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Industrial Crops and Products
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ydrolysis of vegetable oils catalyzed by lipase extract powderrom dormant castor bean seeds
atheus H.M. Avelara, Débora M.J. Cassimiroa, Kádima C. Santosa, Rui C.C. Dominguesa,eizir F. de Castrob, Adriano A. Mendesa,∗
Laboratory of Biocatalysis, Federal University of São João del-Rei, PO Box 56, 35701-970 Sete Lagoas, MG, BrazilDepartment of Chemical Engineering, Engineering School of Lorena, University of São Paulo, PO Box 116, 12602-810 Lorena, SP, Brazil
r t i c l e i n f o
rticle history:eceived 23 July 2012eceived in revised form 4 October 2012ccepted 9 October 2012
eywords:ydrolysisegetable oils
a b s t r a c t
The aim of this work was to verify the ability of lipase extract powder from dormant castor bean seeds(Ricinus communis L.) to the hydrolysis of different vegetable oils for yielding concentrated fatty acids.The enzymatic extract showed higher activity on oils rich on linoleic (C18:2) and linolenic acids (C18:3)such as soybean (86.1 ± 2.3 IU g−1) and canola (81.3 ± 2.4 IU g−1) than on oil rich on oleic acid–oliveoil (61.1 ± 4.5 IU g−1). The enzymatic hydrolysis performed in stirred-tank reactor displayed hydroly-sis degree higher than 80%, with a maximum value attained for canola oil (88.2 ± 6.80%) after 3 h ofreaction. An experimental design was performed to better understand the influence of the independent
lant lipasexperimental design
variables (mass ratio canola oil:buffer, temperature and CaCl2 concentration) and their interactions inthe hydrolysis degree. The main effects were fitted by multiple regression analysis to a quadratic modeland full hydrolysis of canola oil was achieved at 37.5 ◦C without CaCl2 addition and mass ratio oil:buffer(22.1 wt.%). At higher mass ratio oil:buffer (up to 30 wt.%) full hydrolysis could also be attained after2 h at 37.5 ◦C. These results suggest that the use of low-cost lipase from dormant castor bean seeds has
ion of
potential for the product
. Introduction
The production of free fatty acids by the hydrolysis of trigly-erides from several sources is an important component in theconomic exploitation of these naturally produced renewable rawaterials. A significant number of high-value products require fatty
cids in their manufactures, including coatings, adhesives, biofuels,urfactants, specially lubricating oils, shampoos and other personalare products. The best known industrial process to produce freeatty acids in a multi-ton scale is the Colgate-Emery process, whichypically requires operating temperatures of 250 ◦C and a reactionressure of 50 bar. Under these conditions, undesirable reactionsay occur such as oxidation, dehydration, and interesterification
f the triglycerides (Rooney and Weatherley, 2001; Murty et al.,002).
To overcome such limitations, the hydrolysis of triglyceridesatalyzed by lipases is an advantageous approach because it can
e performed under mild conditions (typically at 35 ◦C and atmo-pheric pressure), and exhibits high selectivity, leading to productsith high purity (Shiomori et al., 1995; Villeneuve, 2003; Freitas
et al., 2007; Li and Wu, 2009; Sharma et al., 2009; Mendeset al., 2012). Lipases (triacylglycerol acylhydrolases, EC 3.1.1.3)are carboxylesterases that catalyze the hydrolysis of triglycerides.They are tailored to operate at the interfaces of two-phase sys-tems, a phenomenon known as interfacial activation, in whichthe substrate characteristic is an aggregate of ester molecules,micelles or monomolecular film, interfacing an aqueous medium(Sarda and Desnuelle, 1958; Verger, 1997; Schmid and Verger,1998). In the absence of interfaces, lipases have some elementsof secondary structures (so-called “lid”) covering their active sitesand making them inaccessible to substrates (closed conforma-tion). However, in the presence of hydrophobic interfaces (e.g.oil droplets or gas bubbles), important conformational changestake place yielding the “open structure” of lipases. The lipases alsopossess the ability to catalyze several other types of biotransforma-tions (e.g. esterification, interesterification and transesterification)in environment with low water content. They can catalyze stereo-,enantio- and regioselective reactions, being one of the most widelyused enzymes in industrial processes (Fernández-Lafuente, 2010;Rodrigues and Fernández-Lafuente, 2010a,b; Barros et al., 2010;Sharma et al., 2011; Mendes et al., 2012).
