茶梗

Using tea stalk lignocellulose as an adsorbent for separating decaffeinated tea catechins

J.H.Ye,J.Jin,H.L.Liang,J.L.Lu,Y.Y.Du,X.Q.Zheng,Y.R.Liang *

Zhejiang University,Tea Research Institute,268Kaixuan Road,Hangzhou 310029,China

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

Received 7March 2008

Received in revised form 1July 2008Accepted 2July 2008

Available online 16August 2008Keywords:Antioxidant Caffeine

Pine sawdust Bagasse

Macroporous resin

a b s t r a c t

Lignocelluloses prepared from woody tea stalk,pine sawdust and sugarcane bagasse were used as adsor-bents to isolate decaffeinated catechins from tea extracts and compared with synthetic macroporous resin HPD 600.HPD 600had the highest adsorption capacity to catechins,followed by tea stalk lignocel-lulose while lignocelluloses of pine sawdust and bagasse the least.Tea stalk lignocellulose absorbed pref-erentially tea catechins and showed a good selectivity.HPD 600absorbed caffeine and tea catechins simultaneously.The kinetics data of tea stalk lignocellulose showed a good ?t with the Langmuir iso-therm model.It is considered that tea stalk lignocellulose is an alternative low-cost adsorbent for prepar-ing decaffeinated tea catechins.

ó2008Elsevier Ltd.All rights reserved.

1.Introduction

Tea catechins,a group of abundant polyphenols in tea leaf,have received great attention both from scienti?c community and gen-eral public recently owing to their health bene?ts and functionality such as antimutagenic,anticarcinogenic and anticlastogenic effects (Gupta et al.,2002;Kuroda and Hara,1999).The major compo-nents of tea catechins are (à)-epigallocatechin gallate (EGCg),(à)-epigallocatechin (EGC),(à)-epicatechin gallate (ECg),(à)-epi-catechin (EC),and their epimerization isomers (+)-gallocatechin gallate (GCg),(+)-gallocatechin (GC),(+)-catechin gallate (Cg),and (+)-catechin (C)(Row and Jin,2006).EGCg is regarded as the most important component of tea catechins because of its high concentration in tea and excellent bioactivities.It is reported that EGCg can prevent cancer,cardiovascular disease,obesity as well as diabetes (Dreosti,1996;Johnson and Loo,2000),and also be effective to inhibit HIV reverse transcriptase (Chang et al.,1994).Preparation of catechins complex or pure EGCg will be interesting for further exploiting their bioactivities and related applications.Tea contains two to four percent of caffeine and it is a source for extracting caffeine (Guru and Icen,2004).It was con?rmed that excessive intake of caffeine exerted adverse effects on human health,including sleep deprivation (Hindmarch et al.,2000),abor-tions and miscarriages (Giannelli et al.,2003;Rasch,2003),psycho-social stress and hypersensitivity (Bernhisel-Broadbent,1999;

Nehlig et al.,1992).In order to obtain caffeine-free products of tea and its extracts,many attempts have been made,including the use of organic solvents (Row and Jin,2006),hot water (Liang et al.,2007),supercritical carbon dioxide (Park et al.,2007a,b ),microbial degradation of caffeine (Gokulakrishnan et al.,2005),activated carbon (Ye et al.,2007)and column chromatography (Sakanaka,2003).However,organic solvents extraction is not widely accepted by consumers due to its chemical residue.The hot water treatment is exclusively applicable for treating fresh tea leaf (Liang et al.,2007).Extraction by supercritical carbon diox-ide is still expensive.There is a long way to go for the microbial degradation of caffeine to be industrialized.Column chromatogra-phy is an important method to isolate bioactive ingredients from plant materials,and the selectivity of absorbents is critical to the isolation process.

Lignocellulose prepared using unwanted woody materials such as agricultural byproducts or natural ?bres was con?rmed to be useful absorbents for isolating bioactive components from plants or removing heavy metal ions from waste water (Sciban et al.,2006).Sawdust of North American cedar wood was used to prepare raw decaffeinated tea polyphenol from tea extract (Sakanaka,2003).However,various lignocellulosic materials have different adsorption capacities to individual compounds or metal ions (Shu-kla et al.,2002).In the present paper,lignocellulose materials pre-pared from three plant species were used to decaffeinate tea catechins in comparison with synthetic macroporous resin HPD 600,and the appropriate material was selected for further study on the adsorption mechanism including kinetics and isotherm,as well as column chromatographic conditions.

