Chapter 2

Comparative evaluation and selection of a method for lipid and fatty acid extraction from macroalgae

 

CHAPTER 2  

33  

2.1. Introduction

Macroalgae have been reported to contain more than 2400 natural products of

commercial importance in pharmaceutical, biomedical and nutraceutical industries (Munro

and Blunt, 1999). They have also been extensively utilized as ingredients in human and

animal food preparations owing to their high contents of polyunsaturated fatty acids

(PUFAs), carbohydrates, vitamins, minerals and dietary fibres (Chandini et al. 2008; Kumari

et al. 2010). Nowadays, algal resources have been studied with renewed interest across the

world as an alternative source of renewable energy feedstock that circumvents the

controversy of “fuel versus food.” The attributes for such choice is their relatively higher

production turnovers and amenability for depolymerization of substrate in addition to greater

carbon sequestration potentials than terrestrial feedstock (Lee et al. 2010). Most recently,

Petcavich succeeded in producing hydrocarbon biofuels from transformed kelp Macrocystis

pyrifera with high hydrocarbon producing genes of microalgae, Botryococcus braunii

(Petcavich, 2010).

Fatty acid (FA) analysis has been increasingly gaining importance due to realization

of their beneficial applications in nutritional and health products. Further, they have also

been used for addressing various fundamental and pragmatic research problems in

experimental biochemical, physiological and clinical studies (Iverson et al 2001; Masood et

al. 2005). Further, in biodiesel production, clean burn properties of the fuel are influenced

by FA structural features including chain length and degree of unsaturation (Knothe, 2005).

Thus, a precise quantification of FA can also be used to predict the quality of biodiesel,

which is reduced considerably with the increase in the amount of saturated FAs.

Traditionally, the fatty acid composition of lipid samples is determined by assessing

the corresponding methyl esters via gas chromatography (GC). A large number of analytical

approaches based on initial lipid extraction by solvents, followed by their transmethylation

(i.e., conventional methods) are employed and where FAs are sought, they are extracted and

methylated with one-step procedures wherein methylation reagent is added directly to the

samples without previous extraction (direct transesterification methods). However, both

types of methods have their own advantages and disadvantages that are well illustrated in

the literature (Ruiz-Lỏpez et al. 2003; Meier et al. 2006; Abdulkadir and Tuschiya, 2008;

CHAPTER 2  

34  

Jeannotte et al. 2008; Júarez et al. 2008; Pérez-Palacios et al. 2008; Gómez-Brandón et al.

2008, 2010; Sheng et al. 2011; Martins et al. 2012; Laurens et al. 2012; Ryckebosch et al.

2012; Velasquez-Orta et al. 2012). Conventional methods are time consuming and use toxic

solvents such as chloroform, benzene while direct transesterification methods are simplified,

rapid, and sensitive, minimize the use of solvents and give better recoveries of FAs. Despite

its several disadvantages, conventional methods of lipid extraction are the only means to

study different lipid classes till date (Júarez et al. 2008).

Further, it is only in the last decade that the importance of microalgal biodiesel has

provided an impetus for undertaking research aimed at manipulation of lipid and fatty

extraction techniques to obtain higher yields of lipid and FAs from algae (Mercer and

Armenta, 2011; Sheng et al. 2011; de Boer et al. 2012; Laurens et al. 2012; Ryckebosch et

al. 2012; Samorì et al. 2013). Kim et al. (2011) used ionic liquids for lipid and FA extraction

from Chlorella vulgaris. Cheng et al. (2011) used supercritical CO2 for lipid extraction in

Pavlova sp. and obtained 98.7% yield of TAGs, while Patil et al. (2011) employed

supercritical methanol for direct liquefaction and conversion of wet algal biomass containing

about 90% of water to biodiesel. Chen et al. (2012) obtained 88% recovery rate of total

lipids by the method of subcritical co-solvent extraction (hexane/ethanol, 3/1; v/v) from wet

algal pastes of Nannochloropsis sp. in 50 min. Reep and Green (2011) patented a technology

for extracting lipids from alga without cell sacrifice. They accomplished it by exposing algal

cells in an aqueous medium to an electric field sufficient to cause release of lipids from the

cells. Further, Jones et al. (2012) reported that 2-ethoxyethanol yield >150-200% lipid

recovery as compared to other commonly used extraction solvents such as chloroform,

methanol and hexane in Chlorella sp. Liang et al. (2012a) developed enzyme assisted

aqueous extraction of lipids form microalgae by using cellulase, neutral protease, alkaline

protease, snailase and trypsin and facilitated the lipid extraction process by the application

of sonication. They obtained the highest lipid recovery of 49.82% by a combined sonication-

enzyme treatment at pH 4. The major drawback in this process is the cost of enzyme, which

they proposed, can be partially overcome by the use of immobilized enzymes as

immobilization make enzymes more economical and facilitates separation of enzyme from

product, allows reuse, and improves enzyme stability. Recently, Samorì et al. (2013)

developed a new procedure based on switchable polarity solvents for extracting lipids from

CHAPTER 2  

35  

wet algal samples, thereby circumventing the need for energy intensive drying step and

facilitating easy recovery of lipids from the extraction. These authors used N,N-

dimethylcyclohexylamine (DMCHA) for lipid extraction from two marine microalgae

Nannochloropsis gaditana and Tetraselmis suecia and a freshwater alga Desmodesmus

communis and recovered lipids by adding CO2, thereby switching DMCHA into a hydrogen

carbonate ammonium salt and resulting in the formation of a separate liquid lipid phase.

As far as macroalgae are concerned, they have been extensively explored for their

novel lipids and fatty acids (Khotimchenko and Vas’kovsky, 2004; Al-Fadhli et al. 2006;

Kim et al. 2007; Chang et al. 2008). Although both the conventional and direct

transesterification methods have been employed by researchers to study their lipids and FA

profiles (Khotimchenko et al. 2002; Li et al. 2002; Sanchez-Machado et al. 2004; Matanjun

et al. 2008; Kumari et al. 2010; Galloway et al. 2012; Gosch et al. 2012), Bligh and Dyer

(Bligh and Dyer, 1959) and Folch methods (Folch et al. 1957) based on chloroform/

methanol solvent system have been invariably used as standard methods. However, other

solvents such as dichloromethane/ methanol (Graeve et al. 2002) and diethyl ether (El-

Shoubaky et al. 2008) have also been used, but there are no established criteria for choosing

the most appropriate one. A researcher has to be intelligent enough to be able to chose the

best method and ignore the rest. Moreover, the information regarding comparison and

verification are still required to test which methods of extraction and FA derivatization

function best for these matrices. Since, the determination of FAs in macroalgae is itself a

challenge because of their heterogeneous FAs and high contents of PUFAs, it becomes

imperative to study extraction and derivatization procedures to obtain accurate qualitative

and quantitative results.

In view of the growing demand of macroalgae in development of PUFA-related

dietary supplements and lipid-based fuels, a comparison of different lipid and fatty acid

extraction and derivatization methods was accomplished in three different macroalgal

matrices representing one each from Chlorophyta, Phaeophyta and Rhodophyta. The

purpose of the present work was to systematically validate, by a full factorial categorical

design, the efficacy of three most commonly used solvent systems viz.,

chloroform/methanol (1/2 and 2/1, v/v) and dichloromethane/methanol (2/1, v/v) on lipid

CHAPTER 2  

36  

extraction and thereafter the effect of sonication and buffer on the respective solvent

systems. Further, the fatty acid recoveries obtained by the conventional methods were also

compared with one-step direct transesterification methods in order to select the best lipid

and FA extraction method for different macroalgal matrices.

2.2. Materials and Methods

2.2.1. Chemicals

For the identification and quantification of fatty acids, the following analytical grade

standards were used: 37- component FAME Mix C4-C24 (Supelco, USA), 7-hexadecenoic

acid methyl ester (C16:1, n-7) and stearidonic acid methyl ester (C18:4, n-3) (Cayman

chemicals, USA). The internal standard nonadecanoic acid (C19:0) was purchased from

Sigma. All the solvents used (such as chloroform, methanol, dichloromethane and hexane)

were of HPLC grade and other reagents of analytical grade.

2.2.2. Algal samples

Three macroalgal samples representing three different phyla viz. Ulva fasciata

(Chlorophyta), Gracilaria corticata (Rhodophyta) and Sargassum tenerrimum (Phaeophyta)

were collected in March, 2009 from Veraval coast, Gujarat, India and transported to the

laboratory in wet tissue towels in an ice box. They were immediately cleaned thoroughly to

remove the epiphytes and other undesired foreign matter from the fronds, frozen in liquid

nitrogen and stored at -40ºC until the analysis commenced.

2.2.3. Conventional methods (CM)

The lipid extraction efficiency of Bligh and Dyer (Bligh and Dyer, 1959), Folch

(Folch et al. 1957) and Cequer-Sanchez method (Cequier-Sànchez et al. 2008) was

compared in the present study. Further, the extraction efficacy of each method by

application of sonication and buffer individually were also investigated as a part of

optimization. The step-wise experimental procedure including modifications have been

detailed in a flow-chart diagram in Fig. 2.1. All the analyses were done with 500 mg of

ground algal tissues. Butylated hydroxyl toluene (BHT) at 0.001% was added to each

solvent system to minimize lipid peroxidation during the sample preparation.

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37  

Fig. 2.1 Flow diagram of different lipid and FA extraction methods in macroalgae. Note: BD, Bligh and Dyer;

BDS, Bligh and Dyer with sonication; BDB, Bligh and Dyer with buffer; FM, Folch method; FMS,

Folch method with sonication; FMB, Folch method with buffer; CS, Cequier-Sánchez method; CSS,

Cequier-Sánchez method with sonication; CSB, Cequier-Sánchez method with buffer; GM, Garcia

method; LRC, Lepage and Roy modified by Cohen method; C, Chloroform; M, Methanol; D,

Dichloromethane.

Method1: Bligh and Dyer Method (Bligh and Dyer, 1959)

1A (BD): Macroalgal samples were extracted with 3 ml of chloroform/methanol

(1/2, v/v) by vortexing (1min) and centrifugation at 2057 g for 15 min at room temperature

(RT). The supernatants were collected and residues re-extracted thrice with 2 ml of

chloroform/methanol (1/1, v/v) by centrifugation as stated above. All the supernatants were

pooled together, filtered with Whatman filter No. 1 (Whatman, USA) and washed with 2 ml

of milli-Q water, followed by centrifugation at 2057 g for 5 min. The lower organic phases

were collected, evaporated to dryness under nitrogen and total lipid contents were

determined gravimetrically.