The lipases are found in animals, plants, fungi and bacteria(Fernández-Lafuente, 2010; Rodrigues and Fernández-Lafuente,2010a,b; Barros et al., 2010; Sharma et al., 2011; Mendes et al.,2012). Plant lipases appear to be very attractive owing to their low
Average molecular mass (g mol−1) 278.6 280.9 279.6
ost, their high substrate specificity and the fact that they are widelyvailable from natural sources without requirement for molecularenetic technology to produce them, making it a good alterna-ive for commercial exploitation as industrial enzymes (Villeneuve,003; Pierozan et al., 2009; Barros et al., 2010). These enzymes haveeen isolated from leaves, stems, latex, oils, and seeds of oleaginouslants and cereals (Sanders and Pattee, 1975; Villeneuve, 2003;astmond, 2004; Cavalcanti et al., 2007; Pierozan et al., 2009; Barrost al., 2010; de Sousa et al., 2010; Su et al., 2010). Lipases from latexnd seeds of oleaginous plants are mainly used in biotransforma-ion reactions of oils and fats (Villeneuve, 2003; Barros et al., 2010).t has been reported that lipase activity is absent in ungerminatedor dormant) seeds and increases rapidly when germination startsVilleneuve, 2003; Barros et al., 2010). However, in some cases, theipolytic activity has been found in dormant seeds such as peanutSanders and Pattee, 1975), castor bean (Ory et al., 1962; Cavalcantit al., 2007), physic nut (de Sousa et al., 2010), and African oil bean –entaclethra macrophylla Benth (Enujiugha et al., 2004). Among theipases from dormant seed oils, castor bean lipase has been widelysed in hydrolysis of different glycerides to elucidate its catalyticroperties (Ory et al., 1962; Muto and Beevers, 1974; Cavalcantit al., 2007; Barros et al., 2010; Su et al., 2010). Muto and Beevers1974) reported the presence of two lipases in extracts from castorean endosperm. The first with optimal activity at acid region (pH.5–5.0) which is presented in dormant seeds and displayed highctivity at the first 2 days of germination. The second displays highatalytic activity at alkaline pH (pH 9.0) and is particularly activeuring days 3–5 of germination. This lipase is less active than thecid lipase on the hydrolysis of different mono-, di- and triglyceri-es. The production of free fatty acids from vegetable oils catalyzedy microbial and animal lipases is well documented; however, these of plant lipase from castor bean seeds is still scare in the liter-ture.
The aim of this work consisted in the preparation of lipasextract powder from castor bean seeds to catalyze the hydrolysisf vegetable oils such as canola, soybean and olive in a stirred-tankeactor. The substrate that gave the highest hydrolysis degree waselected to perform an experimental design to evaluate the condi-ions that maximize the production of free fatty acids such as massatio oil: buffer, calcium chloride concentration and temperaturef reaction.
. Materials and methods
.1. Materials
Castor bean seeds were acquired from BRSeeds Ltd. (Arac atuba,P, Brazil). Refined olive oil (Carbonell), soybean oil (Liza), and
anola oil (Liza) were purchased at a local market. The compositionn fatty acids was determined according to the Official Methods andecommended Practices of American Oil Chemists’ Society (2004)Table 1). Gum Arabic and calcium chloride were purchased from
nd Products 44 (2013) 452– 458 453
Synth (São Paulo, SP, Brazil). All other chemical reagents were ofanalytical grade.
2.2. Preparation of lipase extract powder from castor bean seeds
Initially, the endosperm tissues were carefully removed andthe shells of the seedling discarded. The endosperms (20 g) werethen triturated in a knife-mill during 10 min by adding acetone(5 mL). The samples were mixed with chilled acetone (ratio 1:5,w/v) under stirring at 150 rpm and 4 ◦C, according to the method-ology described by Pierozan et al. (2009), with slight modifications.Then, the suspension was filtered under vacuum via a Buchner fun-nel and washed with acetone in excess. The delipidated extractfrom seeds was sieved to obtain particles size less than 1 mm. Theproduct was defined as lipase extract powder and used to hydrolyzevegetable oils.
