influence of extraction solvent system on extractability of lipid components from different...

Algal Research xxx (2013) xxx–xxx

ALGAL-00079; No of Pages 8

Contents lists available at ScienceDirect

Algal Research

j ourna l homepage: www.e lsev ie r .com/ locate /a lga l

Influence of extraction solvent system on extractability of lipidcomponents from different microalgae species

Eline Ryckebosch a,b, Charlotte Bruneel a,b, Romina Termote-Verhalle a,b,Koenraad Muylaert c, Imogen Foubert a,b,⁎a KU Leuven Kulak, Research Unit Food & Lipids, Department of Molecular and Microbial Systems Kulak, Etienne Sabbelaan 53, 8500 Kortrijk, Belgiumb Leuven Food Science and Nutrition Research Centre (LFoRCe), KU Leuven, Kasteelpark Arenberg 20, 3001 Heverlee, Belgiumc KU Leuven Kulak, Laboratory of Aquatic Biology, Biology Department Kulak, Etienne Sabbelaan 53, 8500 Kortrijk, Belgium

Abbreviations: CM, chloroform/methanol; DHA, docoEPA, eicosapentaenoic acid (C20:5n−3); FFA, free fatty aHI, hexane/isopropanol (3:2); NL, neutral lipids; Omega-polyunsaturated fatty acids; PL, phospholipids; RDI, rectriacylglycerol.⁎ Corresponding author at: KU Leuven Kulak, Research

of Molecular and Microbial Systems Kulak, Etienne SabbeTel.: +32 56 24 61 73; fax: +32 56 24 69 97.

E-mail address: [email protected] (

2211-9264/$ – see front matter © 2013 Published by Elsehttp://dx.doi.org/10.1016/j.algal.2013.11.001

Please cite this article as: E. Ryckebosch, etmicroalgae species, Algal Res. (2013), http://

a b s t r a c t
a r t i c l e i n f o
Article history:Received 14 June 2013Received in revised form 29 October 2013Accepted 2 November 2013Available online xxxx

Keywords:Total lipidsLipid classesOmega-3 LC-PUFACarotenoids(Phyto)sterolsFood grade solvents

The purpose of this workwas to evaluate two food grade solvent systems, hexane/isopropanol (3:2; HI) and hex-ane (H), for the extraction of lipids from different omega-3 LC-PUFA rich microalgae: Isochrysis galbana,Nannochloropis gaditana,Nannochloropsis sp. and Phaeodactylum tricornutum. We not only focused on differencesin lipid yield, but also lipid class, omega-3 LC-PUFA, carotenoid and sterol yields. Furthermore, an estimation ofthe feasibility of these microalgae oils as an alternative for fish oil was made. For all tested microalgae species,the highest food grade lipid, lipid class, omega-3 PUFA, carotenoid and sterol yield were obtained with HI, witha general recovery highest from Isochrysis, lowest from both Nannochloropsis species, and intermediate fromPhaeodactylum. Total lipid recovery values between 14 and 76% depending on solvent and species were obtained.It was also shown that the omega-3 fatty acid content of all oils was quite similar, while only the H extract wasenriched in neutral lipids. Carotenoidswere co-extracted in a significant amount, although the content in the var-ious oils was quite different.

© 2013 Published by Elsevier B.V.

1. Introduction

Microalgae are the primary producers of the omega-3 long chain poly-unsaturated fatty acids (omega-3 LC-PUFA), such as eicosapentaenoicacid (EPA, C20:5n−3) and docosahexaenoic acid (DHA, C22:6n−3),and are therefore considered a promising alternative for fish oil as sourceof these fatty acids [1]. The daily intake of omega-3 LC-PUFA is still belowthe recommendeddose [2], although their importance to humanhealth isknownworldwide [3]. The need for new sources of these fatty acids is in-creasing, since fish cannot supply global needs for omega-3 LC-PUFA [4]and global fish stocks are even further declining.

Microalgae can be cultured either heterotrophically or photoauto-trophically. During heterotrophic cultivation, which is usually achievedin closed fermenters, microalgae take up organicmolecules as a primarysource of nutrition, while photoautotrophic production refers to theability of microalgae to manufacture organic material from inorganicmatter (CO2) in the presence of light. The latter is more sustainable,

sahexaenoic acid (C22:6n−3);cid; GL, glycolipids; H, hexane;3 LC-PUFA, omega-3 long chainommended daily intake; TAG,

Unit Food & Lipids, Departmentlaan 53, 8500 Kortrijk, Belgium.

I. Foubert).

vier B.V.

al., Influence of extraction sdx.doi.org/10.1016/j.algal.201

because of the use of CO2 as the only carbon source. Omega-3 LC-PUFA from heterotrophic cultivation are already available on the mar-ket, while photoautrophic microalgae as alternative source are still inthe research phase [5]. In a previous study [5], it was shown that thetotal lipid extract of several autotrophicmicroalgae, including Isochrysis,Nannochloropsis, Pavlova, Phaeodactylum, and Thalassiosira containsomega-3 LC-PUFA in a sufficient amount to make a good alternativefor fish oil. The study also showed that the glyco- and phospholipid frac-tion of these oils contains a significant part of the omega-3 LC-PUFA.This is potentially interesting, since this may lead to an increased ab-sorption of omega-3 LC-PUFA [6] and an increased oxidative stabilityof the oil [7,8] compared to triacylglycerol (TAG) oils. The same studyalso showed that microalgae oils contain carotenoids and phytosterols,nutritionally important compounds that can add value to these oils ina number of ways [5].

Although there is a worldwide increasing interest in EPA and DHAfrom microalgae, there is no standardized method for extraction yet.The unicellular nature of microalgae and the interest in polar lipids,and additionally also in carotenoids and phytosterols, make that stan-dard extraction technologies are probably not the best way to recoveroil from microalgae. Some studies have already been published on thelipid extraction from microalgae, although most of them focused onthe recovery of neutral lipids for biodiesel production. Different solvent(mixtures) were compared in their ability to extract lipids frommicroalgae. A combination of chloroform and methanol was alwaysfound to be best to recover lipids from microalgae [9–12] while

olvent system on extractability of lipid components from different3.11.001

http://dx.doi.org/10.1016/j.algal.2013.11.001

mailto:[email protected]

Unlabelled image


Unlabelled image

http://www.sciencedirect.com/science/journal/


Table 1Total lipid content, lipid class content, omega-3 LC-PUFA content, free fatty acid content, %EPA/DHA in lipid classes, carotenoid content and sterol content of the differentmicroalgaespecies.

Isochrysisgalbana

Nannochloropsisgaditana

Nannochloropsissp.

