Enzyme and high pressure assisted extraction of carotenoids from tomato waste.pdf

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ARTICLE IN PRESSFBP-544; No. of Pages 7food and bioproducts processing x x x ( 2 0 1 4 ) xxxxxx

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Food and Bioproducts Processing

j ourna l ho me page: www.elsev ier .com/ locate / fbp

Enzym ecarote

Irini F. SLaboratory o atioAthens, Iroo

a

En noid

u d lyc

th he n

cess. The increase of extraction yield depended on the solvent. Maximum total carotenoid (127 mg/kg d.w.) and

lycopene (89.4 mg/kg d.w.) extraction yields were obtained in enzyme treated samples extracted with ethyl lactate

(solvent:solid = 10:1 mL:g), corresponding to almost 6-fold and 10-fold increase, respectively, with respect to non

enzyme treated samples. HP assisted extraction led to higher extraction yields (from 2 to 64% increase depending on

the solvent used) compared to conventional solvent extraction process performed at ambient pressure for 30 min.

H

so

am

Ke

pr

1. Int

By-productposal problpromising sindustry gemainly of ting on theValle et al.,particularlyestimated billion in 20

Accordinripening stlycopene thsuch as heposed for t

CorrespoE-mail aReceived

http://dx.do0960-3085/ this article in press as: Strati, I.F., et al., Enzyme and high pressure assisted extraction of carotenoids from tomato waste. Food

P assisted solvent extraction was successfully performed at 700 MPa by using signicantly (P < 0.05) lower ratios of

lvent:solid (6:1 and 4:1 mL:g) and reduced processing time (10 min), compared to solvent extraction performed at

bient pressure, solvent:solid ratio 10:1 mL:g and 30 min extraction time.

2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.

ywords: Tomato waste; Carotenoids; Lycopene; Solvent extraction; Enzyme-assisted extraction; High hydrostatic

essure

roduction

s of plant food processing represent a major dis-em for the industry concerned, but they are also aource of valuable components. Tomato processingnerates signicant amounts of waste, consistingomato skins and seeds, in a proportion depend-

specic process from which it is generated (Del 2006). Tomatoes are a rich source of carotenoids,

lycopene. The global market for carotenoids wasto US$1.07 billion and is projected to top US$1.215 (Global Industry Analysts Inc., 2011).g to Sharma and Le Maguer (1996), at the end of

age, tomato skins contain up to ve times morean the pulp. Conventional food grade solvents,

xane, ethanol, and ethyl acetate, have been pro-he extraction of carotenoids from tomato waste.

nding author. Tel.: +30 210 7723166; fax: +30 210 7723163.ddresses: [email protected], [email protected] (I.F. Strati).

13 January 2014; Received in revised form 22 August 2014; Accepted 28 September 2014

However, the yield in most cases is low possibly due to thedifculty for the solvent molecules to penetrate the tomatopeel tissue and solubilize the pigment, while oxidative degra-dation of carotenoids is also possible (Lavecchia and Zuorro,2008).

In that aspect, enzyme-assisted extraction methods aregaining more attention because hydrolytic enzymes breakdown the structural integrity of cell walls rendering theintracellular materials to be more exposed for extraction.Cellulase and pectinase enzymes were employed by severalresearchers as a pretreatment step of tomato based productsprior to solvent extraction for the recovery of carotenoids, andespecially lycopene (Choudhari and Ananthanarayan, 2007;Lavecchia and Zuorro, 2008; Zuorro et al., 2011; Papaioannouand Karabelas, 2012; Ranveer et al., 2013). The enzymatic treat-ment was followed by extraction with hexane, ethyl acetate,acetone or mixtures of solvents. In all cases, there was an

i.org/10.1016/j.fbp.2014.09.012 2014 The Institution of Chemical Engineers. Published by Elsevier B.V. All rights reserved.e and high pressure assistednoids from tomato waste

trati , Eleni Gogou, Vassiliki Oreopoulouf Food Chemistry and Technology, School of Chemical Engineering, Nn Polytechniou 5, 15780 Zografou, Athens, Greece

b s t r a c t

zyme (EA) and high pressure (HP) assisted extraction of carote

sing various organic solvents was examined. Total carotenoid an

e use of pectinase and cellulase enzymes, when compared to tocess (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.012xtraction of

nal Technical University of

s, especially lycopene, from tomato waste

opene extraction yields were increased by

on enzyme treated solvent extraction pro-


ARTICLE IN PRESSFBP-544; No. of Pages 72 food and bioproducts processing x x x ( 2 0 1 4 ) xxxxxx

increase inples, whichbe attributexperimenextraction.carotenoidbe very imptration abiland Oreopo