0960-8524/$-see front matter ó2008Elsevier Ltd.All rights reserved.doi:10.1016/j.biortech.2008.07.003

*Corresponding author.Tel.:+8657186971260;fax:+8657186971704.E-mail address:yrliang@https://www.360docs.net/doc/4616434497.html, (Y.R.Liang).Bioresource Technology 100(2009)

622–628

Contents lists available at ScienceDirect

Bioresource Technology

jo ur na l h o me pa ge :w w w.e ls ev ie r.c o m/lo c a t e/bi or t e c

h

2.Experimental

2.1.Materials

Woody tea stalk(Camellia sinensis)from tea processing byprod-uct,pine sawdust(Pinus massoniana),bagasse of sugarcane(Sac-charum sinensis)and a synthetic macroporous resin HPD600 were used in this study.The tea stalk was supplied by Shenlong Tea Factory(Hangzhou,China).The pine sawdust was obtained from Hangzhou Lumber Mill(Hangzhou,China)and the bagasse was supplied by the Experimental Farm of Zhejiang University (Hangzhou,China).HPD600macroporous resin(Cangzhou Bon Chemical Co.Ltd.,Cangzhou,China)which is usually used as an absorbent for isolating tea catechins complex was purchased from market.

Green tea extract used was supplied by Orient Tea Development Co.Ltd.(Hangzhou,China).Chemical purity grade ethanol was pur-chased from Sinopharm Chemical Reagent Co.Ltd.(Beijing,China). HPLC reference compounds including(à)-epigallocatechin gallate (EGCg),(à)-epigallocatechin(EGC),(à)-epicatechin gallate(ECg), (à)-epicatechin(EC),(+)-gallocatechin gallate(GCg),(+)-gallocate-chin(GC),(+)-catechin gallate(Cg),(+)-catechin(C)were supplied by National Institute of Japan(Kagoshima,Japan),and caffeine was a Sigma product(Sigma–Aldrich,St.Louis,USA).The other chemi-cal reagents used were of HPLC grade(Jinmei Biotech Corporation, Tianjin,China).The water used in the experiments was de-ionized water.

2.2.Preparation of adsorbents

Lignocullulose preparation was carried out according to modi-?ed method described by Sakanaka(2003).The plant materials were dried at105°C for4h,ground using an EUPA TSK-927S grin-der(Cankun Co.Ltd.,Shanghai,China)and sifted to particle size 0.25–0.30mm,and then drenched in2volumes of0.1N NaOH and stood for72h at room temperature.The NaOH solution was drained and the plant materials were washed with water.The NaOH-treated materials were drenched in2volumes of0.1N HCl overnight and then washed with water to pH7,and?nally sus-pended in2volumes of95%ethanol overnight at room tempera-ture and washed again with water.The prepared lignocelluloses were dried under ambient conditions.The lignocelluloses prepared from tea stalk,pine sawdust and bagasse were used as adsorbents in the tests and they were abbreviated as TSL,PSL and BL,respec-tively.The HPD600was pretreated with0.1N NaOH and0.1N HCl as the same method.

2.3.Test of adsorption capacity

Ten gram of above pretreated adsorbents was added into 500mL beaker?asks containing200mL of tea extract solution (20g Là1).The?asks were shaken at150rpm and25°C for12h using H2S-H water bath shaker(Donglian Electronic Technology Co.Ltd.,Harbin,China)and then the supernatants were sampled for HPLC.The adsorption capacity was calculated by the following Eq.(1):

q?V C0àCT

e

G

e1T

where q was adsorption capacity of the adsorbent(mg gà1)at equi-librium,C0and C were the initial and?nal solute concentrations of the solution(mg mLà1),respectively.V was the solution volume (mL)and G was weight of adsorbent used(g).

Selectivity coef?cient K B

A

characterizes quantitatively the ability of an adsorbent to select one of two solutes present in the same solution and is calculated by the following Eq.(2):K B

A

?

R B=R A

C B=C A

?

C A

C B

?

R B

R A

e2T

K B

A

was selectivity coef?cient.A and B represented caffeine and total catechins in the present paper,respectively.C A and C B were concen-trations(mg mLà1)of caffeine and total catechins in the solution at equilibrium.R A and R B were concentrations(mg gà1)of caffeine and total catechins in the adsorbent phase at equilibrium.The selectiv-ity coef?cient is in proportional to concentration of total catechins in the product isolated by the adsorbent.