Phaeodactylumtricornutum

Total lipidcontenta

27.7 ± 0.5 28.2 ± 0.9 30.2 ± 0.3 17.8 ± 0.3

Lipid class contentb

Neutral lipids(NL)

16.3 ± 0.4 8.2 ± 0.3 14.0 ± 0.2 6.4 ± 0.2

Glycolipids(GL)

5.8 ± 0.2 10.3 ± 0.5 7.0 ± 0.2 6.6 ± 0.1

Phospholipids(PL)

5.5 ± 0.2 9.7 ± 0.4 9.1 ± 0.1 5.9 ± 0.1

Omega-3 LC-PUFA contentc

EPA(C20:5n−3)

50 ± 4 3625 ± 156 3883 ± 85 1640 ± 199

DHA(C22:6n−3)

1482 ± 106 – – 113 ± 15

% EPA/DHA in lipid classesd

Neutral lipids(NL)

0.43 ± 0.06/13.0 ± 0.5

10.7 ± 0.1/− 19 ± 7/− 13.3 ± 0.1/0.90 ± 0.00

Glycolipids(GL)

0.23 ± 0.06/5.2 ± 0.3

39.6 ± 0.4/− 51.1 ± 0.4/− 36.3 ± 0.1/0.53 ± 0.06

Phospholipids(PL)

0.7 ± 0.1/8.8 ± 0.4

18.2 ± 0.3/− 22.8 ± 0.3/− 12.9 ± 0.2/2.47 ± 0.06

Free fatty acid contentb

Total 5.9 ± 0.3 0.8 ± 0.1 0.8 ± 0.2 0.46 ± 0.01

Carotenoid contente

Carotene 89 ± 15 115 ± 8 116 ± 12 48 ± 4Diadinochromef 30 ± 2 – – –

Diadinoxanthin – 144 ± 10 118 ± 10 112 ± 9Diatoxanthin 74 ± 2 – – 50 ± 4Fucoxanthin 974 ± 58 – – 621 ± 49Lutein – 21 ± 1 – –

Neoxanthin – 11 ± 2 – –

Violaxanthin – 445 ± 26 417 ± 34 –

Zeaxanthin – 92 ± 3 20 ± 2 –

Sterol contente

Cholesterol – 376 ± 14 536 ± 6 –

Phytosterols 303 ± 14 336 ± 14 210 ± 3 202 ± 5

a Total lipid content is expressed in g/100 g DW. Mean and standard deviation of 12replicates are reported.

b Lipid class content and free fatty acid content are expressed in g/100 g DW.Mean andstandard deviation of 3 replicates are reported.

c Omega-3 LC-PUFA content is expressed in mg/100 g DW. Mean and standard devia-tion of 3 replicates are reported.

d % EPA/DHA in lipid classes is expressed in % FAME of total FAMEs of that class. Meanand standard deviation of 3 replicates are reported.

e Carotenoid and sterol content are expressed inmg/100 g DW.Mean and standard de-viation of 3 replicates are reported.

f The diadinochrome content was estimated using the calibration curve ofdiadinoxanthin.

2 E. Ryckebosch et al. / Algal Research xxx (2013) xxx–xxx

dichloromethane and methanol or ethanol performed (almost) equallywell [9,12]. However, due to the (potentially) carcinogenic nature, thevolatility and corrosive character of dichloromethane, its use is highlyregulated. Therefore, less toxic, non-halogenated solvents must beconsidered as an alternative. Addition of a polar alcohol to a non-polarsolvent was shown to be necessary to obtain a reasonable extractionefficiency [9,11,12]. Hexane/methanol (3:2), hexane/isopropanol (3:2),and cyclohexane/1-butanol (9:1) proved to be the best non-halogenated solvent mixtures [9,12,13]. However, all previous studiestested solvents on only one species ofmicroalga,mostlyNannochloropsis,thus it is not clear whether solvent (mixtures) perform equally well forother microalgae species. To the best of our knowledge, no researchhas been published yet on comparing different extraction solvents fordifferent species of microalgae in one study.

The purpose of this work was therefore to evaluate two food gradesolvent systems for the extraction of lipids from different omega-3 LC-PUFA rich microalgae: Isochrysis galbana, Nannochloropis gaditana,Nannochloropsis sp. and Phaeodactylum tricornutum. These microalgaewere selected based on Ryckebosch et al. [5], ease of cultivation anddifferences in cell wall composition (see discussion for more details).Hexane/isopropanol (3:2) was evaluated, as in a previous study it wasfound to be the best non-halogenated solvent mixture [12], as well ashexane, which is currentlymost commonly used for commercial extrac-tion of food lipids and also for extraction of omega-3 LC-PUFA contain-ing triglycerides from heterotrophic microalgae. Not only the lipidyield was evaluated, but also the lipid class, omega-3 LC-PUFA, caroten-oid and sterol yield were examined. The main focus was set on thedifferences between different microalgae species. Furthermore, the im-plications of the choice of extraction solvent for the feasibility ofmicroalgae oils as an alternative for fish oil were investigated.

2. Material and methods

2.1. Microalgae biomass

Biomass of omega-3 LC-PUFA producing photoautotrophic micro-algae was obtained from European companies. Nannochloropsisgaditanawas obtained from LGem (Voorhout, The Netherlands), whileNannochloropsis sp. and Isochrysis galbana were from Proviron(Hemiksem, Belgium). Biomass of Phaeodactylum tricornutum Pt1 8.6was obtained fromCCMP2561 in the Provasoli–Guillard National Centerfor Culture of Marine Phytoplankton (East Boothbay, Maine, US). It wasproduced in a high quantity by LGem (Voorhout, The Netherlands). Thetotal lipid, lipid class, omega-3 fatty acid, free fatty acid, carotenoid andsterol content of the different microalgae species was determined ac-cording to the methods described in Sections 2.2. to 2.7. The resultswere summarized in Table 1. The biomass composition of thesemicroalgae was within the range found in literature.

2.2. Extraction of microalgae biomass

The total lipid extract was obtained by extraction with chloroform/methanol 1:1 as described by Ryckebosch et al. [11]. Therefore,100 mg of freeze-dried algae is mixed with 4 mL of methanol, 2 mL ofchloroform and 0.4 mL of water. After vortexing, 2 more mL of chloro-form andwater is added. After another vortexing step and a centrifuga-tion step, the solids are re-extracted with 4 mL chloroform/methanol1:1, again followed by vortexing and centrifugation. This procedure isrepeated two more times. The four combined solvent layers are mixedto obtain the total lipid extract.

The extractions with hexane/isopropanol (HI, 3:2) and hexane (H)were performed somewhat different: during extraction, the microalgaecells were disrupted with a bead beater, twice for 60 s at 30 Hz. Subse-quently, the biomass was extracted four times with the extraction sol-vent (mixture). All the extracts were again combined to form the finalextract.

