Enzyme-assisted oil extraction of lutein from marigold (Tagetes erecta) flowers and stability of lutein during storage

In e na ional Jou nal o Ag icul u al and Food Resea ch ISSN 1929-0969 | Vol. 4 No. 2, pp. 11- 19 * Co esponding au ho : mika.kaimainen@u u. i Enzyme- Assis ed Oil Ex ac ion o Lu ein om Ma igold ( e ec a ) Flowe s and S abili y o Lu ein Mika Kaimainen 1 1 Food Chemis y and Food D e elopmen , Depa men o Biochemis y, Uni e si y o Tu ku, 2 Bio echnology and Food Resea ch, MTT Ag i ood Resea ch Finland, 31600 Jokioinen, Finland Abs ac Ma igold (Tage es e ec a ) lowe s a e a ich sou ce o o ganic sol en s. In his s udy, lu ein was ex ac ed om ma igold lowe s wi h enzyme ex ac ion wi hou using o ganic sol en s. Ex ac ion p oduced oil ha con ained 0.36 mg/ml lu ein (p esen as lu ein es e s bu calcula ed as ee lu ein), and he yield was compa able o sol en ex ac ion. The oil con aining lu ein was used o p oduce oil mix u es as s abilize s and some emulsions we e also s con aining lu ein we e s o ed in he da k a 20 moni o ed du ing s o age. S abili y o lu ein was good in oil (85 emulsions (77–91% ) bu sligh ly wo se in sp ay ex ac ion o lu ein om ma igold lowe s is a po en ial al e na i e o sol en ex ac ion wi h compa able e iciency. In addi ion, he e a e no sol en esidues in showed good s abili y o lu ein in oil o in emulsions s o ed in he da k a oom empe a u e, and hese p epa a ions could be used in di e en ood p oduc s. Keywo ds: lu ein, oil ex ac ion, emulsion, s In oduc ion Ca o enoids a e ed, o ange, o yellow colo ed e penoid compounds p esen in he chlo oplas s o plan s and hey ha e many unc ions, including ligh ha es ing and pho op o ec ion (Ba ley Scolnik 1995, F ank and Cogdell 1996). Ca o en oids ha e i amin A ac i i y, and hey ha e been shown o ha e an ioxida i e p ope ies ela ed o many heal h bene i s as e iewed by Kiokias Go don (2004) and Jomo a and Lu ein is an oxygen-c on aining ca o enoid, and like o he ca o enoids, i has an ioxida i e p ope ies, bu i is also associa ed wi h eye heal h (Bha acha yya e al. 2010, Abdel- Aal e al. 2013). In e na ional Jou nal o Ag icul u al and Food Resea ch 19 (2015) www.science a ge .com mika.kaimainen@u u. i Assis ed Oil Ex ac ion o Lu ein om Ma igold ( ) Flowe s and S abili y o Lu ein du ing S o age 1 * , Eila Jä enpää 2 and Raine Huopalah i e elopmen , Depa men o Biochemis y, Uni e si y o Tu ku, 20014 Tu ku, Finland Bio echnology and Food Resea ch, MTT Ag i ood Resea ch Finland, 31600 Jokioinen, Finland ) lowe s a e a ich sou ce o lu ein pigmen , which is usually ex ac ed wi h o ganic sol en s. In his s udy, lu ein was ex ac ed om ma igold lowe s wi h enzyme ex ac ion wi hou using o ganic sol en s. Ex ac ion p oduced oil ha con ained 0.36 mg/ml lu ein as lu ein es e s bu calcula ed as ee lu ein), and he yield was compa able o sol en ex ac ion. The oil con aining lu ein was used o p oduce oil -in- wa e emulsions wi h di e en polysaccha ide mix u es as s abilize s and some emulsions we e also s p ay d ied. We emulsions, d y emulsions, and oil con aining lu ein we e s o ed in he da k a 20 –22°C o 10 weeks, and he amoun o lu ein es e s was moni o ed du ing s o age. S abili y o lu ein was good in oil (85 % o he ini ial amoun ) and in we ) bu sligh ly wo se in sp ay -d ied emulsions (67–75% ). Enzyme ex ac ion o lu ein om ma igold lowe s is a po en ial al e na i e o sol en ex ac ion wi h compa able e iciency. In addi ion, he e a e no sol en esidues in he lu ein p epa a ion. P elimina y s o age es s showed good s abili y o lu ein in oil o in emulsions s o ed in he da k a oom empe a u e, and hese p epa a ions could be used in di e en ood p oduc s. lu ein, oil ex ac ion, emulsion, s abili y, polysaccha ides, s o age es Ca