1B (BDS; Bligh and Dyer with sonication): The lipids were extracted as mentioned

above for Method 1A, with slight modification, the tissues were sonicated for 2 min on ice

CHAPTER 2  

38  

in a cup sonicator, Branson 2210 (USA) for cell disruption in the extraction medium prior to

centrifugation.

1C (BDB; Bligh and Dyer with buffer): The lipids were extracted with 3 ml of

chloroform/methanol/50mM phosphate buffer (1/2/0.8, v/v/v) by vortexing (1min) and

centrifugation at 2057 g for 15 min at RT. The supernatants were collected and residues re-

extracted thrice with 2 ml chloroform/methanol/buffer (1/1/0.8, v/v/v) by centrifugation as

stated above. All the supernatants were combined, filtered and washed with 2 ml of 50 mM

phosphate buffer, followed by centrifugation at 2057 g for 5 min. The lower organic phases

were collected, evaporated to dryness under nitrogen and total lipid contents were

determined gravimetrically.

Method 2: Folch method (Folch et al. 1957)

2A (FM): Lipids were extracted with 3 ml of chloroform/methanol/ (2/1, v/v) by

vortexing and centrifugation as described for Method 1A. The supernatants were collected,

residues re-extracted thrice with 2 ml chloroform/methanol (1/1, v/v) by centrifugation and

combined supernatants were washed with 2 ml of milli-Q water. The lower organic phases

were collected after centrifugation at 2057 g for 5 min at RT, thereafter, evaporated to

dryness under nitrogen and total lipid contents were determined gravimetrically.

2B (FMS; Folch method with sonication): The lipids were extracted as mentioned

above for Method 2A, with the application of sonication for 2 min on ice for cell disruption

in the extraction medium prior to centrifugation.

2C (FMB; Folch method with buffer): In this method, buffer was added to the

extraction solvent mixture of Method 2A (Folch et al. 1957), and thus the tissues were

extracted initially with chloroform/methanol/50 mM phosphate buffer (2/1/ 0.8, v/v/v),

otherwise all the steps were same as Method 1C.

Method 3: Cequier - Sánchez method (Cequier-Sànchez et al. 2008)

3A (CS): Lipids were extracted with 3 ml of dichloromethane/methanol/ (2/1) by

vortexing (1min) and centrifugation at 2057 g for 15 min at RT. Again, the supernatants

were collected and residues re-extracted thrice with 2 ml dichloromethane/methanol (1/1).

CHAPTER 2  

39  

All the supernatants were combined, filtered and washed with 2 ml of milli-Q water and

centrifuged at 2057 g for 5 min. The lower organic phases were collected, evaporated to

dryness under nitrogen and total lipid contents were determined gravimetrically.

3B (CSS; Cequier - Sánchez with sonication): The lipids and FAs were extracted as

mentioned above for Method 3A (CS), with the application of sonication to tissues for 2 min

on ice, for cell disruption in the extraction medium prior to centrifugation.

3C (CSB; Cequier - Sánchez with buffer): In this method, buffer was added to the

extraction solvent mixture of Method 3A (CS) and thus, the tissues were extracted initially

with dichloromethane/methanol/50 mM phosphate buffer (2/1/ 0.8, v/v/v), otherwise all the

steps were same as Method 1C.

Preparation of FAMEs

The fatty acids were converted to their fatty acid methyl esters (FAMEs) by

transmethylation of lipid samples thus obtained by solvent extraction, with 1ml of 1%

NaOH in MeOH, followed by heating for 15 min at 55°C, adding 2 ml of 5% methanolic

HCl and again heated for 15 min at 55°C then adding 1ml of milli-Q water. 10 µl of 1 mg

ml-1 nonadecanoic acid (C19:0) was spiked in to the lipid samples as internal standard.

FAMEs were extracted by hexane (3 x 1ml) and evaporated to dryness under nitrogen

(Carreau and Dubacq, 1978). They were redissolved in 200 µl of hexane and stored in -40

ºC in glass vials until analyzed by GC-MS.

2.2.4. Direct transesterification (DT) methods

Two direct transesterification methods were also applied to macroalgal samples

whereby, FAs were directly extracted from the tissues without initial lipid extraction as

described below.

Method 4: GM; Garcia method (de la Cruz-Garcỉa et al. 2000)

Tissues (500 mg) were treated with 2 ml of toluene and 3 ml of freshly prepared 5%

methanolic HCl, spiked with internal standard, nonadecanoic acid (10 µl of 1 mg ml-1)

mixed thoroughly by vortexing in oak ridge tubes, closed under nitrogen and heated for 2 h

CHAPTER 2  

40  

in a water bath at 70 ºC. After cooling to room temperature, 4 ml of 6% aqueous K2CO3 and

2 ml of toluene were added and the mixtures were vortexed and centrifuged for 5 min at

2057 g. The organic phases were drawn off and dried with anhydrous Na2SO4 and solvents

were removed under nitrogen. FAMEs were solubilized in 200 µl of toluene and analyzed

by GC-MS.

Method 5: LRC; Lepage and Roy (Lepage and Roy, 1986) as modified by Cohen et al.

(Cohen et al. 1988 )method

Tissues (500 mg) were treated with 5 ml of acetyl chloride/ methanol reagent (1/19;

v/v), spiked with internal standard, nonadecanoic acid (10 µl of 1 mg ml-1) and esterified at

80°C for 1 h. After cooling, 1 ml of water and 2 ml of n-hexane were added to the mixture,

vortexed and centrifuged at 2057 g for 5 min. The organic phases were collected, filtered

and dried with anhydrous sodium sulphate. Solvents were removed under nitrogen and the

methyl esters solubilized in 200 µl of n-hexane for GC-MS analysis.

2.2.5. GC-MS Analysis

The GC-MS analysis of FAME samples was carried out on a QP2010 gas

chromatography mass spectrometer (GC-2010 coupled with GC-MS QP-2010) equipped

with an autosampler (AOC-5000) from Shimadzu (Japan) using a RTX-5 fused silica

capillary column, 30 m x 0.25 mm x 0.25 µm (Rastek). Helium (99.9% purity) was used as

the carrier gas with the column flow rate of 1 ml min-1 and the pre-column pressure of 49.7

KPa. The column temperature regime was 40 °C for 3 min, followed by a 5 °C min-1 ramp

up to 230 °C followed by 40 min at 230 °C. The injection volume and temperature was 0.2

µl and 240 °C and the split ratio was 1/30. The mass spectrometer was operated in electron

compact mode with electron energy of 70 eV. Both the ion source temperature and the

interface temperature were set at 200 °C. FAME peaks were identified by comparison of

their retention times with authentic standards by GC-MS Post run analysis and quantified by

area normalization.

2.2.6. Statistical analysis and experimental design

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41  

All the analytical determinations were performed in triplicate and the mean values

were reported. The yields of fatty acids and total lipids obtained by different methods for

each macroalgal samples were compared by analysis of variance (ANOVA), values

significant at p ≤ 0.05. Further, the effect of different solvent mixtures and modifications

applied on the quantity of TL and FAMEs obtained via conventional methods were analyzed

by experimental design. The studied factors were type of macroalgal matrix (A), with three

levels (Chlorophyta, Phaeophyta and Rhodophyta); extraction method (B), with three levels

(BD, FM and CS), sonication (C), with two levels 0 (= absent), 1 (= present) and buffer (D),

with two levels 0 (= absent), 1 (= present). A full factorial categorical design with

{3x3x2x2} = 36 runs was selected to study both the main effects and the interactions. This

type of design consists of all levels of combinations of two or more non-quantitative factors,

where the user sets the number of levels. The response variables analyzed were the amount

of total lipids (TLs), total FAs (TFAs) expressed as the sum of the concentrations (mg g-1

FW) of all identified FAs; and three other dependent variables calculated as the sum of the

contents of the FAs belonging to the same structural class, i.e., saturated FAs (ΣSFAs),

monounsaturated FAs (ΣMUFAs) and polyunsaturated FAs (ΣPUFAs), expressed in µg g-1

FW. The results obtained were again evaluated by analysis of variance (ANOVA), which

measures whether a factor contributes significantly to the variance of the response. The

experimental design and the data analysis were performed using Unscrambler version 9.8

(Camo, USA).

2.3. Results

The TL and TFA contents of the three macroalgae studied in this study are shown in

Fig. 2.2 and the fatty acid profiles are recorded in Tables 2.1, 2.2 and 2.3. The three

macroalgae exhibited the typical profiles of their respective phyla, U. fasciata being rich in

C18 PUFAs, G. corticata in C20 PUFAs and S. tenerrimum in both. As expected, U.

fasciata reported the highest TL and TFA contents followed by S. tenerrimum and G.

corticata.

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42  

Fig. 2.2 TL (A) and TFA (B) yields (mg g-1 FW) in U. fasciata, G. corticata, and S. tenerrimum.

The efficiency of TL and TFA extraction varied with the macroalgal matrices and

extraction methods. The addition of buffer to the respective solvent systems also rendered

significantly different yields while sonication displayed non significant effects (Table 2.4).

The contents of SFAs, MUFAs and PUFAs were also greatly affected by these three factors

except extraction method, which was found non-significant for MUFAs. Besides

determining the influence of each factor on TL and FAs, second order interactions were also

evaluated (Table 2.4). A significant interaction between extraction method and buffer was

found for all the variables studied except for TL which largely depended on the interaction

of macroalgal matrix and extraction method. Further, all the interaction effects were more

conspicuous for PUFAs yield showing significant positive interactions except for sonication

which showed no significant interaction with any factor.

The modified Folch method, FMB (Method 2C) gave the highest TL yield in U.

fasciata and G. corticata while modified Bligh and Dyer method, BDB (Method 1C)

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43  

rendered the highest TL yield in S. tenerrimum (Fig. 2.2). However, the differences between

the three conventional methods were larger and the simple Folch method, FM (Method 2A)

gave higher TL contents than the simple Bligh and Dyer method, BD (Method1A) and

Cequier-Sánchez method, CS (Method 3A) in U. fasciata (2.3-fold and 3.3-fold

respectively), G. corticata (4.0-fold and 1.5-fold respectively) and S. tenerrimum (2.7-fold

and 1.8-fold respectively). Further, the application of sonication to three established

conventional methods increased the TL yields in all the three matrices except FMS whereby,

the TL yields decreased considerably in the three matrices. The addition of buffer also

improved the lipid extraction in macroalgae except in S. tenerrimum where both the FMB

and CSB methods failed to increase the efficacy of lipid extraction.