Please cite this article as: E. Ryckebosch, et al., Influence of extraction smicroalgae species, Algal Res. (2013), http://dx.doi.org/10.1016/j.algal.201

2.3. Lipid part of the extract

Since the food grade extracts were not washedwithwater, theymaynot only contain lipids but alsomore polar compounds such as carbohy-drates and proteins. To eliminate these non-lipid polar components, thelipid part of the obtained extracts was also determined. To do so, ex-tracts are dissolved in 8 mL chloroform/methanol (1:1) and washedwith 2 mL water. The top layer, containing the non-lipid material, wasremoved, while the bottom layer, containing the lipids, was dried andweighed. The lipid part of the extract was determined in triplicate.

2.4. Analysis of lipid class content

The lipid class content (neutral lipid, glycolipid and phospholipidcontent) of the extracts was determined using silica solid phase



Fig. 1. Extraction yield (in g/100 g DW; mean ± SD; n = 3) obtained with differentextraction solvent (mixtures) (chloroform/methanol, CM; hexane/isopropanol, HI;hexane, H) from the different microalgae species. The non-lipid fraction as well as thelipid fraction is represented.

3E. Ryckebosch et al. / Algal Research xxx (2013) xxx–xxx

extraction (SPE) as previously described in Ryckebosch et al. [11]. Brief-ly, the SPE column is conditionedwith 10 mL chloroform. Then, approx-imately 10 mg lipids in 100 μL chloroform are applied to the column.Elution with 10 mL chloroform yields the non-polar lipids, 10 mL ace-tone gives the glycolipid fraction and 10 mL methanol yields the phos-pholipids. The lipid class content was determined in triplicate.

2.5. Analysis of fatty acid content and composition

To determine the fatty acid content, the lipid extracts were methyl-ated with 1% sulfuric acid in methanol, followed by extraction of the re-quired methyl esters with hexane, according to Christie [14] with slightadjustments previously described by Ryckebosch et al. [11]. The obtain-ed fatty acid methyl esters (FAMEs) were separated by gas chromatog-raphy with cold on-column injection and flame ionization detection(FID) (Trace GC Ultra, Thermo Scientific, Interscience, Louvain-la-Neuve, Belgium). An EC Wax column of length 30 m, ID 0.32 mm,film 0.25 μm (GRACE, Lokeren, Belgium) is used with the followingtime–temperature program: 70 °C–180 °C (5 °C/min), 180 °C–235 °C(2 °C/min), 235 °C (9.5 min). Peak areas are quantifiedwith Chromcardfor Windows software (Interscience, Louvain-la-Neuve, Belgium).FAME standards (Nu-check, Elysian, USA) containing a total of 35 differ-ent FAMEs are analyzed for provisional peak identification, which arethen confirmed by use of GC–MS (Trace GCUltra, ISQ Single QuadrupoleMS, Thermo Scientific, Interscience, Louvain-la-Neuve, Belgium) usingan Rxi-5 Sil MS column of length 20 m, ID 0.18 mm, film 0.18 μm(Restek, Interscience, Louvain-la-Neuve, Belgium). Quantitative andqualitative determination of the fatty acids was performed in triplicate.

2.6. Free fatty acid content

The free fatty acid content was determined following the procedurepreviously described [15] and based on themethod for selective forma-tion of dimethyl amide derivatives of Kangani et al. [16]. The dimethylamide derivatives were analyzed on a gas chromatograph with coldon-column injection and flame ionization detection (FID) (Trace GCUltra, Thermo Scientific, Interscience, Louvain-la-Neuve, Belgium) ac-cording to the method described by Ryckebosch et al. [15]. The time–temperature program was extended to: 100 °C–160 °C (10 °C/min),160 °C–240 °C (2 °C/min), 240 °C (54 min). The determination of FFAcontent was performed in triplicate.

2.7. Analysis of carotenoid composition and content

For the determination of the carotenoid content and composition,2 mg of the extract was dissolved in 10 mL methanol. This solutionand a 1/10 dilution were analyzed by high performance liquid chroma-tography (HPLC) coupled to a photodiode array detector (PAD) (Alli-ance, Waters, Zellik, Belgium) according to Wright et al. (1991) [17].For quantification, calibration curves were composed for each caroten-oid. Alloxanthin, diadinoxanthin, diatoxanthin, lutein, neoxanthin,violaxanthin and zeaxanthin were purchased from DHI (Hørsholm,Denmark). β-carotene was purchased from Sigma-Aldrich (Bornem,Belgium). When the area of a carotenoid exceeded the calibrationcurve, the 1/10 dilution was used to quantify. The analysis was per-formed in triplicate.

2.8. Analysis of cholesterol and phytosterol content

For the determination of the sterol content, 5β-cholestan-3α-ol(200 μg; Sigma-Aldrich, Bornem, Belgium) was, for quantificationpurposes, first added to extracts. Then, saponification was performedaccording to Abidi (2004) [18], with some modifications. Briefly, 10–20 mg of the oil was stirred overnight with potassium hydroxide(1 M) in ethanol (4 mL). Water (4 mL) was added to the reaction mix-ture followed by three sequential extractions with diethylether (8 mL).


The ether extracts were combined and the solventwas removed using arotary evaporator, giving the nonsaponifiable fraction. Finally, the sterolcomponentswere silylated according to Toivo et al. (2000) [19]. For this,anhydrous pyridine (200 μL) and derivatization reagent (200 μL) con-taining BSTFA (99%) and TMCS (1%) were added to the nonsaponifiablefraction. To complete the silylation, solutions were incubated at 60 °Cfor 1 h. Before GC-analysis, the solution was diluted with 600 μL hex-ane. The silylated sterols were separated by gas chromatography withcold on-column injection and flame ionization detection (FID). AnRtx-5 column (length 30 m, ID 0.25 mm, film 0.25 μm) (Restek,Interscience, Louvain-la-Neuve, Belgium) was used with the followingtime–temperature program: 200–340 °C at 15 °C/min, 340 °C(10 min). Peak areas were quantified with Chromcard for Windowssoftware (Interscience, Louvain-la-Neuve, Belgium). Peak identificationwas confirmed by use of GC–MS (Trace GC Ultra, ISQ Single QuadrupoleMS, Thermo Scientific, Interscience, Louvain-la-Neuve, Belgium) usingan Rxi-5 Sil MS column of length 20 m, ID 0.18 mm, film 0.18 μm(Restek, Interscience, Louvain-la-Neuve, Belgium). Cholesterol wasidentified separately. All other peaks showing a sterol backbone weresummed, resulting in the total phytosterol content. The analysis wasperformed in triplicate.

2.9. Statistics

Multiple results were statistically evaluated using one-way analysisof variance (ANOVA) and a post hoc Tukey Test withα = 0.05. Two re-sults were compared using a t-test with α = 0.05. Single results werestatistically compared to 100 using a one-sample t-test with α = 0.05(Sigmaplot 11, Systat Software Inc., Chicago).