On the tural chandamage thmetabolitewhile it hapounds suc(Knorr, 1993ies have beecarotenoidconsumed As an extrin 2004, antion time a2004). Recetive compofrom grapepounds froXi (2006) ahigher yielextraction

Several (Strati and and especiwere highlwe further extractabilition. Tomaenzymes pnon-polar dene the HP processthe initial explored incarotenoid

2. Ma

2.1. Ch

Hexane anfrom Thermethanol, p(SigmaAldp.a., was pChemie Gmanalysis (acof HPLC grGermany). lycopene infrom DSMwas used ftrophotomwas purchapany, St. Lo

lulyvillus bell

46e preonta00 Unark). ted

e buf pect

Pla

o pr culsingre coupo

0.35eniztory berstonteny groium

crit caro

En

oundachium

f thee mcubB 1

baches (pportymed Cethe ated

Afte in s25 0 mLon f, theifugemitenk (extract as

Hig

o wahe aixtuerimhe in extraction yield compared to the untreated sam- varied from 2- to 18-fold. These differences may

ed to different raw material, enzyme preparation,tal conditions and solvents used for the subsequent

The effect of solvent on the extraction yield ofs from enzymatically treated tomato waste mightortant, as different solvents show different pene-ities and solubilizing effect on carotenoids (Stratiulou, 2011).

other hand, HP processing can cause some struc-ges, such as cell deformation or cell membraneat increase cell permeability as well as secondary

diffusion and consequently mass transfer rates,s very little effect on low-molecular-weight com-h as avour compounds, vitamins and pigments; Corrales et al., 2008). In the literature, earlier stud-n published with regards to the effect of HP on total

content and carotenoid availability of commonlyvegetables (McInerney et al., 2007; Plaza et al., 2012).action method, this technology was rst studiedd it was found to effectively shorten the extrac-nd increase the process efciency (Zhang et al.,ntly, HP has been implemented to extract bioac-unds from natural sources, such as anthocyanins

by-products (Corrales et al., 2008), or phenolic com-m Maclura pomifera fruits (Altuner et al., 2012), whilepplied HP on tomato paste waste and obtainedds of lycopene compared to conventional solventperformed at ambient pressure for 30 min.solvents have been examined in a previous workOreopoulou, 2011) for the extraction of carotenoids,ally lycopene, from tomato waste and the resultsy dependent on the solvent. In the present studyinvestigated the possibility to enhance carotenoidty by enzyme pre-treatment or HP assisted extrac-to waste was treated with pectinase and cellulaserior to extraction with various solvents, from the hexane to the highly polar ethanol so as tocombined effect on extraction yield. Additionally,ing, in the pressure range of 100800 MPa with(prior to pressurization) temperature of 25 C was

order to obtain the maximum extraction yields ofs and lycopene from tomato waste.

terials and methods

emicals and enzymes

d ethyl acetate, analytical grade, were purchasedo Fisher Scientic (Fair Lawn, NJ). Acetone and

.a., were purchased from Sigma Chemical Co.rich Company, St. Louis, MO). (-)-Ethyl L-lactate,urchased from Fluka Analytical (SigmaAldrichbh, Munich, Germany). All solvents used for HPLCetonitrile, 1-butanol and methylene chloride) wereade and were obtained from Merck (Darmstadt,REDIVIVO Lycopene 10% FS (10% microcrystalline

corn oil containing a-tocopherol as antioxidant) Nutritional Products (Kaiseraugst, Switzerland)or the preparation of standard solutions for spec-etric measurements. All-trans lycopene standardsed from Sigma Chemical Co. (SigmaAldrich Com-uis, MO).

