ODS JOURNALOFFUNCTIONALFO 5 (2 0 1 3) 4 2 4 - 4 3 3

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Sintesis dengan katalis lipase dan karakterisasi lipid terstruktur berbasis minyak biji kapak

Maryam Khodadadi Sebuah, Sarya Aziz Sebuah, Richard St-Louis b, Selim Kermasha Sebuah,*
Sebuah Departemen Ilmu Pangan dan Kimia Pertanian, McGill University, 21,111 Lakeshore, Ste-Anne de Bellevue, QC, Kanada H9X 3V9

b Département de Biologie, Chimie et Géographie, Université du Québec à Rimouski, 300 Allée des Ursulines, Rimouski, QC, Kanada G5L 3A1

ARTICLEINFO ABSTRAK

Sejarah artikel: Biosintesis lipid terstruktur (SLs) dilakukan dengan menggunakan minyak biji kapak (FO) dan tricaprylin (TC) dalam media pelarut

Diterima 11 Oktober 2012 Diterima organik (OSM), menggunakan lipase komersial terpilih, termasuk Amano DF, Novozym 435, Lipozyme TL-IM dan Lipozyme RM-IM.

dalam bentuk revisi 22 November Rantai asil lemak dari triasilgliserol (TAG) yang disintesis diidentifikasi dengan analisis ionisasi kimia tekanan atmosfer / spektrometri
2012 massa (APCI / MS), sedangkan distribusi posisi asam lemak dari MLM- dan MML-SLs (M-medium dan L-panjang rantai asam lemak)
Diterima 26 November 2012 Tersedia online 1 ditentukan dengan kromatografi cair kinerja tinggi ion perak (Ag + /
Januari 2013

Kata kunci:
HPLC). Pengaruh suhu reaksi ( T r, 30–50 C), konsentrasi enzim
Lipase
( E c, 0,5–4%, b / v), aktivitas air awal ( Sebuah w, 0,05–0,43) dan waktu reaksi ( R t, 0–72 jam) tentang efisiensi enzim, dipelajari. Hasil
Lipid terstruktur
biokonversi (%) dari hasil sintesis
Ketertarikan
MLM- dan MML-SLs dipantau di bawah parameter reaksi yang ditetapkan untuk setiap lipase. Hasil maksimum MLM-SLs diperoleh
Minyak biji rami
dengan urutan, Novozym 435> Lipozyme TL-IM> Lipozyme RM-IM> Amano DF. Selain itu, dengan mempertimbangkan rasio MLM- ke
Hasil biokonversi
MML-SLs yang dihasilkan oleh masing-masing enzim, Novozym 435 dan Lipozyme TL-IM dipilih sebagai enzim yang paling efektif

untuk peminatan FO dan TC.

2012 Elsevier Ltd. Semua hak dilindungi undang-undang.

1. pengantar kelompok asil ke posisi spesifik dari gliserol; di sisi lain, proses kimiawi kurang spesifik dan

karenanya dapat mengarah pada pembentukan produk samping, dengan penurunan hasil

Sifat fungsional dan fisik, nasib metabolisme dan manfaat kesehatan yang diduga dari berbagai selanjutnya ( Lee & Akoh, 1998a ).

lemak dan minyak berbeda karena residu komponen asam lemak (FA) dan posisinya pada

tulang punggung gliserol dari triasilgliserol (TAG) ( Akoh, Lee, & Fomuso, 1998; Goderis dkk., Di antara berbagai jenis SL, SL terstruktur tipe MLM (MLM-SLs), rantai menengah FA

1987 ). Lipid khusus yang mengacu pada lemak dan minyak dengan sifat fungsional atau nutrisi (MCFA) dan rantai panjang penting FA (LCFA), masing-masing ditetapkan, di sn- 1,3 dan sn- 2

khusus, termasuk berbagai macam produk di antaranya lipid terstruktur (SL) sebagai kelas posisi tulang punggung gliserol, telah menjadi subjek yang sangat menarik. Lipase pankreas

utamanya ( Xu & Akoh, adalah a sn- 1,3 enzim regioselektif dan menghidrolisis TAG menjadi sn- 2 monoasilgliserol

(2-MAGs) dan FA bebas, di mana MCFA yang dibebaskan dengan mudah diserap oleh jalur

portal dan dengan cepat teroksidasi di hati yang menyediakan energi ( Odle, 1997 ); 2-MAG dari

2002). Lipid terstruktur, yang menunjukkan perubahan komposisi dan posisi distribusi asam LCFA diserap secara efisien melalui sistem limfatik ( Bugaut, 1987 ). Oleh karena itu, MLM-SL

lemak dalam triasilgliserol, dapat diperoleh baik secara kimiawi maupun enzimatis ( Osborn & dapat digunakan untuk a

Akoh, 2002 ). Melalui transesterifikasi enzimatik, dimungkinkan untuk memasukkan yang

diinginkan

* Penulis yang sesuai. Telp .: +1 514 398 7922; faks: +1 514 398 7977. Alamat email: selim.kermasha@mcgill.ca
(S. Kermasha). 1756-4646 / $ - lihat materi depan
2012 Elsevier Ltd. Semua hak dilindungi undang-undang.

