Molecules 2009, 14, 2573-2581; doi:10.3390/molecules14072573 Article OPEN ACCESS molecules ISSN 1420-3049 www.mdpi.com/journl/molecules Extrction of Oil from Whet Germ by Supercriticl CO 2 Alessndr Pirs 1, Antonell Ros 2, Dnilo Flconieri 1, Silvi Porcedd 1, Mri. A. Dessì 2 nd Bruno Mrongiu 1, * 1 2 Diprtimento di Scienze Chimiche, Università degli Studi di Cgliri, Cittdell Universitri di Monserrto, SS 554, km 4.500 09042 Cgliri, Itly; E-mils: pirs@unic.it (A.P.); dnilo.flconieri@tiscli.it (D.F.); porcedd@unic.it (S.P.) Diprtimento di Biologi Sperimentle, Sezione di Ptologi Sperimentle, Università degli Studi di Cgliri, Cittdell Universitri di Monserrto, SS 554, km 4.500 09042 Cgliri, Itly; E-mils: nros@unic.it (A.R.); dessim@unic.it (M-A.D.) * Author to whom correspondence should be ddressed; E-mil: mronb@unic.it; Tel.: +39 070 6754412; Fx: + 39 070 6754388 Received: 26 My 2009; in revised form: 30 June 2009 / Accepted: 13 July 2009 / Published: 15 July 2009 Abstrct: This study exmined the supercriticl fluid extrction of whet germ oil. The effects of pressure (200-300 br t 40 C) nd extrction time on the oil qulity/quntity were studied. A comprison ws lso mde between the reltive qulities of mteril obtined by SFE nd by orgnic solvent extrction. The extrcts were nlyzed for - tocopherol nd polyunsturted ftty cid content. The mximum whet germ oil yield t bout 9% ws obtined with supercriticl crbon dioxide extrction t 300 br, while ftty cid nd -tocopherol composition of the extrcts ws not remrkble ffected by either pressure or the extrction method. Key words: whet germ oil; vitmin E; ftty cid; supercriticl extrction; crbon dioxide 1. Introduction Fts nd oils ply n importnt role in the food industry nd re essentil prt of humn nutrition. Vegetble oils provide ft-soluble vitmins such s vitmins A, D, E, nd K, nd re lso source of
Molecules 2009, 14 2574 essentil unsturted ftty cids, which cnnot be synthesized by the humn body. In order to meet nutritionl requirements, new vegetble oil resources re being sought s sources of these vitmins nd essentil ftty cids. Whet germ oil hs the highest tocopherol content of ll vegetble oils, up to bout 2,500 mg/kg, nd lso the highest content of -tocopherol, which represents round 60% of the totl content [1]. Whet germ oil is lso highly vlued for its high content of unsturted ftty cids: it hs bout 80%, consisting mostly of linoleic (18:2) nd linolenic (18:3) cids [2], both of which re of gret importnce in humn metbolism nd cnnot be synthesized by the orgnism. They re precursors of group of hormones clled prostglndins, which ply n importnt role in muscle contrctions nd in the proper heling of inflmmtory processes [3]. Furthermore, linoleic cid [4] helps to eliminte cholesterol nd is precursor of cell membrne phospholipids [4]. Whet germ is by-product of the whet milling industry. Germ constitutes bout 2-3% of the whet grin nd cn be seprted in firly pure form from the grin during the milling process [5]. Whet germ contins bout 11% oil [6]. Whet germ oil is used in products such s foods, biologicl insect control gents, phrmceuticls nd cosmetic formultions [7]. Polyunsturted ftty cids nd bioctive compounds re prone to oxidtion nd degrdtion under the conditions used for conventionl edible oil extrction nd refining methods [8]. Solvent extrction is common method of extrction of oils from vegetble mtter. In recent yers supercriticl fluid extrction (SFE) hs received incresed ttention s n importnt lterntive to conventionl