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Analytical Methods

Reverse micellar extraction of lectin from black turtle bean (Phaseolus vulgaris ):Optimisation of extraction conditions by response surface

methodology

Shudong He a ,b ,John Shi a ,b ,?,Elfalleh Walid a ,c ,Hongwei Zhang d ,Ying Ma a ,?,Sophia Jun Xue b

School of Food Science and Engineering,Harbin Institute of Technology,Harbin,Heilongjiang 150090,China b

Guelph Food Research Center,Agriculture and Agri-Food Canada,Guelph,Ontario N1G 5C9,Canada c

Institut des Régions Arides de Médenine,Laboratoire d’Aridoculture et Cultures Oasiennes,4119,Tunisia d

School of Food Science and Engineering,Northeast Agricultural University,Harbin,Heilongjiang 150030,China

a r t i c l e i n f o Article history:

Received 15November 2013

Received in revised form 26May 2014Accepted 30May 2014

Available online 7June 2014Keywords:

Reverse micellar extraction Lectin

Response surface methodology Yield

Puri?cation factor

a b s t r a c t

Lectin from black turtle bean (Phaseolus vulgaris )was extracted and puri?ed by reverse micellar extrac-tion (RME)method.Response surface methodology (RSM)was used to optimise the processing parame-ters for both forward and backward extraction.Hemagglutinating activity analysis,SDS–PAGE,RP-HPLC and FTIR techniques were used to characterise the lectin.The optimum extraction conditions were deter-mined as 77.59mM NaCl,pH 5.65,AOT 127.44mM sodium bis (2-ethylhexyl)sulfosuccinate (AOT)for the forward extraction;and 592.97mM KCl,pH 8.01for the backward extraction.The yield was 63.21±2.35mg protein/g bean meal with a puri?cation factor of 8.81±0.17.The ef?ciency of RME was con?rmed by SDS–PAGE and RP-HPLC,respectively.FTIR analysis indicated there were no signi?cant changes in the secondary protein https://www.wendangku.net/doc/eb18823300.html,parison with conventional chromatographic method con?rmed that the RME method could be used for the puri?cation of lectin from the crude extract.

1.Introduction

Anti-nutritional factors,such as lectins,can interfere with the absorption and utilisation of the biological nutrients present in the legumes (Boniglia et al.,2008).Thus,various cooking and processing methods are often applied to reduce the level of the anti-nutritional factors,and improve the utilisation ef?ciency of legumes (Valdes,Coelho,Michelluti,&Tramonte,2011;Wang,Lewis,Brennan,&Westby,1997).However,legume lectins are known to be associated with various properties including plant protection,recognition of nitrogen-?xing bacteria,immunomodulatory,antifungal,antiviral,antiproliferative,antitumor,and HIV-1reverse transcriptase inhib-itor activities (Charungchitrak,Petsom,Sangvanich,&Karnchanatat,2011;Davidson &Stewart,2004;Peumans &VanDamme,1996;Zhang,Shi,Ilic,Xue,&Kakuda,2009).Thus,legume lectins are cur-rently a subject of intensive studies.

Lectins are non-enzymatic proteins,which bind speci?cally and reversibly to different types of glycoproteins,mono-and oligosaccharides (Gabius,André,Kaltner,&Siebert,2002;Nasi,Picariello,&Ferranti,2009).Lectins have been puri?ed via conven-tional methods such as salt or acid precipitation,aqueous extrac-tion and chromatographic separations,e.g.,ion exchange and size exclusion chromatography,or af?nity chromatography (Ren et al.,2008).The conventional processes are usually too expensive,tedious,and time consuming,and lead to low recoveries of little or no relevance for industrial application (Porto et al.,2011).Thus,new cost-effective bioprocess separation and puri?cation tech-niques are required.