http://dx.doi.org/10.1016/j.jff.2012.11.015
 ODS JOURNALOFFUNCTIONALFO 5 (2 0 1 3) 4 2 4 - 4 3 3
diet seimbang untuk pasien dan bayi dengan kebutuhan khusus ( Lee & Akoh, 1998b ).
Asidolisis dengan katalis lipase, di mana MCFA dan minyak nabati masing-masing
digunakan sebagai sumber donor asil dan LCFA esensial, adalah salah satu pendekatan paling
umum untuk sintesis MLM-SL ( Hamam & Shahidi, 2008; Kim
& Akoh, 2005; Nunes, Pires-Cabral, & Ferreira-Dias, 2011). Namun, minat yang dikatalisis lipase
tampaknya menjadi rute yang lebih tepat daripada asidolisis dengan komplikasi yang lebih
sedikit dalam pemulihan SL ( Osorio, Ferreira-Dias, Gusmão, & Da
Fonseca, 2001). Meskipun reaksi asidolisis, yang digunakan untuk sintesis MLM-SL, telah
dilaporkan secara luas dalam literatur, terdapat sedikit informasi tentang minat katalis lipase
antara TAG atau etil ester dari FA rantai pendek atau menengah dan TAG heterogen, seperti
minyak biji kapak (FO) ( Irimescu, Hata, Iwasaki, & Yamane, 2001; Shin, Akoh,
& Lee, 2010; Yang, Fruekilde, & Xu, 2003). Dengan semakin dikenalnya pentingnya makanan x- 3
FAs, FO dengan kandungan asam linolenat yang tinggi semakin banyak digunakan untuk
memproduksi minyak sehat ( Chen, Ma, Liang, Peng, & Zuo, 2011;
Gunstone & Harwood, 2007).
Kekuatan penyelesaian yang cukup besar dari kromatografi cair kinerja tinggi fase terbalik
(RP-HPLC), digunakan sebagai teknik yang tepat untuk pemisahan TAG. Kesulitan utama, saat
menganalisis campuran kompleks, adalah pemisahan TAG dengan nilai nomor karbon ekuivalen
(ECN) yang sama, di mana isomer posisional tidak dapat ditentukan ( Andrikopoulos, 2002;
Buchgraber, Ulberth, Emons, &
Anklam, 2004). Kromatografi ion perak umumnya digunakan untuk pemisahan isomer TAG, di
mana urutan elusinya bergantung pada peningkatan derajat ketidakjenuhan FA ( Andrikopoulos,
2002 ).
Tujuan dari penelitian ini, yang merupakan bagian dari pekerjaan penelitian yang sedang
berlangsung ( Bai et al., Sedang dicetak ), adalah untuk mengkarakterisasi TAG, disintesis oleh
kation bunga FO dan tricaprylin (TC) dalam media pelarut organik (OSM), dan untuk menyelidiki
efisiensi lipase komersial yang dipilih untuk sintesis MLM- dan MML-SLs. Efeknya
parameter yang dipilih, termasuk suhu reaksi ( T r),
konsentrasi enzim ( E c), pelarut aktivitas air awal
( Sebuah w) dan waktu reaksi ( R t), pada hasil biokonversi (%) diselidiki.
2. material dan metode
2.1. Bahan
Minyak biji rami (FO) adalah hadiah dari Arista Industries, Inc. (Wilton, CT, USA). Lipase amobil
komersial terpilih termasuk, Novozym 435 dari Candida antarctica, Lipozyme RM-IM dari Rhizomucor
meihei dan Lipozyme TL-IM dari Thermomyces lanuginosus, diperoleh dari Novozymes Nordisk
A / S (Bagsværd, Denmark). Lipase DF dari Rhizopus oryzea disumbangkan oleh Amano
Enzyme (Nagoya, Jepang). Lipase pankreas babi, garam empedu, tricaprylin (kemurnian> 99%)
dan saringan molekuler berbentuk batang (3 Å) dibeli dari Sigma-Chemical Co. (St-Louis, MO,
USA). Semua pelarut organik tingkat HPLC dan garam tingkat ACS, digunakan untuk
pra-ekuilibrasi reaksi
aktivitas air awal sedang ( Sebuah w), dibeli dari Fish-
425
er Ilmiah (Fair Lawn, NJ, USA). Standar kromatografi gas-cair (GLC) dibeli dari Nu-Check Prep
(Elysian, MN, USA).
2.2. Komposisi asam lemak dan distribusi posisi
2.2.1. Persiapan FAME
Profil asam lemak (FA) dari FO diperoleh dengan analisis GLC dari FA yang dimetilasi. Metil
ester asam lemak (FAMEs) diperoleh sesuai dengan modifikasi metode Rocha-
Uribe dan Hernandez (2004). Lima sampai sepuluhmilligram minyak biji kapak dilarutkan dalam
0,6 mL heksana dan 60 l L natrium metoksida 2 M dalam 20% metanol. Campuran diinkubasi
pada 65 C dalam bak air pengocok timbal balik (Model 25, Precision Scientifc, Chicago, IL,
USA). Setelah 20 menit inkubasi, 1 mL larutan asam sulfat 10% dalam metanol ditambahkan ke
dalam campuran, diikuti dengan inkubasi selama 30 menit dalam penangas air pada suhu 85 C.
FAME diekstraksi dua kali dengan 4 mL heksana dan dipulihkan untuk analisis.
2.2.2. Analisis GLC dari FAME
FAME dianalisis dengan GLC, menggunakan Chromatograph Seri 6890 Agilent (Agilent
Technologies, Wilmington, DE, USA), dilengkapi dengan kolom HP-INNOWax (30 m ·
ID 30 mm · 0.25 l ketebalan film) yang dibeli dari Agilent Technologies, dengan detektor ionisasi
api (FID) dan helium dengan kemurnian sangat tinggi sebagai gas pembawa dengan laju aliran 1
mL / menit dengan rasio pemisahan 1:20. Suhu injektor dan detektor ditetapkan masing-masing
pada 150 dan 230 C. Suhu kolom awal adalah 150 C selama 1 menit sebelum dinaikkan menjadi
180 C, dengan kecepatan 10 C / menit, diikuti dengan laju peningkatan 1 C / menit menjadi 220
C dalam 40 menit dan ditahan di sana selama 5 menit tambahan. FAME diidentifikasi dengan
membandingkan waktu retensi dengan standar. Asam nonadekanoat (19: 0) digunakan sebagai
standar internal untuk penentuan kuantitatif FA.
2.2.3. sn-2 Analisis posisi
Hidrolisis pankreas digunakan untuk menentukan posisi distribusi FAs dalam minyak biji fl
axseed ( Luddy, Barford, Jamu,
Magidman, & Riemenschneider, 1964). Lima miligram minyak biji kapak dicampur dengan 2 mL
buffer Tris-HCl (1 M,
pH 7,6), 0,5 mL 0,05% garam empedu, 0,2 mL CaCl 2,2% 2
dan 3 mg lipase pankreas. Campuran diinkubasi
penangas air pada suhu 37 C selama 3 menit, kemudian vortex dengan kuat dan diekstraksi 2
kali dengan 3 mL dietil eter. Komponen campuran kemudian dipisahkan dengan kromatografi
lapis tipis preparatif (KLT), menggunakan Silica gel 60 GF plate 20. · 20 cm dengan indikator
fluorescent (Whatman, Fisher Scientifc, Fairfield, NJ, USA), dimana sampel yang diekstraksi
diencerkan dalam 100 l L kloroform dan diendapkan pada pelat KLT. Pemisahan dilakukan
sesuai dengan metode yang dijelaskan oleh Hita dkk. (2007) di ruang pengembangan, berisi
campuran pelarut kloroform / aseton / asam asetat (96: 8: 2, v / v / v). Pita yang dipisahkan
divisualisasikan di bawah UV (235 nm) dalam lemari analisis fluoresensi (Spectroline, Model
CX20, Westbury, NY, USA) dan pita yang sesuai dengan 2-MAGs dikerok dan diekstraksi 3 kali
dengan 4 mL dietil eter. Suspensi dari produk yang diekstraksi disaring melalui filter kaca frit dan
dipekatkan
426 ODS JOURNALOFFUNCTIONALFO 5 (2 0 1 3) 4 2 4 - 4 3 3