2.3. Determination of hydrolytic activity on different vegetableoils
The hydrolytic activity of the lipase powder extract from dor-mant castor bean seeds was determined on the hydrolysis ofemulsified vegetable oils, according to the methodology describedby Soares et al. (1999), with slight modifications. The substrate wasprepared by mixing 50 g vegetable oils (canola, olive and soybean)with 150 g gum Arabic solution (30 g L−1). The reaction mixturecontaining 5 mL emulsion, 5 mL 100 mmol L−1 phosphate buffer(pH 7.0), and 0.1 g of lipase powder extract was incubated for 5 minat 37 ◦C. The reaction was stopped by addition of 10 mL commer-cial ethanol. The fatty acids formed were titrated with 20 mmol L−1
sodium hydroxide solution in the presence of phenolphthalein asindicator. One international unit (IU) of activity was defined as theamount of enzyme that liberates 1 �mol free fatty acid per minute(1 IU) under the assay conditions.
2.4. Enzymatic hydrolysis in stirred-tank reactor
Hydrolysis reactions of canola, olive and soybean oils were car-ried out in 350 mL plastic flasks containing 10 g of vegetable oils,90 mL buffer acetate 100 mmol L−1 (pH 4.5) and 2 g of lipase extractpowder. The mixtures were then agitated with a mechanical stir-rer at a constant speed of 1000 rpm. Reactions were performed at25 ◦C under atmospheric pressure for a maximum period of 3 h.Samples (1 g) were periodically removed from the reactor via asyringe, weighed and transferred to 125 mL conical flask at inter-vals of 30 min. Ten milliliters of commercial ethanol were addedto the sample to denature the enzyme, thus effectively freezingthe reaction. Phenolphthalein indicator was added and the mixturetitrated against standard 20 mmol L−1 sodium hydroxide solution.The hydrolysis degree was defined as the percentage weight offree fatty acids in the sample divided by the maximum theoreti-cal amount and calculated using Eq. (1) (Rooney and Weatherley,2001).
Hydrolysis (%) = VNaOH × 10−3 × MNaOH × MMWt × f
× 100 (1)
where V is the volume of sodium hydroxide solution (NaOH)required during titration; M is the NaOH concentration(20 mmol L−1); MM is the average molecular mass of fattyacids for each vegetable oil (see Table 1); Wt is the weight of thesample taken and f is the fraction of oil at start of reaction.
2.5. Experimental design
A 23 full experimental design with three replicates at the cen-ter point was used to evaluate the reaction parameters in the
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Table 2Experimental design for the hydrolysis of canola oil in a stirred-tank reactor at pH 4.5 (buffer acetate – 100 mmol L−1) by 2 h using 2 wt.% lipase extract powder from castorbean seeds.
Run Coded (actual) Responsehydrolysis (%)
Independent variables
Mass ratio oil:buffer (wt.%) Temperature (◦C) CaCl2 (mmol L−1)
vigorous mechanical stirring (1000 rpm). At industrial scale, thehydrolysis of triglycerides performed without the addition of emul-sifier agents would be more attractive economically since less
nzymatic hydrolysis. Star points were added to the experimentalesign to compose second order models. Seventeen experimentsere performed in a random order. The levels of each independent
ariable were chosen based on the importance of the experiments.he parameters were mass ratio canola oil:buffer acetate (x1), tem-erature (x2) and calcium chloride concentration (x3). The codednd corresponding uncoded values are given in Table 2. The hydrol-sis degree was taken as response of the design experiment. Resultsere analyzed using Statistica version 7.0 (StatSoft Inc., USA) soft-are. The statistical significance of the regression coefficients wasetermined by Student’s test, the second order model equation wasvaluated by Fischer’s test and the proportion of variance explainedy the model obtained was given by the multiple coefficient ofetermination, R2.
.6. Optimization of the enzymatic hydrolysis of vegetable oils
After selecting the experimental conditions that maximize theydrolysis of canola oil, the influence of mass ratio oil:buffer vary-
ng from 22.1 wt.% to 50 wt.% on the hydrolysis degree was furthererified. The reactions were performed in a stirred-tank reactorontaining 50 g of medium (oil + buffer) in absence of CaCl2 at7.5 ◦C for 2 h under vigorous stirring (1000 rpm).