CelAsperg(Colomand pHenzymniger, cof 10,0Denmcalculaacetat5.0 for

2.2.

TomattomatoprocesMoistuwaste 80.48 homoglaboraIdar-Oture cThe dralumin

Thetion of

2.3.

Dry, gr(1.0 g ealumintion oand ththen inbath WSchwaenzymthat refor enzAFP an

At inactiv3 min.placedbath (using 1agitaticedure(Centrcare Li

Blavent eamoun

2.4.

Tomatwith tvent mHP expfrom t this article in press as: Strati, I.F., et al., Enzyme and high pressure assisocess (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.012e AN 3500, a powder enzyme preparation ofniger cellulase, was obtained from Lyven S.A.

es, France), with enzyme activity of 3500 200 U/g (concentration 1%). Pectinex Ultra AFP, a liquidparation produced from Aspergillus aculeatus and A.ining pectin lyase and polygalacturonase activitiesits/mL, was obtained from Novozymes (Bagsvaerd,Both products were stored at 4 C and prior to use,amounts of enzyme preparations were diluted withfer solution of appropriate pH (4.8 for cellulase andinase) to obtain the desired enzyme concentration.

nt material

ocessing waste, composed of skin and seeds oftivar Red Sea, was collected from a Greek tomato

plant (NOMIKOS S.A., Aliartos, Viotia, Greece).ntent was determined at fresh tomato processingn arrival at the laboratory and found to be%. Tomato waste material was air dried at 25 C,ed in a domestic blender and nally ground in amill (Cutting Mill Pulverisette 15, Fritsch Gmbh,ein, Germany) equipped with a 0.5 mm sieve. Mois-t of ground dry tomato waste was 7.65 0.21%.und material was kept in glass jars wrapped with

foil at 20 C before further processing.ical steps involved in the EA and HP assisted extrac-tenoids from tomato waste are described in Fig. 1.

zyme aided extraction of carotenoids

and homogenized tomato waste was distributed) in tightly-closed glass test tubes, covered with

foil. To each test tube, 7.0 mL of enzyme solu- appropriate enzyme concentration were addedixture was vortexed for 3 min. The samples wereated under agitation in a Memmert shaking water-4 with drive SV 1422 (Memmert GmbH + Co. KG,, Germany). The incubation temperature for bothectinase and cellulase) was chosen according to

ed by the supplier and literature review as optimal activity and was 45 C and 55 C for Pectinex Ultrallulyve AN 3500, respectively.end of the incubation period the enzymes were

by immersing the test tubes in boiling water forr ltration, the tomato waste solid residue wascrew-top conical asks in a thermostated water0.1 C) and was subjected to solvent extraction,

of each solvent. The whole system was kept underor 30 min. On completion of the extraction pro-

mixture was centrifuged at 1000 g for 10 min Thermo scientic Heraeus Megafuge 16R, DJB Lab-d, UK), to separate the supernatant.nzyme free) samples were prepared for each sol-tion process, by adding buffer solution in the same

the enzyme solution, prior to solvent extraction.

h pressure treatment

ste samples (2.5 g each) were weighed, mixedppropriate volume of different solvents or sol-res and packed into polypropylene pouches forents. The pouch was sealed after eliminating airside and placed into the HP vessel. HP samplested extraction of carotenoids from tomato waste. Food


ARTICLE IN PRESSFBP-544; No. of Pages 7food and bioproducts processing x x x ( 2 0 1 4 ) xxxxxx 3

Fig. 1 Enzscheme of

preparationorder to avtreatment. tomato waferred in tutotal carotepressure rato pressurizsion, due tSolvent to sexamined.