Sistem SpeedVac (Model AES1010, Savant Instruments; asam kaprilat (COC, CCO, CLnC dan CCLn). Fase gerak, terdiri dari pelarut A, campuran

Holbrook, NY, AS). Produk yang diperoleh kembali dimetilasi, seperti yang dijelaskan toluena / etil asetat / heksana (1: 1: 8, v / v / v) dan pelarut B, campuran heksana / asetonitril

sebelumnya, dan dilakukan analisis GLC. (100: 1, v / v) . Elusi dimulai pada 100% pelarut A dan secara bertahap diubah menjadi 100%

pelarut B dalam waktu 50 menit, kemudian kembali ke 100% pelarut A dalam 20 menit. Pada

2.3. Karakterisasi lipid terstruktur (SLs) akhir proses, kolom diimbangi dengan fase gerak A selama 10 menit. Metode kedua ( Chandler,

2001 ) dilakukan untuk mengukur isomer MLM dan MML asam linoleat dengan asam kaprilat

2.3.1. Analisis spektrometri massa / HPLC fase terbalik (CLaC dan CCLa). Fase gerak terdiri dari pelarut A, campuran toluena / heksana (1: 1, v / v) dan

Pemisahan komponen reaksi dari lipasecatalyzed interesteri fi kation dari FO dengan TC pelarut B, campuran toluena / etil asetat (9: 1, v / v). Elusi dimulai pada 100% pelarut A dan

dilakukan dengan RP-HPLC, menggunakan kolom Zorbax SB-C18 (250 · 4,6 mm, 5 l m) dibeli secara bertahap diubah menjadi 100% pelarut B dalam jangka waktu 20 menit, ditahan selama

dari Agilent Technologies (Wilmington, DE, USA). Sistem elusi terdiri dari campuran metanol / 10 menit sebelum dikembalikan ke 100% pelarut A dalam 10 menit. Pada akhir proses, kolom

asetonitril (5: 7, v / v) sebagai pelarut A dan isopropanol yang mengandung asam format 0,1% diimbangi dengan fase gerak A selama 10 menit.

sebagai pelarut B.Elusi diawali oleh aliran isokratik 100% pelarut A untuk a Periode 5 menit,

diikuti oleh gradien 7 menit hingga 100% pelarut B dan dipertahankan selama 10 menit, diikuti

oleh gradien 6 menit hingga 100% pelarut A dan dipertahankan selama 2 menit. Sampel yang

diinjeksi adalah 10 l L, dengan aliran 0,5 mL / menit.

2.4. Reaksi minat

Komponen reaksi juga menjadi sasaran analisis spektrometri massa / HPLC, menggunakan

sistem spektrometri massa ionisasi kimia tekanan atmosfer (APCI-MS), (ThermoFinnigan, San Reaksi bunga axseed oil (FO) dan tricaprylin (TC) dilakukan dalam 30 mL reactor fl ask.