In this regard,reverse micellar extraction (RME)has been recognised as a novel method for downstream processing (DSP)of biological products (Hebbar,Sumana,Hemavathi,&Raghavarao,2012;Krishna,Srinivas,Raghavarao,&Karanth,2002;Liu,Xing,Chang,&Liu,2006).Reverse micelles (RM)are thermodynamically stable nanometre size droplets,stabilized in an apolar environment,through a monolayer of surfactant mole-cules containing an aqueous solution.The biomolecules could be solubilised in the inner core of a successful RM,and could be re-extracted into a fresh aqueous phase under certain conditions of pH and ionic strength,or using a variety of alternative backward transfer techniques (Gaikaiwari,Wagh,&Kulkarni,2012;Mathew &Juang,2007;Nandini &Rastogi,2010).Nonetheless,the application of RME for the separation and puri?cation of lectin

?Corresponding authors.Addresses:School of Food Science and Engineering,

Harbin Institute of Technology,Harbin,Heilongjiang 150090,China;Guelph Food Research Center,Agriculture and Agri-Food Canada,Guelph,Ontario N1G 5C9,Canada.Tel.:+12262278083;fax:+12262178183(J.Shi).Tel.:+8645186282903;fax:+8645186282906(Y.Ma).

E-mail addresses:john.shi@agr.gc.ca (J.Shi),maying@https://www.wendangku.net/doc/eb18823300.html, (Y.Ma).

was only recently demonstrated in a model system using a com-mercial sample(Nascimento,Coelho,Correia,&Carneiro-da-Cunha,2002),and few studies for natural crude extract(CE) of legumes have been reported(He,Shi,Walid,Ma,&Xue,2013; Hou,Hou,Liu,Qin,&Li,2010).It was reported that the ability of reverse micellar systems to selectively extract the target lectin from a protein mixture can be drastically affected by many vari-ables,such as the ionic strength,pH and surfactant concentration in the forward extraction,and pH and ionic strength in the back-ward extraction(He et al.,2013).Therefore,more systemic and detailed reports on the effect of the processing parameters are becoming more and more signi?cant.

Black turtle bean(Phaseolus vulgaris),is a kind of common beans.It is an important industrial crop in subtropical and tropical countries,and especially popular in Latin American cuisine (Kumar,Verma,Das,Jain,&Dwivedi,2013).In the present study, response surface methodology(RSM)with Box–Behnken Design (BBD)was used to optimise the extraction conditions for reverse micellar extraction of lectin from black turtle bean.The lectin thus extracted,was further puri?ed by one-step ion exchange chroma-tography,and the in?uence of reverse micellar extraction on the protein structure was determined through gel electrophoresis, RP-HPLC analysis,and Fourier Transform Infrared Spectroscopy (FTIR)measurements.

2.Materials and methods

2.1.Materials

Black turtle beans(P.vulgaris)were obtained from a local mar-ket(Harbin,Heilongjiang,China).Sodium bis(2-ethylhexyl)sulfo-succinate(AOT)from Fluka(USA);isooctane(HPLC grade)from China National Medicines Corporation Ltd.(China);enhanced bicinchoninic acid kit from Beyotime Institute of Biotechnology (China);DEAE cellulose(DE52)from Whatman Chemicals(Eng-land).All other chemicals and reagents used were of analytical grade or HPLC grade.

2.2.Crude extract preparation of lectin

For the extraction of lectin,150g of dried black turtle beans were ground to?ne powder,passed through a80-mesh sieve,then mixed with10volumes of phosphate buffered saline(PBS,10mM, pH7.0)by agitation overnight at4°C.The mixture was centrifuged at9000g for60min at4°C,and the supernatant was used as crude extract for further extraction.