3. Results and discussion

3.1. Extraction yield, lipid class content and lipid (class) recoveries

The extraction yield obtained for the different microalgae with thedifferent solvent systems is summarized in Fig. 1, showing the total ex-traction yield and the amount of non-lipids in the extract. For the CMextract, this division in lipids and non-lipids is not indicated since thenon-lipid components were already eliminated during the washingstep of the extraction itself. H extraction leads to the lowest amount oflipids, while HI extraction showed an extraction yield intermediate be-tween CM and H. As expected, extraction with CM yielded the mostlipids. After all, this method has already been shown to be the referencemethod to extract total lipids from microalgae [11]. For this reason, the


image of Fig.�1


Fig. 2. Lipid class content (in % of oil extract; mean ± SD; n = 3) obtained with differentextraction solvent (mixtures) (chloroform/methanol, CM; hexane/isopropanol, HI; hex-ane, H) from the different microalgae species.


percent recovery of lipids due to extraction with HI and H from the dif-ferent microalgae species is defined as the yield obtained with HI and Hrespectively divided by the total yield obtained with the referencemethod (CM—Table 1) multiplied by 100. The values are presented inTable 2. The lipid class content of the different oil extracts from the dif-ferent microalgae species is given in Fig. 2. For each microalga, the neu-tral lipid content (NL) was highest in the H extract and lower in the CMand HI extracts. Consequently, the amount of polar lipids (GL and PL)was lowest in the H extract. The percent recovery of the different lipidclasses on extraction with HI and H from the different microalgae spe-cies, is also shown in Table 2. Lipid recovery with HI and H rangedfrom 53 to 76% and from 14 to 53% respectively depending on themicroalgae species. Recovery of NL with HI ranged from 62 to 102%,while with H only 25–82% of NL were extracted. For the GL and PL, therecovery with HI was between 61–96% and 57–65% respectively, whilewith H it was only 8–13% and 8–22% respectively. The higher totallipid an lipid class recovery on using HI compared to H, and the higherneutral lipid recovery compared to polar lipid recovery for both sol-vents, are in accordance to our previous results on N. gaditana [12].These observations could be explained by a combination of two factors:(1) Some NL can be stored in themicroalgae cell in the form of small oilbodies, which are extractable by a non-polar extraction solvent, such ashexane. (2) Polar lipids as well as some NL however form complexeswith proteins in the cell membrane. The protein–lipid associations can-not be brokenwith a non-polar extraction solvent, and even some polarextraction solvents were shown to be less adequate thanmixtures witha halogenated solvent [12,20].

The non-lipid part of theHI extracts varied between 6 and 18%,whilethe non-lipid part of the H extracts varied between 0.4 and 13%. All HIextracts contained more non-lipids than the corresponding H extractsof the same microalga. This can be explained by the more polar natureof isopropanol, capable to extract more of the non-lipid,more polarma-terial (e.g. proteins, carbohydrates) of the microalgae biomass.

Most importantly for this study however, it was observed that thetotal lipid and lipid class recovery with one solvent (mixture) wasdifferent for the different microalgae species. Generally, Isochrysisshowed the highest recovery for total lipids and for all lipid classes.

Table 2Percent recovery1 of lipids, lipid classes, fatty acids, carotenoids and sterols due to extraction w

Isochrysis galbana Nannochloropsis gaditan

HI H HI H

Lipid recovery 76 ± 4a 53 ± 1w 57 ± 4c 13.9

Lipid class recoveryNeutral lipids (NL) 102 ± 3a,* 82 ± 2w 69 ± 4c 25 ±Glycolipids (GL) 96 ± 4a,* 13 ± 2w 65 ± 5b 11 ±Phospholipids (PL) 65 ± 3a 22 ± 2w 60 ± 4a 13.4

Omega-3 LC-PUFA recoveryEPA (C20:5n−3) 117 ± 15a,* 68 ± 13w 64 ± 5b 14.3DHA (C22:6n−3) 98 ± 9a,* 73 ± 8w – –

Carotenoid recoveryCarotene 109 ± 19a,* 79 ± 13wx,* 78 ± 10a,* 53 ±Diadinochrome$ 75 ± 6 34 ± 4 – –

Diadinoxanthin – – 36 ± 17b 21 ±Diatoxanthin 111 ± 7a,* 34 ± 2w – –

Fucoxanthin 107 ± 9a,* 16 ± 1x – –

Violaxanthin – – 12 ± 1b 6.2 ±Zeaxanthin – – 75 ± 11a 12.4

Sterol recoveryCholesterol – – 68 ± 4a 44 ±Phytosterols 108 ± 5a,* 95 ± 5w,* 68 ± 5b 45 ±

a, bshow significant differences betweenHI extracts of differentmicroalgae (α = 0.05). w, xshowthe recovery is not significantly different from 100 (α = 0.05).

$ The diadinochrome content was estimated using the calibration curve of diadinoxanthin.1 The percent recovery is defined as the yield obtained with HI and H divided by the maxim

deviation of 3 replicates is reported unless in the case of lipid recovery where 12 repetitions w


Phaeodactylum showed intermediate recovery for total lipids and theNL class. Both Nannochloropsis species showed the lowest recovery fortotal lipids and the NL class. Recovery of PL was the same for the HI ex-tracts of all microalgae, while recovery of GLwas the same for theHI ex-tracts of all microalgae except Isochrysis. Recovery of PL in the H extractswas highest for Isochrysis, lower for N. gaditana and lowest forNannochloropsis sp. and Phaeodactylum, while recovery of GL in the Hextracts was highest for Isochrysis, and lower for the other microalgae.Multiple factors may explain these observations. First, the higher neu-tral lipid and free fatty acid content of Isochrysis (Table 1) may lead toa higher lipid recovery as both classes have been shown to be extractedmore easily [12,21,22]. Secondly, the difference in cell wall permeabilitymay also explain differences in recovery between different species.Nannochloropsis species indeed possess a thick rigid cell wall that con-tains an aliphatic, non-hydrolysable biopolymer, called algaenan

ith HI and H from the different microalgae species.

