Article pubs.acs.org/JAFC
Polar Lipid Profile of Nannochloropsis oculata Determined Using a Variety of Lipid Extraction Procedures K. Servaes,† M. Maesen,† B. Prandi,‡ S. Sforza,‡ and K. Elst*,† †
Unit Separation and Conversion Technology, Flemish Institute for Technological Research (VITO), Boeretang 200, 2400 Mol, Belgium ‡ Department of Food Science, University of Parma, Viale delle Scienze 59/A University Campus, 43124 Parma, Italy S Supporting Information *
ABSTRACT: Lipid compositions obtained from microalgae species are affected by both the cultivation conditions and the extraction method used. In this study, the extraction of lipids from Nannochloropsis oculata using traditional and modern extraction technologies with several solvents has been compared. Because important polyunsaturated fatty acids are bound to polar lipids, these polar lipids were the main focus of this study. The dominant compounds in the glycolipid fractions were monogalactosyldiglycerides and digalactosyldiglycerides bearing fatty acid chains containing at least one site of unsaturation. Phosphatidylcholine and trimethylhomoserines were detected in the phospholipid fractions. The fatty acid profile comprised large fractions of C16:0, C16:1, C20:5, and C18:3. Extraction of specific compounds was determined by extraction efficiency as well as differences in the selectivity of the method used. The composition derived from a glycolipid fraction was observed to be affected by the method used to a greater extent than the phospholipid fraction. KEYWORDS: microalgae, extraction, lipids, galactolipids, phospholipids, phosphatidylcholine
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pids).4,10,11 Their presence in the polar lipid fraction is potentially interesting because it may lead to both increased absorption of omega-3 LC-PUFAs and better oxidative stability of the oil compared to triacylglycerol (TAG) oil.10,11 In the literature, significant attention has been paid to the presence of free fatty acids and TAG in algae extracts, primarily related to the production of biodiesel,1,4,8,14−22 whereas information on the occurrence of glyco- and phospholipids is rather scarce.11,12,15,21,22 Phospholipids and glycolipids are important components of cellular membranes.21 Certain bioactive phospholipids are applied in the nutritional and pharmaceutical fields to improve human health and to prevent certain diseases. Moreover, being amphiphilic molecules and natural surfactants, commercial phospholipids are used in food applications to improve baking, enhance wetting, reduce viscosity and prevent crystallization as an additive in chocolate, and stabilize margarine.23,24 Because the quantity of lipids in microalgae is relatively small (15−30% on average depending on the species), the extraction procedure used needs to be as efficient as possible to maximize the amount of lipids extracted. 25 Different extraction technologies and solvent systems have already been described in the literature,2,5,14 although the majority have been performed in the framework of biodiesel production and thus focused only on the recovery of neutral lipids (e.g., TAG). The methods range from classical organic solvent extraction,1,10,11,17,26 Soxhlet extraction,13,20,25,27 pressurized fluid extraction4 (PFE, sold as “Accelerated Solvent Extraction”,
INTRODUCTION Microalgae are a heterogeneous group of cellular organisms that convert carbon dioxide into potential biofuels, food, feed, and high-value bioactives by means of sunlight. Compared to traditional crops, the cultivation of microalgae is characterized by a high growth rate, short growth time, high biomass production, and minimal land use.1,2 Microalgae are a rich source of organic micromolecules, such as lipids, proteins, carbohydrates, and pigments.3,4 Thus, the cultivation conditions applied (medium composition, temperature, illumination intensity, etc.) have a major impact on the productivity, quality, and exact composition of the final products (e.g., fatty acid pattern of lipids). Therefore, by tuning the cultivation conditions, it is possible to control the content of specific types of compounds.5,6 Because of this, microalgae can be an important and sustainable source of feedstock chemicals with numerous applications within the food and feed, cosmetic, pharmaceutical, and fuel industries.7,8 Polyunsaturated fatty acids (PUFAs) and more specifically omega-3 long-chain fatty acids (omega-3 LC-PUFA), such as eicosapentaenoic acid (EPA; C20:5) and docosahexaenoic acid (DHA; C22:6), are known to be beneficial to human health due to their unique pharmaceutical properties. Despite the rising awareness of their importance, their daily intake in most countries is still below the recommended dose.4,8−11 Fish oil is currently the major source of these omega-3 LC-PUFAs. However, with depletion of fish stocks, it will become increasingly difficult to meet the human demand for these omega-3 LC-PUFAs by fish oil alone.10−13 Microalgae are an abundant source of these omega-3 LC-PUFAs and hence can be a valuable alternative for fish oil.10,11,13 Recent studies have shown that a significant portion of these omega-3 LC-PUFAs are associated with polar lipids (i.e., glyco- and phospholi© 2015 American Chemical Society
Received: Revised: Accepted: Published: 3931
July 2, March March March