o enoids a e ed, o ange, o yellow colo ed e penoid compounds p esen in he chlo oplas s o plan s and hey ha e many unc ions, including ligh ha es ing and pho op o ec ion (Ba ley and Cogdell 1996). Ca o en - oids ha e i amin A ac i i y, and hey ha e been shown o ha e an ioxida i e p ope ies ela ed o many heal h bene i s as e iewed by Kiokias and Valko (2013). on aining ca o enoid, and like o he ca o enoids, i has an ioxida i e p ope - ies, bu i is also associa ed wi h eye heal h Aal e al. 2013). Ma igold lowe s (Tage es sp. amoun s o ca o enoids, which a e a y acid es e s and dies e s o lu ein, which makes hese lowe s a good sou ce o lu ein pigmen (Bha acha yya e al. 2010, Hadden e al. 1999, Lapsho a e al. 2013). Lu ein is usually ex ac ed om ma igold lowe s ei he as a y acid es e s and dies e s, o , i he ex ac ion is combined wi h saponi ica ion, i is ex ac ed as ee lu ein. In ei he case, di e en kinds o o ganic sol en s a e used o ex ac ion, and hese ha e o be emo ed comple ely by e apo a ion i he ex ac e lu ein es e s a e o be used in ood applica ions. www.science a ge .com Assis ed Oil Ex ac ion o Lu ein om Ma igold ( Tage es S o age and Raine Huopalah i 1 e elopmen , Depa men o Biochemis y, Uni e si y o Tu ku, Bio echnology and Food Resea ch, MTT Ag i ood Resea ch Finland, 31600 Jokioinen, Finland lu ein pigmen , which is usually ex ac ed wi h o ganic sol en s. In his s udy, lu ein was ex ac ed om ma igold lowe s wi h enzyme -assis ed oil ex ac ion wi hou using o ganic sol en s. Ex ac ion p oduced oil ha con ained 0.36 mg/ml lu ein as lu ein es e s bu calcula ed as ee lu ein), and he yield was compa able o sol en ex ac ion. wa e emulsions wi h di e en polysaccha ide p ay d ied. We emulsions, d y emulsions, and oil o 10 weeks, and he amoun o lu ein es e s was o he ini ial amoun ) and in we ). Enzyme -assis ed oil ex ac ion o lu ein om ma igold lowe s is a po en ial al e na i e o sol en ex ac ion wi h compa able he lu ein p epa a ion. P elimina y s o age es s showed good s abili y o lu ein in oil o in emulsions s o ed in he da k a oom empe a u e, and hese sp. ) con ain high amoun s o ca o enoids, which a e p edominan ly a y acid es e s and dies e s o lu ein, which makes hese lowe s a good sou ce o lu ein pigmen (Bha acha yya e al. 2010, Hadden e al. 1999, Lapsho a e al. 2013). Lu ein is usually ex ac ed om ma igold lowe s ei he as a y acid es e s and dies e s, o , i he ex ac ion is combined wi h saponi ica ion, i is ex ac ed as ee lu ein. In ei he case, di e en kinds o o ganic sol en s a e used o ex ac ion, and hese ha e o be emo ed comple ely by e apo a ion i he ex ac e d lu ein o lu ein es e s a e o be used in ood applica ions. 12 © Kaimainen, Jä enpää and Huopalah i 2015 | Enzyme-Assis ed Oil Ex ac ion Science Ta ge Inc. www.science a ge .com In he Eu opean Union, Di ec i e 95/45/EU de ines he aw ma e ials and he pu i y c i e ia o he ood colo an s (OJ L 22.9.1995). Fo lu ein pigmen s (E161b), he di ec i e lis s he sol en s ha can be used in he ex ac ion o ee and es e i ied lu ein om ce ain plan ma e ials. O hese sol en s, lu ein is mos soluble o dichlo o- me hane and ace one and, o a lesse ex en , o hexanes and alcohols (C a and Soa es 1992) whe e ee lu ein is conce ned. Ba zana e al. (2002) showed ha ee lu ein is less p one o iso- me isa ion and deg ada ion in hexane, which migh be a bene i , al hough he solubili y is lowe . I has o be no ed, ha he solubili y o ee lu ein di e s immensely om he solubili y o lu ein es e s in some sol en sys ems (Ama e al. 2003). Hojnik e al. (2008) conduc ed modeling o he ex ac ion p ocess using sol en s combined wi h alkaline e hanolysis, esul ing in ee lu ein p epa a ions. In hei model, he ex ac ion yield was mos a ec ed by he pa icle