a Nannochloropsis sp. Phaeodactylum tricornutum

HI H HI H

± 0.9z 53 ± 3c 19.6 ± 0.7 y 67 ± 3b 25 ± 1x

2z 62 ± 3c 35 ± 1y 83 ± 4b 56 ± 3x

1wx 61 ± 4b 9.4 ± 0.6x 65 ± 4b 8 ± 1x

± 0.9x 57 ± 3a 10.4 ± 0.6y 65 ± 3a 7.9 ± 0.5y

± 0.9x 56 ± 3b 17 ± 1x 77 ± 11b,* 29 ± 4x

– – 63 ± 13b 28 ± 4x

5x 81 ± 10a,* 56 ± 7x 112 ± 12a,* 97 ± 12w,*– – – –

2x 68 ± 7a 21 ± 2x 79 ± 9a,* 40 ± 8w

– – 77 ± 8b 39 ± 8w

– – 78 ± 8b 31 ± 7w

0.5x 63 ± 8a 8.8 ± 0.8w – –

± 0.6x 80 ± 8a,* 19 ± 2w – –

2w 72 ± 4a 48 ± 2w – –

3y 65 ± 4b 40 ± 2y 77 ± 4b 66 ± 3x

significant differences betweenH extracts of differentmicroalgae (α = 0.05). *shows that

um yield obtained with the CM reference method multiplied by 100. Mean and standardere performed.


image of Fig.�2


Fig. 3.Omega-3 LC-PUFA content (in mg/g oil extract; mean ± SD; n = 3) obtained withdifferent extraction solvent (mixtures) (chloroform/methanol, CM; hexane/isopropanol,HI; hexane, H) from the different microalgae species. a, b show significant differences be-tween extracts of one microalga (α = 0.05).


[23–26], all together explaining low permeability. The cell wall ofPhaeodactylumwas shown to consist of a high concentration of polysac-charides, associated with only a small amount of weakly polymerizedsilicate [27], explaining the high permeability. Contradictory data arefound on the presence [28] or absence [29] of a cell wall surroundingIsochrysis cells. These should explain the high permeability. Lemahieuet al. [30] also observed that omega-3 LC-PUFA from Phaeodactylumand Isochrysis were more easily incorporated into hen eggs when henswere fedmicroalgae, compared toNannochloropsis. The difference in di-gestibilitymay coincide with a difference in extractability, both due to adifferent cell wall. Finally, the amount of lipids in oil bodies compared tolipids complexed with proteins, and the strength of the bonds in theselipid–protein complexes might also be different between microalgaespecies and might explain higher or lower extractability of lipids. Ofcourse, a combination of the factors can also be the basis for the ob-served differences.

3.2. Omega-3 fatty acid content

The omega-3 LC-PUFA content of the microalgae oil extracts isshown in Fig. 3. Lipid extracts (CM, HI and H) with high EPA content(N100 mg/g oil) were obtained from both Nannochloropsis species andPhaeodactylum, while lipid extracts (CM, HI and H) with high DHA con-tent (N50 mg/g oil) were extracted from Isochryris as could be expectedbased on the results of Ryckebosch et al. [5].

The percent recovery of omega-3 LC-PUFA due to extraction with HIand H from the different microalgae species, is shown in Table 2. It isclear that the recovery of the omega-3 fatty acids was quite similar tothe lipid recovery. When a higher lipid recovery was obtained, a higherfatty acid recovery was obtained and vice versa.

3.3. Carotenoid composition and content

The carotenoid content of the different extracts obtained from the dif-ferent microalgae species is shown in Table 3. Carotenoid contents rangewidely from 1 to 43 mg/g oil. The calculated percent recovery (based onCM as referencemethod) is shown in Table 2. The values depend on typeof carotenoid,microalgae species and extraction solvent. Similar to in ourprevious study [12] the recoveries can be explained by a combination oftwo factors. First, a similar polarity of the carotenoid and the solvent(mixture) allows the carotenoid to dissolve in this particular solvent(mixture). This can explain the recovery of the carotenoids in the H ex-tracts of Isochrysis and, to a lesser extent, of Phaeodactylum: fucoxanthin,the most polar carotenoid, is recovered the least, while with decreasingpolarity–diadinochrome, diadinoxanthin, diatoxanthin, and carotene—the carotenoids are more recovered. Secondly, the location of the carot-enoids can be dual and this can also explain different recoveries. Withinthe cell, carotenoids aremostly associatedwith the photosyntheticmem-brane, complexed to proteins [31]. These carotenoid–protein complexescan only be disrupted by polar organic solvents able to form hydrogenbonds. This explains the better extraction recoveries with HI than withH. Secondary carotenoids are however not localized in the photosynthet-ic apparatus, but in oil bodies, which also store TAG. If β-carotene is alsostored in these oil bodies in the microalgae species used in this study, aswas shown for Parietochloris incise [32], this helps to explain the overallhigher recovery of carotene compared to the xanthophylls.

When comparing carotenoid recoveries from the differentmicroalgae, the highest recovery with both solvent (mixtures) was ob-tained from Isochrysis and Phaeodactylum, while both Nannochloropsisspecies showed the lowest recovery. In contrast to lipid recovery, notmuch difference was found between Isochrysis and Phaeodactylum.This can be explained by the fact that the first factor explaining thehigher lipid recovery of Isochrysis, being its higher neutral and freefatty acid content cannot play a role in carotenoid recovery. The mostobvious explanation for differences in carotenoid recovery is thus thecell wall permeability, which seems to be high and more or less


similar in Isochrysis and Phaeodactylum, while it is lower in bothNannochloropsis species. Taking this result back to the explanation ofthe different lipid recoveries, this means that the cell wall permeabilityfactor will probably also play the largest role there. Other potential fac-tors explaining differences in carotenoid recovery might be the amountof carotenoids associated with oil bodies compared to carotenoidscomplexed to proteins, aswell as the strength of the carotenoid–proteinassociations in the different microalgae.

3.4. Sterol content

The sterol content of the microalgae oil extracts is shown in Fig. 4.Cholesterol was only present in the extracts obtained from both


image of Fig.�3


Table 3Carotenoid content1 of the different extracts from the microalgae species.

Carotene Diadinochrome$ Diadinoxanthin Diatoxanthin Fucoxanthin Violaxanthin Zeaxanthin

IsochrysisCM 3.2 ± 0.5b 1.1 ± 0.1a – 2.7 ± 0.1b 35 ± 2b – –

HI 3.8 ± 0.2ab 0.87 ± 0.03b – 3.2 ± 0.2 a 41 ± 2a – –

H 4.5 ± 0.2a 0.7 ± 0.1c – 1.6 ± 0.1c 10.1 ± 0.3c – –

Nannochloropsis gaditanaCM 4.1 ± 0.2b – 5.1 ± 0.3a – – 15.8 ± 0.7a 3.27 ± 0.01a

HI 4.9 ± 0.5b – 3 ± 1b – – 2.8 ± 0.2c 3.8 ± 0.5a

H 13.6 ± 0.7a – 6.9 ± 0.2a – – 6.2 ± 0.3b 2.6 ± 0.1b

Nannochloropsis sp.CM 3.8 ± 0.4c – 3.9 ± 0.3ab – – 14 ± 1a 0.7 ± 0.1b

HI 5.2 ± 0.3b – 4.4 ± 0.2a – – 14 ± 1a 0.89 ± 0.04a

H 9.9 ± 0.6a – 3.78 ± 0.01b – – 5.6 ± 0.1b 0.589 ± 0.003b

PhaeodactylumCM 2.7 ± 0.2c – 6.3 ± 0.5b 2.8 ± 0.2b 34 ± 3a – –

HI 4.2 ± 0.2b – 6.9 ± 0.6b 3.1 ± 0.2b 38 ± 2a – –

H 10.3 ± 0.8a – 10 ± 2a 4.4 ± 0.8a 43 ± 8a – –

a,bshow significant differences (α = 0.05).$ The diadinochrome content was estimated using the calibration curve of diadinoxanthin.1 Carotenoid content is expressed in mg/g oil extract. Mean and standard deviation of 3 replicates is reported.