HP unit tional BV, R45 mL capa1000 MPa anuid was ptional BV, Rwas achievtrolled by asamples in

rate

ratur proc

the re set witild u

e by a reso ap

10 mas inixturo sci

Kinventts as. Thwas ixedequilibtempeing thewithinpressucontacsure busampl300 MP25 C twithinsion wthe mThermUnited

Consolvenresults(2011) were m this article in press as: Strati, I.F., et al., Enzyme and high pressure assisocess (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.012

yme and high pressure assisted extractioncarotenoids from tomato waste.

was performed 5 min prior to compression inoid any further carotenoids extraction before HPSolvent extract was discharged from the solidste immediately after decompression and trans-bes before further use in order to determine thenoid content. HP treatments were conducted at thenge of 100800 MPa for 130 min. Temperature prioration was 25 C and was elevated during compres-o adiabatic heating, approximately 3 C/100 MPa.olid ratios of 10:1 mL:g, 6:1 mL:g and 4:1 mL:g were

used (Food Pressure Unit FPU 1.01, Resato Interna-oden, Holland) comprised a six vessel system ofcity each, with a maximum operating pressure ofd temperature of 90 C. The pressure transmitting

olyglycol ISO viscosity class VG 15 (Resato Interna-oden, Holland). Process temperature in the vesselsed by water circulation in the outer jacket con-

heating-cooling system. Before introduction of the the pressure unit, pressure vessels were already

agitated veture was cescientic HKingdom), subsequen

2.5. Tot

The total cfrom enzymspectrophotrometer, Uaccording tit was exprof dry mate

The lycby high pquantied evaporation111, Switzelene chloridltered thrinjected forlent 1100 Seof a HP 110nary Pumpand a Rheotem was e(250 mm 4tonitrile (Athe followin1.0% C init10 min, 61.and 30% C C in 50 mincolumn tem472 nm. Thout on a Ch19992000,

2.6. Sta

EA and HParate trialsd at the process temperature (25 C). Pressure ande were monitored and recorded (1s intervals) dur-ess by temperature and pressure sensors installedavailable HP vessels equipment; temperature andnsors are positioned inside the 45 mL vessels inh the pressure transmitting uid (polyglycol). Pres-p (15 MPa/s) resulted in compression heating of theapproximately 3 C/100 MPa (i.e. HP processing ofulted in an adiabatic temperature increase fromproximately 34 C) followed by cooling to 25 Cin during pressure holding time (HP decompres-

stantaneous). On completion of the HP processing,e was centrifuged 1000 g for 10 min (Centrifugeentic Heraeus Megafuge 16R, DJB Labcare Limited,gdom), to separate the supernatant.ional extractions were performed with the same

those used in HP extractions, to compare thee procedure described by Strati and Oreopouloufollowed. Briey, 10.0 g of dry, ground raw material

with solvent at a liquid to solid ratio of 10:1 in anssel, kept at 25 C for 30 min. Subsequently the mix-ntrifuged at 1000 g for 10 min (Centrifuge Thermoeraeus Megafuge 16R, DJB Labcare Limited, Unitedto separate the supernatant, which was used fort carotenoid analyses.

al carotenoid and lycopene determination

arotenoid content of the supernatants obtainede- and HP assisted extractions was measured

tometrically (Helios Alpha & Beta UV-Visible Spec-nicam Instruments, Cambridge, United Kingdom),o the procedure of Strati and Oreopoulou (2011) andessed as mg of total extracted carotenoids per kgrial weight (mg/kg d.w.).opene content of the extracts was determinederformance liquid chromatography (HPLC), andusing a reference curve. After complete solvent

in a rotary vacuum evaporator (Rotavapor RErland), the samples were dissolved in 1 mL methy-e and transferred to a vial. The new solution was

ough a 0.45 m membrane lter and 20 L were HPLC analysis (Strati et al., 2012). The HPLC (Agi-ries, Waldbronn, Germany) system was composed0 Series Diode Array Detector, a HP 1100 Quater-, an Agilent 1100 Series Micro Vacuum Degasserdyne model 7010 Sample Injector. The HPLC sys-quipped with a YMC (Tokyo, Japan) C30 column.6 mm I.D., 5 m particle). A mobile phase of ace-), 1-butanol (B), and methylene chloride (C) withg gradient elution was used: 69.3% A, 29.7% B and

ially, increased to 67.2% A, 28.8% B and 4% C in6% A, 26.4% B and 12% C in 20 min, 49% A, 21% Bin 40 min and returned to 69.3% A, 29.7% B and 1%. The ow rate was maintained at 2 mL/min, theperature at 25 C, and detection was carried out at

e analysis of the chromatographic data was carriedemStation for LC 3D software (Agilent TechnologiesWaldbronn, Germany).