2014 19, 2015 24, 2015 24, 2015 DOI: 10.1021/acs.jafc.5b00241 J. Agric. Food Chem. 2015, 63, 3931−3941
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Journal of Agricultural and Food Chemistry ASE) to microwave-assisted28 and ultrasound-assisted26,27 organic solvent extraction and supercritical fluid extraction (SFE).13,16,17 The majority of the methods use organic solvents, such as chloroform, pure alcohols (methanol, ethanol, isopropanol), hexane, dichloromethane, ethyl acetate, and so forth, or mixtures thereof. A combination of chloroform and methanol has been reported in the literature as the best option to recover total lipids from microalgae. These mixtures extract both neutral lipids by chloroform and polar lipids by methanol.10,11,26 A chloroform/methanol mixture, for example, is used in the method of Folch et al.29 for the extraction of total lipids from microalgae. This method, originally optimized for the isolation and purification of total lipids from animal tissues, uses 2:1 chloroform/methanol for lipid extraction and water to remove nonlipid substances from the extract.29,30 Recently, 3:2 hexane/isopropanol has been suggested as an alternative to the chloroform/methanol system.10,14,18 The mixture works in a similar fashion to the chloroform/methanol system with a higher selectivity toward neutral lipids compared to that of chloroform/methanol.14,18 However, these traditional extraction methods have some disadvantages, including the use of hazardous and flammable liquid organic solvents, costly high-purity solvents, and the presence of potentially toxic emissions during extraction. The extraction processes (e.g., Soxhlet) are often slow and timeconsuming. Furthermore, an energy-intensive evaporation step is required for solvent removal. Additionally, these organic solvents can cause adverse health and environmental effects. To minimize these drawbacks, alternative, greener extraction techniques have been investigated5,16 and have led to the development of other extraction methods that are considered to be more sustainable, such as PFE and SFE. Despite the fact that PFE requires temperatures between 50 and 200 °C and pressures of 10−15 MPa, the associated increased lipid solubility, improved cell wall penetration by solvent, relatively low solvent use compared to other methods, and reduced emission of volatile organic compounds (VOC) due to the required pressure all add up to make this technique somewhat greener than the more traditional methods. Further advantages include its ability to be automated and a lack of light and oxygen within the process, which is beneficial when working with bioactive molecules.4,5,31 The vast majority of SFE applications use supercritical CO2 (scCO2) as the primary solvent. The low critical temperature (31.1 °C) and moderate critical pressure (7.4 MPa) make it particularly useful for extractions of thermolabile compounds.5,14,16 Numerous literature examples of scCO2 extractions on different natural sources have been reported.7,8,17,32−34 A common practice in SFE, which has to be mentioned in relation to the physicochemical properties of supercritical fluids, is the use of modifiers (cosolvents). These cosolvents (e.g., methanol and ethanol) are added to the primary fluid to enhance extraction efficiency. For example, the addition of 1− 10% methanol or ethanol to scCO2 expands its extraction range to include more polar lipids. The addition of these modifiers is often required to assist scCO2 in the extraction of highly polar compounds.16 One disadvantage of scCO2 extraction, however, is its high capital cost (CAPEX) due to its pressure-driven nature. It has also been reported in the literature that differences in lipid extraction arise due to the method and solvent used.15,35 Furthermore, the extraction efficiency of a particular solvent (system) cannot generally be extrapolated to microalgae
species, as indicated in the study by Ryckebosch et al.10 The main factor that determines recovery from different microalgae species seems to be the permeability of the cell wall. The purpose of the present study was therefore to compare traditional (Soxhlet, Folch) and modern (PFE, scCO2) extraction techniques for the extraction of lipids from Nannochloropsis oculata. Thus, the main focus was on the differences in the fatty acid profiles of the glyco- and phospholipids. The Soxhlet extraction method was included due to its simplicity in operation, relative safety, and potential for scaling up to an industrial plant level. Chloroform/methanol (2:1) was evaluated, as it is used as the extraction solvent in the Folch method, and hexane was selected as it is currently most commonly applied for both commercial extraction of food lipids and extraction of omega-3 LC-PUFA containing triglycerides from heterotrophic microalgae.10 Ethanol was evaluated due to its low cost, its volatility and strong affinity for membrane-associated lipid complexes because of its ability to form hydrogen bonds.14 On the basis of UPLC-MS data, the nature of glyco- and phospholipids present in the different extracts has been identified. Therefore, this study aims at providing new insights to the existing literature by giving a comprehensive view of the influence of the extraction method used on the lipid profile of the polar lipids at the molecular level.