size o he biomass. Such bene i s can be ob ained using ca e ul g inding bu also enzyme-assis ed ex ac ion. Ba zana e al. (2002) es ed some combina ions o plan cell wall de- g ading enzymes and hexane ex ac ion, and hey epo ed ex ac ion e iciencies o up o 97 pe cen . In ou p e ious s udies we ex ac ed ca o enoids om chopped Ma igold lowe heads and chopped whole plan s using supe c i ical ca bon dioxide. Bo h es e i ied and ee lu ein in he ex ac s o g een pa s and mainly es e i ied lu ein in he ex ac s o lowe heads was ob ained (Jä enpää e al. 2008). The ex ac ion was no e y e icien , and Ma e al. (2008) demons a ed a supe c i ical luid ex ac ion me hod o lu ein whe e ege able oil was used as an ex ac ion enhance . This app oach yields lu ein in oil whe e lu ein is less p one o deg ada ion han in a concen a ed pas a ob ained using pu e ca bon dioxide (Jä enpää e al. 2008). As an example o o e all po en iali y o ob aining ca o enoids om a biomass using new p ocesses, Gou eia e al. (2007) demons a ed he algal oil and ca o enoid eco e y om mic oalgae using ace one, ege able oil, and supe c i ical ca bon dioxide luid. They epo ed he bes eco e y using ege able oil a oom empe a u e. The eco e y co ela ed di ec ly wi h how well he biomass was c ushed and g ound be o e ex ac ion. Encapsula ion o mic oencapsula ion is a e m used o p ocesses in which an ac i e ma e ial is o ally en eloped in a coa ing ma e ial. Thus, he ac i e ma e ial is isola ed and p o ec ed om he en i onmen . Also he physico-chemical p ope - ies, such as solubili y cha ac e is ics, o he encap- sula ed ma e ial can di e om hose o he ma e ial wi hou encapsula ion. Di e en mic o- encapsula ion echniques, such as emulsi ica ion and sp ay d ying, ha e been used o enhance he s abili y o ca o enoids (Rod iguez-Huezo e al. 2004, Khalil e al. 2012). As ca o enoids a e mos ly oil-soluble, simple oil-in-wa e (o/w) emul- sions can be used o inco po a e ca o enoids in o aqueous oods. Emulsi ica ion can also inc ease he bioa ailabili y o lu ein (Vishwana han e al. 2009). In ou p esen s udy, he biomass was i s g ound, bu he elease o ca o enoids om he biomass was aided using a mace a ing enzyme mix u e con aining cellulolysic and pec inolysic ac i i ies. Vege able oil was chosen as an ex ac an in o de o yield ue sol en - ee lu ein p epa a ion use ul o he encapsula ion s udies. Sepa a ion o aqueous and oil phases can be conduc ed using economical echniques also in la ge scale, hus he p ocess could be easily scaled-up. We ha e p e- iously used a pola lipid ac ion om oa as an emulsi ie o o/w emulsions (Kaimainen e al. 2012). In ha s udy we had some p oblems wi h he c eaming o emulsions, bu we ha e since managed o e a d he c eaming by adding small amoun s o s abilizing polysaccha ides in o he emulsion (unpublished da a). The aim o he p esen s udy was o ex ac lu ein es e s om Ma igold lowe s wi hou o ganic sol en s using enzyme-assis ed oil ex ac ion, o p oduce o/w emulsions wi h lu ein in he oil phase and di e en s abilizing polysaccha ides in he wa e phase, and o s udy he s abili y o lu ein du ing s o age. We also sp ay d ied some o he emulsions because d y powde s would be easie o s o e han we emulsions, and d ying could also ha e an e ec on he s abili y o lu ein. Ma e ials and Me hods In all s ages o he expe imen , p ecau ions we e made o p o ec he samples om ligh and oxida ion. These included using dim ligh ing in he labo a o y, using opaque con aine s o co e ing In e na ional Jou nal o Ag icul u al and Food Resea ch | Vol. 4 No. 2, pp. 11-19 13 Science Ta ge Inc. www.science a ge .com anspa en con aine s wi h aluminum oil, and using an an ioxidan du ing he ex ac ion o lu ein. Ma e ials The oa pola lipid ac ion used as an emulsi