tistical analysis

assisted extractions were performed in two sep- and analyses in triplicate. The average of threeted extraction of carotenoids from tomato waste. Food



Fig. 2 Effetotal carote

measurempresented ments. StatSTATISTICAtechnique determine lycopene cdence level

3. Re

3.1. En

3.1.1. DeconditionsPreliminarymine the efenzymes. Hseries. In t240 min, wand equal trespectivelcarotenoidof incubatiogressive reddegradationincubationfrom the pare furtherronment amay lead toIn the seco(180 min) aenzymes vato 350 U/g carotenoidcentrations

3.1.2. Effextraction sThe effectextraction experimen

Effenoide as

reasitistiction he trl carse, wventsellulaby C2013es inynert obato

mase, h--dmaryl opeies a

found in the middle lamella and primary cell wallsct of enzyme pretreatment time on the extractednoid yield from tomato waste.

ents was calculated for each experiment and theresults are the mean values of duplicate experi-istical analysis was carried out using the software

(Stat soft. Inc., 1999). The analysis of varianceand Duncans multiple range tests were used tothe signicant difference in total carotenoid andontent between different treatments at 95% con-

(P < 0.05).

sults and discussion

zyme assisted extraction of carotenoids

termination of improved enzyme incubation

series of experiments were conducted to deter-fective incubation time and concentration for bothexane was used as the extraction solvent in each

he rst series, incubation time varied from 30 tohile the enzyme concentration was kept constanto 70 U/g and 122.5 U/g for pectinase and cellulase,y (Fig. 2). As indicated from the results the highest

Fig. 3 carotehexan

in incno staextracever, tof totacellulaas solthan cwaste et al. (enzymsible swas no

Tomtainingcellulothe 1,4the prifor celactivitpectin this article in press as: Strati, I.F., et al., Enzyme and high pressure assisocess (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.012

yield for both enzymes was observed after 180 minn. Increasing the incubation time resulted in a pro-uction in yields. This suggests that the enzymatic

of cell-wall components occurs within 180 min of. Lycopene and other carotenoids that are releasedrotective chromoplast structures during this time

exposed to the conditions of the external envi-nd can undergo rapid oxidative degradation that

reduction in extraction yield (Ranveer et al., 2013).nd series, the incubation time was kept constantnd the concentrations of pectinase and cellulaseried from 35 to 140 U/g of tomato waste, and 61.25of tomato waste, respectively (Fig. 3). Maximum

yield was obtained at pectinase and cellulase con- of 70 U/g and 122.5 U/g, respectively.

ect of enzyme treatment combined with differentolvents

of enzyme treatment combined with differentsolvents was studied in a subsequent series ofts. Pectinase and cellulase enzymes were effective

(Choudhariis predomideeply emdegradationvent molecand dissolv

Overall,(with acetosolvent) henzyme-trealso increafold (with eenzyme-treethanol weous experibe further dhexane or also report(2011) for treated tomfor lycopenct of enzyme concentration on the total extraction yield from tomato waste usingextraction solvent.

ng total carotenoid and lycopene yield, whileal (P < 0.05) difference was generally observed inyield by using either enzyme (Fig. 4a and b). How-eatment with pectinase increased the extractionotenoids and especially of lycopene, compared tohen hexane, acetone and ethyl lactate were used. Pectinase was also reported as more effectivese for the enzymatic treatment of tomato peel and

houdhari and Ananthanarayan (2007) and Ranveer). A preparation consisting of a mixture of the two

equal volumes was also assessed; however, a pos-gistic effect of the combined use of two enzymesserved (data not shown).peel tissue is a highly structured material con-ny different polysaccharide components, such asemicelluloses, and pectins. Cellulase hydrolyzes

-glycosidic linkages in cellulose, which is present in cell wall; therefore cellulase is mainly responsiblening. Pectinase has pectolytic and hemicellulolyticnd therefore breaks down pectic substances andted extraction of carotenoids from tomato waste. Food

and Ananthanarayan, 2007; Gross, 1984). Lycopenenantly found in the tomato peel chromoplasts,bedded within the membrane structures. By the

of structural components of tomato tissue, sol-ules penetrate more easily the ruptured peel tissuee lycopene (Cuccolini et al., 2013).

