the effect of extraction methods on fatty acid and carotenoid of marine microalgae nannochloropsis...

8/10/2019 The Effect of Extraction Methods on Fatty Acid and Carotenoid of Marine Microalgae Nannochloropsis oculata.pdf

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Pertanika J. Trop. Agric. Sci. 36 (2): 145 - 160 (2013)

ISSN: 1511-3701 Universiti Putra Malaysia Press

TROPICAL AGRICULTURAL SCIENCEJournal homepage: http://www.pertanika.upm.edu.my/

Article history:Received: 18 May 2011Accepted: 2 November 2011

ARTICLE INFO

E-mail addresses :[email protected] (Loh, S. P.),[email protected] (Lee, S. P.)* Corresponding author

The Effect of Extraction Methods on Fatty Acid and CarotenoidCompositions of Marine Microalgae Nannochloropsis oculataand Chaetoceros gracilis

Loh, S. P. 1,2* and Lee, S. P. 11 Department of Nutrition and Dietetics, Faculty of Medicine and Health Sciences, Universiti Putra Malaysia,43400 Serdang, Selangor, Malaysia2 Institute of Bioscience, Universiti Putra Malaysia, 43400 Serdang, Selangor, Malaysia

ABSTRACT

This study was conducted to assess three extraction methods for the determination offatty acid compositions and carotenoids (lutein, zeaxanthin, -carotene, and -carotene)from marine microalgae, Nannochloropsis oculata (NO) and Chaetoceros gracilis (CG ). For this purpose, three different extraction methods for the determination of fatty acids(dichloromethane:methanol, water:propan-2-ol:hexane and direct saponi cation-ethanolKOH) and carotenoids (hexane:ethanol:acetone:toluene, methanol:chloroform andmethanol:tetrahydrofulran) were used. Two derivatization methods using different typesof catalyst (acetyl chloride and boron tri uoride) were also used for the transmethylationof the fatty acids into corresponding methyl esters. The results of the fatty acidcompositions showed that NO had a higher amount of n-3 and n-6 polyunsaturated fattyacid (PUFA), particularly eicosapentaenoic acid (EPA) (C20:5). CG was predominantlyhigh in palmitic acid (C16:0) and palmitoleic acid (C16:1). The extraction method 1(dichloromethane:methanol) and extraction method 2 (water: propan-2-ol: hexane) withacetyl chloride-catalyzed transmethylation were found to be the best methods for thedetermination of fatty acid compositions in NO and CG, respectively. A signi cantlyhigher (P


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Loh, S. P. and Lee, S. P.

146 Pertanika J. Trop. Agric. Sci. 36 (2): 146 - 160 (2013)

Keywords: Carotenoids, Chaetoceros gracilis ,

extraction, fatty acid composition, Nannochloropsis

occulata , microalgae

INTRODUCTION

Microalgae are prokaryotic or eukaryotic photosynthet ic microorganisms unified primarily by the lack of roots, leaves, andstems that characterize higher plants. Theycan be found almost anywhere, with water and

sunlight as their fundamental requirements,including lakes, soils, rivers, hot springs, andthe ocean. Microalgae contain high valuecompounds like fatty acids [- linolenicacid, arachidonic acid, eicosapentaenoicacid (EPA), docosahexaenoic acids(DHA), etc.], pigments (chlorophyll andcarotenoids), vitamins (biotin, vitamins Cand E, and others) (Converti et al., 2009).

Chaetoceros gracillis , a diatom in the classof Bacillariophycae and Nannochloropsisocculata , a unicellular green alga withspherical shape of the Eustigmatophyceaeclass plays an important role in the foodchain system and it is also commonly usedas live feed; thus, it is widely cultivated in

sh hatcheries and shrimp farms (Gwo etal., 2005).

DHA and EPA are constantly an areaof interest in nutrition because they areessential for optimizing human health.DHA is important for the developmentof the brain and eyes in pre-term andyoung infants, as well as for supportingcardiovascular health in adults, whereasEPA is essential for the human metabolismand involved in the blood lipid equilibrium

that prevents hypertriglyceridemia and anti-in ammatory activities (Kroes et al., 2003;

Ward & Singh, 2005; Fajardo et al., 2007).Previously, sh was the principal dietarysource of DHA and EPA. However, due tothe serious environmental consequencesand continuous exploitation, the decliningsources of marine sh stocks and sh oilhave prompted research into new sources of

polyunsatured fatty acids (PUFAs) (Burja etal., 2007). In addition, certain disadvantages

of sh oil, such as the unpleasant odour, possible pollutants, and mixed fatty acid properties have also encouraged the searchfor alternative sources of PUFAs (Pulz &Gross, 2004).

In addit ion, microalgae containa multitude of pigments, particularlychlorophyll and carotenoid. Carotenoidsare essential to human health and important

in commercial applications (Felti et al., 2005). For example, -carotene acts as

pro-vi tamin A and has been proven to prevent xeropthlamia (Puah et al., 2005);astaxanthin acts as a natural colorant formuscle in marine fish and crustaceans;lutein, zeaxantin and canthaxantin forchicken skin coloration, pharmaceutical

purposes, and also as a natural food additive

(Del Campo et al., 2000; Pulz & Gross,2004).