ie was ex ac ed om oa lakes (A ena sa i a) using a supe c i ical luid p ocess desc ibed by A o e al. (2007), and i consis s mainly o di e en glycol- lipids (monogalac osyldiacylglyce ol, digalac osyl- diacylglyce ol and s e yl glucoside) and phos- pholipids (phospha idyl choline) (Kaimainen e al. 2012, A o e al. 2007). Ma igold lowe s (T. e ec a L.) we e g own in Rymä ylä, Finland. Cellulase enzyme Econase CE (ac i i y 2330 U/ml) was supplied by AB Enzymes (Da ms ad , Ge many) and pec inase enzyme Pec inex Ul a SP-L (ac i i y 9500 U/ml) by No ozymes (Bagswae d, Denma k). Ci ic acid monohyd a e and disodium hyd ogen phospha e dihyd a e we e supplied by J.T. Bake (De eu e , Ne he lands), sodium chlo ide by Riedel-de Haën (Seelze, Ge many), and po assium chlo ide by Me ck (Da ms ad , Ge many). All sal s we e o analy ical g ade. F ee lu ein s anda d was supplied by Ex asyn hese (Lyon, F ance). Gua gum (GG) and locus bean gum (LBG) om Ce a onia siliqua seeds we e supplied by Sigma Ald ich (S . Louis, MO, Uni ed S a es). Bu yla ed hyd oxy oluene (BHT) was supplied by Supelco (Belle on e, PA, Uni ed S a es). Mal odex in (MD) C*Spe se 01314 (DE 12.5-15.5) was supplied by Ce es a (Hol e, Denma k). Me hanol and n-hexane we e supplied by J.T. Bake (De eu e , Ne he lands), Ace one by VWR (Fon enay-sous-Bois, F ance), and E hyl Ace a e by Lab-scan (Sowinskiego, Poland). All sol en s we e o HPLC g ade. Wa e used was pu i ied e e se osmosis wa e (Milli-Q Plus ul a- pu e wa e sys em, Millipo e, Molsheim, F ance). Comme cial apeseed oil (Bunge Finland Oy, Raisio, Finland) and xan han gum (XG) (Vuohelan He kkupuo i Ky, Ha ola, Finland) we e bough a a local g oce y s o e. Oil Ex ac ion o Lu ein Es e s om Ma igold Flowe s Ma igold lowe s we e suspended in wa e a p o- po ion 1:9 and ea ed wi h cellulase and pec inase (bo h enzymes 10 ml/l) a oom empe a u e o e nigh (20–25°C, 18–24 hou s) wi h magne ic s i ing. A e he enzyma ic b eakdown o lowe s (obse ed by he loss o solid ma e in he sus- pension), he wa e suspension was mixed wi h apeseed oil a p opo ion 5:1 a oom empe a u e o one hou wi h magne ic s i ing o ex ac lu ein es e s in o he apeseed oil. A e ex ac ion, he oil phase was sepa a ed om he wa e phase by cen i uging aliquo s o he mix u e a 1050 x g o 10 minu es wice and collec ing he sepa a ed oil a e each cen i uga ion. The oil ex ac ion was epea ed wi h esh oil mixed wi h he same wa e suspension. The amoun o lu ein es e s in oil was de e mined by making a 20 mg/ml dilu ion in ace one and analyzing i wi h HPLC using he same me hod as in he s o age es desc ibed below. The oil ex ac s o he epea ed ex ac ions we e combined and s o ed in he da k a 4°C un il used o emulsion p epa a ion and s o age es . P epa a ion o Emulsions and Sp ay D ying The emulsions we e p epa ed simila ly o ou p e ious esea ch (Kaimainen e al. 2012) wi h sligh modi ica ions. We used a pH o 5.8 ins ead o 2.6 because e en hough he oa lipid emulsi ie showed highe solubili y in lowe pH, he poly- saccha ides used as s abilize s a e s able om pH 4 o 10 (D ehe 1999). A pH 5.8 ci a e-phospha e bu e was p epa ed by mixing 0.1 M ci ic acid solu ion, 0.2 M disodium hyd ogen phospha e solu ion, and wa e in p opo ion 197:303:500, espec i ely. Oa lipid emulsi ie (5 mg/ml) and polysaccha ides (3 mg/ml) we e dissol ed in he bu e wi h magne ic s i ing a 50–60°C o wo hou s. Fou di e en polysaccha ide mix u es we e used as s abilize s: gua gum + xan han gum 1:3 and 3:1 and locus bean gum + xan han gum 1:3 and 3:1. Emulsions we e made by adding 20 mg/ml o oil con aining lu ein es e s and homogenizing wi h a high speed mixe . Two di e en equipmen was used o emulsi ica ion: ei he an Ul a-Tu ax® T 25 Digi al dispe se wi h a S 25 N – 10 G