Nannochloropsis species, in an amount ranging from 13 to 39 mg/g ex-tract. Phytosterols were present in the extracts obtained from allmicroalgae species tested, in an amount ranging from 7 to 34 mg/g ex-tract. The percent recovery of sterols due to extraction with HI and Hfrom the different microalgae species, is shown in Table 2. In general,the sterol recovery in the HI extract was always higher than in the H ex-tract, although recovery with the different solvent systems was not sig-nificantly different for Isochrysis. The higher recovery with HI wasalready observed in our previous study [12] and could be explained bythe sterol-membrane associations, which can only be broken usingpolar hydrogen bond forming solvents. The difference in sterol recoverybetween the HI and H extract also depends on the microalgae species.This may have to do with the form in which the sterols are present:free sterols, steryl esters, or steryl glycosides [33]. Free sterols and sterylesters are probably more soluble in H (and even HI) than the steryl gly-cosides, so the fact that Isochrysis mainly contains free sterols [34] andPhaeodactylum contains mainly contains steryl glycosides [33] may ex-plain the higher recovery in the H extract of Isochrysis. This may alsobe the reason for the larger differences for sterol recovery betweenIsochrysis and Phaeodactylum than for the carotenoid recovery. Al-though the amount of sterols in a particular steryl class is also depen-dent on the culturing conditions, such as light and temperature [33].

When comparing the different microalgae species, the sterols werebest recovered from Isochrysis (~100%), while recovery from bothNannochloropsis specieswas lowest,with Phaeodactylum reaching an in-termediate recovery. The most evident explanation for differences insterol recovery from the different microalgae species is again the cellwall permeability. Other possible explanations may be the amount ofsterols in a particular steryl class, as described above. The amount of ste-rols complexed to cell membranes, and the strength of the sterol-membrane associations might also play a role.

3.5. Implications for feasibility of microalgae oils as alternative for fish oil

From our previous study [5] it was clear that some microalgae oils,including those from Isochrysis, Nannochloropsis, and Phaeodactylumshow potential as an alternative for fish oil. The amount of oil to be con-sumed on a daily basis to reach an intake of 250 mg EPA + DHA/daywas shown to be feasible (b2.5 g/day for most species). These resultswere however obtained based on the total lipid extract obtained usingchloroform/methanol. In the current study it was therefore investigatedif the oils obtained with the food grade extraction solvents, HI and H,show the same potential as an omega-3 LC-PUFA alternative.


First of all, and most importantly, it was shown that there was nostriking difference between the omega-3 fatty acid content of the differ-ent extracts of one microalga (Fig. 3). This can be explained by the factthat the recoveries for total lipids and omega-3 fatty acids (Table 2)were similar. When the same calculations would be made as byRyckebosch et al. [5], the amount of oil to be consumed daily to reachan intake of 250 mg EPA + DHA/daywill thus bemore or less indepen-dent of the extraction solvent used and will thus still be sufficiently lowwhen using food grade solvents.

Secondly, it was shown that the GL and PL content was higher in theHI extract than in the H extract for eachmicroalgae (Fig. 2). This may bean advantage of the HI extract since this might lead to an increased ab-sorption of omega-3 LC-PUFA, as it has been suggested that ingestingLC-PUFA from PL rather than TAG leads to a more efficient absorptionin the human body [6]. Furthermore, the increased amount of omega-3 LC-PUFA in polar lipids can also lead to oils that are less susceptibleto oxidation, as it was shown that omega-3 LC-PUFA associated tophospholipids or glycolipids are less easily oxidized than when theywere associated to TAG [7,8]. On the other hand, most commercialomega-3 LC-PUFA oils contain only TAG, which might be beneficial fortheir processability, since polar lipids show emulsifying properties [35].

It was shown previously [5] that co-extracted carotenoids can addvalue tomicroalgae oil when compared tofish oil. It is known that carot-enoids are antioxidants [36], so they can increase the oxidative stabilityof the oil and they can lower oxidative stress in humans [37–39]. Fur-thermore, carotene, diatoxanthin, fucoxanthin and zeaxanthin mayhave other beneficial properties, besides their antioxidant capacity, aswas described by Ryckebosch et al. [5]. Table 3 shows that carotenoidsare also co-extracted with HI and H, although the content in these oilswas in some cases quite different to that in the CMextract. Nevertheless,all microalgae oils extracted with food grade solvents will thus result ina high to very high (for H extracts) intake of carotene (9.6–29 mg/day,compared to the RDI of 4.8 mg/day), which works as provitamin A(European Responsible Nutrition Alliance, ERNA). The clearly high caro-tene content in the food grade oils, especially in the H oils, might giverise to potential overdosing, since the British Expert Committee on Vita-mins and Minerals (EVM) and the Nutrition Societies of German-speaking countries recommend a maximum dose of no more than 7and 10 mg/day respectively, although the European Food Safety Au-thority and the U.S. Food and Nutrition Board did not set a figure for atolerable upper intake level of beta-carotene [40]. The Nannochloropsisoils can also raise human intake levels (1.4–7 mg/day) of zeaxanthin(RDI 6 mg/day), and this was almost independent of the solvent used



Fig. 4. Sterol content (inmg/g oil extract; mean ± SD; n = 3) obtainedwith different ex-traction solvent (mixtures) (chloroform/methanol, CM; hexane/isopropanol, HI; hexane,H) from the different microalgae species. a, b show significant differences between ex-tracts of one microalga (α = 0.05).


to extract the oils. Zeaxanthin protects against eye diseases and skinconditions [41]. Phaeodactylum oils, again independent of the solventused to extract the oils, can also provide high amounts of fucoxanthin(90–96 mg/day), of which was shown that intake of 2.4 mg/day mightalready show antiobesity effect [42]. Isochrysis oils provide even higheramounts of fucoxanthin (136–149 mg/day), except the oil extractedwith H (30 mg/day). The risk of overdose with fucoxanthin from bothPhaeodactylum and Isochrysis oils are definitely not absent, but has notbeen studied yet. Both Phaeodactylum and Isochrysis oils can also delivera considerable amount of diatoxanthin (5–12 mg/day), which mighthave anti-inflammatory properties [43].