Jose, CA, USA), yang terdiri dari kromatografi cair Surveyor Plus yang digabungkan dengan Sebelum setiap reaksi enzimatik, larutan stok dengan konsentrasi FO dan TC yang ditentukan

perangkap ion LCQ Advantage dengan Xcalibur System Control Software (Versi 1.3) untuk disiapkan dengan baik n-

akuisisi dan pemrosesan data. Spektrometer massa dioperasikan dalam mode ion positif,

dengan energi disosiasi induksi tumbukan 10 V. Sumber APCI dioperasikan dengan suhu kapiler heksana. Jumlah yang sesuai dari larutan stok FO dan TC dimasukkan ke dalam permintaan fl

150 C, suhu sumber 400 C, tegangan sumber 6,0 kV, dan sumber untuk memperoleh proporsi akhir dari FO ke TC (40: 120 mM) dalam volume reaksi total 3 mL.

Reaksi enzimatik dimulai dengan penambahan sejumlah lipase yang telah ditentukan (0,5–4%, b

/ v, g / mL), yang telah disetarakan sebelumnya dengan larutan garam jenuh. Aktivitas air awal

arus 5.0 l A, dengan selubung dan gas pembantu N 2 di 35 dan 15, masing-masing. ( Sebuah w) disesuaikan dengan menempatkan lipase dan n-

heksana dalam bejana tertutup, berisi garam jenuh pilihan

solusi karakteristik Sebuah w nilai-nilai, termasuk LiCl

2.3.2. Analisis HPLC ion perak ( Sebuah w = 0,11), K (C 2 H. 3 HAI 2) ( Sebuah w = 0,23), MgCl 2 ( Sebuah w = 0,33) dan K 2 BERSAMA 3

Larutan ratusan mikroliter ditarik dari campuran reaksi pada interval waktu tertentu dan dilakukan ( Sebuah w = 0.43). Keseimbangan aktivitas air diperoleh selama periode minimal 3 hari inkubasi

analisis HPLC. Karakterisasi MLM- dan MML-SL yang disintesis, dilakukan dengan pada suhu 4 C untuk padatan en-

menggunakan kolom ChromSpher 5 Lipid (250 · 4,6 mm, Varian Inc., Lake Forest, CA, USA). zyme dan suhu kamar untuk fase pelarut organik.

Sistem HPLC Beckman (Model 126, Beckman Instruments Inc., San Ramon, CA, USA), Dehidrasi ( Sebuah w = 0,05) dari lipase amobil dan n- heksana dilakukan dengan penambahan

dilengkapi dengan detektor pencar laser evaporatif, ELSD (Model II A, Varex Corporation, langsung dari saringan molekuler

Rockville, MD, USA) dan penanganan data terkomputerisasi sebagai serta sistem analisis (3 Å) ke dalam pelarut, ditempatkan dalam wadah tertutup untuk enzim dan dipelihara

integrasi (Karat 32, Beckman Instruments). Kondisi detektor ELSD adalah tekanan 20 psi, aliran semalaman. Permintaan reaktor diinkubasi dalam ketidakjelasan di bawah vakum, pada suhu

nitrogen 50 mL / menit dan suhu 85 C.Volume sampel yang diinjeksikan adalah 20 l L, yang berbeda dengan getaran terus menerus, menggunakan pengocok orbital (New Brunswick

menggunakan elusi fase gerak gradien dengan laju aliran 1 mL / menit. Puncaknya diidentifikasi Scienti fi c Co., Inc., Edison, NJ, USA) untuk periode waktu yang berbeda. Percobaan dijalankan

dengan penggunaan TAG standar yang disintesis secara chemoenzymatically sesuai dengan secara acak, dengan uji coba kontrol dalam kondisi yang sama, tetapi tanpa lipase.

modifikasi dari metode yang dijelaskan oleh Halldorsson, Magnusson, dan Har-

2.5. Pemilihan katalis

Lipase komersial terpilih, termasuk Lipase DF, Novozym

435, Lipozyme TL-IM dan Lipozyme RM-IM, diselidiki untuk efisiensi mereka untuk

aldsson (2001), dengan langkah pemurnian tambahan menggunakan kolom semipreparatif mengkatalisasi n-

Zorbax SB-C18 (250 · 9,4 mm, 5 l m; Agilent Technologies). Kurva kalibrasi dibuat dari berbagai media heksana minyak biji kapak fl dan tricaprylin. Pengaruh parameter yang dipilih, termasuk

konsentrasi MLM-SL yang mengandung asam kaprilat, oleat, linoleat dan linolenat. suhu (30-50 C), konsentrasi enzim (0,5–4%, w / v) dan aktivitas air awal pelarut (0,05-0,43),

pada hasil biokonversi dari MLM- dan MML- yang disintesis SL terutama diselidiki dengan satu

parameter pada satu desain eksperimental waktu untuk setiap enzim. Kecepatan agitasi (150

Pemisahan dan kuanti fi kasi isomer posisi SL yang disintesis dilakukan, menggunakan dua rpm) dan rasio molar substrat TC / FO (3: 1) dipertahankan konstan untuk semua enzimatis

program elusi gradien. Metode pertama ditujukan untuk menghitung isomer MLM dan MML dari

asam oleat dan linolenat dengan

 ODS JOURNALOFFUNCTIONALFO 5 (2 0 1 3) 4 2 4 - 4 3 3 427

reaksi. Untuk memilih lipase yang paling tepat, hasil biokonversi dari SL yang disintesis dimonitor

di bawah kondisi optimal untuk setiap enzim pada interval waktu yang berbeda.

2.6. Calculation of the bioconversion yield

The bioconversion yield (%) was defined as the molar concentration of the synthesized SLs

divided by the molar concentration of the limiting substrate (FO) in the blank at time t,

multiplied by 100.