dispe sing ool (IKA®, S au en, Ge many) a 24 000 pm o 5 minu es o a Magic LAB® dispe sing sys em wi h mic o-plan assembly and 6F blade (IKA®, S au en, Ge many) a 16 000 pm o 5 minu es. The Magic LAB equipmen was used o p oducing a sligh ly la ge olume o he emulsions o sp ay d ying o wo s abilize composi ions. A e emulsi ica ion o hese la ge ba ches, 200 mg/ml mal odex in (MD) was added o he emulsion, a small sample was aken o s o age es o see he e ec o added MD, and he es o he ba ch was sp ay d ied wi h a Mini Sp ay D ye B290 (Büchi, Flawil, 14 © Kaimainen, Jä enpää and Huopalah i 2015 | Enzyme-Assis ed Oil Ex ac ion Science Ta ge Inc. www.science a ge .com Swi ze land). The d ying condi ions we e as ollows: inle ai empe a u e 150°C, ou le em- pe a u e 84°C, ai low 450 l/h, eed low 7 ml/min, aspi a o 37 m 3 /h and nozzle o i ice 1.50 mm. A summa y o s abilize composi ions used o emulsions and equipmen used o emulsi- ica ion along wi h code le e s is shown in Table 1. Table 1 Sample codes o emulsion samples, composi ion o s abilize s used in hem and he equipmen used o hei emulsi ica ion. GG = gua gum, XG = xan han gum, LBG = locus bean gum, MD = mal odex in. S ample Code S abilize C omposi ion Emulsi ica ion Equipmen A GG + XG 3:1 Ul a-Tu ax® B GG + XG 1:3 Ul a - Tu ax® C LBG + XG 3:1 Ul a - Tu ax® D LBG + XG 1:3 Ul a - Tu ax® E GG + XG 3:1 + MD Magic LAB® F LBG + XG 1:3 + MD Magic LAB® G GG + XG 3:1 + MD, sp ay d ied Magic LAB® H LBG + XG 1:3 + MD, sp ay d ied Magic LAB® S o age Tes and Ex ac ion o Lu ein and Lu ein Es e s A o al o six we emulsion samples, wo d ied emulsion samples (lis ed in Table 1), and one oil sample was used in he s o age es . Samples we e s o ed in da k a 20–22°C in plas ic ubes, and he amoun o lu ein in samples was de e mined in iplica e a e 0, 1, 2, 5 and 10 weeks o s o age. The s o age condi ions we e selec ed based on p elimina y es s made wi h lu ein es e s in oil, which showed ha he s abili y was good in da k and low empe a u e and poo in ligh (unpublished da a). Emulsions we e also inspec ed isually a he same ime poin s. Be o e aking a sample o lu ein ex ac ion, he emulsions we e gen ly mixed by u ning he con aine s a ew imes. Fo he ex ac ion o lu ein and lu ein es e s, 1 ml o emulsion sample, 200 mg o d ied emulsion sample + 1 ml o wa e o 20 mg o oil + 1 ml o wa e we e ans e ed in o a glass ube wi h a sc ew cap. 500 µl o sa u a ed NaCl solu ion and 100 µl o 2 M ci ic acid solu ion was added o b eak he emulsion and help he ex ac ion p ocess, as he s abilizing polysaccha ides a e deg aded i pH is lowe han 3 (D ehe 1999). The ube was ho oughly mixed wi h a o ex mixe , a e which 2 ml o hexane con aining 0.2 mg/ml BHT was added, and he ube was shaken a 500 pm o 10 minu es. The hexane and wa e phases we e sepa a ed by cen i uging a 966 x g o 5 minu es, a e which he hexane phase was ans e ed o ano he ube con aining 2 ml o 8.8 mg/ml KCl solu ion. The second ube was shaken a 500 pm o 2 minu es and cen i uged a 966 x g o 5 minu es, a e which he washed hexane phase was collec ed o a hi d ube. The con en s o he i s ube we e ex ac ed again wi h 2 ml o hexane con aining 0.2 mg/ml BHT, and he p ocedu e was epea ed using he same ube and KCl solu ion in ube 2, and he hexane phases we e combined in ube 3. Hexane was e apo a ed unde a s eam o ni ogen a 25–30°C, and he esidue was dissol ed in 1 ml o ace one and il e ed h ough a 0.45 µm PTFE memb ane. I no analyzed immedia ely a e ex ac ion, he samples we e s o ed a -80°C un il HPLC analysis. HPLC Analysis o Lu ein and Lu ein Es e s HPLC analysis o lu ein con en was pe o med using a combined and modi ied me hod desc ibed by Cano (1991) and Mon eal e al. (1999). A liquid ch oma og aph (Shimadzu co po a ion, Kyo