Similarly, the amount of TFAs and different FA groups were also affected by the

modifications employed in the present study. The method 1C (BDB) demonstrated the

highest TFAs, SFAs, MUFAs and PUFAs yields unanimously for the three different

macroalgal matrices, among the three conventional methods investigated prior to

transmethylation by Carreau & Dubacq method. The application of sonication and buffer

individually to the BD solvent system significantly increased the yields of TFAs, SFAs,

MUFAs and PUFAs in the macroalgal matrices except for MUFAs content in S. tenerrimum.

In contrast, their addition to the FM solvent system (FMS, Method 2B and FMB, Method

2C) considerably decreased the recovery of fatty acids while CS system showed erratic

results. The application of sonication with the CS solvent mixture (CSS, Method 3B) caused

a decrease in FAs yields whereas, buffer (CSB, Method 3C) oppositely increased the yield

in macroalgae studied except in S. tenerrimum.

On comparing with DT methods, the conventional methods for FA extraction prior to

transmethylation yielded significantly less FAs. The LRC (Method 6) rendered the highest

yields in U. fasciata about 2-times higher TFA, SFA, PUFA and 1.1-times MUFA contents

than the most efficient BDB (Method 1C) while GM (Method 5) was more efficient for G.

corticata and S. tenerrimum both showing 1.5- to 2.0-times higher yields. However, LRC

(Method 6) was the only exception in G. corticata and S. tenerrimum to give quite lower

FAs contents than BDB (1C).

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44  

Table 2.1 Fatty acid composition of Ulva fasciata (μg g-1 FW).

Fatty acids Conventional methods Direct transesterification methods

Bligh and Dyer method Folch method Dichloromethane method BD BDS BDB FM FMS FMB CS CSS CSB GM LRC

C14:0 10.11±5.2c 12.22±0.6 c 33 ±1.61 c 17.35±2.84 c 9.51±0.2 5.09±0.05 c 10.98±0.38c 7.13±0.78 c 65.37±3.1 b 221.54±5.4 a 74.27±0.41 b

C16:0 239±6.74 f 322.2±8.6 f 891.2±3.94 c 463.8±7.33 de 350.5±3.83ef 133.86±2.8 f 200.6±7.19ef 207.9±7.58ef 689.93±6.7cd 1845.1±7.2 b 2416.4±8.3 a

C18:0 23.13±1.13 c 19.78±0.18 c 95.66±4.45 b 31.85±1.55 c 20.98±2.5 c 23.13±0.72c 13.35±0.95 c 6.92±0.51 c 97.86±1.9 b 259.25±3.1 a 97.39±0.59 b

C20:0 1.83±0.10 d 4.59±0.6 c 9.28±5.7ab 4.55±0.22cd 2.97±0.2 d 2.06±0.3 d 3.14±0.15 d 1.75±0.15 d 7.01±0.4 b 12.02±0.20 a 11.14±0.03 a

C22:0 8.69±0.45 c 14.27±2.5 c 34.49±2.1 b 19.63±1.7 c 8.56±0.2 c 5.84±0.58 c 8.85±0.28 c 8.81±0.28 c 42.89±4.3 b 59.99±1.53 a 75.13±0.32 a

C16:1(n-7) 8.75±0.55 d 18.02±2.82cd 52.35±2.93 a 20.82±2.4cd 15.61±0.5cd 9.65±0.7 d 11.4±0.32 d 8.91±0.94 d 31.48±3.2bc 47.31±0.44 a 46.87±0.40ab

C16:1(n-9) 3.77±0.5 c 15.35±1.9 c 46.21± 2.93 b 17.03±1.0 c 13.5±2.6 c 2.24±0.2 c 12.63±0.43 c 7.56±1.71 c 29.36±4.3 b 21.42±0.83 c 58.21±0.92 a

C18:1(n-9) 42.32±3.13 c 88.83±3.74 c 237.1±2.57 b 93.16±6.8 c 82.94±4.1c 33.76±1.80 c 21.7±1.26 c 20.06±3.5 c 47.59±1.5 c 333.0±3.07 a 345.14±4.2 a

C18:2(n-6) 114.39±6.7 de 99.72±2.6 de 256.86±9.2bc 180.16±3.8cd 131.74±3.1 de 54.53±2.80 e 72.4±5.18 de 53.03±6.2 e 128.1±4.7 de 328.9±6.77 b 447.52±4.6 a

C18:3(n-6) 8.09±0.45 b 8.93±0.5 b 21.11±0.77 a 15.86±1.8 a 10.45±0.6 b 3.06±0.6 b 31.19±4.16 a 3.95±1.2 b 11.0±2.15 a 18.71±0.15 a 32.67±0.49 a

C18:3(n-3) 382.32±6.7 d 454.37±10.5 d 1219.6±9.0 c 640.20±6.5 d 555.92±6.78 d 249.7±5.80 d 267.1±1.42 d 159.19±3.2 d 652.1±3.9 d 2201.4±7.3 b 3419.53±8.3 a

C18:4(n-3) 54.91±3.3ef 68.05±7.2 def 201.43± 8.36 b 116.88±5.7 de 72.07±2.64edf 30.70±1.37 f 54.38±1.7ef 13.91±0.96 f 196.5±4.2bc 129.67±1.4cd 273.82±6.3 a

C20:3(n-6) 3.55±0.13 c 5.43±1.1 c 7.92± 0.34 c 6.77±0.26 c 3.17±1.0 c 3.48±1.5 c 3.21±0.22 c 2.87±0.75 c 10.3±1.67bc 27.29±1.47 a 15.99±0.6 b

C20:3(n-3) 4.94±0.2 de 6.26±1.4 de 12.70± 0.45cd 8.81±1.49 c 4.43±0.6 de n.d. 3.6±0.14 e n.d. 12.2±2.45 c 18.18±0.38b 26.12±0.17 a

C20:4(n-6) 9.63±0.59bcde 11.8 5±0.6bcd 30.14± 1.5a 16.87±2.62bc 11.26±0.8bcde 5.95±0.33de 9.25±0.85cde 4.46±1.3e 16.9±4.14bc 18.80±0.13b 34.19±0.43a

C20:5(n-3) 5.87±0.30d 8.13±1.8cd 20.21± 0.78a 13.44±3.5bc 8.54±0.5cd 2.68±0.11d 5.43±0.94 d 3.06±1.2 d 13.71±3.6bc 18.90±0.24ab 25.49±0.58 a

C22:6(n-3) 23.94±1.42ef 38.34±1.0 de 73.70± 3.15bc 51.75±2.13 d 23.57±3.1ef 10.26±0.52 f 26.85±0.85ef 16.86±1.97ef 36.02±3.2 de 86.47±1.9 b 170.07±1.21 a

Others 12.07±0.5 c 18.51±3.0bc 73.83± 4.22 a 23.13±0.92bc 17.49±0.5bc 13.1±0.35 c 23.12±0.46bc 17.5±1.26bc 24.44±1.6bc 76.77±1.7 a 41.78±0.18 b

SFA 291.6±5.7 c 384.85±6.2 b 1106.4±9.68 c 554.1±6.5 c 405.4±4.03 c 177.9±4.3 c 241.5±0.94 c 235.76±4.8 c 933.85±8.5b 2469.2±8.9 a 2707.9±9.7 a

MUFA 58.15±3.8 b 129±4.6 b 366.7±4.65 a 137.2±3.03 b 1116.7±10.7 b 50.7±1.13 b 45.77±0.20 b 40.42±0.66 b 114.7±1.9 b 407.2±3.81 a 458.3±5.63 a

PUFA 607.64±7.5 c 701±15.42 c 1843.8±7.64 c 1050.7±12.5 c 109.4±7.21 c 360.4±8.04 c 473.42±5.2 c 257.34±4.8 c 768.2±5.4 c 2848.3±9.2 b 4445.4±10.6 a

n6/n3 0.29±0.05:1ab 0.22±0.02bc 0.21±0.01bcd 0.26±0.05ab 0.23±0.02bc 0.22±0.02bc 0.32±0.12 a 0.33±0.01 a 0.27±0.02ab 0.16±0.05cd 0.14±0.01 d

Others: Summation of the contents of C12:0, C13:0, C15:0, C17:0, C17:1 (n-7) and C20:1 (n-9); a–f : Values in a row without a common superscript are significantly different (p ≤ 0.05); n.d.: not

detected; Note: BD-Bligh and Dyer; BDS-Bligh and Dyer with sonication; BDB-Bligh and Dyer with buffer; FM-Folch method; FMS-Folch method with sonication; FMB-Folch method with buffer;

CS-Cequier - Sánchez method; CSS-Cequier-Sánchez method with sonication; CSB-Cequier -Sánchez method with buffer; GM-Garcia method; LRC-Lepage and Roy modified by Cohen method.

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45  

Table 2.2 Fatty acid composition of Sargassum tenerrimum (μg g-1 FW).

Fatty acids Conventional methods Direct transesterification methods

Bligh and Dyer Method Folch Method Dichloromethane Method BD BDS BDB FM FMS FMB CS CSS CSB GM LRC

C14:0 5.66 ±1.12 g 6.38±0.7 g 63.3±3.2 b 20.43±7.09ef 18.98±5.8ef 27.01±2.7 de 61.52±5.6bc 14.49±1.5fg 36.86±4.3 d 148.34±6.8 a 52.9±0.5 c

C15:0 2.03±1.05 d 2.54±0.3cd 5.59±0.5bc 3.97±0.5cd 3.52±0.9cd 5.26±1.3bc 4.69±0.6bcd n.d. 3.59±0.4cd 24.35±5.7 a 7.6±0.3 b

C16:0 39.03±7.9 f 34.89±4.08 f 286.50±8.8 b 151.76±6.1cd 86.41±4.1ef 94.31±3.5 def 327.83±20.6 b 67.65±4.9ef 113.77±6.8 d 547.17±17.8 a 180±8.8 c

C18:0 3.86±0.39 f 4.45±0.61 f 39.69±3.17 b 9.56±3.7ef 7.59±1.9 f 18.35±3.4 d 32.88±5.3 c 6.6±0.93 f 15.12±3.9 de 92.46±2.8 a 27.26±4.8 c

C20:0 1.17±0.01 c 1.29±0.07 c 2.36±0.34bc 2.07±0.9bc 2.29±0.1bc 1.96±0.3bc 3.2±0.5 b n.d. 4.31±0.57 b 10.35±0.6 a 1.89±0.3bc

C22:0 1.86±0.32 c 2.67±0.12 c 5.45±0.43 b 2.44±0.6 c 3.32±0.8bc 4.18±1.8bc 4.09±0.4 b n.d. 2.93±0.7bc 17.12±4.5 a 5.41±0.4 b