Although microalgae contain important bioactive components (particularly PUFAand carotenoids), the extraction methods,especially for algae, are not well established,as there are no standard extraction methodsfor the determination of the fatty acid contentor carotenoids in microalgae (Wiltshire et


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Fatty Acid and Carotenoids from Two Marine Microalgae

147Pertanika J. Trop. Agric. Sci. 36 (2): 147 - 160 (2013)

al., 2000). Lipids are mainly a mixture ofesters, and therefore, the preparation of

fatty acid methyl esters (FAME) consistsessentially on the conversion of one ester toanother (i.e. transesteri cation) by cleavageof an ester bond via an alcohol; when such analcohol is methanol, the reaction is referredto as methanolysis or transmethylation (Liu,1994). Transmethylation are reversiblereactions which are normally accomplishedin the presence of a catalyst, either an acid or

a base. Reactions involving acidic catalystsrequire heat to accelerate the process.Commonly used acidic catalysts are (theBrnsted-Lowry acid) HCl, H 2SO 4, acetylchloride and (the Lewis acid) BF3. Base-catalyzed methanolysis proceeds much morerapidly under mild temperature conditionsthan acid-catalyzed reactions. However,

bases cannot catalyze the esteri cation of

FFAs.The nutritional value of microalgae

is strongly dependent on its bioactive pro le; therefore, methods that could getthe highest value is preferred. Felti et al. (2005) suggested that the absence of astandard extraction method for carotenoidis actually attributed to the wide spectrumof the analyzed materials (foodstuff, plant,

animal, and human samples) and the widerange of the carotenoids present. As a result,three extractions methods of fatty acidand carotenoids from marine microalgae,

Nannochloropsis oculata and Chaetoceros gracilis were evaluated in the current study.The criteria used in choosing these methodswere maximum extraction ef ciency, ease ofhandling, and use of solvents of low toxicity.

MATERIAL AND METHODS

Microalgae Samples

Both the Nannochloropsis oculata andChaetoceros graci l is samples were

purchased from Reed Mariculture Inc. USA.The samples were collected using non-

probability, convenient sampling method.Both the microalgae were purchased in two

batches and prior to the extraction, thesesamples were freeze-dried, homogenized,and kept at -20 oC until further use.

Oil Extraction

The rst extraction method used was adoptedfrom Cequier-Sanchez et al. (2008). First,500 mg of the samples were extracted bymixing 15 ml of dichloromethane-methanol2:1 (v/v) contained in a beaker. The mixingwas performed with occasional gentle hand

agitation for 2 hours. Subsequently, thesamples were ltered and transferred into anew test tube to which 3.13ml of an aqueoussolution of potassium chloride (0.88%, w/v)was added with strong agitation, followed

by centrifugation (Universal 320/320RBenchtop Centrifuges, Hettich Instruments,Germany) at 350g at 4 0C for 5 minutes. Theaqueous upper phase was discarded and

the organic phase was evaporated using arotary evaporator (Bchi Rotavapor R-200,Switzerland).

The second extraction method wasadopted from Schlechtriem et al. (2003)with a slight modi cation, in which hexanewas used to substitute the cyclohexane. First, 500 mg samples were weighed intothe Falcon tubes and mixed with 10ml of


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propan-2-ol and 12.5 ml of hexane usinga vortex for 30s. The tubes were placed in

an ultrasonic bath at room temperature for15 minutes. Then, 13.75ml of water wasadded to obtain a mixture of water: propan-2-ol:hexane (11:8:10 v/v/v). The mixturewas mixed again using the vortex for 30s.The different phases were separated bycentrifugation at 1800g for 10 minutes andthe organic phase was transferred into a pre-weighed Flacon tube with a dropper. The

organic phase containing the lipid fractionwas separated at the top of the extractionmixture (the hexane phase). Subsequently,the second extraction with 12.5ml of hexanecontaining 13% v/v propan-2-ol was done.The mixture was vortexed and placed intothe ultrasonic bath for another 15 minutes.After centrifugation, the hexane phase wasadded to the rst extract. The tubes were

placed in a water bath (50 0C) for about 15minutes and the solvent was evaporated todryness using a rotary evaporator.

The third extraction method wasadopted from Burja et al. (2007) with aminor modi cation, in which 95% ethanolwas used instead of 96% ethanol. First, the500mg samples were weighed. Then, 38mlof 3mM potassium hydroxide in ethanol

(95%) was added into a 150ml beaker.The beaker was passed under the ow ofnitrogen, and shaken for 1 hour in a water

bath set at 60C. Thereafter, the sampleswere cooled to room temperature and

ltered through lter paper. The biomasswas washed with 10ml of ethanol andtransferred into a new beaker, to which,10ml of water was added. Unsaponi ables

were extracted by adding 20ml of hexaneand gently mixing twice. After the layers

were separated, the pH was adjusted to1 (from pH 13-14) by the addition ofhydrochloric acid:water (1:1, v/v). The toplayer, containing the fatty acid fraction, wasrecovered by two rounds of the addition of10ml of hexane and a gentle mixing. Lastly,the solvent at the top layer was evaporatedto dryness using a rotary evaporator.