methods. Supercriticl fluids hve djustble extrction chrcteristics due to their density, which cn be controlled by chnges in pressure or temperture. In ddition, other properties such s low viscosity, high diffusivity nd low surfce tension enhnce the solute mss trnsfer from inside solid mtrix. Supercriticl crbon dioxide (SC-CO 2 ), being nontoxic, nonflmmble, inexpensive nd esily seprble from the extrcts, hs been the most frequently used extrctnt in the food nd phrmceuticl industries [9-11]. Furthermore, the low criticl temperture of crbon dioxide llows extrction of thermolbile compounds without degrdtion. Use of supercriticl fluid such s crbon dioxide for extrction of whet germ oil hs previously been reported by severl reserch groups [12-18]. Amoung these, Pnfili et l. [12] studied the effect of pressure, temperture, extrction time nd whet germ oil prticle size on oil extrction yields. They found tht ftty cids composition not chnge while the gretest concentrtion of -tocopherol ws found in the frction smple t 75 min (P= 38 MP, T= 55 C, prticle size= 0.35 mm). According to Tniguchi et l. [13] the - nd -tocopherol content of supercriticl crbon dioxide extrcted oil were similr to those of hexne extrcted oil. However, Molero Gomez nd Mrtinez de l Oss [14] reported higher tocopherol content in the SC-CO 2 extrcted mteril, s compred to tht of the hexne extrcted oil. Sho et l. [15] found tht the ftty cid composition of germ oil extrcted by SFE ws similr to the oil extrcted by hexne. Eisenmenger nd Dunford [16] lso showed tht methods used for oil extrction nd refining did not hve remrkble effect on the ftty cid composition of the oil, wheres SC-CO 2 extrcted oil hd higher tocopherol content thn tht of commercilly hexne extrcted oil. Ge et l. [17] reported tht the yield of -tocopherol extrcted by SFE-CO 2 were much higher thn those prepred by chloroform/methnol extrction. However, the solution of chloroform/methnol showed stronger solvent power to extrct -tocopherol form whet
Molecules 2009, 14 2575 germ. Finlly, Zcchi et l. [18] found tht petroleum ether extrcted oil showed the highest vlues for ftty cids nd tocopherol contents. The im of this work is to study the effects of pressure nd extrction time on the extrction efficiency nd oil qulity. 2. Results nd Discussion Figure 1 shows the influence of operting pressure on the extrction yield. An extrction time of 8 h t 300 br gives whet germ oil yield bove 9% (w/w), oil recovery bout 80%. The SC-CO 2 gives n extrction yield not significntly different from orgnic solvent extrction. A comprison is mde between the reltive qulities of the oils produced by SC-CO 2 nd by orgnic solvent extrction (hexne, methnol, chloroform-methnol 2:1 mixure) in Figure 2. The difference is interpreted tht the orgnic solvents re much less selective thn CO 2 in the extrcted oil nd produce oil contining some undesirble compounds. Figure 1. Effect of pressure on the extrction yield of whet germ oil:, 200 br nd 40 C;, 250 br nd 40 C;, 300 br nd 40 C. 10 8 Yield, % 6 4 2 0 0 100 200 300 400 500 Extrction time, min Figure 2. Effect of extrction method on the yield of whet germ oil. Chloroform-Methnol Methnol Extrction method Hexne SFE 300 SFE 250 SFE 200 0 2 4 6 8 10 12 Yield, %