C16:1(n-7) 9.93±2.48 f 9.58±0.81 f 71.83±5.6b 31.64±3.2 d 27.85±2.8 de 27.10±3.5 de 35.48±6.2ef 7.37±1.5 f 16.04±1.9 def 165.37±1.0 a 55.28±2.7 c

C18:1(n-9) 9.74±2.16ef 7.98±0.6 f 82.33±6.9 b 33.7±4.2cd 25.8±5.3 def 27.37±2.1 de 78.03±4.42 b 16.64±6.5 def 24.67±8.4 def 264.36±3.5 a 48.56±2.2 c

C18:2(n-6) 15.4±3.6 f 11.27±1.24 f 117.02±5.73 b 47.40±5.86 d 44.01±2.6 de 43.25±3.3 de 73.47±0.76 c 13.69±1.9 f 26.31±1.15ef 235.35±12.3 a 88.52±8.1 b

C18:3(n-6) 3.61±1.8 de 2.94±0.13 e 13.57±0.7 b 4.32±1.3 de 8.79±4.2 c 4.61±0.8 de 10.55±0.6 c n.d. 5.91±0.22 d 16.61±1.2 a 10.54±0.5 c

C18:4(n-3) 6.03±0.6 f 12.67±0.3ef 57.48±8.3 b 18.32±4.9 de 25.78±8.3cd 25.13±1.8cd 38.28±5.6 c n.d. 11.28±1.6ef 78.4±7.1 a 40.94±2.9bc

C20:2(n-6) 5.1±1.02bc 5.31±0.51bc 8.91±6.5 a 5.72±1.2 b 3.75±0.9 c 5.34±1.8bc 10.21±0.7 a n.d. 5.26±2.1bc 10.35±1.1 a 5.45±0.5bc

C20:3(n-6) 4.48±1.3 d 6.36±0.7 d 12.25±0.67 c 3.81±0.97 d 6.06±2.1 d 4.89±1.1 d 28.72±6.5 a 7.71±1.8 d 13.27±2.9 c 17.54±0.8 b 12.39±0.7 c

C20:3(n-3) 2.5±0.6ef 3.28±0.42 de 9.93±0.36bc 4.22±0.7 d 4.16±0.7 d 3.58±0.9 d 10.05±0.6 b n.d. 2.17±0.6 f 12.38±0.6 a 8.96±0.4 c

C20:4(n-6) 42.36±9.5 e 65.97±5.7 de 346.2±18.2 b 115.4±8.5cde 113.25±5.6cde 110.2±4.1cde 372.32±9.6 b 81.27±8.7 de 142.62±4.71cd 466.9±18.8 a 189.45±8.4 c

C20:5(n-3) 6.02±0.51 f 6.80±0.64 f 56.84±6.5cd 18.22±5.6ef 30.13±7.3 e 110.2±5.4 a 61.21±8.9bc 6.91±0.9 f 11.0±1.7 f 102±5.6 a 46.67±2.5 d

Others 1.23±0.1 f 2.62±0.55 e 44.12±5.2bc 26.75±7.8cde 19.26±1.34 de 18.83±3.5 de 27.97±2.1cd 1.65±0.1 f 18.59±3.5 de 123.15±8.3 a 57.87±2.9 b

SFA 54.84±7.4 f 54.85±6.1 f 405±6.4 b 194.5±7.5 d 126.3±2.8 def 156.7±7.2 de 447.8±11.2 b 90.4±7.4ef 185.6±4.8 d 872.13±22.4 a 285.1±2.8 c

MUFA 19.67±4.6ef 17.56±1.4 f 196±11.7 b 87.8±8.5 d 68.8±1.5 d 67.7±3.6 d 133.6±7.5 c 24.01±0.94ef 54.9±1.62 de 520.5±8.5 a 151.7±10.2 c

PUFA 85.5±8.6 f 114.6±9.5 e 622.2±18.4 b 217.5±8.8 de 235.9±5.8 d 221.7±10.2 de 604.8±15.4 b 109.6±6.31 e 217.8±6.8 de 939.5±18.3 a 402.9±13.7 c

n6/n3 4.85±0.38 c 4.03±0.13cd 4.03±0.33cd 4.33±0.62cd 2.94±0.24 d 3.25±0.62cd 4.5±0.67cd 14.6±3.03 a 7.75±1.5 b 3.88±0.16cd 3.21±0.32cd

Others: Summation of the contents of C12:0, C17:0, C17:1 (n-7), C18:1 (n-9) trans, C20:1 (n-9), C22:1 (n-9); a–f: Values in a row without a common superscript are significantly different (p≤ 0.05);

n.d: not detected; Note: BD-Bligh and Dyer; BDS-Bligh and Dyer with sonication; BDB-Bligh and Dyer with buffer; FM-Folch method; FMS-Folch method with sonication; FMB-Folch method

with buffer; CS-Cequier - Sánchez method; CSS-Cequier-Sánchez method with sonication; CSB-Cequier -Sánchez method with buffer; GM-Garcia method; LRC-Lepage and Roy modified by

Cohen method.

CHAPTER 2  

46  

Table 2.3 Fatty acid composition of Gracilaria corticata (μg g-1 FW).

Fatty acids Conventional methods Direct transesterification methods

Bligh and Dyer Method Folch Method Dichloromethane Method BD BDS BDB FM FMS FMB CS CSS CSB GM LRC

C12:0 1.9±0.1f 2.22±0.2ef 7.2±0.5 b 4.08±0.8cd 3.14±0.3 de 2.38±0.9ef 2.04±0.3 f 2.07±0.7ef 4.5±0.5 c 14.36±1.2 a 5.02±0.6 c

C14:0 5.17±0.3 c 8.45±0.6 c 20.74±3.9 c 18.24±0.65 c 16.85±2.5 c 11.6±0.35 c 13.92±0.6 c 12.56±0.5 c 16.41±1.9bc 26.3±1.98ab 33.48±2.7 a

C15:0 2.0±0.3 e 3.92±0.5 d 5.61±0.2 c 7.17±0.28 b 5.47±0.4 c 3.68±0.8 d 2±0.4 e 1.42±0.1 e 3.88±0.6 d 12.3±0.4 a 5.84±0.5bc

C16:0 133.54±2.6 g 166.50±2.85fg 741.79±8.2 b 306.26±7 de 306.21±4 de 259±6.2 def 203.5±7.4efg 137.5±8.1 dg 346.83±4.0 d 1045.5±7.2 a 550.85±6.8 c

C17:0 2.98±0.2 c 2.45±0.4 c 4.94±1.0 c 5.27±0.39 c 3.35±0.4 c 25±3.98 b 1.66±0.1 c 1.5±0.01 c 42.05±3.9 a 3.74±0.4 c 3.56±0.7 c

C18:0 11.38±0.2 de 20.19±0.4cde 46.84±4.6ab 17.12±1.02 de 8.90±0.3 e 25±2.03cd 15.64±3.7 de 11.17±1.1 de 48.06±4.2 a 56.5±3.5 a 32.98±1.1bc

C20:0 1.09±0.1 e 1.43±0.41 de 4.57±0.6 a 2.45±0.02 c 2.28±0.3 c 2.49±0.4 c 1.53±0.3 de 1.07±0.1e 3.72±0.5 b 4.59±0.3 a 1.95±0.7cd

C22:0 1.61±0.2 de 2.52±0.4cd 2.24±0.1cde 2.96±0.12bc 3.79±0.2ab 1.47±0.1 de 1.53±0.3 de 1.12±0.1 e 2.15±0.6cde 4.38±0.3 a 2.87±1.6bc

C16:1(n-7) 5.12±0.3 de 2.32±0.3 e 12.88±1.1bc 18.95±1.27 b 11.8±0.7cd 9.99±0.5cd 7.47±0.7cde 5.63±0.1 de 11.27±1.0cd 33.37±3.4 a 7.33±2.8cde

C17:1(n-7) 0.98±0.1 d 1.09±0.1 d 4.33±0.9 a 1.75±0.5cd 2.38±0.4bc 2.17±0.3bc 1.13±0.1 d 1.01±0.1 d 2.72±1.0 b 4.37±0.2 a 1.06±0.2 d

C18:1(n-9) trans

2.13±0.2 e 4.0±0.3 de 14.5±1.4 b 10.51±0.44 c 6.79±0.2 d 10.28±0.5 c n.d. n.d. n.d. 13.82±1.3 c 18.16±2.7 a

C18:1(n-9) 4.73±0.2 g 14.10±0.9 e 45.1±4.1 a 28.72±1.13cd 29.6±2.8 c 33.47±2.2 b 12.75±0.9 e 10.81±1.1ef 13.83±1.7 e 39.92±2.4ab 22.41±2.2 d

C18:2(n-6) 4.83±0.7 f 7.03±0.91ef 13.16±2.3 c 13.52±0.24 c 11. 2±0.3cde 8.39±1.2 def 8.43±0.8 def 6.05±1.1 f 10.2±1.1cdef 52.17±4.0 a 21.70±4.8 b

C20:3(n-6) 2.70±0.5 f 8.28±0.6cde 11.53±0.7cd 23.94±0.86 a 13.73±0.5bcd 8.8±1.1cde 11.83±0.6cd 10.76±0.9cd 12.1±0.7bcd 4.62±0.2 e 17±4.5 b

C20:4(n-6) 13.83±1.1 h 39.46±1.12gh 207.33±6.1b 102.81±1.7de 75.27±5.6ef 49.3±1.9fg 118.54±7.6 d 82.29±5.7ef 169.6±8.37 c 275.8±5.1 a 125±6.2 d

C20:5(n-3) 3.10±0.6cd 3.44±0.7cd 26.58±3.1 a 8.64±0.17 b 9.31±0.6 b 3.81±0.7cd 3.77±0.9cd 2.19±0.1 d 4.0±1.4cd 4.96±0.3 c 4.93±0.3 c

SFA 159.66±3.8 i 207.7±5.12ghi 833.9±9.3 b 363.5±7.8de 350±8.31 def 331±9.8efg 245.3±5.0fgh 170.5±9.2 hi 471.3±9.68 d 1167.7±11.2 a 636.5±9.8 c

MUFA 12.96±0.7 e 21.5±1.6 de 76.8±7.5 b 59.9±2.8 c 50.68±0.4 c 55.91±3.3 c 25.8±3.0 de 19.6±2.2 de 31.7±4.7 d 112.1±7.1 a 48.97±0.78 c

PUFA 24.47±2.9 i 58.2±1.34 hi 258.6±3.6 b 148.9±1.2 d 109.5±3.3ef 70.3±3.3gh 144.5±8.7 de 103.1±7.6fg 197.9±3.7c 337.6±9.4 a 168.6±3.2cd

n6/n3 6.95±0.5 e 15.9±3.7 d 8.7±3.7 e 16.54±2.5 d 10.7±0.7 e 17.93±3.8 d 24.12±1.1 c 24.62±3.9 c 32±1.68 b 67.1±2.3 a 33.5±7.54 b

a–f: Values in a row without a common superscript are significantly different (p ≤ 0.05); n.d.: not detected; Note: BD-Bligh and Dyer; BDS-Bligh and Dyer with sonication; BDB-Bligh and Dyer

with buffer; FM-Folch method; FMS-Folch method with sonication; FMB-Folch method with buffer; CS-Cequier - Sánchez method; CSS-Cequier-Sánchez method with sonication; CSB-

Cequier-Sánchez method with buffer; GM-Garcia method; LRC-Lepage and Roy modified by Cohen method.