Preparation of Fatty Acid Methyl Esters(FAMEs)

T h i s a c e t y l c h l o r i d e - c a t a l y z e dtransmethylation method was adoptedfrom Carvalho and Malcata (2005). Thelipid extracts (2mg) were subjected to acid-catalyzed transesteri cation by dissolvingthem in 2ml of a freshly prepared mixtureof acetyl chloride and methanol at a ratio of

5:100 (v/v), together with 1mg of tricosanoicacid as an internal standard. The reagentswere placed in Te on-capped Pyrex tubes,and the reaction continued at 100C for1 hour under pure nitrogen and darkness.After cooling to 30~40C, 1ml of theextracting solvent (isooctane containing0.01% butylated hydroxytoluene, BHT)was added, and the FAME solvent solution

was mixed using a vortex for between 5 to30s. The puri cation of the solution wasachieved by adding 1ml of water, causingthe formation of two immiscible phases,which were then allowed to separate.Subsequently, the upper extracted solvent

phase was recovered and stored in sealedglass vials at -20 0C until GC analysis.

The BF3-catalyzed transmethylation


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method was adopted from Carvalho andMalcata (2005), which was modi ed by the

use of 10% (v/v) of BF3 in methanol insteadof 12% (v/v) of BF3 in methanol. First, allthe lipid extracts (2mg) were subjected to a

preliminary alkaline hydrolysis with 0.5Msodium hydroxide at 100 0C for 5 minutes.Subsequently, it was dissolved in 2ml of10% (v/v) BF3 in methanol, together with1mg of tricosanoic acid as an internalstandard. The reagents were placed in the

Te on-capped Pyrex tubes, and the reactionwas allowed to continue at 100C for 30minutes under pure nitrogen and darkness.The subsequent procedure was similar to theacetyl chloride-catalyzed transmethylation

procedure, as described above.

Gas Chromatography Analysis

The assay of FAME was analyzed using gaschromatography (Agilent 6890, ISA AgilentTech, USA) equipped with a split/splitlessinjector, and Hewlett Packard EL-980 ameionization detector (FID). The FID systemwas used to separate and quantify eachFAME component. FAME was separatedusing DB-23 column (60m x 0.25mm ID,and 0.15 m). The chromatography datawere recorded and integrated using the

chemistation software (version 6). The oventemperature was programmed to hold at50 0C for 1 min, before it was increased to175 0C with 25 0C/min, held for 4 minutes,and lastly increased to 230 0C with 4 0C/minand held for 5 mins. The temperature forthe injector and detector was set at 250 0Cand 280 0C, respectively. One microlitre ofthe sample volume was injected with a split

ratio of 1:50l and a column temperature of110 0C. The carrier gas was helium gas (1.0

ml/min) which was controlled at 123.4kPa/Hz and the air used for FID was held at275.6kPa.

Calculation of Fatty Acid

The identification of the fatty acidcompositions for the sample was made

by comparing the retention time of thesample FAMEs with those of Supelco37 component FAMEs mixture (Sigma-aldrich, USA) for each chromatography

peak. The quanti cation of the fatty acidwas done using tricosanoic acid (C23:0)as an internal standard. The amount of theindividual fatty acid was calculated usingthe expression: C i = C p (A i/A p), where A isthe chromatographic area units and C is theamount of fatty acid. Subscript p representsthe internal standard and i refers to any fattyacid. The percentage of the individual fattyacid in the total amount of fats used wascalculated as C i/total amounts of fat x 100%.

Carotenoids Extraction

The rst extraction method was adoptedfrom Inbaraj et al. (2006). First, 400mg offreeze-dried microalgae was mixed with12 ml hexane-ethanol-acetone-toluene(10:6:7:7 v/v/v/v) in a volumetric flask.After shaking for 1 hour, 0.8ml 40%methanolic potassium hydroxide was addedand the solution was saponi ed at 25 0C inthe dark for 16 hours. Then, 12ml of hexanewas added to partition the carotenoids. Themixture was shaken for 1 min and 10%sodium sulfate solution was added. After


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shaking for 1 minute, the upper layer wascollected and the lower layer was repeatedly

extracted twice with hexane. Finally, theupper extracts were pooled and evaporatedto dryness using a rotary evaporator.