. Results and discussion
The catalytic activity of the lipase extract powder was deter-ined on the hydrolysis of three vegetable oils emulsified with
um Arabic solution. Lipase from castor bean extract preferred toydrolyze oils having long-chain polyunsaturated fatty acids suchs soybean (86.1 ± 2.3 IU g−1) and canola (81.3 ± 2.4 IU g−1) oils. Asan be seen in Table 1, these oils have high concentrations of linoleicC18:2) and linolenic acids (C18:3). However, a slight decrease on itsydrolytic activity was detected using olive oil (61.1 ± 4.5 IU g−1),
triglyceride with high concentration of oleic acid (C18:1).Hydrolysis of the three different vegetable oils (canola, olive
nd soybean) was performed in a stirred-tank reactor for 3 h at5 ◦C without adding emulsifier agents at pH 4.5 buffer acetate
00 mmol L−1. The lipase exhibits optimum activity at pH 4.5 and
s inactivated at pH values above 6.0 at 30 ◦C (Eastmond, 2004). Theinetics profiles of the hydrolysis of the vegetable oils are displayedn Fig. 1. Under these conditions, 50% of hydrolysis was observed
5.5 (0) 99.45.5 (0) 1005.5 (0) 98.3
for all vegetable oils after 1 h of reaction. Hydrolysis degree for oliveand soybean oils was around 83% after 2 h of reaction. Among thetested vegetable oils, maximum hydrolysis degree was verified forcanola oil at 3 h (88.2 ± 6.8%). Hence, this oil was selected for theoptimization of hydrolysis reactions by experimental design.
A 23 full factorial design was performed to attain the opti-mal conditions of mass ratio oil:buffer (x1), temperature (x2) andcalcium chloride concentration (x3) for the hydrolysis of canolaoil. The assays were conducted without adding emulsifier agents.Hydrolysis reactions mediated by lipases from several sourceshave been carried out in the presence of these compounds toincrease the lipid–water interfacial area, which, in turn, enhancesthe observed rates of lipase-catalyzed reactions (Tiss et al., 2002).In this work, the emulsification of the systems was attained by
M.H.M. Avelar et al. / Industrial Crops and Products 44 (2013) 452– 458 455
Table 3Estimated effects, standard errors and p-values for the hydrolysis degree of canolaoil obtained in the experimental design.
Variable Effect Standard error p-Value
Mean 99.589 ±1.146 0.0000x1 −3.511 ±1.442 0.0409x2
1 −1.152 ±1.766 0.5326x2 22.322 ±1.448 0.0000x2
2 −16.881 ±1.802 0.0000x3 0.520 ±1.508 0.7393x2
3 −2.214 ±2.106 0.3238x1·x2 5.600 ±1.767 0.0132
oi
1iftcqof
tctE
H
wa
wfie
SttswpipTtidy(
Fig. 2. Response surface plot of hydrolysis degree predicted from the quadraticmodel. The effect of the mass ratio oil:buffer and temperature and their reciprocalinteraction on the hydrolysis degree of canola oil.
peration steps would be need. The experimental design is shownn Table 2, together with the experimental results.
Results indicated that the hydrolysis degree varied from 68.4% to00% and total hydrolysis degree was observed for different exper-
mental conditions (assays 3, 4, 7, 8, 10, 12, 13 and 16). Resultsrom Table 2 were used to estimate the main variable effects andheir interactions (Table 3). The statistical analysis showed signifi-ant linear effect for mass ratio oil:buffer (x1) and temperature (x2),uadratic effect for temperature (x2
2) and the interaction mass ratioil:buffer and temperature (x1·x2). The most significant effect wasound for the variable temperature at 95% of confidence level.
From these results, statistical model could be composed withhe coefficients correspondent to the significant effects. Theoefficients related to non-significant effects were excluded fromhe model. The best fitting response function can be described byq. (2).
here x1 and x2 represent the coded values for mass ratio oil:buffernd temperature, respectively.
The statistical analysis of the model (Table 4) indicated that itas significant at 95% confidence level, without significant lack oft (p > 0.05). Moreover, the R2 value indicated that the model canxplain more than 97.8% of the experimental variability.