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MATERIALS AND METHODS
The microalgae species Nannochloropsis oculata was used in this study to perform the different extractions. Lyophilized algae were kindly provided by Proviron Industries NV (Hemiksem, Belgium; lot: CCAP 849/1). The algae were first concentrated in a microfiltration unit and subsequently centrifuged, resulting in approximately 9.44 L with a dry weight of ∼12.3%. They were then subjected to a freeze-drying process without delay by pouring concentrated microalgae mixtures onto 600 mL trays (∼1 cm thickness). The freeze-drying process was repeated once to completely dry the algae. Finally, the freeze-dried algae were vacuum packed. The analysis of Proviron Industries NV performed using the procedure described by Ryckebosch et al.1 specified a total lipid fraction of Nannochloropsis oculata amounting to ∼31% with the following breakdown of the different lipid classes: 34% neutral lipids, 24% glycolipids, and 42% phospholipids. This data, obtained by the procedure of Ryckebosch et al.1 (i.e., extraction with 1:1 chloroform/ methanol), will be considered as the reference. GC-grade chloroform, methanol, ethanol, and hexane were purchased from VWR International (Leuven, Belgium). All weight determinations were carried out on a calibrated analytical balance. Extraction Conditions. All extractions were performed in triplicate using 7.5 g of freeze-dried Nannochloropsis oculata after homogenizing them with a mortar and pestle. For the Folch extraction, however, only 1 g of freeze-dried Nannochloropsis oculata was used. For the PFE and Soxhlet extraction, three solvents were selected: hexane, ethanol, and 2:1 chloroform/methanol. Conventional Folch extraction was performed with 2:1 chloroform/methanol, and the SFE extractions were performed with scCO2 or scCO2 plus 30% ethanol as a cosolvent. More detailed description of the different extraction procedures is given below. After each replicate extraction experiment, with the exception of the Folch extraction, the extract was split into two fractions, and these fractions were accurately weighed. Both fractions were dried under nitrogen atmosphere until visibly dry and stored in a vacuum oven at 23 °C overnight. Soxhlet Extraction. For Soxhlet extraction, the freeze-dried algae were placed in a 22 × 80 mm extraction thimble (Whatman, VWR, Leuven) and extracted with 80 mL of solvent (hexane, ethanol, 2:1 chloroform/methanol) for 6 h in a 30 mL Soxhlet apparatus. Pressurized Fluid Extraction. Lipids were extracted from the lyophilized and homogenized algae using an automated Dionex 3932
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Journal of Agricultural and Food Chemistry ASE200 Accelerated Solvent Extraction (ASE) system. The algae (60%) were mixed with Celite 545 (40%) (0.02−1 mm, Merck, VWR, Leuven) prior to being loaded into Dionex standard 33 mL stainless steel extraction thimbles. After a small amount of Celite was added to the extraction cell, the sample mix (12.5 g) was loaded. Sea sand (Merck, PA, USA; VWR, Leuven, Belgium) was finally used to fill any extra space in the cell. Extractions were performed with hexane, ethanol, and 2:1 chloroform/methanol as the extraction solvent. The same conditions were used for all of the solvents: 1500 psi (103 bar) and 125 °C with a 5 min static extraction (after 5 min preheating) and 4 static cycles. Upon completion of the extraction, the thimble was flushed with solvent (50%) and purged with nitrogen for 120 s. The solvent was collected in 60 mL vials with Teflon septa. Conventional Folch Extraction. One gram of freeze-dried algae was weighed and put into glass vessels. One volume of chloroform, 1 volume of methanol, and 1 volume of chloroform were added in sequence to the algae sample. The mixture was shaken manually after each solvent addition. After addition of the three solvents, the mixture was shaken for 15−20 min at room temperature at high speed. Subsequently, phosphate buffered saline (PBS, pH 7−7.5, 0.8 volume) was added to obtain a final concentration of 1:20 (sample weight/ solution volume; g/mL). This PBS was added to increase the ionic strength of the upper phase to improve phase separation.29 The mixture was then centrifuged at 1000g for 1 min to allow the organic and aqueous layers to separate. After the organic layer was removed and collected, the aqueous layer was extracted twice more by adding only 2 volumes of chloroform followed by manual shaking and centrifugation to induce phase separation. The chloroform phases were collected in a tared glass vial. The extract was dried under nitrogen atmosphere until visibly dry and put in a vacuum oven at 23 °C overnight. Supercritical Extraction. An ISCO SFX 220 Supercritical Fluid Extractor equipped with an ISCO Model 260 D Syringe pump, ISCO Restrictor Temperature Controller and an ISCO SFX 200 Controller (BRS) was used. Supercritical extraction with either CO2 or CO2/30% ethanol (cosolvent) was conducted at a pressure of 400 bar and a temperature of 40 °C with a 240 min dynamic extraction. The flow rate of scCO2 and scCO2/30% ethanol was set to 3 and 2.1 mL/min, respectively. The microalgae powder was placed in a high-pressure extraction cell. Carbon dioxide was liquefied by a cooler and pressurized to the preferred pressure using a high-pressure pump. Pressurized CO2 was then pumped into the heated extraction cell during the SFE process. The extract was collected in a glass tube by venting scCO2. The temperature of the restrictor (2 mL/min TCR 0.75) was set at 90 °C. Analysis of Total Lipid Content in the Lipid Part of the Extract. With the exception of the Folch extraction, two fractions were obtained from each replicate extraction. One of these fractions was stored at −20 °C under nitrogen. Because it is known that some solvents (mixtures) may not only extract lipids but also nonlipid polar compounds, such