Molecules 2009, 14 2576 Figure 3 shows the concentrtion of -tocopherol (expressed s g/mg oil) mesured in whet germ oil smples obtined by SC-CO 2 (200, 250, nd 300 br, temperture of 40 C, extrction time of 8 h) nd orgnic solvent extrction (hexne, methnol, nd chloroform-methnol). There ws no considerble vrition in -tocopherol content mong oil smples obtined by different extrction procedures, except for oil from MeOH extrction, tht exhibited the lowest -tocopherol mount. Figure 3. -Tocopherol mount (expressed s g/mg oil) mesured in whet germ oil smples obtined by SC-CO 2 (200, 250, nd 300 br) nd orgnic solvent extrction (hexne, methnol, nd chloroform-methnol). -Tocopherol ( g/mg oil) 1,2 1,0 0,8 0,6 0,4 0,2 0,0 Methnol Hexne Chlor/Meth SFE 200 SFE 250 SFE 300 Extrction method Quli-quntittive informtion on the individul ftty cids tht compose the lipid clsses of whet germ oils were obtined by GC nd HPLC nlyses. Tble 1 shows the ftty cids composition, expressed s percentge of totl ftty cids, of oil smples obtined by using orgnic solvents nd SC- CO 2. In ll smples the min ftty cids were plmitic (16:0), oleic (18:1 n-9), linoleic (18:2 n-6), linolenic (18:3 n-3), nd eicosenoic (20:1 n-9). Tble 1 lso shows results for sturted (SFA), monounsturted (MUFA), nd polyunsturted (PUFA) ftty cids. PUFA were the min group of ftty cids in ll nlysed smples, rnging from 57-60%, nd vlues of bout 18% nd 21-23% were observed for SFA nd MUFA, respectively. A one-wy ANOVA showed significnt differences (p<0.05) mong different extrction procedures in prticulr for oleic, linoleic, linolenic, rchic (20:0), nd eicosenoic ftty cids. It lso showed significnt differences mong different extrction procedures for monounsturted nd polyunsturted ftty cids. Methnol extrct showed the lowest vlue for MUFA nd the highest vlue for the PUFA frction. The mounts, expressed s g/mg oil, of the min unsturted ftty cids (18:1, 18:2, nd 18:3 n-3) in ll whet germ oils nd re reported in Figure 4. Significnt differences (p<0.05) were observed mong different extrction procedures. Whet germ oil from MeOH extrction showed the lowest mounts of unsturted ftty cids (101.7, 230.3, nd 36.4 g/mg for 18:1 n-9, 18:2 n-6, nd 18:3 n-3, respectively, s men vlues over six smples), wheres oil obtined t 250 br exihibited the highest levels, showing vlues of 188.4, 409.1, nd 59.5 g/mg for 18:1 n-9, 18:2 n-6, nd 18:3 n-3, respectively.
Molecules 2009, 14 2577 Tble 1. Ftty cids composition (expressed s % of totl ftty cids), mesured by gs chromtogrphy-flme ioniztion detection (GC-FID) method, of whet germ oil obtined by different extrction procedures. Ftty cid Chloroform/Methnol Methnol Hexne SFE 200 SFE 250 SFE 300 14:0 0.11 ± 0.00 0.14 ± 0.00 0.12 ± 0.00 0.15 ± 0.05 c 0.16 ± 0.02 b,c 0.12 ± 0.01 16:0 16.98 ± 0.06 17.12 ± 0.73 17.53 ± 0.23 17.49 ± 0.83 17.15 ± 0.09 16.49 ± 0.60 b,d 16:1 n-7 0.25 ± 0.01 0.37 ± 0.14 0.30 ± 0.00 0.26 ± 0.04 0.29 ± 0.05 0.25 ± 0.01 18:0 0.90 ± 0.06 0.85 ± 0.04 0.91 ± 0.01 0.92 ± 0.10 0.98 ± 0.07 0.90 ± 0.03 18:1 n-7 0.79 ± 0.08 0.91 ± 0.01 0.87 ± 0.08 0.88 ± 0.16 0.87 ± 0.05 0.79 ± 0.05 18:1 n-9 20.06 ± 0.18 18.87 ± 0.16 20.45 ± 0.03 20.15 ± 0.69 19.97 ± 0.09 20.41 ± 0.22 18:2 n-6 51.90 ± 0.03 52.57 ± 0.66 50.82 ± 0.01 51.00 ± 1.03 51.55 ± 0.18 52.13 ± 0.51 b,d 18:3 n-3 7.02 ± 0.05,b 7.62 ± 0.11 6.53 ± 0.01 6.40 ± 0.16,c 6.42 ± 0.09,c 6.48 ± 0.07,c 20:0 0.19 ± 0.04,b 0.11 ± 0.00 0.30 ± 0.00 0.13 ± 0.03 b 0.36 ± 0.04,c,d 0.29 ± 0.06,c,d,e 20:1 n-9 1.29 ± 0.01 1.02 ± 0.02 1.39 ± 0.01 1.19 ± 0.16,b 1.39 ± 0.01,d 1.43 ± 0.01,c,d SFA 18.20±0.08 18.25±0.77 18.87±0.21 18.00±0.70 18.67±0.04 17.82±0.70 b MUFA 22.39±0.09 21.17±0.02 23.00±0.06 22.53±0.91 22.53±0.00 22.88±0.22 PUFA 58.92±0.18 60.20±0.77 57.35±0.02 57.27±0.70,c 57.97±0.27,c 