The response surfaces and the contour plots were obtained usingoftware Statistica version 7.0 as displayed in Figs. 2–4. Fig. 2 showshe interaction between the variables mass ratio oil:buffer andemperature and their effects on the response variable (hydroly-is degree). This indicates that the hydrolysis degree was improvedith the increase of the temperature and a slight reduction on thisarameter was found for temperature higher than 50 ◦C due to the
nactivation of the enzyme at high temperature. Optimum tem-erature of hydrolysis was found to be in the range 37.5–50 ◦C.he increase of the concentration of canola oil at low tempera-ure also reduced the hydrolysis degree of canola oil due to the
ncrease of the emulsion viscosity by the agglomeration of oilroplets (McClements and Weiss, 2005). Hence, maximum hydrol-sis degree can be attained at the highest mass ratio oil:buffer22.1 wt.%) and temperature varying from 37.5 to 50 ◦C.
able 4nalysis of variance (ANOVA) for the model that represents the hydrolysis degree of cano
Source Sum of squares Degree of freedom
x1 37.08 1
x2 1484.869 1
x22 548.45 1
x1·x2 62.72 1
Lack of fit 15.19 5
Pure error 50.006 8
Cor. total 2220.623 17
Fig. 3. Response surface plot of hydrolysis degree predicted from the quadraticmodel. The effect of the mass ratio oil:buffer and calcium chloride and their recip-rocal interaction on the hydrolysis degree of canola oil.
The effect of the mass ratio oil:buffer and calcium chloride con-centration, and their interaction on the hydrolysis degree of canolaoil is shown in Fig. 3. The addition of calcium ions in hydrolysis reac-tions of triglycerides has been performed due to the formation of
la oil.
Mean square F-value p (prob > F)
37.08 5.93 0.04081484.869 237.55 0.0000
548.45 87.74 0.000062.72 10.03 0.0132
3.03 2.58 2.63166.25
456 M.H.M. Avelar et al. / Industrial Crops and Products 44 (2013) 452– 458
F del. To
iatAf
rchatfhhv
ihvotpvolmGbi
et al., 2011). Moreover, high oil concentration may increase thefatty acids concentration at interface oil/water leading to changesof the ionization state of the enzyme which affects its activity andselectivity. Under optimized conditions, full hydrolysis of canola
22.1 25 27 .5 30 35 40 45 500
20
40
60
80
100
Hyd
roly
sis
(%)
ig. 4. Response surface plot of hydrolysis degree predicted from the quadratic mon the hydrolysis degree of canola oil.
nsoluble Ca-salts of the fatty acids released during hydrolysis, thusvoiding product inhibition (Li et al., 2011). However, its effect onhe hydrolysis degree was not significant at 95% confidence level.s can be observed, the mass ratio oil:buffer also was not significant
or the hydrolysis of canola oil, as well as, its interaction.Fig. 4 displays the effect of the temperature and calcium chlo-
ide concentration on the hydrolysis degree. The response surfacelearly shows that calcium ions were not significant to increaseydrolysis degree of canola oil. However, in agreement with Fig. 2
strong influence of the temperature was verified. Increasing theemperature from 25 to 37.5–50 ◦C increased the hydrolysis degreerom 68% to 100%. Here, it possible to observe that for temperatureigher than 50 ◦C the lipase undergoes inactivation, thus reducingydrolysis degree of canola oil. The interaction between these twoariables was found to be no significant at 95% confidence level.
The results also showed that the production of fatty acids can bencreased for higher mass ratio oil:buffer. Hence, the influence ofigher mass ratio oil:buffer varying from 22.1 wt.% to 50 wt.% waserified on the hydrolysis degree of canola oil maintaining fixed thether variables. The results showed that up to 30 wt.% oil:buffer aotal hydrolysis of canola oil was detected (Fig. 5). For higher oilroportion, reduction on the hydrolysis degree of canola oil waserified. This effect could be attributed to the aggregation of theil droplets at high oil concentration that eventually resulted inarger droplets size, hence reducing the contact between enzyme
olecules and oil droplets (McClements and Weiss, 2005; Sun andunasekaran, 2009). High oil concentration also provides the inhi-ition of the enzyme by an excess of superficial substrate molecules
n comparison to adsorbed enzyme molecules (Saktaweewong
he effect of the calcium chloride and temperature and their reciprocal interaction
Mass ratio oil:buffer (% wt. )
Fig. 5. Influence of the mass ratio oil:buffer on the hydrolysis degree of canola oil.The reactions were performed at pH 4.5 (buffer acetate – 100 mmol L−1) by 2 h undervigorous mechanical stirring (1000 rpm) in a stirred-tank reactor.