2.3.Reverse micellar extraction from crude extract

The reverse micellar system was carried out by dissolving a pre-determined amount of AOT in isooctane(25–225mM),and the water content(W o)was adjusted to the value of27([H2O]/[surfac-tant])by distiled water.The aqueous phase was developed by diluting the crude extract with various buffer solutions ranging from pH4–7;i.e.,citric acid–disodium hydrogen phosphate buffer and phosphate saline buffer,each containing sodium chloride (15–125mM)at1mg/mL.The forward extraction was performed by mixing1L of the aqueous phase with equal volume of reverse micellar system at200rpm for15min at25±0.1°C.The upper organic phase rich in lectin was recovered by centrifuging at 3000g for10min at the room temperature.In the backward extraction,the organic phase was mixed with equal volume of fresh aqueous solutions(phosphate saline buffer and sodium car-bonate-bicarbonate buffer)at various pH’s(6.5–9.5)and KCl con-centration(200–1000mM)at200rpm for15min at25±0.1°C.After centrifugation at3000g for10min at the room temperature, to separate the phases,the sub-layer aqueous phase was used for further analysis.

2.4.Experimental design

In order to optimise the extraction conditions and verify the in?uence of the synergy of variables,the Box–Behnken experimen-tal design(BBD)was carried out with?ve variables at each of three levels(Supplementary Table1).The most important key indepen-dent variables and their optimisation ranges,such as NaCl concen-tration(X1,15–125mM),pH of the aqueous phase(X2,4–7),AOT concentration(X3,25–225mM)for the forward extraction,as well as pH of the new aqueous phase(X4,6.5–9.5),KCl concentration (X5,200–1000mM)for the backward extraction were selected as experimental factors.Each experiment was replicated at least three times and the yield(mg protein/g bean meal,Y1)and the puri?cation factor of lectin(Y2)were taken as the responses of the experiments,and the parameters were expressed as:

Yieldemg protein=g bean mealT

Total protein after backward extractionemgT

Total bean meal for the lectin extractionegT

e1TPurification factor

The hemagglutinating activity after backward extraction The hemagglutinating activity of crude extract

e2T

A second order polynomial equation model was used to?t the yield and puri?cation factor data with interaction terms as given below:

Y i?A0tA i

x itA ij

x i x jtA ii

ei–jTe3T

where Y i were the predicted response values;A0,A i,A ij and A ii were the regression coef?cients of the model for the intercept,linear, cross product terms and quadratic,respectively;x i were the vari-ables studied(Bera,Panesar,Panesar,&Singh,2008).

Once the regression equation model was developed,the response surface methodology(RSM)was employed to optimise the?ve most signi?cant key variables,and identify the behaviour of the system within the optimised ranges.The experimental design,regression and graphical analysis were obtained using the Design Expert Version8.0.6(Stat-Ease Inc.,Minneapolis,USA).

2.5.Protein content determination

The protein content was determined by the bicinchoninic acid (BCA)method using the Enhanced bicinchoninic acid kit(He et al.,2013;Nascimento et al.,2008).The lectin concentrations of the extracted samples were determined by RP-HPLC analysis.

2.6.Hemagglutinating activity measurement

The hemagglutination assay was carried out by measuring the maximal visible agglutination on2%suspension of rabbit red blood cells at4°C,and the hemagglutination activity(HU/mg)was expressed as the number of hemagglutination titer per mg protein (Ren et al.,2008).

2.7.Gel electrophoresis

Sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE)was performed with12%separating polyacrylamide gel and5%stacking polyacrylamide gel(10cm length, 1.5mm thickness)using a Bio-Rad electrophoresis system(Powerpac

94S.He et al./Food Chemistry166(2015)93–100

Universal,Bio-Rad Laboratories,Inc.,USA).Fifty microlitres(15l L) of each sample were loaded on each well,and run approximately for95min using Tris–glycine–SDS running buffer(25mM Tris, 192mM glycine,0.1%SDS,pH8.3)at constant voltages of80V for stacking gel and120V for separating gel,respectively.After separation,the gel was stained with Coomassie Brilliant Blue R-250in a5:4:1solution of methanol–water–acetic acid for1h, and destained extensively with several volumes of12%methanol and7%acetic acid.The destained gel was next subjected to the gel imaging system(Universal Hood II model,Bio-Rad Laboratories, Inc.,USA),and analysed by the Quantity One4.6.2software.