o, Japan) consis ing o a GT-154 acuum degasse , wo LC-10AT VP sol en deli e y modules, an SIL-10A Au o injec o wi h sample coole , a CTO- 10A column o en, an SPD-M10AVP diode a ay de ec o , and an SCL-10 A VP sys em con olle was used wi h a e e sed phase column Luna 5u C18 (leng h 150 mm, inne diame e 3.00 mm, pa icle size 5 µm and po e size 95Å, Phenomenex, To ance, CA, Uni ed S a es). The sys em was compu e -con olled wi h a Labsolu ions e sion 1.23 SP1 so wa e (Shimadzu Co po a ion, Kyo o, Japan). A g adien mix u e o me hanol and wa e (75:25) as eluen A and e hyl ace a e as eluen B was used, aising pe cen age o B om 0% o 70% du ing 0–10 minu es, ollowed by a u he ise o 100% du ing 10–14 minu es, a e which he ini ial condi ions o 0% B we e es o ed du ing 14–20 minu es, and he sys em was equilib a ed o he ollowing un du ing 20–35 minu es. The column was ope a ed a 40°C, low a e was kep cons an a 1.0 ml/min, injec ion olume was 10 µl, and samples we e kep a 8°C in he sample coole . In e na ional Jou nal o Ag icul u al and Food Resea ch | Vol. 4 No. 2, pp. 11-19 15 Science Ta ge Inc. www.science a ge .com De ec ion wa eleng h was 445 nm, and ee lu ein and lu ein es e s we e quan i ied as ee lu ein using a calib a ion cu e made using di e en concen a ions o an ex e nal s anda d o ee lu ein in ace one. Quan i ica ion could be done wi h ee lu ein s anda d, as he es e i ied a y acids do no a ec he mola abso p i i y o lu ein. S a is ical Analysis The ex e nal lu ein s anda d calib a ion cu e was made wi h eigh concen a ion poin s anging om 0.70 o 15.6 µg/ml, and each poin was calcula ed as mean alue o i e eplica e analyses. The co ela ion coe icien o he calib a ion cu e was 0.9996. The amoun o lu ein in he samples a each ime poin du ing he s o age es was calcula ed as mean alue o iplica e analyses and compa ed o he ini ial amoun o each sample. Due o he di e en sample ma ices (we emul- sion, sp ay d ied emulsion o plain oil), he quan i- a i e amoun s o lu ein a ied be ween samples, and compa ison o he ini ial amoun allowed o easie compa ison o lu ein s abili y be ween samples. S a is ical di e ences be ween samples and ime poin s we e analyzed wi h one-way analysis o a iance (ANOVA) oge he wi h Tukey’s - es . Di e ences be ween ime poin s we e also e i ied wi h epea ed measu es ANOVA oge he wi h leas signi ican di e ence es . IBM® SPSS® S a is ics e sion 21 so wa e (IBM Co po a ion, A monk, NY, Uni ed S a es) was used in he s a is ical analyses. Resul s and Discussion The oil ex ac ion o lu ein es e s om ma igold lowe s was success ul, wi h a lu ein yield (p esen as lu ein es e s bu calcula ed as ee lu ein) o 1.1 mg/g o ma igold lowe s ( esh weigh ). The lu ein con en s o oil ex ac s we e 0.50 mg/ml o he i s ex ac , 0.23 mg/ml o he second ex ac , and 0.36 mg/ml o he combined ex ac . The yield o lu ein was qui e simila o wha we ex ac ed om he same ma igold lowe s using ace one (unpublished da a). In li e a u e, he lu ein con en o ma igold lowe s is usually epo ed o d y weigh , and i a ies widely depending on he a ie y. Examples o epo ed lu ein con en s (pe d y ma e ) o di e en a ie ies o Tage es e ec a a e 0.77–14.4 mg/g (Khalil e al. 2012), 1.61–6.11 mg/g (Li e al. 2007), 0.18–3.0 mg/g (Piccaglia e al. 1998), and 10.3–17.0 mg/g (Deineka e al. 2007). Deineka e al. (2007) also measu ed he lu ein con en o esh weigh , and i was 2.10– 2.97 mg/g wi h app oxima ely 5–6 old di e ence o con en pe d y weigh . We do no ha e he a ie y da a o ou ma igold lowe s, bu compa ing o hese p e iously epo ed lu ein con en s, he lu ein yield o 1.1 mg/g lowe s ( esh weigh ) is qui e simila . Ini ial appea ances o he emulsions mixed by di e en equipmen we e simila . Sp ay d ying o emulsions p oduced sligh