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47  

Table 2.4 Main effects of the type of macroalgal samples (Ulva fasciata, Gracilaria corticata, and Sargassum tenerrimum); the extraction methods and

modifications (sonication and buffer) on total lipid (TL), total FAs (TFAs), saturated FAs (SFAs), monounsaturated FAs (MUFAs) and polyunsaturated

FAs (PUFAs).

Variables Model

Main effects Interactions

Macroalgal matrix (A)

Extraction method

(B)

Sonication (C)

Buffer (D)

AB AC AD BC BD CD

TL F-ratio 4.05 5.75 9.41 0.07 7.18 6.07 0.02 2.73 2.85 1.63 0.59 p-value 0.003 0.013 0.002 0.78 0.016 0.003 0.97 0.095 0.087 0.22 0.45

TFAs

F-ratio 5.16 20.45 4.21 0.605 13.02 1.83 0.083 1.88 0.26 11.6 0.14 p-value 0.0009 0.00 0.034 0.45 0.002 0.17 0.92 0.18 0.77 0.0008 0.71

SFAs F-ratio 6.25 14.76 6.003 0.82 28.75 1.76 0.148 2.41 0.23 17.5 0.11 p-value 0.0003 0.0002 0.011 0.38 0.0001 0.18 0.86 0.12 0.79 0.0001 0.74

MUFAs

F-ratio 4.92 25.8 3.36 0.51 4.98 2.51 0.28 1.25 0.33 7.85 0.16p-value 0.0011 0.00 0.06 0.48 0.04 0.08 0.75 0.31 0.72 0.004 0.69

PUFAs F-ratio 6.73 15.5 6.5 0.07 21.96 5.06 0.694 2.8 0.32 16.95 0.22 p-value 0.0002 0.0002 0.008 0.79 0.0002 0.008 0.51 0.09 0.72 0.0001 0.64

The second-order interactions are also shown. Values in bold are significant at p ≤ 0.05; Note: TL-Total lipid, TFAs-Total fatty acids, SFAs-Saturated fatty acids, MUFAs-Monounsaturated fatty

acids, PUFAs-Polyunsaturated fatty acids.

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48  

Table 2.5 Fatty acid composition of Ulva fasciata (wt/wt% FW).

Fatty acids

Conventional methods Direct transesterification methods

Bligh and Dyer method Folch method Cequier - Sánchez method

BD BDS BDB FM FMS FMB CS CSS CSB GM LRC

C14:0 1.1 ± 0.11b 1.0 ± 0.14b 1.0 ± 0.12 b 1.0 ± 0.2 b 0.7 ±0.05 b 0.9 ±0.2 b 1.4 ±0.08 b 1.4 ±0.16 b 3.1 ±0.15 a 4.0 ±1.55 a 1.0 ± 0.1 b

C16:0 25.4 ± 1 e 26.6 ± 0.7de 26.6 ± 3cd 26.8 ± 2cd 26.1±1.8 e 23 ± 1.4 e 26.5±1.6 de 39 ± 5 a 32.6 ± 0.7bc 32.8 ± 5 b 31.7 ± 0.8bcd

C18:0 2.6 ± 0.36 b 1.7 ± 0.46 c 2.8 ± 0.32 b 1.8 ± 0.33 c 1.6 ± 0.09 c 3.9 ± 0.27 a 1.8 ±0.1 c 1.4 ± 0.2 c 4.6 ± 0.42 a 4.6 ± 0.46 a 1.3 ± 0.12 c

C20:0 0.2 ± 0.02bc 0.4 ± 0.02 a 0.3± 0.09 a 0.3± 0.05 a 0.2 ± 0.01bc 0.4±0.05 a 0.4 ±0.02 a 0.3 ± 0.04 a 0.3 ± 0.01 a 0.2 ±0.07bc 0.1 ± 0.02 c

C22:0 0.9 ± 0.1 c 1.2 ± 0.01bc 1.0 ± 0.24 c 1.1 ± 0.04bc 0.6 ±0.04 c 0.9±0.75 c 1.2±0.06 b 1.7 ± 0.26ab 2.0 ± 0.08 a 1.0 ±0.18 c 1.0 ± 0.11 c

C16:1(n-7) 0.9 ± 0.17bcd 1.4 ± 0.4ab 1.5 ± 0.31 a 1.3 ± 0.33abc 1.2 ±0.06abc 1.7 ± 0.4 a 1.5±0.09 a 1.7 ± 0.21 a 1.5 ± 0.02 a 0.8 ±0.08cd 0.6 ± 0.04 d

C16:1(n-9) 0.6 ± 0.44bcd 1.3± 0.23abc 1.3 ± 0.8abc 1± 0.5abcd 1±0.13abcd 0.4±0.08 d 1.7 ±0.11 a 1.44 ± 0.12ab 1.38 ± 0.1abc 0.4±0.09 d 0.8±0.02bcd

C18:1(n-9) 3.8 ± 2.8cd 7.1 ± 1.9 a 6.9 ± 0.9ab 5.7 ± 1.4abc 6.2 ±0.3abc 5.8±0.4abc 2.9 ±0.19 d 3.9 ± 0.3cd 2.2 ± 0.34 d 5.9 ±0.9abc 4.5 ± 0.17bcd

C18:2(n-6) 11.8± 1 a 8.4 ± 1.4bc 8.0 ±1.2bcd 10 ± 1.5ab 9.8±0.64ab 9.1±0.72 b 9.5 ±0.43ab 10.2 ± 0.46ab 6.0 ± 0.85cd 5.6 ±1.8 d 5.9 ± 0.3cd

C18:3(n-6) 0.8 ± 0.08 a 0.8 ± 0.18 a 0.7 ± 0.05a 0.9 ± 0.11 a 0.8±0.05 a 0.5±0.12 a 3.9 ±0.5 a 0.7 ± 0.06 a 0.5 ± 0.05 a 0.3 ±0.04 b 0.4 ± 0.1 b

C18:3(n-3) 39.6 ± 2.3ab 37.3 ± 2.3bc 37.6 ± 3.2abc 36.9 ± 3.2bc 41.3 ±2.4ab 42.7±1.6ab 35.2 ± 2bc 30.5± 1.7 c 30.9 ± 3 c 37.8 ±7.8abc 44.9 ±1.4 a

C18:4(n-3) 6.0 ± 1.5bc 5.7 ± 0.6bc 6.1 ± 0.2bc 6.5 ± 0.8bc 5.4 ±0.75bcd 5.0 ±1cd 7.1 ± 0.63 b 2.7 ± 0.42 e 9.2 ± 1.2 a 2.3 ±0.19 e 3.6±0.37 de

C20:3(n-6) 0.4 ± 0.14ab 0.4 ± 0.02ab 0.2± 0.02 b 0.4 ± 0.03ab 0.2±0.02 b 0.6±0.11 a 0.4 ± 0.01 a 0.5 ± 0.02 a 0.5 ± 0.04 a 0.5 ±0.35 a 0.2 ± 0.02 b

C20:3(n-3) 0.6 ± 0.17 a 0.5 ± 0.02 a 0.4 ± 0.03ab 0.5 ± 0.15 a 0.3 ±0.05 b n.d. 0.5 ± 0.03 a n.d. 0.6 ± 0.06 a 0.3 ±0.04 b 0.3 ± 0.03 b

C20:4(n-6) 1.0 ± 0.17ab 1.0 ± 0.13ab 0.9± 0.1ab 1.0 ± 0.08ab 0.8 ±0.05bc 1.0± 0.4ab 1.2 ± 0.08 a 0.8 ± 0.05bc 0.8 ± 0.13bc 0.3 ±0.05 d 0.5 ± 0.01cd

C20:5(n-3) 0.6 ± 0.07 a 0.7± 0.04 a 0.6 ± 0.05 a 0.8 ± 0.08 a 0.6 ±0.02 a 0.4±0.08 a 0.7 ± 0.09 a 0.6 ± 0.09 a 0.6 ± 0.11 a 0.3 ±0.1 b 0.3 ± 0.03 b

C22:6(n-3) 2.5 ± 0.4bcde 3.1± 0.3abc 2.2 ± 0.17 def 2.9 ± 0.3abcd 1.8 ±0.11ef 1.7 ±0.5ef 3.5 ± 0.22 a 3.3 ± 0.4ab 1.7± 0.03ef 1.5 ±0.28 f 2.3± 0.18cde

Others 1.4 ± 0.46def 3.3± 0.53ab 1.6 ± 0.45cdef 2.3 ± 0.09bcde 1.7 ±0.13cdef 2.7±0.02bc 2.5 ±0.03bcd 1.4 ± 0.11 def 1.2 ± 0.04ef 4.2 ±0.05 a 0.6±0.06 f

SFA 31.2 ± 1.9 c 31.8 ± 1.1c 32.9 ± 3.7 c 31.9 ± 2.5 c 30.2 ±1.9 c 30.4±1.2 c 31.9 ±1.9 c 44.4 ± 4.5 b 51.6 ± 2.2 a 43.9 ±6.9 b 35.6 ±0.8 c

MUFA 5.7 ± 1.5 c 10.4 ± 2 a 10.6 ± 2 a 8.4± 1.9abc 8.7 ±0.2ab 8.7 ±0.5ab 6.0 ± 0.4bc 7.8 ± 0.6abc 6.3 ± 0.2bc 7.2 ±0.9bc 6.0 ±0.2bc

PUFA 63.3 ± 1.5 a 57.9± 1.8 a 56.8 ± 4ab 59.9± 2.7 a 61.2 ±1.9 a 61.1±1.3 a 62.2 ± 2.1 a 49.4 ± 2.5bc 42.1 ± 1.9 c 49.0 ±7.8bc 58.4±1 a

n6/n3 0.29 ± 0.ab 0.2 ± 0.04bcd 0.2 ± 0.01bcd 0.26 ±0.05abcd 0.24±0.02abcd 0.22±0.02abcd 0.3 ± 0.12 a 0.3 ± 0.01 a 0.3± 0.02 a 0.16 ±0.05cd 0.1 ±0.01 d Others: Summation of the contents of C12:0, C13:0, C15:0, C17:0, C17:1 (n-7) and C20:1 (n-9) FAs; a-f: Values in a row without a common superscript are significantly different (p ≤ 0.05); n.d:

not detected; Note: BD-Bligh and Dyer; BDS-Bligh and Dyer with sonication; BDB-Bligh and Dyer with buffer; FM-Folch method; FMS-Folch method with sonication; FMB-Folch method with

buffer; CS-Cequier -Sánchez method; CSS-Cequier-Sánchez method with sonication; CSB-Cequier -Sánchez method with buffer; GM-Garcia method; LRC-Lepage and Roy modified by Cohen

method.