2.8.RP-HPLC analysis

HPLC analysis was performed on an Agilent1100Series HPLC Value System(Agilent Technologies,Pittsburg,PA,USA)with a Jupiter C4reversed phase column(250?4.6mm,300?-sized pores,5-l m sized particles)(Phenomenex,Torrance,CA,USA). The solvents were0.1%TFA in Milli-Q water(A)and0.1%TFA in acetonitrile(B).RP-HPLC conditions used with UV detection at 280nm were:30–50%B in40min,50–100%B in2min,100%B in1min,100–30%B in3min.The?ow rate was1mL/min and the injection volume was20l L.

2.9.Fourier Transform Infrared Spectroscopy

FTIR spectroscopic measurements were carried using a Perkin-Elmer FTIR spectrometer(Spectrum one B,PerkinElmer Inc., USA),through the KBr pressed disc method.The second-derivative Y1?61:61t2:16x1t9:26x2t1:59x3t3:35x4à0:60x5t2:65x1x2t3:43x1x3à0:73x1x4à0:17x1x5t0:51x2x3

à3:18x2x4à2:40x2x5t0:54x3x4à2:16x3x5t2:58x4x5

à5:36x2

à8:11x2

à5:76x2

à1:79x2

à5:18x2

e4TY2?8:75t0:93x1t0:75x2t0:19x3t0:15x4à0:06x5t0:35x1x2à0:36x1x3à0:58x1x4t0:01x1x5t0:19x2x3

à0:12x2x4à0:56x2x5à0:27x3x4à0:85x3x5t0:84x4x5

à3:47x2

à4:15x2

à3:57x2

à3:25x2

à3:69x2

e5TThe analysis of variance(ANOVA)for the response surface model equations4and5are shown in Supplementary Table2. According to the results,the F-values(40.29and280.92,respec-tively)and the low P-values(both P-values<0.0001)suggested that both models were highly signi?cant.Meanwhile,the non-signi?-cant lack of?t for the two models(P>0.05)and the well relation-ship between predicted and actual values(Supplementary Fig.1) indicated that the factors in each model were well correlated. The high values of R-Squared(0.9699for Y1and0.9956for Y2)as well as adjusted R-Squared(0.9458for Y1and0.9920for Y2)for the model?tting also indicated that the models?tted the experi-mental data very well.Hence there were only5.42%of the total variation for the yield(Y1)and0.80%for the puri?cation factor (Y2)that could not be explained by the models.Therefore the regression models were successful and could accurately represent the variables chosen in the experimental region.

Three-dimensional(3D)response surface curves for the effects of the pH of new aqueous phase(X4)and KCl concentration(X5)in the backward extraction

puri?cation factor(b),when the forward extraction was?xed at the center point level.

S.He et al./Food Chemistry166(2015)93–10095

phase (X 4)and KCl concentration (X 5)was investigated (Fig.1).When the processing parameters in the forward extraction were ?xed at the center point levels,the yield increased with increasing pH and KCl concentration initially,then reached to a potential equilibrium state at a higher pH after 0level (pH 8.00)like the previous study (He et al.,2013),but dropped after 0level of the KCl concentration (600mM).Meanwhile,the puri?cation factor rose at ?rst,but decreased later,with increasing pH and

KCl

Three-dimensional (3D)response surface curves for the effects of NaCl concentration (X 1),pH of aqueous phase (X 2),AOT concentration (X 3)in the forward (a,c,e)and puri?cation factor (b,d,f),when the backward extraction was ?xed at the optimum level.

concentration.In the backward extraction,the proteins in the organic phase could be freed from the reverse micelles by either electrostatic interaction or size exclusion,through adjusting the pH and KCl concentration(Hebbar et al.,2012;Tonova& Lazarova,2008).However,as the results obtained from the puri?-cation factor,a higher pH might lead to the lectin denaturation at higher KCl concentrations.According to the RSM analysis,the opti-mal yield and puri?cation factor predicted were obtained by the following backward extraction condition:pH0.05level and KCl concentrationà0.018level.Thus,the values above were chosen as the basis for further RSM analysis in the forward extraction.