as carbohydrates and proteins, the other half was treated with 10 mL 2:1 chloroform/methanol to separate the nonlipid and lipid fractions in the extract.11,15 The redissolved extract was shaken for 2 min and then centrifuged for 5 min at 3000g. The supernatant was removed, and the residue was washed twice more with the chloroform/methanol mixture. The lipid fractions of the three washing steps were collected, dried under nitrogen atmosphere until visibly dry, and finally stored in a vacuum oven overnight. The residue was accurately weighed to determine the total lipid content. After weighing, the extract was resuspended in 10 mL 2:1 chloroform/ methanol and divided into different fractions: one was used for analysis of the phosphorus content and the other for fractionation of the lipid classes. After being dried and weighed, the fraction for fractionation and the additional sample were stored at −20 °C under nitrogen to prevent lipid oxidation. Separation of Lipid Classes. To determine the influence of the different extraction methods and conditions on the lipid class composition, the lipid class content of the extracts was determined using silica solid-phase extraction (SPE) performed according to the procedure described by Ryckebosch et al.1
Briefly, a 500 mg/6 mL Grace Pure SPE Silica column (Grace, Lokeren, Belgium) was first conditioned by elution with 10 mL of chloroform. Approximately 10−20 mg of lipids dissolved in 500 μL chloroform was brought onto the silica column. Elution with 10 mL of chloroform yielded the neutral lipids (NL), and 10 mL of acetone was used for elution of the glycolipids (GL). Finally, the phospholipids (PL) were eluted with 10 mL of methanol. The yield of each class was determined gravimetrically in triplicate. Characterization of the Lipid Extracts and Fractions. The phosphorus concentration was determined in the total lipid extract after washing in 2:1 chloroform/methanol by means of inductively coupled plasma emission spectrometry following DIN EN 14107. A known quantity of each fraction (NL, GL, and PL) was dissolved in 90:10 isopropanol/acetonitrile with 0.1% formic acid. Analysis was based on ultra performance liquid chromatography (UPLC)−mass spectrometry (MS). A Waters Acquity UPLC system (Waters, Milford, MA, USA) was used, equipped with an Acquity UPLC BEH300 C18 column (150 × 2.1 mm; 1.7 μm). The column temperature was maintained at 35 °C. Optimum separation was obtained with a binary mobile phase constituted of 50:50 acetonitrile/water (eluent A) and 90:10 isopropanol/acetonitrile (eluent B) with both solvent mixtures buffered with 0.1% formic acid. The gradient elution program was 0−1 min from 100% A to 45% A, 1−25 min from 45% A to 30% A, 25−26 min from 30% A to 0% A, 26−30 min with 100% B, 30−31 min from 0% A to 100% A, and 31−40 min with 100% A (returned to initial conditions and equilibration of the column). The flow rate of the mobile phase was 0.2 mL/min. Different injection volumes were used to be in the linear range for quantification. The UPLC system was coupled to a Waters Single Quadrupole mass spectrometer (Waters, Milford, MA, USA), which was operated in the positive electrospray ionization mode (ESI+) in full scan acquisition. The parameters of the mass spectrometer were as follows: electrospray source block and desolvation temperatures of 150 and 300 °C, capillary voltage of 4.5 kV and cone voltage of 85 V. Positive identification of the compounds was based on chromatographic retention times, molecular weights, and their characteristic m/ z ions generated by in-source fragmentation. An overview of the retention times and m/z ions of the different compounds is given in the Supporting Information (Table S1). The ion chromatogram of each m/z ratio, corresponding to a particular compound, was derived from the total ion chromatogram using the MassLynx Mass Spectrometry Software (Waters). The area of the resulting single peak was then integrated. Finally, quantitative determination of the phospholipids was made using an analytical standard. Thus, a phospholipid mixture for HPLC from Glycine max (soybean) in chloroform, containing phosphatidylcholine (PC), lyso-phosphatidylcholine, phospatidylethanolamine (PE), and phosphatidylinositol (PI), was purchased from SigmaAldrich (Bornem, Belgium). The phospholipid standard was dried under nitrogen to remove chloroform and reconstituted with 90:10 isopropanol/acetonitrile with 0.1% formic acid (eluent B). Five dilutions were made in duplicate to obtain the following PC concentrations: 25, 50, 100, 150, and 200 μg/mL. These calibration solutions were injected into the UPLC-MS system using the instrumental conditions described above. The calibration curve was made by plotting the total peak area of PC components in function of the total PC concentration reported on the certificate of analysis of the analytical standard. Linear calibration curves were constructed with a squared regression coefficient R2 of 0.99. The limit of detection (LOD) and limit of quantification (LOQ) of the applied method were calculated considering the peak-to-peak signal-to-noise ratio in the chromatogram of the standard solution with the lowest concentration of PC (25 μg/mL). Because various PCs bearing different fatty acids were present in the analytical standard, the signal-to-noise ratio of the lowest chromatographic peak was determined (worst case scenario). The LOD and LOQ were defined as the concentration that would give a signal-to-noise ratio of 3 and 10, respectively. Extrapolating from the signal-to-noise ratio observed for the lowest peak, LOD and LOQ were calculated to be 4 and 13 μg/mL, respectively. 3933
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Figure 1. Total amount of lipids and lipid class composition obtained by the different extraction methods from Nannochloropsis oculata (mean ± SD; n = 3; CHL: chloroform; MeOH: methanol; EtOH: ethanol).