total carotenoid extraction yields were 1.5-foldne as solvent) to 6-fold (with ethyl lactate as

igher when compared to those from the nonated tomato waste sample (Fig. 4a). Lycopene yieldsed by 2.5-fold (with ethyl acetate as solvent) to 10-thyl lactate as solvent) when compared to the nonated samples (Fig. 4b). The yields obtained withre very low in all cases, in agreement with previ-ments (Strati and Oreopoulou, 2011) and will notiscussed. The highest increase was observed withethyl lactate as solvents. A similar increase wased by Lavecchia and Zuorro (2008) and Zuorro et al.lycopene extraction with hexane, from enzyme-ato peel or waste. Hexane is an excellent solvente but, as non polar cannot penetrate easily in the



Fig. 4 (a) from tomatcombined cellulase ensignicant(a < b < c < d

wet materihydrolysis,therefore ilactate on tof tomato cOreopoulouvent that cand conseqthe untreatthe rupturean easier calmost qua

The lowacetone. Acof the plan2005), thereoffered a sment was fapproximamaterial, wPapaioannoZuorro (200obtained w

Resomatt: heFig. 5 from t(solven this article in press as: Strati, I.F., et al., Enzyme and high pressure assisocess (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.012

Total carotenoid and (b) lycopene yield extractedo waste samples using different solventswith enzyme pretreatment using pectinase andzymes. Values with different superscripts differ

ly (P < 0.05) < e < f < g < h < i < j < k < l < m).

al. The rupture of the plant cells by enzymatic renders the lycopene much more accessible, and,ncreases considerably the extraction yield. Ethylhe other hand, which is also an excellent solventarotenoids (Ishida and Chapman, 2009; Strati and, 2011), is a water and hydrocarbon miscible sol-an penetrate much more easily the wet material,uently extract higher amounts of lycopene fromed samples, compared to hexane. Also in this case,

of the plant cells and the release of lycopene allowontact of the pigment with the solvent and anntitative extraction.est increase of extraction yield was observed withetone is a strong, basic solvent that causes swellingt tissue (Mantanis et al., 1995; Obataya and Gril,fore further hydrolysis obtained by the enzymes

mall increase of recovery. When enzymatic treat-ollowed by ethyl acetate extraction, the yield wastely 2.4-fold higher compared to the non-treatedhich is in agreement with the results reported byu and Karabelas (2012), although Lavecchia and8) reported a higher increase. Finally, the increaseith the mixture of hexane and ethyl acetate was

with Sigma

in betweenexpected.

These rebe obtainedmatic treat

3.2. Hig

3.2.1. DettreatmentIn order toextraction oconducted higher extrthan 400 Ma signicansolvent extThe highesat process in processiextraction HP processprocessing

3.2.2. EffTotal carotprocessing,(reported aTable 1. In total caroteextraction wstatisticallya few casesand hexaneat shorter pprocessing ambient prThe reducetion indicatponse surface graph for total carotenoid yieldo waste at different HP processing conditionsxane, solvent to solid ratio = 10:1). Graph createdplot 10.0, Systat Software, Inc.

the values observed by the individual solvents, as

sults suggest that sufcient carotenoid yields can from the wet industrial tomato waste by enzy-

ment followed by extraction with ethyl lactate.

h pressure treatment of tomato waste

ermination of improved conditions of HP

select the most effective HP conditions for thef total carotenoids, preliminary experiments were

as described in Section 2.4. The results showed thataction yields were observed at pressures higherted extraction of carotenoids from tomato waste. Food

Pa; increasing pressure up to 700800 MPa led tot increase in extraction yield when compared toraction performed at ambient pressure (0.1 MPa).t total carotenoid extraction yield was observedtime treatments of 10 min, while further increaseng time did not result to further increase of theyield. According to the results depicted in Fig. 5,ing at 700 MPa and 10 min were selected as theparameters enabling the highest extraction yield.

ect of HP treatment on extraction yieldenoid and lycopene extraction yields under HP

as compared to extraction at ambient pressures conventional solvent extraction) are presented ingeneral, HP assisted solvent extraction increasednoid and lycopene extraction yield, compared toith the same solvents at ambient pressure, while,

signicant (P < 0.05) differences were observed in of solvents; ethanol, hexane, hexaneethyl acetateethyl lactate. However, these yields were achievedrocessing time (10 min), compared to the requiredtime of conventional extraction techniques atessure, which usually ranges from 30 to 60 min.d processing times of HP assisted solvent extrac-e that under HP processes the mass transfer rates



Table 1 tional and high pressure (HP) assisted extraction.