2.4.Test of adsorption isotherm

According to the adsorption performances in the above test,TSL had a high adsorption capacity to catechins and a good selectivity, and so it was chosen as preferential material for further studies on adsorption mechanism.

The TSL(1.5g)was shaken with50mL tea extract solution at concentrations ranging from0.25g Là1to3.5g Là1in H2S-H water bath shaker at150rpm for12h at temperatures of288K,298K and308K,respectively.The supernatants were sampled and cen-trifuged as above before https://www.360docs.net/doc/4616434497.html,ngmuir isotherm model(Lang-muir,1918)was employed in this work.

2.5.Column chromatography

2.5.1.Gradient elution

One milliliter of the tea extract solution(100g Là1)was loaded on the TSL column(8mm i.d.?160mm).The column was washed with30mL of water and then eluted sequentially with20mL eth-anol solution by increasing the concentration from10%to80%(v/v) at?ow rate of0.5mL minà1.The ef?uent was collected in2mL each fraction for HPLC.

2.5.2.Loading volume study

To test the effect of loading volume on isolation and recovery rate of catechins,tea extract solution(100g Là1)was loaded at volumes of 1.0, 1.5, 2.5and 3.0mL on TSL columns(8mm i.d.?160mm),respectively,then sequentially washed with 30mL of water and20mL of10%(v/v)ethanol at?ow rate of 0.5mL minà1.The ef?uents were collected and combined.The col-umn was?nally eluted with30mL of80%(v/v)ethanol at?ow rate of0.5mL minà1.The eluates were dried under reduced pressure at 70°C in weighted glass dishes,respectively.The powder obtained was weighted.Ten mg of the powder was dissolved in10mL50% (v/v)ethanol for HPLC.

2.6.Analysis of catechins and caffeine

Concentrations of catechins and caffeine were analyzed by modi?ed method described in previous paper(Liang et al.,2007). The HPLC conditions were:Injection volume10l L,phenomenex C18column(4.6mm?150mm),column temperature28°C,mo-bile phase A=acetonitrile/acetic acid/water(6:1:193,v),mobile phase B=acetonitrile/acetic acid/water(60:1:139,v),linear gradi-ent elution:from70%(v)A and30%(v)B to15%(v)A and85%(v)B during early33min and then15%(v)A and85%(v)B till38min, Flow rate1mL minà1,Shimadzu SPD ultraviolet detector at 280nm.

2.7.Data statistics

Tests in the present paper were carried out in triplicate and the mean values of the triplicate tests were presented.Statistics was carried out on software of the SAS System for Windows version 8.1(SAS Institute Inc.,Cary,NC,USA).

J.H.Ye et al./Bioresource Technology100(2009)622–628623

3.Results and discussion

3.1.Adsorption capacities of various absorbents

Both adsorption capacity and selectivity coef?cient K B

A

at equi-

librium are important parameters to evaluate the performance of adsorbents.Table1showed that the adsorption capacity of total catechins for HPD600was156.3mg gà1,being the highest among the tested absorbents,followed by TSL.PSL and BL had least adsorption capacity to catechins.However,adsorption capacity to caffeine for HPD600was the highest(18.8mg gà1),followed by BL and PSL.TSL had the least adsorption capacity to caffeine.The

selectivity coef?cients K B

A

of total catechins over caffeine for all the lignocellulose absorbents were signi?cantly higher than HPD600.For example,it was3.35for TSL,being more than six times that of HPD600(Table2).Furthermore,the selectivity coef?-

cient K B

A

of gallated catechins(GCs)over non-gallated catechins (NGCs)was the highest for TSL.This suggests that TSL had the best adsorption selectivity among the tested adsorbents and it was a promising adsorbent for decaffeination and puri?cation of tea cat-echins.Although HPD600had the strongest adsorption capacity to catechins among the tested adsorbents,it is not suitable for isolat-ing decaffeinated tea catechins because it had strong af?nity to both caffeine and catechins.