When the same calculations would be made as by Ryckebosch et al.[5], the amount of sterols co-ingested due to consumption ofmicroalgaeoils to reach a daily intake of 250 mg EPA + DHA is quite similar for theHI extracts (16–51 mg/day) as for the CMextracts (14–46 mg/day), butis higher for theH extracts (32–73 mg/day). Nevertheless, a daily intakeof 1.6–2 g of plant sterols or stanols showed to be necessary to reduceserum cholesterol significantly. It is clear that this amount of sterols can-not be achieved due to consumption of microalgae oils for omega-3 LC-PUFA (2–4 g/day).

4. Conclusion

This study showed that the highest lipid yield was obtained withhexane/isopropanol (HI) when compared to hexane (H) for all testedmicroalgae species. However, only 53–76% of the present lipids and56–100% of the present omega-3 LC-PUFA were extracted. Generally,the recovery of the different components was highest from Isochrysis,lowest from both Nannochloropsis species, and intermediate forPhaeodactylum, although the magnitude of the difference betweenIsochrysis and Phaeodactylum was dependent on the component takeninto account. The main factor that determines recovery from differentmicroalgae species seems to be the permeability of the cell wall, al-though for certain components other factors may play a role. It canalso not be excluded that differences in growth and processing condi-tions between the different species have influenced the results.

Furthermore, the omega-3 fatty acid content of the oils obtainedwith both food grade solvent systems was quite similar to the totallipid extract. The amount of oil to be consumed daily to reach an intakeof 250 mg EPA + DHA/day is thus more or less independent of the ex-traction solvent used. The H extract was enriched in neutral lipids com-pared to the total lipid and HI extracts, which may lead to a decreasedabsorption and oxidative stability of the oil. Carotenoids were also co-extracted in a significant amount with HI and H, although the contentin the oils was quite different. The high carotene content, especially inH oils, might give problems with overdosing, as well as the high fuco-xanthin content in the oils from Phaeodactylum and Isochrysis.

When taking the higher lipid and omega-3 yield, the lower neutrallipid enrichment and the lower carotene content into account, it lookslike hexane/isopropanol is more promising for extraction of omega-3LC-PUFA rich oils from microalgae than hexane.

Acknowledgments

The research presented in this paper was financially supported bythe Flanders' Food-IWT (Omega-OIL Project), a Research Grant of theResearch Foundation–Flanders (FWO) (1.5.122.09N) and KU LeuvenKulak. We acknowledge the companies of the FF' Omega-OIL projectfor the fruitful discussions. A special acknowledgment to Proviron andLGem for the samples of the microalgae. We also acknowledge CedrickVeryser for his drive and accurate work during his holidays.

References

[1] I. Khozin-Goldberg, U. Iskandarov, Z. Cohen, LC-PUFA from photosyntheticmicroalgae: occurrence, biosynthesis, and prospects in biotechnology, Appl.Microbiol. Biotechnol. 91 (2011) 905–915.

[2] B.J. Meyer, Are we consuming enough long chain omega-3 polyunsaturated fattyacids for optimal health? Prostaglandins, Leukotrienes Essent, Fat. Acids 85 (2011)275–280.

[3] U. Gogus, C. Smith, n−3 Omega fatty acids: a review of current knowledge, Int. J.Food Sci. Technol. 45 (2010) 417–436.

[4] M.A. Crawfield, Presentation at the Omega-3 Summit: global summit on nutrition,health and human behavior, March 3–4 2011. (Bruges, Belgium).

[5] E. Ryckebosch, C. Bruneel, R. Termote-Verhalle, K. Muylaert, I. Foubert, Nutritionalevaluation of omega-3 LC-PUFA rich microalgae oils as an alternative for fish oil,Food Chem. (2013)(under review).

[6] J.P. Schuchardt, I. Schneider, H. Meyer, J. Neubronner, C. von Schacky, A. Hahn, Incor-poration of EPA and DHA into plasma phospholipids in response to differentomega-3 fatty acid formulations—a comparative bioavailability study of fish oil vs.krill oil, Lipids Health Dis. 10 (2011) 145.


http://refhub.elsevier.com/S2211-9264(13)00094-5/rf0005



















image of Fig.�4



[7] A.-M. Lyberg, E. Fasoli, P. Adlercreutz, Monitoring the oxidation of docosahexaenoicacid in lipids, Lipids 40 (2005) 969–979.

[8] T. Yamaguchi, R. Sugimura, J. Shimajiri, M. Suda, M. Abe, M. Hosokawa, K. Miyashita,Oxidative stability of glyceroglycolipids containing polyunsaturated fatty acids, J.Oleo Sci. 61 (2012) 505–513.

[9] R.K. Balasubramanian, D.T.T. Yen, J.P. Obbard, Factors affecting cellular lipid extrac-tion from marine microalgae, Chem. Eng. J. 215–216 (2013) 929–936.

[10] S.J. Lee, B.-D. Yoon, H.-M. Oh, Rapid method for the determination of lipid from thegreen alga Botryococcus braunii, Biotechnol. Tech. 12 (1998) 553–556.

[11] E. Ryckebosch, K. Muylaert, I. Foubert, Optimization of an analytical procedure forextraction of lipids from microalgae, J. Am. Oil Chem. Soc. 89 (2012) 189–198.

[12] E. Ryckebosch, S.P. Cuéllar Bermúdez, R. Termote-Verhalle, K. Muylaert, R.Parra-Saldivar, I. Foubert, Influence of extraction solvent system on the extractabil-ity of lipid compounds from the biomass ofNannochloropsis gaditana, J. Appl. Phycol.(2013), http://dx.doi.org/10.1007/s10811-013-0189-y(accepted for publication).

[13] R. Long, E. Abdelkader, Mixed-polarity azeotropic solvents for efficient extraction oflipids from Nannochloropsis microalgae, Am. J. Biochem. Biotechnol. 7 (2011)70–73.

[14] W.W. Christie, Lipid Analysis: Isolation, Separation, Identification and StructuralAnalysis of Lipids, third ed. The Oily Press, Bridgwater, 2003.

[15] E. Ryckebosch, K. Muylaert, M. Eeckhout, T. Ruyssen, I. Foubert, Influence of dryingand storage on lipid and carotenoid stability of the microalga Phaeodactylumtricornutum, J. Agric. Food Chem. 59 (2011) 11063–11069.

[16] C.O. Kangani, D.E. Kelley, J.P. Delany, New method for GC/FID and GC–C-IRMS anal-ysis of plasma free fatty acid concentration and isotopic enrichment, J. Chromatogr.B 873 (2008) 95–101.

