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
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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
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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
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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. (2007), “The cha ac e i-
za ion o oa lipids p oduced by supe c i ical
luid echnologies”, Jou nal o Ce eal Science,
ol. 45, no. 1, pp. 116-119
Ba ley, G.E. and Scolnik, P.A. (1995), “Plan
ca o enoids: pigmen s o pho op o ec ion,
isual a ac ion, and human heal h”. Plan Cell,
ol. 7, no. 7, pp. 1027-1038
Ba zana, E., Rubio, D., San ama ia, R.I., Ga cia-
Co ea, O., Ga cia, F., Ridau a Sanz, V.E. and
López-Munguía, A. (2002), “Enzyme-Media ed
Sol en Ex ac ion o Ca o enoids om Ma i-
gold Flowe (Tage es e ec a)”, Jou nal o
Ag icul u al and Food Chemis y, ol. 50, no.
16, pp. 4491-4496
Bha acha yya, S., Da a, S., Mallick, B., Dha , P.
and Ghosh, S. (2010), “Lu ein Con en and in
Vi o An ioxidan Ac i i y o Di e en Cul i-
a s o Indian Ma igold Flowe (Tage es pa ula
L.) Ex ac s”, Jou nal o Ag icul u al and Food
Chemis y, ol. 58, no. 14, pp. 8259-8264
Boon, C.S., McClemen s, D.J., Weiss, J. and
Decke , E.A. (2010), “Fac o s In luencing he
Chemical S abili y o Ca o enoids in Foods”.
C i ical e iews in ood science and nu i ion,
ol. 50, no. 6, pp. 515-532
Cano, M.P. (1991), “HPLC Sepa a ion o Chlo o-
phyll and Ca o enoid Pigmen s o Fou Kiwi
F ui Cul i a s”, Jou nal o Ag icul u al and
Food Chemis y, ol. 39, pp. 1786-1791
C a , N.E. and Soa es, J.H. (1992), “Rela i e
solubili y, s abili y, and abso p i i y o lu ein
and β-ca o ene in o ganic sol en s”, Jou nal o
Ag icul u al and Food Chemis y, ol. 40, no.
3, pp. 431-434
Deineka, V.I., So okopudo , V.N., Deineka, L.A.
and T e 'yako , M.Y. (2007), “Flowe s o ma i-
gold (Tage es) species as a sou ce o xan ho-
phylls”, Pha maceu ical Chemis y Jou nal,
ol. 41, no. 10, pp. 540-542
D ehe , M. (1999), “Food Sou ces and Uses o
Die a y Fibe ” in Cho, S.S., P osky, L. and
D ehe , M. (eds.) Complex Ca bohyd a es in
Foods, Ma cel Dekke Inc., New Yo k, USA,
pp. 327-371
18
© Kaimainen, Jä enpää and Huopalah i 2015 | Enzyme-Assis ed Oil Ex ac ion
Science Ta ge Inc. www.science a ge .com
Fe a i, C.C., Ge me , S.P.M., Al im, I.D.,
Visso o, F.Z. and de Agui e, J.M. (2012),
“In luence o ca ie agen s on he physico-
chemical p ope ies o blackbe y powde
p oduced by sp ay d ying”, In e na ional
Jou nal o Food Science and Technology, ol.
47, no. 6, pp. 1237-1245
F ank, H.A. and Cogdell, R.J. (1996),
“Ca o enoids in pho osyn hesis”, Pho ochem-
is y and pho obiology, ol. 63, no. 3, pp. 257-
264
Gha sallaoui, A., Roudau , G., Chambin, O.,
Voilley, A. and Sau el, R. (2007), “Appli-
ca ions o sp ay-d ying in mic oencapsula ion
o ood ing edien s: An o e iew”, Food
Resea ch In e na ional, ol. 40, no. 9, pp.
1107-1121
Gou eia, L., Nob e, B.P., Ma celo, F.M., M ejen,
S., Ca doso, M.T., Pala a, A.F. and Mendes,
R.L. (2007), “Func ional ood oil colou ed by
pigmen s ex ac ed om mic oalgae wi h supe -
c i ical CO
2
”, Food Chemis y, ol. 101, no. 2,
pp. 717-723
Hadden, W.L., Wa kins, R.H., Le y, L.W.,
Regalado, E., Ri adenei a, D.M., an B eemen,
R.B. and Schwa z, S.J. (1999), “Ca o enoid
Composi ion o Ma igold (Tage es e ec a)
Flowe Ex ac Used as Nu i ional Supple-
men ”, Jou nal o Ag icul u al and Food
Chemis y, ol. 47, no. 10, pp. 4189-4194
Hojnik, M., Ške ge , M. and Knez, Ž. (2008),
“Ex ac ion o lu ein om Ma igold lowe
pe als – Expe imen al kine ics and modelling”,
LWT - Food Science and Technology, ol. 41,
no. 10, pp. 2008-2016
Jä enpää, E., Tas ula, P., A o, T., Ros iala, N.,
Hie aniemi, V. and Huopalah i, R. (2008),
S abili y o lu ein and lu ein es e p epa a ions
ob ained using supe c i ical luid ex ac ion o
Tage es e ec a”, P oceedings o he 5 h In e -
na ional Cong ess on Pigmen s in Food - Fo
Quali y and Heal h, Helsinki, pp. 133-136
Jomo a, K. and Valko, M. (2013), “Heal h
p o ec i e e ec s o ca o enoids and hei in e -
ac ions wi h o he biological an ioxidan s”,
Eu opean jou nal o medicinal chemis y, ol.