Puri?cation by adsorption chromatography was reported to be based on differences in molecular weight,polarity,or shape of molecule of the sorbates,which led to differentiated af?nity with the adsorbent(Seader and Henley,1998).The difference in molecular structure between caffeine and catechins is that catechins are rich in phenyl and hydroxy groups.Lignocellulose is a complex substance containing p-hydroxyphenylpropanoid units connected by C–C and C–O–C links(Adler,1997),com-posed by lignin,cellulose and hemicelluloses(Sj?str?m,1981). Hemicelluloses establish close associations between lignin and cellulose.Although part of hemicelluloses still remained in the solid fraction of the pretreated materials,plenty of hemicellu-loses were hydrolyzed or removed in the process of NaOH–HCl treatment.The adsorption capacities of tea catechins by lignocel-lulosic materials mainly depend on the contents of lignin and cellulose.Lignin,a highly branched macromolecule,is composed of several types of aromatic compounds such as guaiacyl lignin, syringyl lignin and p-hydroxyphenyl lignin,and has a three-dimensional structure.Cellulose,a linear polymer substance, consists of about10,000–14,000D-glucose residues in b-D(1,4) linkage.Some of the residues are strongly bonded and crystal-lized in a bundle form,thus forming a complex composite with lignin and another polysaccharide.The relatively low solubility and the presence of signi?cant amounts of different aromatic groups and functional groups,such as carbonyl,methoxyl,phe-nolic hydroxyl and aliphatic hydroxyl,in a matrix structure of cross-linked lignin are prerequisites for sorping non-polar organ-ic compounds from aqueous solutions by mechanisms such as physical adsorption,hydrogen bonding as well as coordination and covalent linking(Chakar and Ragauskas,2004;Dizhbite et al.,1999).Cellulose,without aromatic compounds and a three-dimensional structure,might have little effect on the adsorption of catechins,which was con?rmed through the comparison be-tween lignocellulose and cellulose powder(Sakanaka,2003). Therefore lignin is the primarily functional component of ligno-cellulose on adsorption of catechins,which might aggregate cat-echins through hydrogen bonding between hydroxy groups of catechins and nucleophilic radicels on the lignin as well as the interaction of p-orbits.

Bagasse from sugarcane contains74.2–79.7%holocellulose and the hollocelluloses had2–4%Klason lignin(Hassan et al.,2000). Klason lignin contents in woody materials such as pine wood and tea stem ranged from25%to35%(Alves et al.,2006;Zaprometov et al.,1993).Low adsorption capacity of BL(Table1)may be as-cribed to its lower content of lignin.Lignin of tea stem tissues is consisted of not only guaicyl and syringyl structures but also of p-hydroxyphenyl structures(Zaprometov et al.,1993).The compo-sition of tea stalk lignin in TSL may lead to its stronger af?nity to catechins than BL and PSL.The mechanism remains to be further investigated.

3.2.Adsorption isotherm

Langmuir isotherm model is usually used to describe the distri-bution of sorbates between liquid phase and solid absorbent phase. It is expressed by(Langmuir,1918):

1

q

e

?

1

q

m

K bc

t

1

q

m

e3T

where q e was the amount(mg gà1)of sorbates(catechins or caf-feine)adsorbed onto TSL adsorbent at equilibrium.C was equilib-rium sorbate concentration(mg mLà1)in the solution.K b was the association constant and q m the adsorption capacity,which were calculated from the slope and intercept of plot of1/q versus1/C, respectively.Coef?cients of determination R2of caffeine and total catechins onto TSL were statistically signi?cant(Table3),suggest-ing that the adsorption systems showed excellent?t with the Lang-muir isotherm model.The calculated Langmuir parameters also showed that adsorption capacity(q m)and association constant (k b)decreased with increase in temperature,implying the adsorp-tion process was not favourable at raised temperature.However, both q m and K b for total catechins were higher than those for caf-feine at the three tested temperatures.For example,at temperature 288K,q m for total catechins was209.409mg gà1,being3.55times of that for caffeine.It indicates that TSL had a stronger af?nity to catechins than to caffeine and it is suitable for removing caffeine from catechins complex.

Table1

Adsorption capacity of different materials at equilibrium(mg gà1)a

Composition TSL PSL BL HPD600

GC7.4b 4.5c 6.2bc 4.1c

EGC16.4a11.0b12.2b11.5b

C 2.1b 1.5c 2.0b 2.7a

EC12.0ab8.4c9.8b14.6a

EGCg44.5c53.1b40.4d69.7a

GCg27.7a14.6bc17.9b12.8c

ECg35.1ab26.4b29.1b39.3a

Cg 1.6a 1.0b 1.1b 1.7a

Total catechins146.7b120.5c118.6c156.3a

Caffeine 3.9d 4.6c 6.1b18.8a

a Initial concentration of tea extract20g Là1;data followed by different alpha-

betic letters in a same row were signi?cantly different at q=0.05.