[17] S.W. Wright, S.W. Jeffrey, R.F.C. Mantoura, C.A. Llewellyn, T. Bjornland, D.Repeta, N. Welschmeyer, Improved HPLC method for the analysis of chloro-phylls and carotenoids from marine-phytoplankton, Mar. Ecol. Prog. Ser. 77(1991) 183–196.

[18] S.L. Abidi, Capillary electrochromatography of sterols and related steryl esters de-rived from vegetable oils, J. Chromatogr. A 1059 (2004) 199–208.

[19] J. Toivo, A.-M. Lampi, S. Aalto, V. Piironen, Factors affecting sample preparation inthe gas chromatographic determination of plant sterols in whole wheat flour,Food Chem. 68 (2000) 239–245.

[20] R. Halim, M.K. Danquah, P.A. Webley, Extraction of oil from microalgae for biodieselproduction: a review, Biotechnol. Adv. 30 (2012) 709–732.

[21] J. Guckert, K. Cooksey, L. Jackson, Lipid solvent systems are not equivalent for anal-ysis of lipid classes in the microeukaryotic green alga, Chlorella, J. Microbiol.Methods 8 (1988) 139–149.

[22] E. Molina Grima, A. Robles Medina, A. Giménez Giménez, J.A. Sánchez Pérez, F.Garcia Camacho, J.L. García Sánchez, Comparison between extraction of lipidsand fatty acids from microalgal biomass, J. Am. Oil Chem. Soc. 71 (1994)955–959.

[23] P. Blokker, Cell wall-specific ω-hydroxy fatty acids in some freshwater greenmicroalgae, Phytochemistry 49 (1998) 691–695.

[24] F. Gelin, I. Boogers, A.A.M. Noordeloos, J.S. Sinninghe Damsté, R. Riegman, J.W. DeLeeuw, Resistant biomacromolecules in maline microalgae of the classesEustigmatophyceae and Chlorophyceae: geochemical implications, Org. Geochem.26 (1997) 659–675.


[25] F. Gelin, J. Volkman, C. Largeau, S. Derenne, J. Sinninghe Damsté, J. De Leeuw, Distri-bution of aliphatic, nonhydrolyzable biopolymers in marine microalgae, Org.Geochem. 30 (1999) 147–159.

[26] A. Skrede, L.T. Mydland, Ø. Ahlstrøm, K.I. Reitan, H.R. Gislerød, M. Øverland, Evalua-tion of microalgae as sources of digestible nutrients for monogastric animals,J. Anim. Feed Sci. 20 (2011) 131–142.

[27] B. Tesson, M.J. Genet, V. Fernandez, S. Degand, P.G. Rouxhet, V. Martin-Jézéquel, Sur-face chemical composition of diatoms, ChemBioChem 10 (2009) 2011–2024.

[28] E.C. Wootton, M.V. Zubkov, D.H. Jones, R.H. Jones, C.M. Martel, C.A. Thornton, E.C.Roberts, Biochemical prey recognition by planktonic protozoa, Environ. Microbiol.9 (2007) 216–222.

[29] C.J. Zhu, Y.K. Lee, Determination of biomass dry weight of marinemicroalgae, J. Appl.Phycol. 9 (1997) 189–194.

[30] C. Lemahieu, C. Bruneel, R. Termote-Verhalle, K. Muylaert, J. Buyse, I. Foubert, Impactof feed supplementation with different omega-3 rich microalgae species on enrich-ment of eggs of laying hens, Food Chem. 141 (2013) 4051–4059.

[31] G. Britton, Functions of intact carotenoids, in: G. Britton, S. Liaaen-Jensen, H. Pfander(Eds.), Carotenoids, Volume 4: Natural Functions, Birkhäuser Verlag, Basel, 2008,pp. 189–212.

[32] A.E. Solovchenko, Physiological role of neutral lipid accumulation in eukaryoticmicroalgae under stresses, Russ. J. Plant Physiol. 59 (2012) 167–176.

[33] B. Véron, J.-C. Dauguet, C. Billard, Sterolic biomarkers in marine phytoplankton. I.Free and conjugated sterols of Pavlova lutheri (Haptophyta), Eur. J. Phycol. 31(1996) 211–215.

[34] B. Véron, J.-C. Dauguet, C. Billard, Sterolic biomarkers in marine phytoplankton. II.Free and conjugated sterols of seven species used in mariculture, J. Phycol. 34(1998) 273–279.

[35] R. Rombaut, K. Dewettinck, Properties, analysis and purification of milk polar lipids,Int. Dairy J. 16 (2006) 1362–1373.

[36] S. Paiva, R. Russell, β-carotene and other carotenoids as antioxidants, J. Am. Coll.Nutr. 18 (1999) 426–433.

[37] A.C. Butalla, T.E. Crane, B. Patil, B.C.Wertheim, P. Thompson, C.A. Thomson, Effects ofa carrot juice intervention on plasma carotenoids, oxidative stress, and inflamma-tion in overweight breast cancer survivors, Nutr. Cancer 64 (2012) 331–341.

[38] S. Kiokias, M.H. Gordon, Dietary supplementation with a natural carotenoid mixturedecreases oxidative stress, Eur. J. Clin. Nutr. 57 (2003) 1135–1140.

[39] R. Martínez-Tomás, E. Larqué, D. González-Silvera, M. Sánchez-Campillo, M.I.Burgos, A. Wellner, S. Parra, et al., Effect of the consumption of a fruit and vegetablesoup with high in vitro carotenoid bioaccessibility on serum carotenoid concentra-tions andmarkers of oxidative stress in young men, Eur. J. Nutr. 51 (2012) 231–239.

[40] Nutri-facts, http://www.nutri-facts.org/eng/carotenoids/beta-carotene/safety/April2013.

[41] E.J. Johnson, The role of carotenoids in human health, Nutr. Clin. Care 5 (2012)56–65.

[42] M. Abidov, Z. Ramazanov, R. Seifulla, S. Grachev, The effects of Xanthigen in theweight management of obese premenopausal women with non-alcoholic fattyliver disease and normal liver fat, Diabete Obes. Metab. 12 (2010) 72–81.

[43] I. Konishi, M. Hosokawa, T. Sashima, T. Maoka, K. Miyashita, Suppressive effects ofalloxanthin and diatoxanthin from Halocynthia roretzi on LPS-induced expressionof pro-inflammatory genes in RAW264.7 cells, J. Oleo Sci. 57 (2008) 181–189.













http://dx.doi.org/10.1007/s10811-013-0189-y












































































http://www.nutri-facts.org/eng/carotenoids/beta-carotene/safety/










influence of extraction solvent system on extractability of lipid components from different...

Documents