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49  

Table 2.6 Fatty acid composition of Sargassum tennerrimum (wt/wt% FW).

Fatty acids

Conventional methods Direct transesterification methods

Bligh and Dyer method Folch method Cequier - Sánchez method

BD BDS BDB FM FMS FMB CS CSS CSB GM LRC

C14:0 3.5 ± 0.4e 3.4 ±0.3 e 5.2 ±0.21cd 4.1 ±0.5 de 4.3 ±0.36 de 6.1 ±0.15bc 5.3 ±0.53cd 6.7 ±1.22 b 8.0 ±0.54 a 6.4 ±0.41 b 6.3 ±0.1bc

C15:0 1.2 ± 0.5ab 1.4 ±0.17 a 0.5 ±0.01cd 0.9 ±0.25bcd 0.8 ±0.03bcd 1.2 ±0.24ab 0.4 ±0.03 d n.d. 0.8 ± 0.2bcd 1.0 ±0.15ab 0.9 ±0.05abc

C16:0 24.4 ± 2bcd 18.6 ±0.6 c 23.4 ±1.4bcd 30.1 ±4.6 a 20.1±0.5de 21.3 ±1.5cde 27.8 ±0.8ab 30.6 ±1.7 a 25.2 ± 2.4bc 23.4 ±0.9bcd 21.5 ±0.1cde

C18:0 2.5 ± 0.7 de 2.4 ±0.17de 3.2 ±0.03 c 1.9 ±0.23 e 1.8 ±0.06 e 4.1 ±0.34 a 2.8 ±0.12cd 3.0 ±0.42cd 3.3 ± 0.3bc 4.0 ±0.24ab 3.2 ±0.3 c

C20:0 0.7 ± 0.14 a 0.7 ±0.02 a 0.2 ±0.01 a 0.4 ±0.08 a 0.6 ±013 a 0.4 ±0.09 a 0.3 ±0.02 a n.d. 0.8 ± 0.93 a 0.4 ±0.02 a 0.2 ±0.03 a

C22:0 1.2 ± 0.3ab 1.4 ±0.06 a 0.4 ±0.01 de 0.5 ±0.07 de 0.8 ±0.02cd 0.9 ±0.36bc 0.3 ±0.03 e n.d. 0.7 ± 0.13cd 0.7 ±0.12cd 0.6 ±0.01cde

C16:1(n-7) 6.2 ± 0.6abc 5.1 ±0.09 c 5.9 ±0.08bc 6.2 ±1abc 6.4 ±0.3ab 6.1 ±0.4abc 3.0 ±0.06 3.4 ±0.46 d 3.6 ± 0.72 d 7.1 ±0.3 a 6.6 ±0.3ab

C18:1(n-9) 6.1 ± 0.58bc 4.3 ±0.06c 6.7 ±0.09bc 6.6 ±1bc 6.0 ±0.23bc 6.2 ±0.4bc 6.7 ±0.96d 7.2 ±1.6b 5.3 ±0.33c 11.3 ±0.09a 5.8 ±0.27bc

C18:2(n-6) 9.6 ± 0.75a 6.0 ±0.14b 9.6 ±0.37a 9.4 ±1.4 a 10.1 ±0.6 a 9.7 ±0.5a 6.3 ±0.55 a 6.3 ±0.93 b 5.7 ±1.1 b 10.1 ±0.43 a 10.5 ±0.1 a

C18:3(n-6) 2.4 ± 1.5 a 1.6 ±0.12ab 1.1 ±0.04ab 0.9 ±0.22 b 2.0 ±0.6ab 1.0 ±0.11ab 0.9 ±0.2b n.d. 1.3 ±0.13ab 0.7 ±0.03 b 1.3±0.05ab

C20:2(n-6) 3.3 ± 1.3 a 2.8 ±0.13 a 0.7 ±0.01b 1.2 ±0.36 b 0.9 ±0.02 b 1.2 ±0.35 b 0.9 ±0.11 b n.d. 1.1 ±0.13 b 0.4 ±0.01 b 0.6 ±0.01 b

C20:3(n-6) 2.8 ±0.45bc 3.4 ±0.11 a 1.0 ±0.04 de 0.8 ±0.21 d 1.4 ±0.22 d 1.1 ±0.26 de 2.4 ±0.17 c 3.5 ±0.14 a 3.0 ±0.36ab 0.8 ±0.05 e 1.5 ±0.05 d

C20:3(n-3) 1.6 ± 0.13 a 1.7 ±0.07 a 0.8 ±0.04bc 0.9 ±0.29 b 1.0 ±0.0 b 0.8 ±0.16bc 0.9 ±0.11 b n.d. 0.5 ±0.01 c 0.5 ±0.03 c 1.1 ±0.05 b

C20:4(n-6) 26.4 ± 2.5bcd 35.3 ±0.96 a 28.4 ±1.3bcd 23.2 ±6.5cd 26.4 ±0.9bcd 24.7 ±1.3bcd 30.9 ±3.8ab 35.8 ±3.7 a 30.9 ±1.75ab 20 ±0.5 d 22.6 ±0.73cd

C20:5(n-3) 3.8 ±0.35cd 3.6 ±0.04cde 4.6 ±0.17bc 3.7 ±1.1cd 7.0 ±0.68 a 5.5 ±0.6 b 5.2 ±0.4 b 3.2 ±0.54de 2.5 ±0.5 e 4.4 ±0.07bcd 5.5 ±0.4 b

C18:4(n-3) 3.8 ±0.58cde 6.8 ±0.46 a 4.7 ±0.3bcd 3.8 ±1cde 5.9 ±0.6ab 5.5 ±1.4ab 3.3 ±0.2 de n.d. 2.5 ±0.42 e 3.4 ±0.12 de 4.8±051bcd

Others 0.7 ±0.47 e 1.4 ±0.27 e 3.6 ±0.14 d 5.5 ±0.65 b 4.6 ±0.89 a 4.3 ±1ab 1.3 ± 0.13 e 0.8 ±0.16 e 1.0 ±0.18 e 5.4 ±0.62 c 5.9±0.18 c

SFA 34.3 ±2.3bcd 29.3 ±1.3 d 33.1 ±1.2cd 38.8 ±5.4ab 29.4 ±0.6 d 35.2 ±1.4bc 38 ±1.4ab 41.1 ± 3.5 a 40.8 ±1.8 a 37.4 ±0.7abc 34 ±0.2bcd

MUFA 12.2 ±1.2 d 9.4 ±0.1 e 16 ±0.1bc 17.4 ±2.3bc 16 ±0.3bc 15.2 ±0.9 c 11.4 ±1.2 de 10.5 ± 1.4 de 12.0 ±0.7 d 22.3 ±0.2 a 18.1 ±0.3 b

PUFA 53.6 ±3.4bc 61.3 ±1.3 a 50.9 ±1.2bc 44 ±7.8 de 54.6±0.9ab 49.5 ±2bcd 50.7±2.6bcd 48.7 ±2bcd 47.4 ±1.5cd 40.3 ±0.8 e 48 ±0.1bcd

n6/n3 4.9 ±0.4 c 4.0 ±0.1 c 4.0 ±0.3 c 4.3 ±0.6 c 2.9 ±0.2 c 3.2 ±0.6 c 4.5 ±0.7 c 14.6 ±3 a 7.8 ±1.5 b 3.9 ±0.2 c 3.2 ±0.3 c Others: Summation of the contents of C12:0, C17:0, C17:1 (n-7), C18:1 (n-9) trans, C20:1 (n-9), C22:1 (n-9); a-f: Values in a row without a common superscript are significantly different

(p≤0.05); n.d.: not detected. Note: BD-Bligh and Dyer; BDS-Bligh and Dyer with sonication; BDB-Bligh and Dyer with buffer; FM-Folch method; FMS-Folch method with sonication; FMB-

Folch method with buffer; CS-Cequier -Sánchez method; CSS-Cequier-Sánchez method with sonication; CSB-Cequier - Sánchez method with buffer; GM-Garcia method; LRC-Lepage and

Roy modified by Cohen method.

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Table 2.7 Fatty acid composition of Gracilaria corticata (wt/wt % FW).

Fatty acids

Conventional methods Direct transesterification methods

Bligh and Dyer method Folch method Cequier - Sánchez method

BD BDS BDB FM FMS FMB CS CSS CSB GM LRC

C12:0 1.0 ± 0.04a 0.8 ±0.07bc 0.6 ±0.05cde 0.7 ±0.09bcd 0.6 ±0.01cde 0.5 ±0.08 e 0.5 ±0.03 e 0.7 ±0.18cd 0.7 ±0.08cde 0.9 ±0.02ab 0.6 ±.06 de

C14:0 2.6 ± 0.05bcd 3.0±0.26abcd 1.8 ±0.15 d 3.3 ±1.3abc 3.3 ±0.23abc 2.5 ±0.17cd 3.4 ±0.5abc 4.3 ±0.36 a 2.4 ±0.26cd 1.6±1.2 d 4.0 ±0.55ab

C15:0 1.0 ± 0.12ab 1.4±0.06 a 0.5 ±0.04c 1.3 ±0.6 a 1.1 0.07ab 0.8±0.1bc 0.5 ±0.01 c 0.5 ±0.03 c 0.6 ±0.07 c 0.8 ±0.02bc 0.7 ±0.09bc

C16:0 67.8 ± 1.2 a 57.9±1.7cd 63.5 ±0.5abc 53.7±7.1 de 60±0.66bcd 56.9 ±1.4 d 48.8 ±2.2ef 47.3 ±2.8 f 49.6 ±1.5ef 64.7 ±0.5 a 64.5 ±1.2ab

C17:0 1.5 ± 0.1 b 0.9±0.02bc 0.4 ±0.04 c 0.9±0.6bc 0.7 ±0.02bc 5.4±0.93 a 0.4 ±0.06 c 0.5 ±0.03 c 6.1 ±0.6 a 0.2 ±0.02 c 0.4 ±0.01 c