Table2

Selectivity coef?cients K B

A

of different adsorbents a

B/A b TSL PSL BL HPD600

TC/CA c 3.35a 2.49b 1.70c0.54d

GCs/NGCs d 4.10a 3.42a 2.11b0.79c

a Initial concentration of tea extract20g Là1;data with different alphabetic let-

ters in a same row were signi?cantly different at q<0.05.

b B/A Selectivity coef?cients K B

A

of solute B to solute A present in the same

solution.

c TC,total catechins;CA,caffeine.

d GCs,gallated catechins;NGCs,non-gallated catechins.

624J.H.Ye et al./Bioresource Technology100(2009)622–628

3.3.Thermodynamic parameters

Temperature dependent adsorption properties are correlated to thermodynamic parameters,such as standard Gibbs free energy change (D G 0),enthalpy change (D H 0)and entropy change (D S 0).The D G 0was calculated based on the following relationships (Kula et al.,2008):

D G 0?àRT ln Kc

e4T

where R was the gas constant;Kc was equilibrium constant,i.e.,the ratio of sorbate concentration on solid adsorbent phase (CBe )to sor-bate concentration in the solution phase (CAe )at equilibrium.Table 4shows that Kc values for total catechins were more than 4times those for caffeine,implying that more catechins were ab-sorbed on TSL,comparing to caffeine.The values of D G 0for total catechins and caffeine were negative,indicating the adsorption pro-cess was spontaneous one.The free energy change is related to the

adsorption properties,i.e.,from à20kJ mol à1to 0kJ mol à1for physisorption and from à80kJ mol à1to à400kJ mol à1for chemi-sorption (Atkins,1990).The D G 0values in the Table 4suggested that the adsorption processes of caffeine and catechins onto TSL were spontaneous physical processes and did not gain external en-ergy.The negative D G 0value was in proportional to the driving force.The D G 0values for total catechins were more negative than those for caffeine,suggesting that the adsorption of catechins were more spontaneous.Enthalpy change (D H 0)and entropy change (D S 0)were calculated from Kc through Van’t Hoff equation:

ln Kc ?

D S 0R àD H 0

RT

e5T

Enthalpy change (D H 0)is the amount of heat released or absorbed when the reaction occurs at constant pressure.Entropy change (D S 0)is a measure for degree of freedom of the adsorbed species or the disorder of a system.The D H 0and D S 0were obtained from the slope and intercept of the Van’t Hoff plot of ln Kc versus 1/T (Fig.1)and the obtained D H 0for total catechins was à15.114kJ mol à1in comparison with D H 0for caffeine à11.625kJ mol à1while the D S 0for total catechins was à7.941J mol à1K à1in comparison with D S 0for caffeine à8.246J mol à1K à1.A higher negative value of D H 0for total cate-chins than for caffeine indicated that the adsorption of catechins onto the TSL was more exothermic than caffeine.The negative entropy change (D S 0)values for caffeine and total catechins testi-?ed a decrease in degree of freedom of the caffeine and catechins when they were adsorbed onto the TSL.

Table 3

Langmuir parameters of caffeine and total catechins onto TSL at different temperatures a Temperature

Caffeine Total catechins

K b

q m R 2Langmuir equation

K b q m R 2Langmuir equation

288K 0.89858.9430.993*q ?52:931C

1t0:898C 2.830209.4090.997*q ?592:627C

1t2:830C 298K 0.63453.3570.990*q ?33:828C 1t0:634C 2.434186.1570.995*q ?453:106C 1t2:434C 308K

0.395

49.377

0.965*

q ?19:504C 1t0:395C

2.077

167.031

0.988*

q ?346:923C 1t2:077C

a

Initial concentrations of tea extract used were 0.25–3.5g L à1.

*Statistically signi?cant at p <0.01.