58.61±0.57,b,d SFA, sturted ftty cids; MUFA, monounsturted ftty cids; PUFA, polyunsturted ftty cids. Men nd stndrd devition of 6 smples (n = 6). p< 0.05, = versus methnol, b hexne, c chloroform-methnol, d 200 br, e 250 br. Figure 4. Vlues (expressed s g/mg oil) of oleic (18:1), linoleic (18:2), nd linolenic (18:3 n-3) cids mesured by high-performnce liquid chromtogrphy (HPLC-DAD) method in whet germ oil smples obtined by SC-CO 2 (200, 250, nd 300 br) nd orgnic solvent extrction (hexne, methnol, nd chloroform-methnol). p< 0.05, = versus methnol, b hexne, c chloroform-methnol, d 200 br, e 250 br; (n = 6). 500 18:1 18:2 18:3 n-3 cd Ftty cid g/mg oil) 400 300 200 100 b d bc e d d 0 Methnol Hexne Chlor/Meth SFE 200 SFE 250 SFE 300 Extrction method
Molecules 2009, 14 2578 The oxidtion sttus of ftty cids ws lso mesured by HPLC detection of conjugted dienes ftty cids hydroperoxides (HP). The level of the oxidtive products HP in the oils extrcted by SC-CO 2 rnged from 12 to 14 nmol/mg oil, vlues comprble to those mesured in oils obtined by solvent extrction (10-15 nmol/mg oil), nd prevlently derived from 18:2 n-6 degrdtion, due to the gret content of this ftty cid nd the high stbility of its hydroperoxides [19]. Our results confirm previous reports indicting no significnt qulity differences in SC-CO 2 extrcted whet germ oil compred to solvent extrcted oil. In conclusion, supercriticl crbon dioxide extrction hs immedite dvntges over trditionl extrction techniques: it is flexible process due to the possibility of continuous modultion of the solvent power/selectivity of the SC-CO 2, llows the elimintion of polluting orgnic solvents nd of the expensive post-processing of the extrcts for solvent elimintion. 3. Experimentl 3.1. Mterils All solvents used, of the highest vilble purity, were purchsed from Merck (Drmstdt, Germny). Triolein, trilinolein, ftty cids nd ftty cids methyl esters stndrds, nd desferl (deferoxmine mesylte slt) were purchsed from Sigm Aldrich (Miln, Itly). The regent 20% BF 3 in MeOH ws purchsed from Supelco (Bellefonte, PA, USA). cis,trns-13-hydroperoxyoctdecdienoic cid (c,t-13-hpode) nd cis,trns-9-hydroperoxyoctdecdienoic cid (c,t-9- HPODE) were purchsed from Cscde (Cscde Biochem. Ltd., London, U.K.). All other chemicls used in this study were of nlyticl grde. Flky food-grde whet germ ws supplied from Sos Ortos (Ozieri, Itly). Before using the vegetble mtter ws ground with Mlvsi mill (Bologn, Itly) nd the prticle sizes were in the 180-250 m rnge. 3.2. Solvent Extrction Totl lipids were extrcted from liquots (100 mg) of whet germ by ddition of CHCl 3 /MeOH (12 ml, 2/1, v/v) solution [20], or MeOH (8 ml) t room temperture, in order to void ftty cids oxidtion. After filtrtion, liquots of the CHCl 3 or MeOH phse (250 L) were collected nd totl lipids were quntified by the method of Ching, Gessert nd Lowry [21]. Solvent extrction ws lso performed with hexne in Soxhlet pprtus for 16 h, s stndrd nd reference method for the extrction of fts nd oils from food mtrices. 3.3. Apprtus for SFE Supercriticl CO 2 extrctions were performed in lbortory pprtus [22,23], equipped with 400 cm 3 extrction vessel nd two seprtor vessels of 300 nd 200 cm 3 respectively connected in series. Extrction ws crried out in semi btch mode: btch chrging of vegetble mtter nd continuous flow solvent. The extrction of the whet germ oil ws obtined working t 40 C t 200, 250 nd 300 br in the extrction vessel nd by using only one seprtor (t 20 br nd 15 C ) to recover the extrct.