The second extraction method wasadopted from Reboul et al. (2006). First,400mg of frozen-dried microalgae wasadded into 8ml of methanol containing0.57% magnesium carbonate. Subsequently,the samples were homogenised for 30s

using a vortex. Then, 8ml of chloroform,containing 0.005% butylated hydroxyltoluene (BHT), was added. The sampleswere homogenized for 30s more in thevortex blender. After a rest of 15 minutes,8ml of distilled water was added into thesamples and centrifuged (2000g for 10minutes). The lower phases of the sampleswere collected and the remaining upper

phases were extracted by the addition of6ml of tetrahydrofuran. After that, themixture was vortexed for 30s, and 6 ml ofdichloromethane was also added. It wasthen vortexed for another 30s, after which4ml of distilled water was added and themixture was further re-vortexed for 30s.After centrifugation (2000g for 10 minutesat room temperature), the lower phase was

collected and pooled with the previouslycollected phase. Lastly, the collected lower

phase solvent was evaporated to drynessusing a rotary evaporator.

The nal extraction method was adoptedfrom Marinova and Ribarova (2007).First, the pigments were extracted from a400mg sample to which 0.04 g magnesiumcarbonate was added, with 6ml extraction

solvent methanol:tetrahydrofuran (1:1,v/v) containing 0.1% butyl hydroxytoluene

(BHT). The mixture was vortexed forabout 3 minutes, and then centrifuged for3 minutes at 1400g and the supernatantwas collected. The pellet was re-extractedfollowing the same procedure until thesupernatant became colourless. Thecombined supernatants were evaporated todryness using a rotary evaporator.

All the extraction procedures were

performed under subdued light to avoiddegradation loss of the pigments. The residuewas dissolved in methanol at a concentrationof 100mg/ml. Prior to the HPLC analysis,the sample solution was ltered using aWhatman polytetrafluroethylene (PTFE)0.22m syringe lter and the ltrate wasinjected into a HPLC valve with a 1mlsyringe.

Analysis of Carotenoids

The carotenoids were analyzed by usingHPLC (Agilent Series 1100, Model G1313A, Agilent Technologies, Germany)equipped with degasser, quaternary pump,auto sampler and photodiode array detector.The carotenoids were separated by HPLCusing a 150 4.6 mm, 3 m C30 analytical

column (Waters Co., Milford, MA, USA).The mobile phase system comprisedmethanolmethyl tert-butyl ether (MTBE)

water (81:15:4 v/v/v) (A) and methanol/MTBE (10:90 v/v) (B) in the followinggradient conditions 100% of A and 0% B, to50% A and 50% B in 45 minutes, followed

by 100% B within 15 minutes. The columntemperature was set at 25 0C. The volume


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injected into the HPLC was set as 20l andthe ow rate during the separation was set

as 1ml/min. The wavelength used for the photodiode array detector in measuring thecarotenoids was 450nm. The elution timewas 45 minutes for a sample and the posttime was 5 minutes.

The standard for the carotenoidswere prepared from a stock solution of -carotene, -carotene, zeaxanthin andlutein. The identi cation of carotenoids was

made by comparing with these standards,and the spiking test was also carried out tocon rm the identi cation of certain peaks.The carotenoids were quantified using acalibration curve that was prepared using

pure standards in the range of 0.025-5g/ml.

Data Analysis

The computer software Statistical Packagefor Social Sciences version 16 (SPSS 16)was used to analyze the data in this study.The analysis was done in triplicates and theresults were expressed as mean standarddeviation. Meanwhile, the two-way ANOVAwas used to compare the differences in themean amounts of fatty acid and carotenoidfrom the microalgae, Nannochloropsis

oculata and Chaetoceros gracilis usingvarious extraction methods and also two

different transmethylation methods (fattyacid only). The analysis was considered ata signi cance value of p 0.05).

No signi cant differences were observed between NO and CG .


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a primary alcohol with the most activehydroxyl group (highly polar), which could

stimulate the disruption of hydrogen bonds between lipid carbonyl, hydroxyl, and theamino groups, and the compounds of thenonextractable residue (Ruiz-lopez et al., 2003).

Fatty Acid Composition of Nannochloropsis Oculata (NO) andChaetoceros Gracilis (CG) Using

Different Extraction Methods

In terms of the extraction ef ciency of thefatty acid content (weight %), extractionmethod 1 (dichloromethane: methanol)coupled with acetyl chloride catalyzedtransmethylation appeared to be the mostef cient method for NO , as compared toother methods (Table 2). This was becausethis particular method could generate higher

fatty acid content than other methods using both the acetyl chloride and BF3-catalyzedtransmethylation methods. Since NO consists of a polysaccharide cell wall, thesolubility of its cell matrix towards the non-

polar solvent may enable the penetrationof solvents into it and subsequently allowthe oil to dissolve and be extracted fortransmethylation. In addition, this methodis also simpler and easier in its procedureas compared to two other methods.Indeed, a previous study has shown thatdichloromethane, a less hazardous solvent,was an effective extraction solvent for fattyacid research (Cequier-Sanchez, 2008).