Table 4

Equilibrium constant (Kc )and Gibbs free energy change (D G

0)of caffeine and total catechins onto TSL

288K

298K 308K Caffeine Kc

47.60940.47634.735D G 0(kJ mol à1)à9.250à9.169à9.085Total catechins

Kc

212.186171.521140.836D G 0(kJ mol à1)

à12.828

à12.746

à12.669

J.H.Ye et al./Bioresource Technology 100(2009)622–628

625

3.4.Column chromatography

3.4.1.Gradient elution

When tea extract was loaded on the TSL column and washed sequentially with ethanol solutions of increasing concentrations, composition of the eluates changed with the increase in ethanol concentration(Fig.2).Caffeine and non-gallated catechins(NGCs) including EGC,EC,GC and C,were most eluted by water and10% (v/v)ethanol,but gallated catechins(GCs)including EGCg,GCg, ECg,and Cg were eluted by ethanol solutions at concentrations more than20%(v/v).EGCg showed bimodal distribution at ethanol concentrations40%(v/v)and60%(v/v)(Fig.2D and E).ECg had peak at40%(v/v)ethanol ef?uent(Fig.2D).GCg and Cg were eluted by60%(v/v)ethanol(Fig.2E).Partial EGCg was eluted by80%(v/v)ethanol(Fig.2F).The results were consistent with selectivity coef-?cients in Table2.It suggests that decaffeinated product rich in GCs can be obtained by TSL column chromatography.

EGCg is the most important component among tea catehins.It is of great signi?cance to isolate pure EGCg.Fig.2D and E showed that EGCg was relatively abundant and the other catechins were at low levels in fractions collected in the late stage of40%(v/v)eth-anol ef?uent and early stage of60%(v/v)ethanol ef?uent,i.e.,from fraction40to fraction45.It implies that EGCg enriched product could be obtained by TSL column chromatography.HPLC pro?le of fraction41(a part of81and82th mL,2mL per fraction)con-?rmed that EGCg was predominant component,accompanying low levels of ECg,Cg and GCg,suggesting TSL is a promising absor-bent for isolating puri?ed EGCg.

Table5

Effect of loading on decaffeination and recovery of tea catechins(mg)a

Loading volume b1mL 1.5mL 2.5mL 3.0mL

Composition of loaded tea extract Caffeine 4.20±0.08 6.30±0.1210.49±0.2012.59±0.24

EGCg14.78±0.1422.17±0.2136.96±0.3544.35±0.42

Total catechins39.69±0.4359.53±0.6499.22±1.07119.06±1.29

TC/C c9.45±0.169.45±0.169.45±0.169.45±0.16

Mixture of ef?uents of30mL water and20mL10%(v/v)ethanol Caffeine 3.97±0.07 5.71±0.098.67±0.0910.67±0.10 Recovery of Caffeine94.52%90.63%82.65%84.75% EGCg0.37±0.02 3.23±0.06 6.81±0.0815.15±0.11 Recovery of EGCg 2.50%14.57%18.43%34.16% Total catechins16.97±0.1729.34±0.2156.07±0.8975.42±0.96 Recovery of total catechins42.76%49.29%56.51%63.35%

TC/C c 4.28±0.03 5.14±0.04 6.47±0.047.07±0.16

Ef?uent of30mL80%(v/v)ethanol Caffeine0.19±0.010.44±0.02 1.53±0.04 1.51±0.03

Recovery of Caffeine 4.52% 6.98%14.58%12.02%

EGCg14.16±0.1618.64±0.1728.73±0.1928.11±0.21

Recovery of EGCg95.8%84.08%77.73%63.39%

Total catechins21.85±0.3528.89±0.2341.71±0.3441.25±0.38

Recovery of total catechins55.05%48.53%42.04%34.65%

TC/C c115.22±4.2265.77±2.4727.29±0.9427.33±0.79

a TSL column:8mm i.d.?160mm;data were expressed as mean±standard deviation.

b Initial concentration of tea extract was100g Là1.

c TC/CA:ratio of total catechins to caffeine.

626J.H.Ye et al./Bioresource Technology100(2009)622–628

3.4.2.Effect of loading volume on decaffeination of catechins by stepwise elution

Fig.2shows that caffeine was eluted by water and10%(v/v) ethanol and EGCg was partially eluted by80%(v/v)ethanol.Decaf-feinated tea catechins complex might be obtained by a simpli?ed stepwise elution process,i.e.,sequential elution by water,10%(v/ v)ethanol and then80%(v/v)ethanol.Table5showed that recov-ery rate of total catechins in the80%ethanol ef?uent decreased with increase in loading volume.The recovery rate of EGCg in the80%ethanol(v/v)ef?uent decreased from95.80%at loading volume of1mL tea extract containing14.78mg EGCg to63.39% at loading volume of3mL tea extract containing44.35mg EGCg. On the other hand,caffeine remained in the80%ethanol(v/v)ef?u-ent increased with the loading volume.The ratio of total catechins to caffeine(TC/C)in the80%ethanol(v/v)ef?uent decreased with increase in loading volume,suggesting the purity of the catechins obtained declined.If recovery of EGCg was set to95%,the loaded EGCg should not be exceeded1.85mg per cm3TSL adsorbent based on the results in Table5.