3. Results and discussion

3.1. Fatty acid profile of flaxseed oil

Fatty acid (FA) composition and positional distribution of the flaxseed oil (FO), used throughout

the study, is shown in Ta-

Fig. 1 – High-performance liquid chromatography analysis of the synthesized diacylglycerols and
ble 1. The results indicate that FO contains 50.28, 17.87 and
triacylglycerols, through the interesterification reaction of flaxseed oil and tricaprylin; The peaks
19.59 mol% of linolenic, linoleic and oleic acids, respectively, as well as saturated FAs, including
are characterized as dicaprylin (peak
4.56 and 7.27 mol% of stearic and palmitic acids, respectively. These results are in agreement

with those reported in literature ( Aguedo et al., 2008;

# 1), caprylyl-linolenyl glycerol (peak #2), tricaprylin (peak

# 3), dicaprylyl-linolenyl glycerol (peak #4), dicaprylyllinoleyl glycerol (peak #5), dicaprylyl-oleyl
Shin et al., 2010). glycerol (peak

The positional distribution of FA residues, at the TAGs, is also of great interest, since it
# 6), caprylyl-dilinolenyl glycerol (peak #7), caprylyllinolenyl-linoleyl glycerol (peak #8),
could provide information regarding the behaviour of the catalyst towards different types of TAGs caprylyl-linolenyl-oleyl glycerol (peak #9), caprylyl-linoleyl-oleyl glycerol (peak #10) and
and to predict the composition of the final product ( Irimescu et al., 2001 ). The experimental caprylyl-oleyl-oleyl glycerol (peak #11).

findings showed that 24.84, 21.86 and 53.18 mol% of oleic, linoleic and linolenic acids,

respectively, were found to be located at the sn- 2 position of the TAGs. The positional

distributions of these FAs are comparable to those reported in literature ( Gunstone & Harwood,

2007; Mu, Xu, & Høy, 1998 ).

spectrometry analyses of the eluted peaks. APCI technique is often used for the analysis of

TAGs throughout HPLC-MS analyses, because of the relatively simple mass spectra and the

possibility of identifying the positional isomers from the relative intensities of the diacylglycerol

3.2. Structural characterization of synthesized SLs fragment ions ( Fauconnot, Robert, Villard, & Dionisi, 2006; Jakab, Héberger,

3.2.1. HPLC/APCI-MS analyses

Fig. 1 shows the HPLC elution profile for the reaction components of lipase-catalyzed & Forgács, 2002). In positive ion APCI mass spectra, the molecular weight of TAG species can

interesterification of FO and TC. The synthesized TAGs were identified through HPLC/APCI-MS be determined from the pseudomolecular [M+H]+ ion, where the intensity increases with the

increase in unsaturation ( Dugo, Kumm, Crupi, Cotro-

neo, & Mondello, 2006), and from the adducts of formic acid [M+HCOOH]+. The characteristic

fragment ions of the type [M RCOO]+ or so-called [DAG]+, originating from loss of one acyl

Table 1 – Composition and positional distribution of fatty acids (FAs) in flaxseed oil (mol%). a moiety [RCOO]+, enabled the identification of all the acyl chains indicated in Fig. 1 ( Fauconnot et

al., 2006; Van Den

FA sn- 1 + sn- 2 + sn- 3 sn- 1 + sn- 3 sn- 2

Berg, Vermist, Carlyle, Holčapek, & Boon, 2004). The m/z values of the characterized ions are
16:0 7.27 (0.23) b 9.67 (1.37) b Tr c
listed in Table 2 ; the TAGs, hence, have been identified according to their diacylglycerol ions
18:0 4.56 (0.35) b 6.65 (0.67) b Tr
([DAG]+) as well as to the pseudomolecular ions ([M+H]+) and [M+HCOOH]+, obtained by the
18:1 19.59 (0.19) b 18.54 (0.98) b ( 57.73) d 24.84 (1.14) b ( 42.27) d
APCI mass spectrum. The reaction mixture contained two intermediate DAGs and a range of
18:2 17.87 (0.54) b 15.43 (0.69) b ( 59.22) d 21.86 (2.24) b ( 40.78) d

18:3 50.28 (2.04) b 49.44 (1.74) b ( 64.74) d 53.18 (0.47) b ( 35.26) d TAGs with 0–6 double bonds and 36–44 equivalent carbon numbers (ECNs). Peaks # 1 and 2 ( Fig.

a Values are mean values ± relative percentage standard deviation (RSD, n = 3). 1 ) were identified as dicaprylin and caprylyl-linolenyl glycerol, respectively. Peak # 3 ( Fig. 1 ) was

characterized as TC and the predominant peaks # 4, 5 and 6 were identified as the main SLs,
b Relative percentage standard deviation was defined as the standard deviation of fatty acid triplicate obtained by the interesterification reaction between FO and TC. The minor peaks # 7, 8, 9, 10
samplings divided by their respective means, multiplied by 100. and 11 were

c< 0.05 mol%.

d Values in parenthesis indicate the percentage of the subject fatty acid located either at the sn- 1 + sn- 3 or at

the sn- 2 position.

428 JOURNALOFFUNCTIONALFOODS5 (2013)424–433

Table 2 – Equivalent carbon numbers (ECNs) and m/z values of characteristic ions of the identified triacylglycerols (TAGs) by HPLC/APCI-MS.