Fig.2indicates the variations of the responses(Y1and Y2)in accordance with the changes of two of the three processing param-eters in the forward extraction,by setting the other one at zero level.

At the highest level(+1)of the pH of the aqueous phase,the yield and puri?cation factor increased to the highest extent with increase of the NaCl concentration,and only a slight decrease of the yield was found with further increase in NaCl concentration, compared with a downward trend in the puri?cation factor (Fig.2a and b).Meanwhile,the yield and puri?cation factor increased initially before decreasing,while NaCl concentration continuously increased at the lowest level(à1)of the pH of the aqueous phase(Fig.2a and b).This suggested that the favourable interaction between targeted lectin and surfactant head groups was enhanced through the addition of NaCl,which was also condu-cive to the formation of the reverse micelles(Hebbar,Sumana,& Raghavarao,2008;Nandini&Rastogi,2009).Furthermore,the lec-tin might be excluded at a higher NaCl concentration,due to the Debye length reduction and squeezing-out effect(Krishna et al., 2002;Mazzola et al.,2008;Tonova&Lazarova,2008).

The in?uence of pH of aqueous phase and AOT concentration on the yield and puri?cation factor(Fig.2c and d)are shown in Fig.2a and b.Initially the yield and puri?cation factor were found to increase with pH,followed by only a slight decrease in yield at the highest AOT level(+1),while the other parameters all decreased signi?cantly with further increase of pH(Fig.2c and d).In the investigation of AOT-isooctane reverse micellar system, solubilisation of protein occurred when pH was below the isoelec-tric point(pI)of the targeted protein,due to the strong electro-static interaction(Chen,Su,&Chiang,2006;Nandini&Rastogi, 2009;Tonova&Lazarova,2008).However,pH values above the pI of the proteins might lead to a decrease in charge density,result-ing in the yield loss of the lectin.

At all levels of NaCl concentration,it was evident that the yield and puri?cation factor initially increased with increase in AOT con-centration,and then decreased with further addition of AOT (Fig.2e and f).At?rst,the increase of AOT may result in an increase of reverse micelles,which could in turn enhance lectin extraction. However,further increase of AOT may cause a change in the micel-lar shape and clustering,leading to a decrease in the solubilisation capacity for the lectin(Krishna et al.,2002;Tonova&Lazarova, 2008).3.3.Veri?cation of predictive models and optimisation of process

In order to optimise the reverse micellar extraction process,the ?rst partial derivatives of the regression models were used to get the maximum recovery of lectin.The optimum process conditions for NaCl concentration(X1),pH of the aqueous phase(X2),AOT con-centration(X3)for the forward extraction,as well as the pH of the new aqueous phase(X4),KCl concentration(X5)for the backward extraction were77.59,5.65,127.44,8.01and592.97mM,respec-tively(Table1).Under these optimum process conditions,the yield and the puri?cation factor of lectin predicted by RSM were 62.75mg protein/g bean meal and8.86,respectively.

The experimental rechecking was carried out under the optimum process conditions,in order to validate the models. According to Table1,the experimental values of yield and puri?ca-tion factor were63.21±2.35mg protein/g bean meal and 8.81±0.17,respectively.And no signi?cant differences(P>0.05) were obtained between the predicted and experimental response values.Therefore the process conditions for lectin reverse micellar extraction achieved by RSM were practical.Hence all the process-ing parameters in the predictive models were useful in prediction to make the whole lectin extraction more economical and ef?cient.

3.4.Con?rmation of lectin extraction using reverse micelles

After the reverse micellar extraction,the aquous phase contain-ing lectin were dialysed and lyophilized.Then the concentrated sample were subjected to the12%SDS–PAGE gel.In the SDS–PAGE results(Fig.3a),only two polypeptide bands were observed for the reverse micellar extraction https://www.wendangku.net/doc/eb18823300.html,pared with the crude extract sample,the reduction in the number of bands indicated the effectiveness of the reverse micellar process for the extraction of lectin.As shown in Fig.3b,two peaks were detected by RP-HPLC for the lectin sample extracted by reverse micelles,which is consis-tent with the SDS–PAGE results.Therefore the optimum reverse micellar extraction process could be easily used to obtain a high purity lectin for further research.