ly yellowish powde s, which had mois u e con en s o 60 mg/g. Visual inspec ion o we emulsions e ealed only one di e ence du ing he s o age es . The emulsion wi h GG + XG 3:1 + MD (sample E) had sligh c eaming a e 5 and 10 weeks. The c eaming could be due o MD modi ying he s uc u e o he wa e phase o inc easing he densi y di e ence be ween he oil and wa e phases. I could also be an e ec o lowe speed o mixing o he emulsions made wi h MD. The isual appea ance o he o he samples did no change du ing he 10 week s o age pe iod. The HPLC analysis o he samples e ealed peaks o ee lu ein and lu ein es e s in all o he samples. The amoun o ee lu ein was 0.5–1.0% o he amoun o lu ein es e s, and he a io emained cons an du ing he whole s o age pe iod. The amoun o lu ein dec eased in all samples du ing he 10 week s o age pe iod, as can be seen in Table 2. Only in sample B was he di e ence be ween ini ial amoun and amoun a e 10 weeks no s a is ically signi ican , bu e en in ha sample, he amoun a e 5 weeks was signi ican ly lowe han he ini ial amoun . Re en ion o lu ein du ing 10 weeks a ied be ween 67% and 91%. The e could be some analysis e o in he measu emen s o samples B, D, and G, as he amoun o lu ein a e one week is nea ly 12%, 9% o 6% g ea e han in he beginning, espec i ely. The e o could be in he measu emen s o he one week samples bu also in he ini ial amoun . I he e o is in he ini ial amoun , hen i is ca ied on o o he ela i e alues as well. The e en ion o lu ein was wo s in he sp ay d ied samples, 75% o sample G and 67% o sample H, al hough sample G did no di e signi ican ly om he majo i y o he samples. 16 © Kaimainen, Jä enpää and Huopalah i 2015 | Enzyme-Assis ed Oil Ex ac ion Science Ta ge Inc. www.science a ge .com Table 2 Re en ion o lu ein du ing 10 week s o age pe iod wi h s anda d de ia ions o iplica e measu emen s p esen ed as% lu ein le compa ed o ini ial amoun . Sample 0 weeks 1 week 2 weeks 5 weeks 10 weeks A 100 ± 1% a,b 99 ± 2% a,1,2 97 ± 1% b,1 83 ± 3% c,1,2,3 77 ± 3% d,1,2 B 100 ± 5 % a,b 112 ± 6 % c,d,4 99 ± 6 % a,c,e,1 91 ± 5 % e, ,3,4 91 ± 7 % b,d, ,3 C 100 ± 2 % a 101 ± 3 % b,1,2,3 97 ± 2 % a,b,1 87 ± 3 % c,2,3,4 82 ± 3 % d,1,2 D 100 ± 3 % a,b 109 ± 3 % a,3,4 94 ± 3 % b,1,2 83 ± 3 % c,1,2 77 ± 3 % d,1 E 100 ± 2 % a 98 ± 3 % a,1,2 91 ± 2 % b,1,2 81 ± 3 % c,1,2 80 ± 2 % c,1,2 F 100 ± 3 % a 98 ± 4 % a,b,1 97 ± 3 % a,1 81 ± 3 % c,1,2 82 ± 6 % b,c,1,2 G 100 ± 5 % a,b 106 ± 5 % a,2,3,4 94 ± 7 % b,1,2 79 ± 8 % b,c,1,2 75 ± 5 % c,1,4 H 100 ± 1 % a 99 ± 3 % a,1,2 88 ± 5 % b,2 79 ± 2 % b,1 67 ± 4 % c,4 oil 100 ± 1 % a 101 ± 2 % a,1,2,3 96 ± 2 % b,1 91 ± 1 % c,4 85 ± 1 % c,2,3 Time poin s no sha ing a leas one same supe sc ip le e (a- ) a e s a is ically di e en om each o he o each sample (p ≤ 0.05, compa ison by ows) and samples no sha ing a leas one same supe sc ip numbe (1-4) a e s a is ically di e en om each o he in each ime poin (p ≤ 0.05, compa ison by columns). See able 1 o sample codes. Wi h hese emulsion composi ions, sp ay d ying had a nega i e e ec on he s abili y o lu ein. The eason o his is no clea , bu i is possible ha he condi ions in he sp ay d ying p ocess may ha e ini ia ed lu ein deg ada ion eac ions, which hen con inued du ing s o age. This is in spi e o he ac ha he empe a u e o he d ople o pa icle usually does no ise much du ing he sp ay d ying p ocess, as ime in he ho ai is e y sho and mos o he ene gy is used o e apo a e he wa e (Gha sallaoui e al. 2007). Fu he easons may a ise om he e ec o emulsion and powde s uc u e on he p oximi y o oxygen species and lu ein. The su ace o he emulsion is cons i u ed o oa lipids, LBG/GG, XG, and MD, and i could be po ous a e sp ay d ying and hus, mo e easily pene a ed by oxygen han he coa ing in we emulsions. The wall