Neverthe less , ex trac tion method 2(water:propan-2-ol:hexane), coupled withacetyl chloride catalyzed transmethylation,appeared to be the most suitable method

for the determination of fatty acid for CG , particularly C16 and C18 fatty acids (Table

3). The use of additional cell disruptiontreatment (ultrasonic bath) in this extraction

procedure was notably useful in CG , agenus of diatoms, as they have the uniquecharacteristic of a silica-based rigid cell wall,which may be dif cult to break (Scala et al., 2002). The use of an ultrasonic bath wasrelated to the destruction of cell walls and theenhancement of mass-transfer through the

cell wall due to the collapse of the bubbles produced by cavitation (Macias-Sanchezet al., 2009). In this way, its extractionef ciency could be enhanced. Moreover,the use of this particular combination ofsolvents was also highly recommended interms of its safety, low toxicity, and low cost(Smedes, 1999). Although hexane was usedto substitute cyclohexane in this method,

their almost similar properties would notcreate much difference in the result.

Among the three extraction methodsused, method 3, which involved a directsaponi cation using ethanolic potassiumhydroxide, gave the least number and amount(weight %) of fatty acid compositions in

both the acetyl chloride and BF3-catalyzedtransmethylation methods. This result

disagrees with previous study which claimedthat this method was an ef cient techniqueto increase the extraction of fatty acid from

biomass (Burja et al., 2007). However, astudy by Wang et al. (2000) found a lowerconcentration of fatty acids on chickenegg yolk by using the direct saponi cationextraction method compared to othermethods (direct-methylation, chloroform-


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T A B L E 2

F a t t y a c

i d c o m p o s i

t i o n o f

N a n n o c h

l o r o p s i s o c u l a t a

( N O ) u s

i n g

d i f f e r e n

t e x t r a c

t i o n a n

d d e r i v a

t i z a t

i o n

t r a n s m e t

h y l a t i o n ) m e t

h o d s

F a t t y A c i

d C o m p o s i

t i o n

N a n n o c h

l o r o p s

i s o c u l a t a

( N O )