Conventional HP

Total carot(mg/kg dw)

3.63 0.15a 9.30 0.16b19.98 0.50a 17.88 1.48a18.15 0.49a 22.34 0.52b22.03 0.32a 23.34 0.86a21.06 0.90a 23.24 1.24a23.68 0.44a 24.78 0.72a26.98 0.21a 44.19 0.99b57.20 0.40a 64.50 0.60b

147.69 2.33a 165.27 2.73b

Lycopene c(mg/kg dw)

2.47 0.08a 7.04 0.11b13.87 0.09a 14.57 0.05a15.14 0.30a 17.84 0.59b15.94 0.10a 16.01 0.08a14.53 0.31a 15.04 0.16a16.62 0.05a 17.70 0.16a17.14 0.64a 25.01 0.48b46.27 0.04a 50.40 0.73b82.48 1.33a 83.59 4.63a

Values with 5).

of bioactiveare increasmass transrates durindiffusivity mainly attrmembraneability thusinto the tomtion, denatby pressuretomato andunder HP t(2006) repopuree afterthey attributhereafter extracting scomponenability, theincreases, anents are imtimes withsequently t

Also, Hbridges anbranes lescompound(Barbosa-Creported thand producare located(2006) repoHP assistedconcentrattemperaturby 30 min u38.5 kHz, potion of actiaid of scanelectron mhigh pressu

embrane and organelles) and consequently improvedass transfer of the solvents into the leaves and the

contion o notne (Xition

olumer toaroterfor

highe, helven

e to :g. Action solvTotal carotenoids and lycopene content obtained by conven

Solvents

enoids

Ethanol Hexaneethanol (50:50) Hexane Hexaneacetone (50:50) Acetone Ethyl acetate Hexaneethyl acetate (50:50) Hexaneethyl lactate (50:50) Ethyl lactate

ontent

Ethanol Hexaneethanol (50:50)HexaneHexaneacetone (50:50) Acetone Ethyl acetate Hexaneethyl acetate (50:50) Hexaneethyl lactate (50:50)Ethyl lactate

different superscripts within the same row differ signicantly (P < 0.0

compounds from the tomato waste to the solvented, conrming the theory that HP can favour thefer phenomena leading to increased mass transferg the extraction processes due to changes in thecoefcient. Diffusivity coefcient changes can beibuted to pressure induced changes in the plant cell

leading to an increased cell membranes perme- facilitating permeation of the extraction solventato waste cells (Tangwonchai et al., 2000). In addi-

uration of the carotenoid-binding protein induced could be also involved in the cell wall damage of, therefore, facilitate the extraction of carotenoidsreatment (Snchez-Moreno et al., 2009). Qiu et al.rted an increase in extractable lycopene in tomato

processing at 500 MPa, at 20 C for12 min, whichted to the fact that HP can rupture the tissue and

favour lycopene release. High pressure impels theolvent to come into cells to integrate with bioactive

ts, the pressurized cells show increased perme- solubility of extracts is improved as the pressure

wall, mthe msolubleextracthat dlycope

Addvent vin ordtotal cwas penableacetatcic sovolum4:1 mLextraclower this article in press as: Strati, I.F., et al., Enzyme and high pressure assisocess (2014), http://dx.doi.org/10.1016/j.fbp.2014.09.012

nd thus the leaching-out rates of bioactive compo-proved (Xi, 2013). This leads to shorter processing