3.5.Structural comparative studies with conventional method

In subsequent lectin puri?cations using one-step ion exchange chromatography with an AKTA Fast Protein Liquid Chromatogra-phy(FPLC)system(GE Healthcare Life Sciences,USA)with a XK 16/60DE52column(16?60mm),only a few impurities were observed with the proteins in the reverse micellar sample.The lyophilized protein sample obtained from reverse micellar extrac-tion was dissolved with the PBS buffer(10mM,pH8.5)to a?nal concentration of1mg/mL,and then loaded onto the DE52column, which had been pre-equilibrated with10mM PBS buffer(pH8.5). The adsorbed proteins were eluted with a linear salt gradient of 0–1.0M NaCl in the PBS buffer(pH8.5)at a constant?ow rate of 1mL/min for360min.After the puri?cation,the purity of lectin

Table1

Predicted and experimental response values under the optimum process conditions.

Optimum process conditions Predicated yield

(mg protein/g bean meal)Experimental yield

(mg protein/g bean meal)

Predicated puri?cation

factor of lectin

Experimental puri?cation

factor of lectin

Coded Uncoded

X10.13877.59

X20.098 5.65

X30.024127.4462.7563.21±2.358.868.81±0.17

X40.0068.01

X5à0.018592.97

1Where in the?rst column,X

:NaCl concentration,X2:pH of the aqueous phase,X3:AOT concentration for the forward extraction,X4:the pH of the new aqueous phase,X5: KCl concentration for the backward extraction.

2The data in the?fth and seventh columns(mean±standard deviation)related to at least3replicates.

S.He et al./Food Chemistry166(2015)93–10097

obtained from the ion exchange chromatography step was con-?rmed by electrophoresis(Fig.3a)and RP-HPLC analysis (Fig.3b).The effects of the reverse micellar extraction on the sec-ondary structure of lectin were analysed by protein secondary structure prediction using FTIR spectroscopic measurements.The control lectin was puri?ed using ion exchange chromatography and size exclusion chromatography,after ammonium sulphate precipitation(20–80%fraction),over a period of at least3days.

As reported,the shape of the amide I band was used to deter-mine the protein secondary structure,located at1600–1700cmà1

Carbonaro&Nucara,2010;Goormaghtigh,Ruysschaert,&Raussens, Fig.4showed that the contents of secondary structural compositions of lectin extracted from the reverse micelles changed slightly:i.e.,a-helix(1650–1660cmà1)increased from10.23%

12.32%;b-sheet(1610–1642cmà1)increased from25.66%

27.35%,b-turn(1660–1680cmà1)decreased from33.89%to

b-antiparallel(1680–1700cmà1)increased from8.26%to

and the random coil(1642–1650cmà1)decreased from

to20.78%.Consequently,there were no signi?cant changes

secondary structural compositions of lectin from reverse micellar extraction.For the typical b structural compositions,the change of both lectin samples was only in0.90%.Thus the FTIR spectrograms predicted that the protein structure of lectin might be still be retained after the reverse micellar extraction, and the recovered lectin showed similar structures with the sam-ple obtained from the conventional method.

3.6.Summary of lectin puri?cation by conventional method and reverse micellar extraction

The yield(mg protein/g bean meal)and lectin concentration (mg lectin/mg lyophilized powder)puri?ed by different protocols are shown in Table2.The?nal yields(1.37±0.12mg

bean meal)and lectin concentration(0.85±0.08mg lectin/mg lyophilized powder)for reverse micellar extraction exchange chromatography were much higher than the

tional method,0.65±0.13and0.50±0.04,respectively.Therefore, the lectin concentration(0.15±0.09mg lectin/mg lyophilized powder)for the reverse micellar extraction was lower because the competitive binding of non-lectin proteins into the micelles,but the total lectin content was highest(ca.9.48

tin/g bean meal).The content of lectin in P.vulgaris bean

ies signi?cantly due to bean type,and it is usually reported around12mg lectin/g bean meal by immunological