A c e

t y l c h l o r

i d e - c a

t a l y z e

d t r a n s m e t h y l a t

i o n

B F 3 - c a

t a l y z e

d t r a n s m e t

h y l a t i o n

M e t h o d 1

M e t

h o d 2

M e t

h o d 3

M e t

h o d 1

M e t

h o d 2

M e t

h o d 3

C 8 : 0

2 . 7 8 0 . 7 2 a

N D

N D

1 . 6 0 0 . 0 6 b

1 . 7 8 0 . 3 1 b

N D

C 1 2 : 0

2 . 2 5 0 . 1 4

N D

N D

N D

N D

N D

C 1 3 : 0

9 . 0 4 0 . 1 1 a

7 . 5 2 0 . 0 3 b

1 . 8 0 0 . 1 1 c

1 . 9 1 0 . 0 7 d

1 . 1 0 0 . 0 2 e

N D

C 1 4 : 0

2 5 . 6 2

0 . 2 3 a

1 2 . 1

3 0 . 0 9 b

N D

7 . 4 0 0 . 0 4 c

2 . 9 8 0 . 1 0 c d

1 . 4 3 0 . 0 7 d

C 1 4 : 1

N D

N D

N D

N D

N D

3 . 2 2 0

. 0 5

C 1 6 : 0

1 5 9

1 . 5 6 a

7 6 . 1

2 0 . 2 3 b

1 6 . 4

7 0 . 1 7 c

6 0 . 7

8 0 . 4 8 d

1 8 . 9

1 0 . 1 e

9 . 6 2 0 . 0 5 c

C 1 6 : 1

1 9 8

0 . 6 1 a

8 1 . 3

2 1 . 3 2 b

1 4 . 1

3 0 . 1 2 c

7 1 . 6

1 0 . 4 7 d

1 6 . 7

9 0 . 2 e

3 1 . 3

6 0

. 3 9 f

C 1 7 : 0

4 . 4 4 1 . 5 1 a

4 . 9 9 0 . 1 8 a

N D

N D

N D

N D

C 1 7 : 1

6 . 6 7 0 . 2 6 a

2 . 5 0 0 . 0 6 a

N D

2 . 0 4 0 . 0 8 b

N D

N D

C 1 8 : 0

7 . 2 6 0 . 2 8 a

8 . 9 1 0 . 2 3 b

2 . 6 5 0 . 3 1 c

4 . 2 4 0 . 1 5 d

1 . 9 6 0 . 0 8 e

N D

C 1 8 : 1 n 9

t r a n s

N D

N D

N D

N D

N D

N D

C 1 8 : 1 n 9 c i s

5 9 . 8 6

0 . 8 3 a

4 2 . 7

1 0 . 2 9 b

5 . 6 7 0 . 1 3 c

1 9 . 1

6 0 . 2 3 d

7 . 5 1 0 . 4 0 e

3 . 2 7 0 . 1 0 f

C 1 8 : 2 n 6 c i s

4 3 . 8 6

0 . 5 8 a

3 0 . 1

2 0 . 1 2 b

3 . 7 9 0 . 2 0 c

1 4 . 8

4 0 . 1 1 d

6 . 6 5 0 . 1 7 e

2 . 9 6 0 . 1 0 f

C 1 8 : 3 n 6

6 . 7 6 0 . 3 9 a

0 3 . 0

4 0 . 0 3 b

N D

N D

N D

6 . 2 7 0 . 1 8 c

C 1 8 : 3 n 3

2 . 2 3 0 . 3 7 a

N D

N D

N D

N D

N D

C 2 0 : 4 n 6

3 4 . 1 5

0 . 5 4 a

1 2 . 1

0 . 4 0 b

N D

1 0 . 8

6 0 . 0 3 c

1 . 3 6 1 . 1 8 d

0 . 8 8 0 . 7 6 d

C 2 0 : 5

3 5 1

3 . 3 7 a

1 5 2 0 . 1 1 b

1 9 . 2

7 0 . 2 3 c

1 0 6 0 . 8 1 d

3 4 . 2

8 0 . 2 4 e

1 5

. 5 9 0 . 1 5 c

M e t

h o d 1 : ( d i c h l o r o m e t

h a n e : m e t

h a n o l

) , M e t

h o d 2 : ( w a t e r : p r o p a n -

2 - o l : h e x a n e

) , M e t

h o d 3 : ( D i r e c

t s a p o n

i c a t

i o n - e t

h a n o

l i c K O H ) .

E a c

h v a

l u e

i s t h e m e a n

s t a n

d a r d

d e v i a t

i o n o f

t r i p l i c a t e s e x p r e s s e

d a s g k g -

1 o f

t h e

t o t a l o i l

.

W i t h i n a r o w , m

e a n s

f o l l o w e d

b y t h e s a m e

l e t t e r a r e n o

t s i g n i

c a n

t l y d i f f e r e n

t ( p >

0 . 0 5 ) .

N D = n o

t d e t e c

t e d


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T A B L E 3

F a t t y a c

i d c o m p o s i

t i o n o f

C h a e t o c e r o s g r a c

i l i s

( C G ) u s

i n g

d i f f e r e n

t e x t r a c t

i o n a n

d d e r i v a

t i z a t

i o n

( t r a n s m e t

h y l a t i o n

) m e t

h o d s

F a t t y A c i

d C o m p o s i

t i o n

C h a e t o c e r o s g r a c

i l i s

( C G )