ma e ials can a ec he po osi y o he mic ocapsules, and mo e po ous mic opa icles con ain mo e oxygen a ailable o deg ada ion eac ions (Tonon e al. 2010, Fe a i e al. 2012). Howe e , he po osi y o ou sp ay d ied powde s should be assessed o e i y his hypo- hesis. Ca o enoids a e sensi i e o oxida ion ini ia ed by oxygen, ligh , and hea (Boon e al. 2010). The a mosphe e o he sample ubes was no con olled, and he e was ai o e he samples in he ubes, so oxygen was a ailable in he s o age condi ions. As ou samples we e s o ed in da k a 20–22°C, ligh and hea a e no p obable causes o he loss o lu ein in ou samples. The e ec o wa e ac i i y (a w ) on ca o enoid s abili y has been s udied o d ied la e (Po phy a) (Oh e al. 2013), eeze- d ied ca o s (La elli e al. 2007), o sp ay d ied mul iple emulsions (Rod iguez-Huezo e al. 2004). Oh e al. (2013) epo ed inc easing ca o enoid deg ada ion wi h inc easing s o age a w , whe eas La elli e al. (2007) epo ed minimum ca o enoid deg ada ion a s o age a w be ween 0.31 and 0.54 wi h inc easing deg ada ion abo e o below hese alues. Rod iquez-Huezo e al. (2004) epo ed maximum ca o enoid deg ada ion a e a a w 0.628 wi h smalle and p ac ically cons an deg ada ion a e a a w abo e o below his alue. The e ec o a w on ca o enoid s abili y seems o a y wi h di e en sample ma e ials, bu i could be one eason o he di e ence in s abili ies o sp ay d ied emulsions and we emulsions. Al hough we did no measu e he a w o he samples and did no con ol he a w o he s o age condi ions, i seems easonable o expec e y low a w alues o sp ay d ied samples and high a w alues o emulsion samples. The a w o he oil sample is also p obably qui e low. These p elimina y obse a ions wa an u he s udies ocusing on he usabili y o oil and emulsions con aining lu ein in a ious ood appli- ca ions. Fu he s udies on emulsions should also con ain cha ac e iza ion o emulsions (d ople size and d ople size dis ibu ion), which was no done in his s udy. In e na ional Jou nal o Ag icul u al and Food Resea ch | Vol. 4 No. 2, pp. 11-19 17 Science Ta ge Inc. www.science a ge .com Conclusions Enzyme-assis ed oil ex ac ion o lu ein wi hou o ganic sol en s was qui e e ec i e and p oduced oil wi h an adequa e amoun o lu ein. This me hod can be used o ood applica ions, as no o ganic sol en s we e used, and so no e apo a ion s ep is needed, and he e is no isk o sol en esidues. Lu ein s abili y du ing 10 weeks in o/w emulsions s abilized wi h di e en polysaccha ides and in oil was qui e good. This allows inco po a ion o lu ein in bo h aqueous and a y ma ices. Sp ay d ying o hese emulsions wi h mal odex in as wall ma e ial seemed o weaken lu ein s abili y du ing s o age. Di e en wall ma e ials could p oduce be e esul s, bu also he e ec on emulsion s uc u e need o be conside ed. Fo example, inc easing he amoun o long chain polysaccha ides used as s abilize s will g ea ly inc ease he iscosi y o he emulsion, o e en p oduce a gel, which will also a ec he sp ay d ying p ocess. Acknowledgemen s Financial suppo om Raisio Plc Resea ch ounda ion is g a e ully acknowledged. The au ho s wish o hank Ka i Val a and Ma i Jaan o o p o iding he Ma igold lowe s. Con lic o In e es The au ho s decla e no con lic o in e es . Re e ences Abdel-Aal, E.M., Akh a , H., Zahee , K. and Ali, R. (2013), “Die a y sou ces o lu ein and zeaxan hin ca o enoids and hei ole in eye heal h”, Nu ien s, ol. 5, pp. 1169-1185 Ama , I., Ase in, A. and Ga i, N. (2003), “Solubiliza ion Pa e ns o Lu ein and Lu ein Es e s in Food G ade Nonionic Mic o- emulsions”, Jou nal o Ag icul u al and Food Chemis y, ol. 51, no. 16, pp. 4775-4781 A o, H., Jä enpää, E., Könkö, K., Huopalah i, R. and Hie aniemi, V. 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