Molecules 2009, 14 2579 3.4. Preprtion of -tocopherol nd ftty cids Seprtion of ftty cids nd -tocopherol ws chieved by mild sponifiction [24] s follows: dried liquots (3 mg) of the CHCl 3, MeOH, nd hexne extrcts nd the fixed oil (3 mg) were dissolved in EtOH (5 ml) nd then Desferl solution (100 L, 25 mg/ml of H 2 O), queous scorbic cid solution (1 ml, 25% w/v), nd 10 N KOH (0.5 ml) were dded. The mixtures were left in the drk t room temperture for 14 h. After ddition of n-hexne (10 ml) nd H 2 O (7 ml), smples were centrifuged in 4237R refrigerted centrifuge (ALC, Miln, Itly) for 1 h t 900g. The hexne phse with vitmin E ws collected, the solvent ws evported in rotry evportor (Rotvpor R-114, Büchi Lbortechnik, Flwil, Switzerlnd) under reduced pressure, the residue ws dissolved in MeOH (500 L) nd liquots of the smples were injected into the HPLC system. After ddition of further n- hexne (10 ml) to the mixtures, smples were cidified with 37% HCl to ph 3-4 nd then centrifuged for 1 h t 900g. The hexne phse with free ftty cids nd hydroperoxides ws collected nd the solvent ws evported. A portion of the dried residue ws dissolved in CH 3 CN (500 L) with 0.14% CH 3 COOH (v/v) nd liquots of the smples were injected into the HPLC system. Aliquots of dried ftty cids ws methylted with 14% BF 3 in MeOH (1 ml) [25] for 30 min t room temperture. After ddition of n-hexne (4 ml) nd H 2 O (2 ml) smples were centrifuged for 20 min t 900g. The hexne phse with ftty cids methyl esters ws collected, the solvent ws evported, the residue ws dissolved in n-hexne (100 L) nd liquots of the smples were injected into the GC system. The recovery of ftty cids during sponifiction ws clculted by using n externl stndrd mixture. All solvents evportion ws performed under vcuum. 3.5. HPLC nlysis Anlysis of unsturted ftty cids ws crried out with n Agilent Technologies 1100 liquid chromtogrph. Anlyses of unsturted ftty cids nd conjugted diene ftty cid hydroperoxides (HP)[24], detected t 200 nd 234 nm respectively, were crried out with Chrompck column, Inertsil 5 ODS-2 (150 mm 4.6 mm, 5 m prticle size) with mobile phse of CH 3 CN/H 2 O/CH 3 COOH (70/30/0.12, v/v/v) t flow rte of 1.5 ml/min. The identifiction of the peks ws mde using stndrd compounds nd second derivtive s well s conventionl UV spectr, generted using the Agilent Chemsttion A.10.02 softwre. The mount of -tocopherol ws mesured by electrochemicl detection [24], using Thermo Seprtion Products (Miln, Itly) P1000 pump equipped with n electrochemicl detector INTRO (Antec Leyden, Leiden, The Netherlnds). An utomtic injector, Trithlon (Sprk Hollnd BV, AJ Emmen, The Netherlnds) ws used. A C-18 Hewlett Pckrd ODS Hypersil column, 5 m prticle size, 100 2.1 mm, ws used with mobile phse of MeOH/CH 3 COON 0.05M ph 5.5 (95/5, v/v) t flow rte of 0.3 ml/min. The electrochemicl detector ws set t n oxidizing potentil of 0.6 V. Dt were collected nd nlysed using the Agilent Chemsttion A.10.02. softwre. Quntifiction of vitmin E ws performed using stndrd reference curve.