The final total lipid fractions after washing with 2:1 chloroform/methanol extracted by the different methods from Nannochloropsis oculata are shown in Figure 1. Chloroform/methanol (2:1) as the extraction solvent with PFE, Soxhlet, or Folch extraction resulted in comparable total lipid fractions in the range of 26−32%. These total lipid fractions correspond well with the reference value of 31% obtained by Ryckebosch et al.1 via extraction with 1:1 chloroform/ methanol. The 2:1 chloroform/methanol is the most frequently used solvent mixture for lipid extraction from any living tissue and is applied in methods such as Folch and Bligh and Dyer.1,14,29,30 This mixture is able to extract both nonpolar lipids by chloroform and polar lipids by methanol. For this reason, mixtures of chloroform/methanol tend to be more efficient for the extraction of total lipids.26 The highest total lipid fraction is extracted from Nannochloropsis oculata using PFE extraction with ethanol (49%). It can be assumed that, even though a washing step has been performed, coextracted (polar) matrix components, such as phytochemicals, are still present in the extract as observed by Mulbry et al.36 On the basis of the total lipid fraction results, it can be concluded that the polarity of the extraction solvent clearly influences the crude lipid yield using both classical extraction methods (Soxhlet, Folch) and modern techniques (PFE, SFE). Nonpolar solvents can only extract very nonpolar lipids, whereas more polar solvents (mixtures) extract lipids of a broader polarity range, resulting in a higher lipid yield. With either PFE or Soxhlet extractions, the following order of total lipid fraction was observed: ethanol > 2:1 chloroform/ methanol > hexane. The addition of 30% ethanol as cosolvent to pure scCO2 increased the total lipid yield relative to the extraction with pure scCO2. Extractions that use only scCO2 usually give good recoveries of nonpolar lipids. Polar lipids, however, have low solubility in scCO2 and are thus not extracted. Adding ethanol expands the extraction range of scCO2 to include the more polar lipids, resulting in a higher extraction yield. These observations are in accordance with literature data on the influence of solvent polarity on the extraction of lipids from microalgae10,11,20,34,36,37 For example, McNichol et al. observed
Calculations and Statistical Analysis. The presence of PC was assessed using the phosphorus content of the different lipid fractions. The phosphorus content was converted to a PC concentration expressed as g PC/100 g algae using the molecular weight of phosphorus (30.97 g/mol) and of oleoyl-palmitoyl-phosphatidylcholine (PC 16:0/18:1) (760 g/mol). PC 16:0/18:1 was used for this purpose because Wang et al. observed that C16:0 and C18:1 are the major fatty acids in PC in Nannochloropsis lipids.15 The total lipid fraction, as well as the NL, GL, and PL fractions, were calculated on a weight basis as g/100 g algae, and the results are expressed as a percentage. The chromatographic peak area of each compound belonging to a certain class was determined and related to the amount injected. This enabled the various extraction methods to be directly compared on the basis of their effects on each single component. An assessment of the total amount of the different lipid classes present was made by the sum of all chromatographic peak areas in the UPLC chromatogram belonging to the lipid class under consideration. The total peak area per mass algae extracted was then calculated by taking into account the relative contribution of that particular lipid class to the total lipid fraction. Finally, the peak area ratio of one specific compound relative to the total peak area of the lipid class under consideration yields the relative lipid composition (area in %). The PC concentration (μg/mL) was converted to w/w % (mass PC/mass algae) using the sample volume and the weight of the PL fraction. All results are an average of three replicate measurements. Each replicate measurement was obtained from an independent extraction and fractionation. In addition, standard deviations were calculated and are indicated by error bars.
