolysaccharideswereprecipitated by adding 3 times volume of anhydrateethanol for 10 h,and then dissolved in distilledwater6.After the redundant ethanol was removed bytherotaryevaporator,theobtainedcrudepolysaccharides were stored in refrigerator for nextstep.1.3Determination of total sugarPhenol-sulfuric acid method was employed todetermine the content of total sugar according toZhang(1987)16.Glucose was used as the standard.TheobtainedregressioncurveequationisY=0.0066x+0.0067,and the regression coefficient(R2)is0.9995.1.4Selection and preparation of resinThe characteristics of adsorption and desorptionof 6 resins including AB-8,NKA-9,D101,D152,S-8,X-5 were thoroughly compared according to theparameter of adsorption capacity,and the mostsuitable resin for extraction and purification ofpolysaccharides from jujube was selected for nextoptimization of technology.The adsorption capacitywas calculated as the following equation:01()Adsorption rate(mg/g)CCVM（1）Where,C0is the concentration of polysaccharidesof extract before adsorption,mg/mL;C1is theconcentration of polysaccharides of extract afteradsorption,mg/mL;V is the volume of extract,mL;Mis the wet weight of resin,g.The desorption rate(%)is calculated as follows:100Desorption rate(mg/g)CVW（2）Where W is total adsorption,mg;V is the totalvolume of elution,mL;C is the concentration ofpolysaccharides in extracts,mg/mL;Finally,theadsorption kinetic curve is drawn.The yield of polysaccharides(%)and the purityof polysaccharides(%)are calculated as the followingequations:1Yield of polysaccharides(%)100mm（3）Purity of polysaccharides(%)100abmmm（4）Where,m1is the mass of raw material,g;m is themass of raw polysaccharides,g;mais the mass of totalsugars in raw polysaccharides,g;mbis the mass ofreduced sugars in raw polysaccharides,g.1.5Optimization of adsorption condition ofpolysaccharides from jujube using AB-8 resinBased on the results of single factor experiments,sampling flow rate,feed concentrations and filtrate pHvalue were selected as the independent variables forthe response surface design by the software ofDesign-Expert17-18.After Box-Behnken design andthoroughly experiments on the three variables,thedynamic adsorption conditions of AB-8 resin wereoptimized.1.6Optimization of desorption condition of AB-8resinAfter single factor experiments,hydrochloricacidconcentration,ethanolcontentineluent,concentration of NaCl and flow rate of elution wereinvestigated by orthogonal experimental design tooptimize the desorption condition of AB-8 resin.1.7Verification testThe theoretical results are testified byoptimaladsorption and desorption condition ofAB-8 resin.2Results and analysis2.1Static adsorption and desorption performance ofsix resinsTotally six macroporous adsorption resins werecompared according to the performance of staticadsorption and desorption of polysaccharides fromjujube(Table 1).Macroporousresinadsorptionseparation technology,is a new extraction-purificationprocess to selectively remove the ineffective ingredientsusing a special solution,and to adsorb the activeportion from the adsorbent.The amount of adsorptionon the adsorbent depends on the polarity of solute andsolvent,different types of resin showed significantadsorption performance differences19.As shown inTable 1,resin AB-8,D101,X-5 and S-8 showedrelatively stronger adsorption capacity of polysaccharidesfrom Xinzheng Huizao,all with more than 10.0 mg/gof absorbance.AB-8 resin showed the highest staticadsorption,X-5 was the second;NKA-9 and D152were the minimum.The results showed that D101 hadthe highest resolution rate of 85.13%.Although AB-8第 21 期王娜等：灰枣多糖大孔径树脂提纯工艺优化289had a slightly lower desorption rate of 83.87%,it hadhigher adsorption rate,AB-8 macroporous resin wasstill selected for the purification of polysaccharidesfrom jujube cv.Xinzheng Huizao.Table 1Static adsorption and desorption performance of6 resins for polysaccharidesResin namePropertyAdsorption capability/(mg g-1)Desorption rate/%AB-8Nonpolar15.95 0.35e83.87 0.26eD101Nonpolar11.64 0.30c85.13 0.35fX-5Nonpolar13.24 0.38d79.35 0.41dS-8Polar10.89 0.75c62.70 0.40cNKA-9Polar6.57 0.32b48.64 0.78bD152Nonpolar4.87 0.26a18.38 0.33aNote:Data superscripted by common letter are not significantly different atP0.05.2.2Adsorption properties of AB-8 macroporousresin for polysaccharides from Xinzheng Huizao2.2.1Adsorption kinetics of AB-8 macroporous resinAs shown in Fig.1,the adsorption rate of AB-8resin declined sharply with the increase of adsorptiontime,and then saturate gradually until adsorptionequilibrium after 12 h,which suggested physicaladsorption was the main manner for polysaccharidesadsorption of AB-8 resin,and was considered as akind of chronic adsorption.Fig.1Kinetic curve of static adsorption ofAB-8 resin forpolysaccharides from jujube cv.Xinzheng HuizaoFig.2Adsorption balance equation ofAP onAB-8 resin forpolysaccharides from jujube cv.Xinzheng