70, pp. 102-110
Kaimainen, M., Ah enainen, S., Kaa is e, M.,
Jä enpää, E., Kaasalainen, M., Salomäki, M.,
Salonen, J. and Huopalah i, R. (2012), “Pola
lipid ac ion om oa (A ena sa i a): cha ac-
e iza ion and use as an o/w emulsi ie ”, Eu o-
pean Food Resea ch and Technology, ol. 235,
pp. 507-515
Khalil, M., Raila, J., Ali, M., Islam, K.M.S.,
Schenk, R., K ause, J.-P., Schweige , F.J. and
Rawel, H. (2012), “S abili y and bioa ailabili y
o lu ein es e supplemen s om Tage es lowe
p epa ed unde ood p ocessing condi ions”,
Jou nal o Func ional Foods, ol. 4, pp. 602-
610
Kiokias, S. and Go don, M.H. (2004), “An ioxi-
dan P ope ies o Ca o enoids In Vi o and In
Vi o”, Food Re iews In e na ional, ol. 20, no.
2, pp. 99-121
Lapsho a, M.S., Deineka, V.I., Deineka, L.A.,
Blino a, I.P. and T e 'yako , M.Y. (2013),
“Iden i ica ion o xan hophylls in a ma igold
pe al ex ac ”, Jou nal o Analy ical Chemis y,
ol. 68, no. 11, pp. 1014-1019
La elli, V., Zanoni, B. and Zaniboni, A. (2007),
“E ec o wa e ac i i y on ca o enoid deg a-
da ion in dehyd a ed ca o s”, Food Chemis y,
ol. 104, no. 4, pp. 1705-1711
Li, W., Gao, Y., Zhao, J. and Wang, Q. (2007),
“Phenolic, Fla onoid, and Lu ein Es e Con en
and An ioxidan Ac i i y o 11 Cul i a s o
Chinese Ma igold”, Jou nal o Ag icul u al and
Food Chemis y, ol. 55, no. 21, pp. 8478-8484
Ma, Q., Xu, X., Gao, Y., Wang, Q. and Zhao, J.
(2008), “Op imisa ion o supe c i ical ca bon
dioxide ex ac ion o lu ein es e s om ma i-
gold (Tage es e ec L.) wi h soybean oil as a
co-sol en ”, In e na ional Jou nal o Food
Science and Technology, ol. 43, no. 10, pp.
1763-1769
Mon eal, M., De Ancos, B. and Cano, M.P. (1999),
“In luence o C i ical S o age Tempe a u es on
Deg ada i e Pa hways o Pigmen s in G een
Beans (Phaseolus ulga is C s. Pe ona and
Boby)”, Jou nal o Ag icul u al and Food
Chemis y, ol. 47, pp. 19-24
Oh, S., Shin, M., Lee, K. and Choe, E. (2013),
“E ec s o wa e ac i i y on pigmen s in d ied
la e (Po phy a) du ing s o age”, Food Science
In e na ional Jou nal o Ag icul u al and Food Resea ch
| Vol. 4 No. 2, pp. 11-19 19
Science Ta ge Inc. www.science a ge .com
and Bio echnology, ol. 22, no. 6, pp. 1523-
1529
OJ L 22.9.1995, “Commission Di ec i e 95/45/EC
o 26 July 1995 laying down speci ic pu i y
c i e ia conce ning colou s o use in ood-
s u s”, O icial Jou nal o he Eu opean
Communi ies, ol. 226, pp. 1-45
Piccaglia, R., Ma o i, M. and G andi, S. (1998),
“Lu ein and lu ein es e con en in di e en
ypes o Tage es pa ula and T. e ec a”,
Indus ial C ops and P oduc s, ol. 8, no. 1, pp.
45-51
Rod iguez-Huezo, M.E., Ped oza-Islas, R., P ado-
Ba agan, L.A., Be is ain, C.I. and Ve non-
Ca e , E.J. (2004), “Mic oencapsula ion by
sp ay d ying o mul iple emulsions con aining
ca o enoids”, Jou nal o Food Science, ol. 69,
no. 7, pp. E351-E359
Tonon, R.V., B abe , C. and Hubinge , M.D.
(2010), “An hocyanin s abili y and an ioxidan
ac i i y o sp ay-d ied açai (Eu e pe ole acea
Ma .) juice p oduced wi h di e en ca ie
agen s”, Food Resea ch In e na ional, ol. 43,
no. 3, pp. 907-914
Vishwana han, R., Wilson, T.A. and Nicolosi, R.J.
(2009), “Bioa ailabili y o a nanoemulsion o
lu ein is g ea e han a lu ein supplemen ”,
Nano Biomedicine and Enginee ing, ol. 1, pp.
57-73