A c e

t y l c h l o r

i d e

t r a n s m e t

h y l a t i o n

B F 3 - c a

t a l y z e

d t r a n s m e t

h y l a t i o n

M e t h o d 1

M e t

h o d 2

M e t

h o d 3

M e t

h o d 1

M e t

h o d 2

M e t

h o d 3

C 8 : 0

3 . 1 2 0 . 6 2 a

N D

N D

1 . 4 1 0 . 2 7 b

0 . 6 7 0 . 5 9 b

0 . 8 7 0 . 7 6 b

C 1 2 : 0

2 . 0 3 2 . 0 4

N D

N D

N D

N D

N D

C 1 3 : 0

5 . 8 3 1 0 3 a

1 4 . 4

6 0 . 0 7 b

0 . 9 3 0 . 8 1 c

2 . 2 6 0 . 0 9 d

1 . 5 0 0 . 2 2 d

N D

C 1 4 : 0

7 7 . 0 3

0 . 1 0 a

8 0 . 7

0 0 . 5 5 a

1 1 . 4

5 5 . 5 7 b

1 9 . 7

3 0 . 1 0 c

4 7 . 3

4 0 . 1 9 d

4 . 0 4 0 . 1 1 e

C 1 4 : 1

2 . 4 2 0 . 1 1 a

2 . 3 3 0 . 0 2 a

N D

0 . 9 3 0 . 0 4 b

1 . 6 0 0 . 1 0 c

N D

C 1 6 : 0

6 0 . 6 0

1 . 7 8 a

1 2 9 0 . 4 7 b

1 3 . 5

2 7 . 6 0 c

1 2 . 1

3 0 . 1 2 d

7 0 . 3

8 0 . 1 3 e

8 . 5 0 0 . 1 0 c

C 1 6 : 1

1 0 7

0 . 2 8 a

1 0 3 0 . 8 8 a

1 9 . 2

9 7 . 3 8 b

2 5 . 7

1 0 . 5 5 c

9 2 . 7

0 0 . 0 9 d

6 . 5 4 0 . 1 7 e

C 1 7 : 0

2 0 . 3 8

0 . 3 4 a

1 8 . 8

9 0 . 2 9 a b

3 . 5 0 1 . 5 9 c

4 . 8 0 0 . 0 6 d

1 7 . 9

5 0 . 0 7 b

1 . 3 3 0 . 1 4 e

C 1 7 : 1

2 8 . 7 2

0 . 2 2 a

2 5 . 5

5 4 . 3 4 a

6 . 2 8 2 . 3 6 b

6 . 5 5 0 . 2 5 c

2 2 0 . 2 0 b

2 . 1 4 0 . 0 6 c

C 1 8 : 0

8 . 1 3 0 . 0 4 a

2 0 . 9

5 0 . 4 3 b

N D

1 . 6 4 0 . 0 3 c

1 2 . 5

2 0 . 1 2 d

N D

C 1 8 : 1 n 9

t r a n s

N D

5 . 6 8 0 . 2 8 a

N D

N D

6 . 6 2 0 . 1 4 b

N D

C 1 8 : 1 n 9 c i s

1 1 . 8 0

0 . 1 8 a

1 7 3 0 . 2 9 b

5 . 7 9 0 . 7 0 c

N D

3 . 3 8 0 . 1 9 d

2 . 4 2 0 . 0 4 e

C 1 8 : 2 n 6 c i s

7 . 7 6 0 . 0 9 a

1 2 1 0 . 2 1 b

3 . 0 8 0 . 1 8 c

1 . 0 5 0 . 0 2 d

5 . 1 3 0 . 1 5 e

1 . 5 9 0 . 1 1 d

C 1 8 : 3 n 6

N D

N D

N D

N D

1 . 2 2 0 . 0 5 a

N D

C 1 8 : 3 n 3

N D

4 . 6 4 0 . 1 0 a

N D

N D

N D

N D

C 2 0 : 4 n 6

7 . 8 3 8 . 7 4 a

N D

N D

N D

2 . 4 4 0 . 1 1 b

N D

C 2 0 : 5

1 8 . 0 5

0 . 3 5 a

1 4 . 9

3 0 . 2 8 a b

3 . 8 2 3 . 3 2 c

3 . 5 2 0 . 0 6 d

1 7 . 4

5 0 . 1 2 b

N D

M e t

h o d 1 : ( d i c h l o r o m e t

h a n e : m e t

h a n o l

) , M e t

h o d 2 : ( w a t e r : p r o p a n -

2 - o l : h e x a n e

) , M e t

h o d 3 : ( D i r e c

t s a p o n

i c a t

i o n - e t

h a n o

l i c K O H ) .

E a c

h v a

l u e

i s t h e m e a n

s t a n

d a r d

d e v i a t

i o n o f

t r i p l i c a t e e x p r e s s e

d a s g k g -

1 o f

t o t a l o i l

.

W i t h i n a r o w , m

e a n s

f o l l o w e d

b y t h e s a m e

l e t t e r a r e n o

t s i g n i

c a n

t l y d i f f e r e n

t ( p >

0 . 0 5 ) .

N D = n o

t d e t e c

t e d .


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methanol extraction, and postextractionsaponi cation), although the reason was

unknown.A c e t y l c h l o r i d e - c a t a l y z e d

transmethylation generated a higher amount(weight %) of fatty acid compared toBF3-catalyzed transmethylation in boththe microalgae. This might be due to thehighly basic condition of acetyl chloride,which could cause severe disruption tocell integrity, making in situ methyl ester

derivation efficient (Tran et al., 2009).Furthermore, the use of acetyl chloride-catalyzed transmethylation procedure hasseveral advantages as compared to themost commonly performed methanolic BF3method, such as longer shelf-life (withoutthe need for refrigeration), lower cost, andsmaller amount of catalyst required (5%acetyl chloride versus 10% BF3) (Carvalho

& Malcata, 2005).

Percentage of Extraction Yields(Carotenoids)

Table 4 shows that extraction method 1 (hexane:ethanol:acetone:toluene) generated the

highest extraction yields for both NO (74.54 4.75%) and CG (69.28 14.71%). This

was probably due to the longer period ofcontact time (1 hour) between the cellularcomponent to be extracted and the solventmixtures in extraction method 1 as comparedto the other two methods (Henriques et al. ,2007). The two-way ANOVA showedsigni cant differences between the samples( p = 0.022) on the extraction yields. Overall,it could be seen that all the extraction

methods used generated higher extractionyields in NO than in CG . However, thedifference in the extraction yields wassmall among these microalgae, particularly

between extraction methods 2 and 3.

Carotenoids Concentration of the Different Extracts of Nannochloropsis Oculata (NO)and Chaetoceros Gracilis (CG)

As shown in Table 5, -carotene wasfound to be the highest, followed byzeaxanthin, -carotene, and lutein in the

NO using different extraction methods . However, extraction methods 1 and 2 werethe only methods that could detect lutein

TABLE 4Extraction yield of carotenoids using different extraction methods from microalgae Nannochloropsisoculata (NO) and Chaetoceros gracilis(CG)

Extraction methods Nannochloropsis oculata (NO) Chaetoceros gracilis (CG)

Method 1 (Saponi cation)(hexane:ethanol:acetone:toluene)

745 48 a 693 147 a

Method 2 (No saponi cation)(methanol:chloroform)

682 25 a 515 12 b

Method 3 (No saponi cation)(methanol:tetrahydrofuran)

636 39 a 502 25 a

Each value is the mean standard deviation of triplicates expressed as g kg -1dry weight.

Within a row, means followed by the same letter are not signi cantly different ( p> 0.05). No signi cant differences were observed between the 3 extraction methods.