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RESULTS AND DISCUSSION Total Lipid Fraction and Lipid Class Composition. The total lipid fractions were obtained after the washing step with 2:1 chloroform/methanol. Data on the influence of this washing step on the total lipid fraction are shown in the Supporting Information (Figure S1). In general, the total lipid fractions originating from extractions with more polar solvents (e.g., ethanol) were the most affected by the washing step with 2:1 chloroform/methanol, resulting in a larger mass loss compared to nonpolar extractions. The polar extractions are indeed more prone to the coextraction of nonlipid, more polar matrix materials (e.g., proteins and carbohydrates). 3934
DOI: 10.1021/acs.jafc.5b00241 J. Agric. Food Chem. 2015, 63, 3931−3941
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Figure 2. Total ion current (TIC) chromatogram of the GL fraction for PFE extraction with 2:1 chloroform/methanol. In the separate graph, the total chromatographic peak area is shown (mean ± SD; n = 3; CHL: chloroform; MeOH: methanol; EtOH: ethanol). The chemical structure of the compounds identified in the GL fractions is shown. The ESI+ mass spectrum of a compound is given as an example.
that hexane extracted ∼3% of lipids from Nannochloropsis granulate, whereas ethanol and 2:1 chloroform/methanol extracted >25% of lipids on a dry weight basis.20 In a study by Balasubramanian et al., the use of hexane alone gave only 16% lipid yield whereas the addition of a polar solvent improved the yield.37 Furthermore, no significant difference in the lipid extraction efficiency was observed between the different extraction methodologies (sonication, PFE, homogenization, Soxhlet) all using 2:1 chloroform/methanol as the extraction solvent. To gain insight into the lipid class composition, we fractionated the different extracts into three portions using SPE to determine the percentages of NL, GL, and PL (Figure 1).1 The lipid class composition of the 2:1 chloroform/ methanol extracts obtained by Soxhlet, PFE, or Folch is in accordance with the specifications on Nannochloropsis oculata applying the reference method (1:1 chloroform/methanol). The distribution of the lipids among the different classes is as follows: phospholipids > neutral lipids > glycolipids. Phospholipids are predominantly present in these extracts in the range of 40−45% of total lipids. Low polarity solvents are best for the extraction of neutral lipids, including waxes and pigments, which are characterized by a very low polarity. In contrast, lipids from the chloroplast and membrane, which contain GL and PL, respectively, are more effectively extracted by more polar organic solvents, such
as ethanol or methanol.10,37,38 The affinity of polar lipids (GL and PL) for more polar solvents (mixtures) is also observed in the present study. The PL fraction in the ethanol extracts is the most abundant, whereas the yield of PL using hexane is almost negligible due to the apolar character of the solvent. For SFE, the same trend is observed. Using pure scCO2, the lipid fraction consists mainly of NL, which demonstrates the selectivity of scCO2 toward nonpolar molecules.16,39 Its nonpolar nature makes pure scCO2 incapable of interacting with either polar lipids or neutral lipids that form complexes with polar lipids.14 Through the addition of ethanol, the affinity toward GL and PL is enhanced, although the NL fraction is still predominant. The findings in this study are consistent with literature data. In the study by Ryckebosch et al. comparing the extraction efficiency of 3:2 hexane/isopropanol and hexane for the extraction of lipids from different microalgae species, the NL content was higher in the hexane extract and lower in the 1:1 chloroform/methanol and 3:2 hexane/isopropanol extracts for each microalga.10 Hence, the amount of polar lipids (GL and PL) was lowest in the hexane extract. Ryckebosch et al. also compared dichloromethane/ethanol with nonhalogenated solvent systems for the extraction of lipids from Nannochloropsis gaditana.11 Dichloromethane/ethanol extracted the three lipid classes with essentially the same efficiency, whereas all nonhalogenated solvent mixtures more easily extracted NL than the polar lipids (GL and PL). Of the nonhalogenated 3935
DOI: 10.1021/acs.jafc.5b00241 J. Agric. Food Chem. 2015, 63, 3931−3941
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Figure 3. Relative distribution (% of total peak area) of the different MGDGs and DGDGs in the GL fraction. The results are the average of three replicate measurements (mean ± SD; CHL: chloroform; MeOH: methanol; EtOH: ethanol).