rops a
or
tHrafcartpsuiiomil1pfitoo(tlAblayu
4
eppchfUmdehtmtdhtii
A
0s
M.H.M. Avelar et al. / Industrial C
il may be achieved without adding CaCl2 at 37.5 ◦C, 30 wt.% massatio oil:buffer during 2 h under vigorous stirring (1000 rpm).
The hydrolysis of triglycerides has been performed preferen-ially by commercial lipase preparations from several sources.owever, the lipase extract from castor bean seeds presented
esults more attractive in the hydrolysis of triglycerides than lipasesvailable commercially from microbial and animal tissues. Lipaserom Candida rugosa was immobilized by covalent attachment onhitosan beads prepared by binary activation with glutaraldehydend 1-ethyl-3-(3-dimethylaminopropyl) carbodiimide hydrochlo-ide (Ting et al., 2008). The biocatalyst prepared was then used inhe production of biodiesel by an enzymatic/acid-catalyzed hybridrocess (so-called hydroesterification) using soybean oil as feed-tock. Maximum hydrolysis degree was found to be 88% after 5 hnder optimal conditions. Pinheiro et al. (2008) tested free and
mmobilized lipase from C. rugosa by entrapment in sol–gel matrixn the hydrolysis of three different vegetable oils such as canola,live and soybean by experimental design. Both oils presentedaximum hydrolysis degree around 7% after 4 h of reaction using
mmobilized lipase. However, for the reactions performed by freeipase, the highest hydrolysis degree was found for olive oil (up to2%). Padilha and Augusto-Ruiz (2010) proposed a protocol for theroduction of polyunsaturated fatty acids (PUFA) by hydrolysis ofsh oil catalyzed by free porcine pancreatic lipase, followed by crys-allization between −18 and 5 ◦C. The fish oil hydrolysis was carriedut at 38 ◦C in buffer ammonium pH 8.0 for 60 min, yielding 46 ± 2%f hydrolysis. The production of essential fatty acids concentratelinoleic and linolenic acids) from soybean oil was carried out usinghree different lipase preparations from C. rugosa, Thermomycesanuginosus and porcine pancreatic lipase (Freitas et al., 2007).lthough the highest hydrolysis degree was attained with micro-ial lipases, up to 70% after 24 h of reaction, porcine pancreatic
ipase showed to have the most satisfactory specificity for linoleicnd linolenic acids. Thus, this lipase was selected for further hydrol-sis reactions. Maximum hydrolysis degree (35%) was obtainedsing 3.0 wt.% of lipase and 0.08 wt.% of NaCl in buffer phosphate.
. Conclusion
Enzymatic hydrolysis of vegetable oils was performed by lipasextract from castor bean seeds in a stirred-tank reactor. This lipaseresented high hydrolytic activity on vegetable oils having highercentage of polyunsaturated fatty acids such as soybean andanola oils. The maximum hydrolysis degree was obtained in theydrolysis of canola oil (88.2 ± 6.8%). Therefore, this oil was selected
or the optimization of hydrolysis reactions by experimental design.nder optimized conditions (without adding CaCl2, at 37.5 ◦C andass ratio oil:buffer 30 wt.%), total hydrolysis of canola oil was
etected after 2 h of reaction. This study demonstrated that thexperimental design is appropriate for the maximization of theydrolysis of canola oil. This methodology also makes it possibleo determine a desirable working region where a better perfor-
ance for the hydrolysis reaction can be achieved. According tohese results, the application of a low-cost lipase extract fromormant castor bean seeds showed satisfactory results on theydrolysis of vegetable oils. This strategy is economically attrac-ive for the production of free fatty acids from triglycerides, anmportant class of intermediates compounds for the oleochemicalndustry.
cknowledgements
The authors are grateful to FAPEMIG (Process number APQ-1527-10), CNPq, CAPES and FINEP (Brazil) for their financialupport.
nd Products 44 (2013) 452– 458 457
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