Acyl-FAs a DB b ECNs [M+HCOOH]+ [M+H]+ [M RCOO]+

CCC – 36 516 471 27

CCLn 3 36 650 605 327,461

CCLa 2 38 652 607 327,463

CCO 1 40 654 609 327,465

CLnLn 6 36 784 739 461,595

CLnLa 5 38 786 741 463,597

CLnO 4 40 788 743 461,465,599

CLaO 3 42 790 745 463,467,601

COO 2 44 792 747 465,603

a Fatty acid components of the triacylglycerols: C = caprylic, O = oleic, La = linoleic and Ln = linolenic acid.

b Number of double bonds.

Fig. 2 – Atmospheric pressure chemical ionization-mass spectrometry (APCI-MS) analysis of the major synthesized structured triacylglycerols obtained by lipase-catalyzed interesterification of flaxseed

oil (FO) and tricaprylin (TC). (A) Dicaprylyl-linolenyl glycerol; (B) dicaprylyl-linoleyl glycerol; (C) dicaprylyl-oleyl glycerol; (D) caprylyl-dilinolenyl glycerol; (E) caprylyl-linolenyl-linoleyl glycerol; (F)

caprylyl-linolenyl-oleyl glycerol; (G) caprylyl-linoleyl-oleyl glycerol; (H) caprylyl-oleyloleyl glycerol.

 JOURNALOFFUNCTIONALFOODS5 (2013)424–433 429

characterized as the TAGs, containing 2 unsaturated acyl moieties along, with a substituted

caprylic acid.

The fragmentation patterns of the structured TAGs, obtained by the interesterification

reaction, are presented in Fig. 2. The fragmentation of Peak #4 ( Fig. 1 ) resulted in a

characteristic pattern of dicaprylyl-linolenyl glycerol ( Fig. 2 a), with an abundant molecular ion of

[M+HCOOH]+ at m/z 648.4 and a protonated ion of [M+H]+ at m/z 605.4 as well as

fragmentation ions of [M C8:0]+ and [M C18:3]+ at m/z 461.4 and

327.3, characterized as caprylyl-linolenyl glycerol and dicaprylyl glycerol, respectively. Peak #5 ( Fig.

1 ) indicates the presence of abundant fragment ions at m/z of 463.4 and 327.3 ( Fig. 2 b)

corresponding to [M C8:0]+ and [M C18:2]+ and molecular ions of [M+HCOOH]+ and [M+H]+ at m/z

values of

652.4 and 607.4, respectively, representing dicaprylyl-linoleyl glycerol. Peak #6 ( Fig. 1 ) was

characterized as dicaprylyl-oleyl glycerol, with abundant fragment ions of [M C8:0]+ and [M

Fig. 3 – Chromatogram of silver-ion exchange highperformance liquid chromatography (HPLC)
C18:1]+ at m/z of 465.4 and 327.3 ( Fig. 2 c), respectively, along with a molecular ion of
analysis of structured
[M+HCOOH]+ at m/z of 654.3. Peak #7 ( Fig. 1 ) produced two abundant molecular ions of [M+H]+
lipids obtained by lipase-catalyzed
and [M+HCOOH]+ at m/z of 739.6 and 784.4, respectively, corresponding to caprylyl-dilinolenyl
interesterification of tricaprylin (TC) and flaxseed oil (FO) using a gradient elution of, solvent A:
glycerol as well as fragment ions of [M C18:3]+, [M C8:0]+ and [MAG]+ at m/ z values of 461.5,
toluene/ethyl acetate/ hexane (1:1:8, v/v/v), and solvent B: hexane/acetonitrile (100:1, v/v),
595.6 and 335.3, representing caprylyl-linolenyl glycerol, linolenyl-linolenyl glycerol and linolenin,
monitored by evaporative laser scattering detector (ELSD); Peaks characterized as TC (peak
respectively ( Fig. 2 d). The fragmentation of peak #8 ( Fig. 1 ) resulted in fragment ions of [M
#1), 1,3dicaprylyl-2-oleyl glycerol (peak #2), 1(3),2-dicaprylyl-3(1)oleyl glycerol (peak #3),
C18:3]+ and [M C8:0]+ at m/z of
1(3),2-dicaprylyl-3(1)-linolenyl glycerol (peak #4), and 1,3-dicaprylyl-2-linolenyl glycerol (peak

#5), respectively.

463.4 and 597.6 ( Fig. 2 e) corresponding to caprylyl-linoleyl glycerol and linoleyl-linolenyl glycerol,

respectively, with molecular ions of [M+HCOOH]+ and [M+H]+ at m/z of 786.4 and 741.6,

respectively, suggesting the presence of caprylyllinolenyl-linoleyl glycerol. Peak #9 ( Fig. 1 ) was ( Mondello et al., 2005 ). Overall, the stability of the silver iondouble bond complex is influenced by

characterized as caprylyl-linolenyl-oleyl glycerol, with abundant molecular ions ( Fig. 2 f) of the spatial arrangement of the overlapping orbitals, the basicity of electronic pairs in the olefinic

[M+HCOOH]+ and [M+H]+ at m/z ratio of 788.4 and 743.6, respectively, as well as fragment ions molecule and the solvent effect ( Nonadek,

of [M C18:1]+, [M C18:3]+ and [M C8:0]+ at m/z of 461.4, 465.4 and 599.6 corresponding to

caprylyl-linolenyl glycerol, caprylyl-oleyl glycerol and linolenyl-oleyl glycerol, respectively. The 1992).

fragmentation of peak #10 ( Fig. 1 ) resulted in fragment ions of [M C18:2]+, [M C8:0]+ and [M Figs. 3 and 4 illustrate the chromatograms of silver-ion RPHPLC, obtained using the two

C18:1]+ at m/z of different gradient elutionmethods mentioned in Section 2.3.2 . The applied gradient methods

enabled the separation of asymmetric SLs (CCO, CCLa and CCLn) from their symmetric

positional isomers (COC, CLaC and CLnC), obtained by the enzymatic interesterification of FO

and TC, and to determine the ratio of MLM-SLs to their corresponding positional MML isomers.