enhancement of the phosphorus concentration comparable to the content in the lipid extract of PFE with hexane. Compound Identification in the Polar Lipid Fractions GL and PL. GL Fractions. An overview of the total chromatographic peak area integrated for the GLs in the different extracts is given in Figure 2. This data also indicates that by increasing the polarity of the solvent, more glycolipids are being extracted as indicated by the higher total chromatographic peak area. For all of the extraction methods used, the majority of the GLs (i.e., highest total peak area/mass algae extracted) is detected in the corresponding fractions. Analysis of the different PL fractions, however, revealed that quite a persistent proportion of GL is also present in these fractions (Figure 2). Thus, all extraction methodologies lead to higher yields of GL than the analysis of the GL fractions indicate. Similarities in chemical structure, and consequently polarity, between GL and PL lead to the observed incomplete separation, especially when using SPE. Glycolipids and more specifically monoglycodiglycerides and diglycodiglycerides were detected as sodium adducts in the GL and PL fractions (Figure 2). Because galactolipids are the predominant lipids in photosynthetic membranes, they could
solvents, the highest recovery of NL was obtained with 3:2 hexane/isopropanol and ethyl acetate/hexane. Also for the GL, 3:2 hexane/isopropanol proved to be the most efficient solvent, whereas for PL, ethanol was best.11 Similar results have also been observed by Balasubramanian et al.37 and Mendes34 and co-workers in which the latter uses solvent polarities to explain these observations. The phosphorus content of the lipid extracts gives insight into the presence of phospholipids. According to the literature, PC was reported to be the major component of phospholipids in many microalgae, though its content might vary due to species-specificity or culture conditions.8,11 The phosphorus content of the different crude lipid extracts (Supporting Information, Figure S2) is in accordance with the lipid class composition. The extraction of PL is favored by a more polar extraction solvent. Indeed, the highest phosphorus content is observed for ethanol, followed by 2:1 chloroform/ methanol, and then hexane. Furthermore, as pure scCO2 is not polar enough to extract the phospholipids, none were detected above the LOQ in the scCO2 extract. The increase in polarity by the addition of 30% ethanol, however, resulted in 3936
DOI: 10.1021/acs.jafc.5b00241 J. Agric. Food Chem. 2015, 63, 3931−3941
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Figure 4. Total ion current (TIC) chromatogram of the PL fraction for PFE extraction with 2:1 chloroform/methanol. In the separate graph, the total chromatographic peak area is shown (mean ± SD; n = 3; CHL: chloroform; MeOH: methanol; EtOH: ethanol). The chemical structure of the compounds identified in the PL fractions is shown. The ESI+ mass spectrum of a compound is given as an example.
it is stated that PUFAs of microalgae are predominantly linked to polar lipids, especially glycolipids (MGDG, DGDG, and sulphoquinovosyldiacylglycerol (SQDG)).4,10,11 Also within every class, the abundance of one (or more) MGDG or DGDG compound(s) with a particular fatty acid is favored depending on the extraction method and solvent used (Figure 3). For example, DGDG 16:0/16:1 is the principle DGDG extracted with 2:1 chloroform/methanol as the solvent using either Soxhlet or PFE. The SFE-scCO2 extraction, on the other hand, preferentially extracts MGDG 20:5/20:5. These observations again demonstrate that a more polar solvent mixture extracts more polar lipids (i.e., more sugars and shorter fatty acid chains), whereas the use of a less polar solvent yields less polar lipids (i.e., single sugars and longer fatty acid chains). Wang et al. characterized the lipid profile of Nannochloropsis oculata extracted with ethanol (80 °C, 30 min) followed by separation of the lipid and nonlipid fractions using the Folch’s procedure.15 DGDG was the major GL class, accounting for 28.4% of all quantified polar lipids. The contribution of MGDG amounted to 2.1%. Hence, Wang et al. obtained an excess of DGDG in the ethanol extract, whereas MGDG and DGDG were more equally distributed in the ethanol extracts (PFE/ Soxhlet) in the present study. According to Wang et al., the major fatty acids bound were C16:0 (52.3%), C14:0 (14.0%), and C18:1 (10.8%) in MGDG and C16:0 (51%) and C16:1 (19.1%) in DGDG.15 In this study, however, different types of fatty acids are linked to MGDG and DGDG. Whereas C16:0
be assumed to be monogalactosyldiglycerides (MGDG) and digalactosyldiglycerides (DGDG). The structures of MGDG and DGDG are given in Figure 2. In these natural compounds, the galactopyranose moiety is linked to the glycerol backbone, bearing two long fatty acid chains. As can be seen in Figure 3, the methodology used has a significant effect on the extraction yield of the galactolipids, shifting more toward MGDG or DGDG. SFE with pure scCO2 and the Soxhlet extraction with hexane predominantly yield MGDGs with 98% and 92% of the total peak area, respectively, belonging to MGDG. The additional galactose moiety makes DGDG sufficiently polar to prevent its extraction with a nonpolar solvent. In contrast, the PFE extraction with hexane does extract DGDGs (53% MGDG and 47% DGDG), which can be explained by the higher extraction temperature and pressure. The extraction yield of DGDG is enhanced by increasing the solvent polarity with DGDG contributing >60% for the PFE or Soxhlet extraction with 2:1 chloroform/ methanol. Linked to MGDG and DGDG are different types of fatty acid (Figure 3). All of the galactolipids identified contain at least one site of unsaturation with a strong presence of C20:5. Among the saturated fatty acids, palmitic acid (C16:0) is the most abundant. PUFAs such as C20:5 are prone to oxidation,30 and the incidence of C20:5 in the GL fractions indicates that the extraction methods used are able to preserve it intact. This occurrence of C20:5 is in accordance with literature data, where 3937
DOI: 10.1021/acs.jafc.5b00241 J. Agric. Food Chem. 2015, 63, 3931−3941
Article
Journal of Agricultural and Food Chemistry
Figure 5. Relative distribution over different DGTS and lyso-DGTS expressed as % of total peak area of DGTS and lyso-DGTS, respectively. Results are the average of three replicate measurements (mean ± SD; CHL: chloroform; MeOH: methanol; EtOH: ethanol).