C18:0 5.8 ± 0.12abc 7.1±1.02 a 4.0±0.24bcd 3.0±1.5cd 1.8±0.16d 4.9 ±0.8abc 3.8 ±0.4cd 3.8 ±0.25cd 6.9 ±0.11ab 3.5 ±0.19cd 3.8 ±0.56 d

C20:0 0.6 ±0.01a 0.5±0.06ab 0.4±0.02 c 0.4±0.03bc 0.4±0.03bc 0.6 ±0.06 a 0.4 ±0.03cd 0.4 ±0.02cd 0.5 ±0.06 a 0.3 ±0.01 de 0.2 ±0.05 e

C22:0 0.8 ±0.06 a 0.9±0.02 a 0.2±0.02 d 0.5 ±0.17 b 0.7 ±0.05 a 0.3 ±0.09bcd 0.4 ±0.03bc 0.4 ±0.03bc 0.3 ±0.04cd 0.3±0.02cd 0.3 ±0.15cd

C16:1(n-7) 2.6 ±0.0ab 0.8±0.03 c 1.1 ±0.04bc 3.4±2.3 a 2.3 ±0.2abc 2.3 ±0.56abc 1.8 ±0.15abc 1.9 ±0.2abc 1.7 ±0.21bc 2.1±0.12abc 0.8 ±0.18 c

C17:1(n-7) 0.5 ±0.02 a 0.4±0.04ab 0.4 ±0.04bc 0.3±0.09 c 0.5 ±0.04a 0.5 ±0.08 a 0.3 ±0.04 c 0.3 ±0.02 c 0.4 ±0.07bc 0.3±0.01 c 0.1 ±0.001d

C18:1(n-9) 2.4 ±0.03de 5.0 ±0.48bc 3.9±0.11 c 5.1±1.9bc 5.8 ±0.3 b 7.7 ±1.8 a 3.1±0.38 d 3.7 ±0.2cd 2.0 ±0.19 e 2.1 ±0.09 e 2.7 ±0.3 de

C18:1(n-9) trans

1.1 ±0.05 d 1.4 ±0.14bcd 1.2±0.03cd 1.9 ±0.8abc 1.3 ±0.11cd 2.4 ±0.61 a n.d. n.d. n.d. 2.5±0.08 a 2.1 ±0.17ab

C18:2(n-6) 2.4 ±0.3 c 2.5 ±0.09 c 1.1±0.07 h 2.4 ±0.6cd 2.2 ±0.2de 1.9 ±0.25 f 2.1 ±0.21ef 2.1 ±0.16ef 1.5 ±0.17 g 3.2 ±0.17 a 2.7 ±1.2 b

C20:3(n-6) 1.4 ±0.2 f 2.9 ±0.25 c 1.0±0.08 g 4.3 ±1.8 a 2.7±0.23 c 2.0 ±0.35 d 2.9 ±0.37 c 3.7 ±0.18 b 1.8 ±0.27 e 0.3 ±0.01 h 2.0 ±0.22 de

C20:4(n-6) 7.0 ±0.4 f 13.6±1.8 de 17.7±0.61 c 18.1 ±1.2 c 14.7±0.6cde 11.4 ±3.2ef 28.7 ±1.2 a 27.9 ±3.6ab 24.5 ±0.9 b 17.1 ±0.8cd 14.5 ±1.1cde

C20:5(n-3) 1.6 ±0.24bc 1.2±0.08cd 2.3 ±0.09 a 1.5 ±0.2bc 1.8 ±0.11 b 0.9 ±0.43 de 0.9 ±0.07 de 0.8 ±0.04 e 0.6 ±0.07 e 0.3 ±0.01 e 0.6 ±0.17 e

SFA 81.1 ±1.1 a 72.3±1.5 b 71.4 ±0.6 b 63.9 ±7.6cd 68.6±0.4bc 72 ±5.9 b 58.9 ±1.9 d 58.6 ±3.6 d 67.6 ±1bc 72.3 ±0.9 b 74.5 ±1.3 a

MUFA 6.6 ±0.1bcd 7.6 ±0.7bcd 6.6 ±0.1bcd 10.8 ±5.2ab 10 ±0.5abc 12.9 ±3 a 6.3 ±0.4cd 6.7 ±0.4bcd 4.6 ±0.4 d 6.9 ±0.2bcd 5.8 ±0.3cd

PUFA 12.4 ±1.1 f 20.1 ±1.8 de 22.1 ±0.7cd 26.4 ±1.4bc 21.5±1 d 16.2 ±4.2ef 35.1 ±1.7 a 35 ±2.5 a 28.6 ±1.3 b 20.9 ±0.9 d 19.8 ±1 de

n6/n3 6.9 ±0.5 e 15.9 ±0.4 d 8.7 ±0.4 e 16.5 ±2.5 d 10.7 ±0.7 de 17.9 ±3.8cd 24.1 ±1.1 c 24.6 ±3.9 c 32 ±1.7 b 67.1 ±2.3 a 33.5 ±7.5 b a-f: Values in a row without a common superscript are significantly different (p≤0.05); n.d: not detected; Note: BD-Bligh and Dyer; BDS-Bligh and Dyer with sonication; BDB-Bligh and Dyer with

buffer; FM-Folch method; FMS-Folch method with sonication; FMB-Folch method with buffer; CS-Cequier-Sánchez method; CSS-Cequier-Sánchez method with sonication; CSB-Cequier-Sánchez

method with buffer; GM-Garcia method; LRC-Lepage and Roy modified by Cohen method.

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51  

2.4. Discussion

Reliable methods for quantitative extraction of lipids and FAs are of paramount

importance owing to their biochemical, physiological, clinical and nutritional applications.

The accuracy of different lipid extraction methods depend on the solubility of their

constituent lipid classes in the solvents employed and the nature of sample matrix as both

could influence the extent of lipid extraction. According to Christie (Christie, 1993),

extraction solvents/mixtures should be polar enough to remove lipids from their associating

cell constituents, but not too polar that the solvents do not readily solubilize all the

triacylglycerols (TAGs) and other non-polar lipids.

The present study also displayed significant variations in lipid and FA contents,

owing to the matrix effect and solubilising ability of different solvent systems employed to

completely disrupt the cellular membranes and dissolve the entrapped lipids from

macroalgae. Similar findings have also been exemplified earlier by many researchers in

various matrices including marine tissues (Meier et al. 2006), marine animals (Abdulkadir

and Tuschiya, 2008) meat (Júarez et al. 2008; Pérez-Palacios et al. 2008), plant tissues

(Ruiz-Lỏpez et al. 2003), soils (Gómez-Brandón et al. 2008, 2010), microalgae (Griffiths et

al. 2010; Laurens et al. 2012; Ryckebosch et al. 2012) and thus, modified the methods for

obtaining the best results, signifying the need of method optimization and validation for

every sample matrix. Ruiz-Lopez et al. (2003) and Abdulkadir and Tuschiya (2008)

validated and suggested the use of direct transesterification methods for fatty acid extraction

in marine animals and plant tissues respectively. Meier et al. (2006) optimized one-step

extraction/methylation method for FA determination in marine tissues using 23 full factorial

categorical design and studied the effect of reaction time, temperature and presence of non-

polar solvent on FA recoveries employing derivatization reagent of anhydrous methanol

containing 2.5 M HCl and further compared them with Folch method. This study clearly

emphasized that the two methods showed similar FA composition when the values were

expressed on wt/wt% although the one-step method gave higher recoveries than the

traditional Folch method. Perez-Palacios et al. (2008) compared standard and continuous

soxhlet method (both with and without previous acid hydrolysis), Folch, Bligh and Dyer

method and reported the efficiency of Folch method for TL recoveries in meat while Bligh

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52  

and Dyer method gave the lowest yield. In another comprehensive study, Juarez et al.

(2008) recommended the use of application specific method, for example saponification

method for PUFA analysis due to higher recoveries and acceptable variation values, in situ

method for fast analysis of FAs while the classic method for lipid classes determination in

meat and their products. Further, Jeannotte et al. (2008) successfully modified Bligh and

Dyer method (chloroform/methanol/phosphate buffer; 1/2/0.8 by volume) for FA extraction

from soil samples. Subsequently, Gomez-Brandon et al. (2008, 2010) recommended a

combination of Folch method followed by derivatization by trimethylsulfonium hydroxide

(TMSH) for obtaining higher recoveries of FAs from soil and solid organic samples. In

microalgae, Johnson and Wen (2009) tested biodiesel production from algae Schizochyrtium

limacinum SR21 using different solvents (methanol, chloroform, hexane and petroleum

ether). They obtained a maximum 68% of FAMEs yield when chloroform or hexane were

added to methanol using 1.5 mol of sulphuric acid and 132:1 mol of methanol and solvent at

90°C for 40 min. Griffiths et al. (2010) while comparing the effectiveness of direct

transesterification (DT) with those of Bligh and Dyer, Smedes, and Askland methods for FA

extraction in three species of microalgae found that the Folch method was the most effective

method, but comparison with DT revealed that all extraction methods were incomplete.

Sheng et al. (2011) compared the the efficiency of extracting lipids from Synechocystis

using 15 different solvent mixtures and concluded that gravimetric extraction yields are

highly dependent on the polarity of the solvents used and the composition of the algal lipids.

They also found that the Bligh and Dyer and Folch method based on chloroform/methanol

solvent system gave the highest lipid recoveries. Ryckebosch et al. (2012) studied the effects

of different solvents (chloroform/methanol, 1:1, chloroform/methanol, 2:1,

dichloromethane/ethanol, 1:1, hexane/isopropanol, 3:2, acetone, diethyl ether and methyl-

tert-butyl ether/methanol,10:3), pretreatment (lyophilisation, inactivation of lipases and

addition of antioxidants) and cell disruption techniques (liquid nitrogen, sonication and bead

beating) on total lipid content, lipid class and FA composition of different microalgae. They

also found that chloroform/methanol (1:1) was the best solvent mixture for the extraction of

lipids from microalgae.

FA values in the present study are reported in mg g-1 FW due to discrepancies

deciphered by Meier et al. (2006) and O’Fallon et al. (2007) in expressing the values in

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53  

wt/wt%, since the latter could mask the inadequacies of the method and could also be wrong

in the case of differential extraction and synthesis of FAMEs by a particular method.