The characterization of the positional isomers of SLs via silver-ion chromatography has been

467.4, 601.6 and 463.5 ( Fig. 2 g), with an abundant molecular ion of [M+HCOOH]+ at m/z of reported for the transesterification products of single-cell oil, containing docosahexaenoic acid

790.5 suggesting the presence of caprylyl-linoleyl-oleyl glycerol. Similarly, the fragmentation of (DHA) and docosapentaenoic acid (DPA), with caprylic acid ( Iwasaki, Han,

peak #11 ( Fig. 1 ) led to major fragment ions of [M C18:1]+ and [M C8:0]+ at m/z of 465.4 and

603.6 ( Fig. 2 h), corresponding to caprylyl-oleyl glycerol and diolein, respectively, as well as the

molecular ions of [M+HCOOH]+ and [M+H]+, with an m/z of 792.5 and 747.6, respectively,

characterized as caprylyl-oleyl-oleyl glycerol.

Narita, Rosu, & Yamane, 1999), triolein with decanoic acid ( Chandler, 2001 ), tripalmitin with oleic

acid ( Nagao et al.,

2001) and tripalmitin-rich fraction with ethyl oleate ( Lee,

Son, Akoh, Kim, & Lee, 2010).

3.2.2. Silver-ion HPLC analysis With reference to the chemoenzymatically synthesized standards, in which their structure

Silver-ion chromatography, has proven to be a powerful tool for the separation and quantification and purity have been confirmed by HPLC/APCI-MS analyses, the MLM- and MML-SLs have

of TAG isomers as well as those mixtures containing a wide range of FAs with different chain been identified. As illustrated in Fig. 3 , using the first gradient method, tricaprylin (peak #1) was

lengths ( Adlof, 1997 ). The retention of TAGs or other olefinic compounds by silver-ion eluted first, followed by 1,3-dicaprylyl-2-oleyl glycerol (peak #2), 1(3),2-dicaprylyl-3(1)-oleyl

chromatography is due primarily to the interaction of the silver ions and the olefinic p glycerol (peak #3), 1(3),2-dicaprylyl-3(1)linolenyl glycerol (peak #4) and 1,3-dicaprylyl-2-linolenyl

glycerol (peak #5). Fig. 4 shows that in the second applied gradient method, tricaprylin (peak #1)

was eluted first,

electrons of the biomolecules ( Adlof & List, 2004 ). The elution sequence is dependent on the

degree of unsaturation and on the position/configuration of the double bond, within each FA
430 JOURNALOFFUNCTIONALFOODS5 (2013)424–433

25

A
20

15

10

0
20 B

15

10

Bioconversion Yield (%)

Fig. 4 – Chromatogram of silver-ion exchange highperformance liquid chromatography (HPLC)

analysis of structured lipids obtained by lipase-catalyzed interesterification of tricaprylin (TC) and 5

flaxseed oil (FO) using a gradient elution of, solvent A: toluene/hexane (1:1, v/v) and solvent B:

toluene/ethyl acetate (9:1, v/v), monitored by evaporative laser scattering detector (ELSD);
0
Peaks characterized as TC (peak #1), 1,3-dicaprylyl-2-linoleyl glycerol 20 C

15

(peak # 2), and 1(3),2-dicaprylyl-3(1)-linoleyl

10
glycerol (peak #3), respectively.

0
20
Table 3 – Optimum reaction conditions obtained for selected lipases in the D
interesterification of flaxseed oil and tricaprylin.

15

Lipase type Optimum reaction conditions

c 10
R ta T rb Ec aw

Amano DF 72 40 2 0.23
5
Lipozyme TL-IM 4 50 2 0.23

Novozym 435 24 50 1 0.23

Lipozyme RM-IM 8 30 2 0.05 0

0 15 30 45 60 75 90
a Reaction time (h).

b Reaction temperature ( C). Reaction Time (h)

c Enzyme concentration (% w/v).
Fig. 5 – Time course corresponding to the bioconversionyield of

1,3-dicaprylyl-2-linolenyl glycerol (CLnC), 1(3),2-

dicaprylyl-3(1)-linolenyl glycerol (CCLn), 1,3-dicaprylyl-2linoleyl

followed successively by 1,3-dicaprylyl-2-linoleyl glycerol (peak #2) and glycerol (CLaC), 1(3),2-dicaprylyl-3(1)-linoleyl

1(3),2-dicaprylyl-3(1)-linoleyl glycerol (peak #3). glycerol (CCLa), 1,3-dicaprylyl-2-oleyl glycerol (COC) and 1(3),2-dicaprylyl-3(1)-oleyl glycerol

(CCO), in the interesterification of tricaprylin and flaxseed oil catalyzed by lipase Novozym 435

3.3. Lipase-catalyzed interesterification of SLs (A), Lipozyme TL-IM (B), Lipozyme RM-IM (C) andAmanoDF (D); CLnC ( j), CCLn ( h), CLaC (.),

CCLa ( 4), COC ( d) and CCO ( s).