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and carotene in NO, respectively, whilezeaxanthin was not detected in extraction

method 2. For zeaxanthin and -carotenecontents in NO, extraction method 1, whichinvolved the saponi cation step, generatedthe highest concentration (g/100g dryweight) as compared to the other methods.The functions of saponification includehydrolyzing the carotenoid esters andremoving the chlorophyll and unwantedlipids on microalgae, which may interfere

with chromatographic separation (Howeet al. , 2006). Since microalgae were highin their lipid content, saponi cation wasnecessary to achieve better results (betteridenti cation and higher concentration) as

compared to the other two methods, whichdo not employ saponi cation.

Just like NO, CG was found to be thehighest in the amount (g/kg dry weight)of -carotene, followed by lutein andzeaxanthin using different extractionmethods . However, -carotene was notdetected in CG with either of these extractionmethods. This does not indicate the absenceof -carotene in CG because the failureto detect it might be due to other possible

reasons such as the presence of light andoxygen while handling the samples orstoring that would have contributed to itsdegradation. As shown in Table 6, extractionmethod 2 was the only method that could not

TABLE 5Carotenoid concentrations (g kg -1 dry weight) of Nannochloropsis oculata (NO) using different extractionmethods

Carotenoids

Method 1

( Saponi cation)(hexane:ethanol:acetone: toluene)

Method 2

(No saponi cation)(methanol : chloroform)

Method 3

(No saponi cation)(methanol: tetrahydrofuran)

Lutein 1.55 0.01 ND ND

Zeaxanthin 3.74 0.03 a ND 2.67 0.08 b

-carotene 9.58 0.002 a 9.46 0.05 b 8.69 0.04 c

-carotene ND 2.16 0.004 ND

Each value is the mean standard deviation of triplicates expressed as g kg -1 dry weight.Within a row, means followed by the same letter are not signi cantly different ( p> 0.05).

ND = not detected.

TABLE 6: Carotenoid concentrations (g kg -1dry weight) of Chaetoceros gracilis (CG) using different

extraction methods

CarotenoidsMethod 1

( Saponi cation)(hexane:ethanol:acetone: toluene)

Method 2(No saponi cation)

(methanol:chloroform)

Method 3(No saponi cation)

(methanol: tetrahydrofuran)

Lutein 1.33 0.003 a ND 1.57 0.02 b

Zeaxanthin 0.58 0.01 a 0.75 0.003 b 8.68 0.02 c

-carotene 7.945 0.002 a ND 8.08 0.01 b

-carotene ND ND ND

Each value is the mean standard deviation of triplicates expressed as g kg -1dry weight.Within a row, means followed by the same letter are not signi cantly different ( p> 0.05).

ND = not detected.


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detect the presence of lutein and -carotenein CG . Hence, extraction method 2 was less

suitable for the determination of carotenoidfor CG as compared to extraction methods1 and 3. However, extraction method 3could generate a higher concentration ofcarotenoids (g/kg dry weight) compared toextraction method 1. In the present study, thesaponi cation step in extraction method 1might not have much impact on carotenoidsdetermination of CG since the absence of the

saponi cation step in extraction method 3yielded a better result for carotenoids.

Moreover, the different cell matrix ofthese microalgae might have contributedto the difference in the concentrations ofcarotenoid in them. As described earlierin the determination of the fatty acidcomposition, the cellular structure of NO was distinctly different to CG . Hence,

CG with its unique characteristic of asilica-based rigid cell wall might causeincomplete extraction of the biochemicalcompounds by the solvents alone, withoutany additional treatment (e.g. ultrasound

bath, enzymes, microwave-assisted, etc.).This is in accordance with a publishedstudy, whereby an efficient disruptiontreatment of the membrane was required

in order to achieve the ef cient extractionof carotenoids as there was no standardtechnique can guarantee a maximization ofthe extraction yield (Valduga et al. , 2009).Unlike CG , the polysaccharide cell wall of

NO might also be easier to penetrate usingcompatible solvents, and subsequently allowthe extraction of the desired biochemicalcompounds.

CONCLUSION

The comparison of various extractionmethods on both fatty acids and carotenoidsrevealed that they produced extractswith different characteristics as well asquantitative differences. For fatty aciddetermination, the utilization of method1 (dichloromethane:methanol) appearedto be the most efficient method for

NO . Nevertheless, extraction method 2(water:propan-2-ol:hexane), which involvedadditional treatment (ultrasonic bath),appeared to be a more suitable methodfor fatty acid determination in CG . Asfor carotenoids, extraction method 1,which uses the saponification step toremove chlorophyll, unwanted lipids andthe involvement of more solvent mixtures (2

polar and 2 non-polar solvents), generatedthe highest concentration (g/100g dryweight) in NO. However, extraction method3 generated the highest concentrations inCG. Overall, this study has shown that usingthe right extraction method, high amountsof fatty acids and carotenoids could beobtained from the microalgae.

This work was supported by Agri-ScienceFund Grant from the Ministry of Agriculture,Malaysia (Project No. 5450384).

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the effect of extraction methods on fatty acid and carotenoid of marine microalgae nannochloropsis...

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