O’Fallon et al. (2007) reported similar discrepancy observed among FAME conversion

methods resulting in higher C20:4 (n-6) percentages due to differential methylation by BF3

as compared to other methods. Similar differences were observed in the present FA data sets

when expressed in wt/wt% (Table 2.5, 2.6, 2.7). As such there was a good correspondence

between FA composition of three seaweeds obtained by different methods but expressing

the data in wt/wt% tone downed the significance of different methods. For example, FA

contents expressed in wt/wt% showed either similar values or a slight 1.1-1.6-fold

increase/decrease in SFA contents in three seaweeds by all the conventional methods

whereas the actual recoveries were 1.0-7.4-fold higher (Tables 2.1, 2.2, 2.3). Moreover, the

actual SFA yields increased with the application of sonication and buffer by BDS and BDB

(Method 1B and 1C respectively) in G. corticata and S. tenerrimum while the values

reported in wt/wt% oppositely showed a decrease in their respective contents (Table 2.5, 2.6,

2.7). Similar ambiguities were found with the MUFAs and PUFAs values also and thus we

preferred to consider the root values of FA (in µg g-1) better for comparing the results and to

avoid any irregularities.

The lipid determination by the gravimetric method gave considerably higher values

than TFAs reported by GC-MS for both the conventional and direct transesterification

methods. Such higher values have also been reported by Ruiz-Lopez (2003), Meier et al.

(2006), Abdulkadir and Tsuchiya (2008) and Laurens et al. (2012). However, such

disparities may be attributed to the co-extraction of glycerol, phosphate, alcohol containing

groups from phospholipids, polyphenols, pigments, cholesterol and their derivatives along

with FAs. For further direct comparison of total FAs with total lipids, information about the

lipid class composition is needed (Meier et al. 2006; Laurens et al. 2012). Among the three

conventional methods studied, higher recoveries for TL and FAs obtained with simple FM

(Method 2A) as compared with simple BD (Method 1A) could be related to the limited

solubility of the predominantly non polar lipids (triacylglycerols) in the relatively polar

solvent solution (chloroform/methanol 1/2, v/v) employed in BD method (Iverson et al.

2001; Jeannotte et al. 2008).

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54  

In order to enhance the release of lipids from cellular matrices and the access of

extracting solvents to FAs, pre-treatments often become a pre-requisite to ensure effective

lipid extraction. Accordingly, the application of sonication and addition of buffer to the three

solvent systems studied were evaluated. Although sonication enhanced the TL contents by

1.5-fold in U. fasciata, 1.1-1.6-fold in G. corticata and 1.2-2.0-fold in S. tenerrimum in BDS

(Method 1B) and CSS (Method 3B), it did not worked well with the FMS method (Method

2B) in all the three matrices (Fig. 2.2). This contradictory behaviour may be explained by

conformational changes of lipids induced by heat from prolonged sonication, indicating that

sonication time may vary with the extraction method and thus, the solvent (mixture) used.

Later to analyze this contradictory behaviour, the effect of sonication time on total lipid

contents was studied. The analysis revealed a significant increase in the TL yields with the

increase in sonication time in BD (Method1) and CS (Method 3) up to 120 sec and thereafter

decreased while the optimum sonication time was 90 sec in FM (Method 2). This may be the

reason for slightly lower yields obtained for all the species investigated in the present study

by FMS method but the lipid contents obtained after 90 sec sonication was also lower and

non significant. Thus, it is recommended to standardize the sonication time at the

preliminary stage to avoid false negative results. Dunstan et al. (1993) and Ametaz et al.

(2003) also emphasized to maintain a balance between cell wall breakdown and minimum

degradation from ultrasound power effect (cavitational collapse and bulk heating) to achieve

the maximum release of cellular components.

The methods employing buffer rendered the highest TL and better FA recoveries but

not without exceptions. The use of a buffered solvent system is generally recommended for

samples containing large amounts of salts to prevent ionic adsorption effects otherwise,

phospholipids may be ionized and consequently retained during phase separation in the

aqueous phase and not recovered in organic phase (Christie, 1993). Thus, the highest TL

values obtained with buffered solvent system in the present study could be attributed to

higher amounts of inorganic salts in macroalgae. The ambiguity in the highest TL yields

reported by different methods, FMB in U. fasciata and G. corticata while BDB in S.

tenerrimum could be due to different nature of matrices and their chemistry with different

solvent mixtures. Recently, Martins et al. (2012) compared three extraction methods, Bligh

and Dyer (BD), AOAC official method (AOM) and extraction with methanol combined with

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55  

sonication (EMU) and two transesterification methods (7% BF3 in methanol and 5% HCl in

methanol) in three Brazilian macroalgae, Hypnea musciformis, Sargassum cymosum and

Ulva lactuca. They also reported that the optimum extraction method varied for the three

macroalgae, with AOM-HCl being the best method for H. musciformis, BD-BF3 for S.

cymosum and BD-BF3/BD-HCl for U. lactuca. They attributed these variations in lipid

extraction methods to the matrix effect as these species differ in the content of their

carbohydrates and proteins that might interact with triglycerides. Further, despite the above

differences in lipid extraction, BDB (Method 1C) reported the highest FAs recoveries in all

the three macroalgae in the present study. indicating that higher TL values reported by

chloroform/methanol/buffer system (2/1/0.8, v/v/v) of FMB as compared with

chloroform/methanol/buffer system (1/2/0.8, v/v/v) of BDB in U. fasciata and G. corticata

may be due to higher content of non-polar lipids such as TAGs that would not have been

completely converted to methyl esters. In addition, the failure of buffered solvent systems of

FMB and CSB in S. tenerrimum in both the lipid and fatty acid extraction may be due to

hindrance posed by their high organic contents especially polyphenols. These considerations

still deserve further investigations in macroalgae. The efficiency of

chloroform/methanol/buffer system (1/2/0.8, v/v/v) has also been reported by Jeanotte et al.

(2008) for soil samples.

The higher FAs recoveries reported by DT methods (LRC in U. fasciata and GM in

G. corticata and S. tenerrimum) as compared with conventional methods prior to

transmethylation can be explained by incomplete conversion of lipids to FAMEs by Carreau

and Dubacq method and need further modifications for optimizing the yield, unfortunately

this factor has not been the part of the present study. Since, the latter method incorporates

only 15 min of incubation time for saponification followed by another 15 min for

transmethylation, such short incubation periods may be insufficient for complete

derivatization of complex lipids of macroalgae as compared with 1 h and 2 h incubation

times in LRC and GM methods respectively. Nevertheless, such greater FA recoveries have

also been reported previously from biological samples with the direct transesterification

method against classical multi-step techniques (Meier et al. 2006; Abdulkadir and Tuschiya,

2008; Griffiths et al. 2010).

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56  

Apart from this, in the view to avoid the use of hazardous and toxic solvents such as

chloroform, dichloromethane was tested as an alternative option but the dichloromethane-

methanol system (Method 3) adapted from Cequier-Sánchez et al.(2008) along with their

modifications (Method 3B and 3C) failed to compete with the potential of chloroform/

methanol/ buffer system. Actually, chloroform and methanol in combination exhibits strong

dissolving power for the entire range of polarity found in lipids, as well as the ability to

break up membrane and denature (-lipo)proteins (Schreiner, 2006) and buffer added in the

present study helped to overcome the ionic adsorption effects of salt that may hinder lipid

extraction in macroalgae. Similarly, Lee et al. (1998), Sheng et al. (2011) and Ryckebosch et

al. (2012) who compared a range of different polar and non-polar solvents for lipid

extraction reported the higher efficiencies of chloroform/methanol based solvent system for

the extraction of lipids from different microalgae. However, recently, Suganya et al. (2012)

compared the efficiencies of twelve different solvent systems including n-hexane, methyl

tertbutyl ether, chloroform/methanol (1/1), n-hexane/ether (3/1), chloroform/methanol (2/1),

1% diethyl ether and 10% methylene chloride in n-hexane, chloroform:2-propanol (2/1),

hexane/2-isopropanol (3/2), dichloromethane/methanol (1/1), dichloromethane/ethanol (1/1),

acetone/dichloromethane (1/1) and hexane/ethyl alcohol (1/1) and six pre-treatments for

extraction of algal oil from shade-dried U. lactuca. They obtained a maximum yield of

10.88% (g/g) of oil at 5% moisture content, 0.12 mm particle size, 500 rpm stirrer speed, 55

°C temperature, 140 min time and solvent-to-solid ratio as 6:1 with 1% diethyl-ether and

10% methylene chloride in n-hexane solvent mixture. Samorì et al. (2013) reported that the

lipid and FA extraction with N, N-dimethylcyclohexylamine (DMCHA) gave higher lipid

and FA yields as compared to the chloroform/methanol based solvent system. They

proposed that the higher efficiency of DMCHA could be attributed to the fact that DMCHA

could have access to lipids which were not extractable by chloroform/methanol. At present,

DMCHA has not been used for the extraction of lipids/FAs in macroalgae and therefore any

prediction in this regard would be elusive.

Despite the variations among the FA yields of each method, the three macroalgae

showed the typical profiles corresponding to their respective phyla, i.e. U. fasciata being a

green alga was rich of C18 PUFAs while G. corticata being a red alga was rich with C20

PUFAs and S. tenerrimum being brown alga was rich of both C18 and C20 PUFAs (Tables

CHAPTER 2  

57  

2.1, 2.2 and 2.3). Such trends have already been established earlier in several studies

(Khotimchenko et al. 2002; Li et al. 2002; Kumari et al. 2010; Galloway et al. 2012).

Further, standard FAME-mix (Supelco) was analyzed on inter/intra-day basis to

ensure instrument response and data accuracy and a five -point calibration was carried out to

ensure the linearity (r2 = 0.995). At last, regardless of the method used, quantitative

estimation of lipids and FAs require researchers to take precautions at every step involved,

to avoid variations in the results.

In conclusion, this comparative study enabled us to test the efficiency of different

methods for extraction of lipids and FAs in different macroalgal matrices and experimental

design further proved to be an efficient strategy to identify its key factors. The macroalgal

matrix, extraction method and buffer were the key determinants of optimum lipid and FA

recoveries. FMB (Method 2C) was the most efficient method of lipid extraction in U.

fasciata (Chlorophyta) and G. corticata (Rhodophyta) while BDB (Method 1C) in S.

tenerrimum (Phaeophyta) and DT methods gave optimum FA yields in all the three

macroalgae investigated; LRC (Method 6) in U. fasciata and GM (Method 5) in G.

corticata and S. tenerrimum. Thus, it’s ideal to choose an extraction method according to the

desired purpose, DT methods for FA research and conventional methods for the extensive

study of lipid classes as the former does not separate the lipid fractions with extraction and

methylation being accomplished in a single step. Further, care should be taken while

selecting the method for macroalgae, according to the group they belong to, otherwise there

would be a risk of obtaining erratic and inaccurate results.