HP extraction compared to conventional and con-o a more time-effective processing technique.P deprotonates charged groups and disrupt saltd hydrophobic bonds making the cellular mem-s and less selective, thereby rendering thes more accessible to extraction up to equilibriumanovas et al., 1998). Furthermore, Smelt (1998) hasat HP can affect membranes in vegetable cellse disruption of chromoplasts where carotenoids, inducing a better release of these compounds. Xirted that the yield of lycopene by using 500 MPa of

extraction of tomato paste waste with 75% ethanolion in water (1:5 g/mL solid/liquid ratio, at roome) for 1 min was higher (17%) than that obtainedltrasound-assisted extraction (using frequency ofwer of 810 W, at 25 C). Similarly, during HP extrac-ve ingredients from green tea leaves and with thening electron microscopy (SEM) and transmissionicroscopy (TEM), Xi et al. (2011) indicated that ultra-re resulted in the disruption of the leaf tissue (cell

Fig. 6 HPdifferent so10:1 mL:g) superscripstituents into the solvents. Moreover, HP assistedis performed at ambient temperature (2530 C)

favour degradation of carotenoids and especiallyianquan et al., 2005).ally, in the present work, the reduction of sol-e used during HP assisted extraction was studied

investigate the ability of extracting lycopene andenoids using lower solvent volumes. The studymed for selected solvents that were found toer extraction yields, namely: hexane, hexaneethylxaneethyl lactate and ethyl lactate. The spe-ts were used in three different ratios of solventdry tomato waste mass; 10:1 mL:g, 6:1 mL:g, andcording to the results depicted in Fig. 6, HP assistedof lycopene and carotenoids can be performed atent to solid ratio, without signicantly (P < 0.05)ted extraction of carotenoids from tomato waste. Food

assisted extraction of carotenoids performed atlvent to solid ratio values (4:1 mL:g, 6:1 mL:g andfor selected solvents. Values with differentts differ signicantly (P < 0.05) (a < b < c < d < e).



reducing thcan facilitaextraction slow solventtransfer phconvention

4. Co

The exploitbioactives The resultslycopene rincreased blulolytic acsolvent extvent extracsolvent exresults withpared to thtreatment cextraction The advantwet industrmaterial.

Additionextraction of carotentomato prohigher extrtion procesextraction reduced pryields.

Acknowle

The authorfor kindly (Bagsvaerdthe enzymeA. Batrinouand Ch. Tse

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ation of enzymes in industry for extracting plantfor their application in food is a promising eld.

of this study indicate that total carotenoid andecovery from tomato processing waste can bey the use of enzymes with pectinolytic and cel-tivities. The increase of extraction yield by EAraction compared to the non enzyme treated sol-tion process depended on the solvent used. EAtraction using ethyl lactate presented the best

a 10-fold increase in lycopene recovery when com-e non enzyme treated tomato waste. Thus, enzymean be proposed as a pretreatment step to solvent

process in order to improve the extraction yields.age of EA extraction is that it can be performed onial tomato waste, omitting the cost for drying the

ally, the current study showed that HP assistedcan be used to obtain high extraction yieldsoids and lycopene, from waste derived by thecessing industry. HP assisted extraction led toaction yields when compared to solvent extrac-s. Moreover, it was demonstrated that HP assistedcan be performed using lower solvent volume atocessing times without affecting the extraction

dgements

s thank NOMIKOS S.A. (Aliartos, Viotia, Greece)providing the tomato waste and Novozymes

, Denmark) and Lyven S.A. (Colombelles, France) for preparations. The authors are also grateful to Dr.

for her substantial suggestions and to S. Katsoukou for their assistance in the experimental work.

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Enzyme and high pressure assisted extraction of carotenoids from tomato waste1 Introduction2 Materials and methods2.1 Chemicals and enzymes2.2 Plant material2.3 Enzyme aided extraction of carotenoids2.4 High pressure treatment2.5 Total carotenoid and lycopene determination2.6 Statistical analysis

3 Results and discussion3.1 Enzyme assisted extraction of carotenoids3.1.1 Determination of improved enzyme incubation conditions3.1.2 Effect of enzyme treatment combined with different extraction solvents

3.2 High pressure treatment of tomato waste3.2.1 Determination of improved conditions of HP treatment3.2.2 Effect of HP treatment on extraction yield

4 ConclusionsAcknowledgementsReferences

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Enzyme and high pressure assisted extraction of carotenoids from tomato waste.pdf