HuizaoTo quantitatively evaluate the adsorption rate ofAB-8 resin for purification of polysaccharides,thefollowing Langmuir layer adsorption model was usedto fit the equilibrium rate constant of AB-8 resin20.LneetQktQQorLn(1)teQktQ（5）Where,Qtis the absorbance of resin at time t;Qeistheabsorbanceofresinduringadsorptionequilibrium,and K is the adsorption rate constant.Asthe results shown in Fig.2,the Langmuir layeradsorption model was well fit to the purificationbehavior of the AB-8 resin.Adsorption rate constant kis an important constant that reflects the adsorptionrate of resin,the smaller the constant,the longerperiod to achieve equilibrium,which was consideredas a chronic adsorption.2.2.2ResponsesurfaceoptimizationofAB-8macroporous adsorption resin dynamic conditionsAccording to the aforementioned single-factortest,the AB-8 macroporous adsorption resin dynamicconditions were determined and optimized usingresponse surface methodology.Test program andresults are shown in Table 2.Table 2Optimization of dynamic adsorption onAB-8 resinTestnumberIn-flux rate/(mL min-1)(A)Feedconcentration/(mg mL-1)(B)pH value(C)Adsorptioncapacity/(mg g-1)10(1.5)1(2.5)1(6.0)17.252-1(1.0)10(5.0)17.0530-1(1.5)115.46401-1(4.0)17.695-10(2.0)-116.13600019.2571(2.0)0118.0681-1016.319-10116.96100-1016.461100018.641200018.831300018.921400019.081511019.031610-117.39170-1-115.09Table 3Variance analysis for responses of dynamicadsorption on AB-8 resinVariance sourceSSDFMSF valueP valueModel27.5493.0630.290.0001Residual0.7170.10Lack of Fit0.4930.162.990.1588Error0.2240.055Total28.2516Note:R2=0.9750Adj.R2=0.9428Pred.R2=0.7109Adeq.Precision=17.2404.UsingDesign-Expertsoftwaretofittheexperimental data,a quadratic multiple regressionmodel can be obtained as follows:农业工程学报2012 年290Y=18.94+0.18 A+0.96 B+0.52 C 0.20 A B0.040 A C+0.53 B C 1.32 A2 1.25 B2 0.48 C2Where Y is the dynamic adsorption capability;Ais in-flux rate,B is the feed concentration;C is feedpH value.The significance test results of this model isshown in Table 4,which suggests a strong enoughresponse signal model that can be used to analyze andpredict the dynamic polysaccharides adsorption resultsofAB-8resin.Moreinterestingly,anonlinearcorrelationship can also be indicated from differentvariables as the results shown in Table 4.Table 4Significance of test result for regressioncoefficientVariablesRegressioncoefficientMeansquareDegree offreedomF valueP valueIn-flux rate(A)0.180.2612.530.1557feed pH(B)0.967.41173.360.0001feedconcentration(C)0.522.19121.720.0023AB-0.200.1611.620.2432AC-0.046.400 10-310.0630.8085BC0.531.13111.230.0122A2-1.327.39173.120.0001B2-1.256.55164.81DCA,which means theethanol content played the greatest role in desorptionrate,flow rate took the second,and the concentrationof NaCl was the last.It was also indicated that thehighest extraction efficiency could be obtained whenthe concentration of ethanol was 60%.Table 5Optimization of dynamic desorption performanceof AB-8 resinsTestnumberNaClconcentration(mol L-1)(A)Ethanolconcentr-Ation(%)(B)HClconcentr-ation/(mol L-1)(C)Flow rate/(mL min-1)(D)Desorp-tion rate/%1A1(0.2)B1(40)C1(0.1)D1(1.0)54.262A1B2(50)C2(0.2)D2(1.5)82.073A1B3(60)C3(0.3)D3(2.0)73.564A2(0.3)B1C2D371.245A2B2C3D162.396A2B3C1D276.897A3(0.4)B1C3D265.638A3B2C1D374.669A3B3C2D174.99K169.96363.7168.60363.880K270.17373.0476.10074.863K371.76075.14767.19373.153R1.79711.4378.90710.893Table 6Orthogonal analysis of variance testSource of varianceSum ofsquareddeviationsFreedom F ratioF criticalvalueSignificanceNaCl concentration(A)5.79021.00019.000Ethanolconcentration(B)222.284238.39119.000*HCl concentration(C)137.517223.75119.000*Flow rate(D)209.552236.19219.000*Considering all of the test results,the optimizedtechnologyfordynamicdesorptionofdatapolysaccharides using AB-8 macroporous resin couldbe concluded as A3B3C2D2.2.4Verification testThe theoretical results were testified and the第 21 期王娜等：灰枣多糖大孔径树脂提纯工艺优化291results are shown in Table 7.From a workableconsideration,adsorption condition of AB-8 resin wasverified that the adsorption capability was 19.52 mg/gby 1.5 mL/min of in-flux rate,2.2 mg/mL of feedconcentrationand5.6pHvalue.Meanwhile,desorption condition of AB-8 resin was verified byorthogonal optimization results,which represented 0.4mol/L of NaCl,60%of ethanol,0.2 mol/L of HCl and1.5 mL/min of flow rate.Under these conditions,thebest dynamic desorption rate was 85.21%.Table 7Verification test of adsorption and desorptioncondition of AB-8 resinTest numberAdsorption capacity/(mg g-1)Desorption rate/%120.3882.87219.1486.23319.7885.56418.8787.91519.4283.47Average value19.5285.21RSD/%3.012.42Note:RSD is the relative standard difference.2.4Dynamic elution curve of AB-8 resinsThe AB-8 resin was saturated with the solutionconcentration of 2.5 mg/mL and sample flow rate of1.5 mL/min.The neutral polysaccharide was elutedwith 200 mL of distilled water and flow rate o