Molecules 2009, 14 2580 3.6. GC nlysis Ftty cid methyl esters were mesured on Hewlett-Pckrd HP-6890 gs chromtogrph (Hewlett-Pckrd, Plo Alto, CA, USA) equipped with flme ionistion detector nd cynopropyl methyl-polysiloxne HP-23 FAME column (30 m x 0.32 mm x 0.25 m) (Hewlett-Pckrd) [26]. Nitrogen ws used s crrier gs t flow rte of 2 ml/min. The oven temperture ws progrmmed from 45 C to 175 C t rte of 80 C/min nd held for 45 min; injector temperture ws set t 250 C, nd detector temperture t 300 C. The ftty cid methyl esters were identified by compring the retention times with those of stndrd compounds. The percentge composition of individul ftty cids were clculted using clibrtion curve with components injected t different concentrtions, using the Hewlett-Pckrd A.05.02 softwre. 3.7. Sttisticl nlyses Grph Pd INSTAT softwre (GrphPd softwre, Sn Diego, CA, USA) ws used to clculte the mens nd stndrd devitions of three independent experiments involving duplicte nlyses for ech smple/condition (n=6). Mens of results were compred by one-wy nlysis of vrince (One-wy ANOVA) using the Tukey-Krmer Multiple Comprisons Test. Probbility vlues of p < 0.05 were considered s significnt. References 1. Shuler, P. Nturl ntioxidnts exploited commercilly. In: Food Antioxidnts; Hudson, B.J.F, Ed.; Elsevier Applied Science: London, UK, 1990; Chpter 4. 2. Wng, T.; Johnson, L. Refining High-free Ftty Acid Whet Germ Oil. JAOCS 2001, 78, 71-76. 3. Coultte, T. Food, the chemistry of its components; The Royl Society of Chemistry: London, UK, 1989; Chpter 4. 4. Slins, R. Alimentos Grsos y Nutrición, Bromtologí Aplicd l Slud; El Ateneo: Buenos Aires, Argentin, 1993; Chpter 7. 5. Krings, U.; Berger, R.G. Antioxidnt ctivity of some rosted foods. Food Chem. 2001, 72, 223-229. 6. Dunford, N.T.; Zhng, M.Q. Pressurized solvent extrction of whet germ oil. Food Res. Int. 2003, 36, 905-909. 7. Khlon, T.S. Nutritionl implictions nd uses of whet nd ot kernel oil. Cerel Food Worls. 1989, 34, 872-875. 8. Krings, U.; El-Shrty, Y.S.; El-Zeny, B.A.; Pbel, B.; Berger, R.G. Antioxidnt ctivity of extrcts from rosted whet germ. Food Chem. 2000, 71, 91-95. 9. Reverchon, E.; Mrrone, C. Modeling nd simultion of the supercriticl CO 2 extrction of vegetble oils. J. Supercrit. Fluid. 2001, 19, 161-175. 10. Lng, Q.; Wi, C.M. Supercriticl fluid extrction in herbl nd nturl product studies- prticl review. Tlnt 2001, 53, 771-782. 11. Hutl, W.H. Advnces with supercriticl fluids-review. Chemosphere 2001, 43, 123-135.
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