SDS–PAGE analysis of reverse micellar extraction lectin and subsequent puri?cation https://www.wendangku.net/doc/eb18823300.html,ne1:molecular weight marker;lane2:lectin extracted

2mg/mL;lane3:lectin further puri?ed by one-step ion exchange chromatography,1mg/mL;lane4:crude extract of black turtle bean,2mg/mL.(b)

of reverse micellar extraction lectin and subsequent puri?cation lectin.Chromatogram1:lectin extracted by reverse micelles;chromatogram2:lectin

by one-step ion exchange chromatography.Column:Phenomenex Jupiter C4reversed phase column(250?4.6mm,300?-sized pores,5-l m sized particles); mobile phase:Milli-Q water containing0.1%TFA and acetonitrile containing0.1%TFA.

FTIR spectrograms and curve-?tting results of amide I band for lectins extracted from the reverse micelles(a)and obtained from the conventional 1600–1700cmà1.

(Zhang et al.,2009).According to the previous report,about 2.24mg lectin was puri?ed from1g red kidney bean(P.vulgaris) meal using a one-step Af?l-Gel blue af?nity gel(Ren et al.,2008). Since the protein recovery would be reduced by the extra steps in the puri?cation process,the yield obtained in the present study by reverse micellar extraction was about4times higher than that achieved previously.Further,the puri?cation by both reverse micellar extraction and ion exchange chromatography within 1day was more ef?cient and less tedious than the conventional method that requires3–4days to perform.

4.Conclusions

The optimum reverse micellar extraction conditions for lectin extraction from black turtle bean were obtained for the?rst time through this study using response surface methodology and Box–Behnken experimental design.The effects of processing parame-ters,such as NaCl concentration(X1),pH of the aqueous phase (X2),AOT concentration(X3)for the forward extraction,as well as pH of the new aqueous phase(X4),KCl concentration(X5)for the backward extraction,were successfully evaluated by the regres-sion models and the three-dimensional(3D)response surface curves.The reverse micellar extraction process proved to be ef?-cient based on the purity determination of lectin using SDS–PAGE and RP-HPLC analysis.The FTIR spectrum results indicated that the lectin extracted by reverse micellar extraction had similar struc-ture similar to lectin by the conventional method.Therefore,it is suggested that the reverse micellar extraction could be a valuable protocol for use in the laboratory and industrial processes for the puri?cation of lectin from black turtle bean.

Acknowledgements

The authors would like to thank Ms.Shengxin Zhao,School of Municipal and Environmental Engineering,Harbin Institute of Technology,China for her assistance in the FTIR spectroscopic mea-surements.The authors are also deeply grateful to Dr.Gra?a Cunha, Department of Biochemical,Universidade Federal de Pernambuco –UFPE,for the valuable technical suggestions provided,and to Prof.Benjamin K.Simpson,Department of Food Science and Agri-cultural Chemistry,Macdonald Campus,McGill University,for his valuable suggestions and language revision.

Appendix A.Supplementary data

Supplementary data associated with this article can be found,in the online version,at https://www.wendangku.net/doc/eb18823300.html,/10.1016/j.foodchem.2014.

05.156.

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Table2

Summary of lectin puri?cation by conventional method and reverse micellar extraction.

Sample Puri?ed by conventional

method Puri?ed by reverse micellar

extraction

Puri?ed by both reverse micellar extraction and ion

exchange chromatography

Yield(mg protein/g bean meal)0.65±0.13b63.21±2.35a 1.37±0.12b

Lectin concentration(mg lectin/mg

lyophilized powder)

0.50±0.04b0.15±0.09c0.85±0.08a

1The data(mean±standard deviation)related to3replicates as described in materials and methods.

2The difference between the means with the same superscript letter in the same row was signi?cant at95%con?dence interval.

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