When the80%ethanol(v/v)ef?uents were dried at70°C under reduced pressure,it showed that the concentration of total cate-chins and EGCg decreased with increase in loading volume though total yield increased(Table6).If the loading was up to1.5mL or 7.5mg total catechins per cm3TSL,the concentration of total catechins of product obtained was909.2mg gà1,with caffeine 8.8mg gà1.The absorption selectivity of TSL column was better than that of lignocellulose column prepared from North American cedar wood(Sakanaka,2003).For example,the ratio of caffeine to EGCg in products prepared by TSL column was0.009–0.037(TC/ C=115.22–27.33)(Table5),being lower than0.313obtained by North American cedar wood lignocellulose column(Sakanaka, 2003).This study con?rmed that lignocellulosic materials prepared from various plant species have different adsorption selectivities to catechins and caffeine.

4.Conclusion

This study illustrated that adsorption capacity to catechins for tea stalk lignocellulose(TSL)was next to synthetic macroporous resin HPD600,but higher than lignocelluloses prepared from pine sawdust and bagasse.The adsorption capacity to caffeine for TSL was signi?cantly lower than resin HPD600.TSL is a promising alternative adsorbent for isolating decaffeinated catechins.

The kinetics data showed that the selectivity coef?cient of cat-echins over caffeine onto TSL was signi?cantly higher than that onto resin HPD600,supporting that TSL was a better adsorbent for preparing decaffeinated catechins than resin HPD600.The kinetics of the system‘catechins and caffeine/TSL’showed a good ?t with Langmuir isotherm model.

The TSL column chromatography of tea extract showed that tea components were roughly separated into two portions by gradient elution with increasing ethanol solution from0%to80%(v/v).The portion eluted by0–10%(v/v)ethanol contained high concentra-tion of caffeine and partial non-gallated catechins(NGCs)and the portion eluted by ethanol solutions i th concentration more than 20%(v/v)had high level of gallated catechins(GCs).Product abun-dant EGCg was collected in late fractions of40%(v/v)ethanol and early fractions of60%(v/v)ethanol ef?uent.When tea extract loaded on the TSL column was sequentially washed using water, 10%(v/v)ethanol and80%(v/v)ethanol,decaffeinated tea cate-chins complex was collected in80%(v/v)ethanol fraction.How-ever,the decaffeination ef?ciency and recovery rate of catechins decreased with the increase in the loaded tea extract volume. When the loading volume was set up to7.5mg total catechins per cm3TSL column,the concentration of total catechins in the 80%(v/v)ethanol eluates was above90%(w/w)on dry base,with EGCg more than60%(w/w)and caffeine below1%(w/w). Acknowledgements

This study was?nanced by the‘‘Key Technology R&D Pro-gram”(Project No.2006B AD27B06)of the Ministry of Science and Technology of China.The authors also thank Dr.Takeda for kind supply of the HPLC reference catechins.

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Table6

Effect of loading volume on yield and composition of80%(v/v)ethanol eluates(on dry

weight)a

Loading volume 1.0mL 1.5mL 2.5mL 3.0mL

Yield%(w/w)b15.5±0.514.7±0.713.5±0.411.5±0.4

Total catechins(mg gà1)973.2±13.3909.2±8.2884.3±6.6859.2±7.4

EGCg(mg gà1)695.0±7.9627.1±5.7588.0±3.9585.5±4.7

Caffeine(mg gà1) 6.4±0.38.8±0.425.6±0.631.5±0.6

a TSL column:8mm i.d.?160mm;The80%(v/v)ethanol eluates were dried at

70°C under reduced pressure and the data were expressed as mean±standard

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b Yield%=(dry solids weight obtained by drying the80%(v/v)ethanol eluates/

loaded tea extract)?100%.

J.H.Ye et al./Bioresource Technology100(2009)622–628627

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