The selected commercial lipases, used throughout this study, include a free one, Amano DF, and

immobilized ones, Novozym 435, Lipozyme RM-IM and Lipozyme TL-IM. Before monitoring the

time course for the synthesis of MLM- and MML-SLs, the efficiency of each lipase to catalyze the

interesterification reaction between tricaprylin (TC) and TAGs of FO was investigated with one

parameter at a time method, ters were used for investigating the bioconversion yield of MLM- and MML-type SLs, including

COC, CCO, CLaC, CCLa, CLnC and CCLn, obtained at a constant substrate molar ratio of 3:1

(120:40 mM) of TC to FO, with 150 rpm agitation speed.

using different levels of temperature ( T r, 30–50 C), enzyme concentration ( E c, 0.5–4.0%, w/v) and

initial water activity The investigated lipases catalyzed the incorporation of caprylic acid into the TAGs of FO at

( a w, 0.05–0.43). The optimum conditions, selected for each enzyme, are listed in Table 3 . The various ranges and different rates ( Fig. 5 ). The maximum incorporation degree was

established reaction parame-

 JOURNALOFFUNCTIONALFOODS5 (2013)424–433 431

Table 4 – Maximum bioconversion yield ratio of the positional structured lipid isomers, obtained by the interesterification of tricaprylin (TC) and flaxseed oil (FO), catalyzed by selected lipases.

Lipase type Bioconversion yield ratio a

COC b / CCO c CLaC d / CCLa e CLnC f / CCLn g

Amano DF (72 h) 0.30 0.32 4.78

Lipozyme TL-IM (4 h) Novozym 435 0.60 0.58 2.11

(24 h) Lipozyme RM-IM (8 h) 0.49 0.42 2.09

0.49 0.41 2.00

a Bioconversion yield ratio is defined as the ratio of the bioconversion yield (%) of the synthesized MLM to the corresponding MML isomer.

b COC is 1,3-dicaprylyl-2-oleyl glycerol.

c CCO is 1(3),2-dicaprylyl-3(1)-oleyl glycerol.

d CLaC is 1,3-dicaprylyl-2-linoleyl glycerol.

e CCLa is 1,3-dicaprylyl-2-linoleyl glycerol.

f CLnC is 1,3-dicaprylyl-2-linolenyl glycerol.

g CCLn is 1(3),2-dicaprylyl-3(1)-linolenyl glycerol.

obtained in the order of Novozym 435 > Lipozyme TLIM > Lipozyme RM-IM > Amano DF. Similar denaturalization of free enzymes in organic solvents, hence, leading to a lower catalytic activity ( Zhao

results were reported by Kim et al. (2001) , who indicated a higher incorporation of conjugated et al., 2007 ). On the other hand, the immobilization of enzymes would increase the contact

linoleic acid into TC by Novozym 435 as compared to that by Lipozyme IM. Zhao, Lu, Bie, Lu, surface area of lipase with the substrates and improve the enzyme stability in organic solvents,

and Liu resulting in enhancing its activity ( Sheldon, 2007 ).

(2007) screened selected lipases and reported that the highest incorporation of capric acid into

lard was obtained by Lipozyme TL-IM. Hamam, Daun, and Shahidi (2005) reported that among The ratio of the catalyzed MLM- to MML-SLs of each unsaturated FA was compared ( Table

selected lipases, screened for the acidolysis of high-laurate canola oil with eicosapentaenoic acid 4 ). The results indicate that the ratio was in the order of Amano DF > Lipozyme TLIM > Novozym

(EPA), lipase PS-30 from Pseudomonas sp. contributed to the highest incorporation, followed by 435 P Lipozyme RM-IM for the major CLnC and CCLn isomers.

Novozym 435, from C. antarctica,

that showed a higher incorporation as compared to that of Lipozyme IM, from R. meihei. Moreover, 4. Conclusion
Hamam and Shahidi

(2006) investigated the incorporation of EPA, docosapentaenoic acid (DPA) and The overall results demonstrated that the synthesis of structured lipids (SLs) could be achieved
docosahexaenoic acid (DHA), in highlaurate canola oil, and reported that there was no significant by the interesterification of flaxseed oil and tricaprylin in organic solvent medium (OSM). The
difference in the EPA incorporation when lipases Novozym 435 from C. antarctica, AP-12 from Aspergillus
structure of the synthesized TAGs, as well as the positional isomers of the MLM- and MML-SLs
niger and Lipozyme IM from R. meihei were employed; however, were determined. The selected investigated commercial lipases catalyzed the interesterification

reaction at different ranges and rates, where the maximal incorporation degree was obtained in

the order of Novozym 435 > Lipozyme TL-IM > Lipozyme RM-IM > Amano DF. Furthermore, the
Lipozyme IM incorporated the highest amount of DPA, whereas Novozym 435 incorporated the experimental data indicated that the MLM/MML ratio was in the order of that obtained
higher amount of DHA as compared to that with Lipozyme IM. Kim, Kim, Lee,

Chung, and Ko (2002) reported that there was no difference in caprylic acid incorporation by the

acidolysis of perilla oil, using Lipozyme RM-IM and Lipozyme TL-IM.

by Amano DF > Lipozyme TL-IM > Novozym

In contrast to the present results, Carrin and Crapiste 435 P Lipozyme RM-IM for the major synthesized isomers.
(2008) reported that Novozym 435 showed much lower activity than that of Lipozyme RM-IM and

TL-IM for the incorporation of a mixture of palmitic and stearic acids into sunflower oil. For the
Acknowledgement
interesterification of butterfat, flaxseed oil and palm stearin, Shin et al. (2010) indicated a higher

interesterification degree, within the first 6 h, by Lipozyme RM-IM, as compared to that by

This research was supported by a Discovery Grant from the Natural Sciences and Engineering
Novozym 435; however, similar results were obtained for both enzymes after 24 h of acidolysis. Yang,
Research Council of Canada (NSERC).

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