the sn-1 and sn-2 positions (Figure 4). A positively charged trimethylammonium group and a negatively charged carboxyl group give them zwitterionic character at neutral pH. At least three types of betaine lipids have been described with differing permethylated hydroxyamino acids linked to diacylglycerols through an ether bond: DGTS, 1,2-diacylglyceryl-3-O-2′(hydroxymethyl)-(N,N,N-trimethyl)-β-alanine and 1,2-diacylglyceryl-3-O-carboxy-(hydroxymethyl)choline. Of these, the first is by far the most common in nature.40,41 There is evidence in the literature that supports betaine lipids (as well as glycolipids) being used to replace part of the membrane phospholipids in cases of phosphorus starvation as observed in the photosynthetic bacterium Rhodobacter sphaeroides42 and plant Arabidopsis thaliana.43 DGTS can also be present in the lysated form due to the loss of a fatty acid residue, referred to as lyso-DGTS. The total peak area of polar lipids observed in the PL fractions of the different algae extracts is given in Figure 4 with a distinction between PC, DGTS, and lyso-DGTS. DGTS and PC are the major contribution to the total peak area of polar lipids. The highest total peak area was found for the PFE extraction with ethanol, followed by the conventional Folch extraction, with the highest affinity for DGTS. As for the GL fractions, a trend of increasing total peak area with increasing solvent polarity is observed, both for the traditional solvent systems and scCO2.
and C16:1 are common in both studies, the present study reveals the predominance of particular PUFAs, including C18:2, C18:3, and C20:5. This difference in fatty acid profile of the galactolipids can be explained by the cultivation, harvest (sedimentation, chemical flocculation, etc.), and storage (freeze, dry, freeze-dry, wet, etc.) conditions of the microalgae used. The extent of the production of biomass, lipids, carotenoids, and carbohydrates as well as the exact composition of the final products (e.g., fatty acid pattern of lipids) can vary considerably depending on the prevailing conditions in the cultivation medium.5,6 In addition, differences in the fatty acid profile15,35 of the extracts also arise due to the extraction methodology used (i.e., traditional vs PFE must also be taken into account), which can result in a different fatty acid profile. PL Fractions. A typical chromatogram of a PL fraction is shown in Figure 4. In the PL fractions, two different classes of polar lipids were detected: trimethylhomoserines (DGTS; 1,2diacylglyceryl-O-(N,N,N-trimethyl)-homoserine) and phoshatidylcholines (PC). The chemical structure of both classes is given in Figure 4. DGTS belongs to the betaine lipids, a recently discovered group of complex lipids primarily found in the plant kingdom.40 Ether-linked glycerolipids containing a betaine moiety occur naturally in algae, bryophytes, fungi, and in some primitive protozoa and photosynthetic bacteria. These lipids contain a polar group linked by an ether bond in the sn-3 position of the glycerol moiety with the fatty acids esterified in 3938
DOI: 10.1021/acs.jafc.5b00241 J. Agric. Food Chem. 2015, 63, 3931−3941
Article
Journal of Agricultural and Food Chemistry
Table 1. Total Concentration of Phosphatidylcholine (PC) and Relative Distribution among Different Combinations of Fatty Acid Chains in the Different Polar Lipid Fractionsa PFE extraction method
hexane
total PC (% w/w) 0.14 relative distributionc (%) among PC 16:0/16:1 24.6 PC 16:0/18:1 19.0 PC 16:0/18:2 PC 16:0/18:3 16.9 PC 16:0/20:5 6.6 PC 16:1/16:1 7.8 PC 16:1/18:0 PC 16:1/20:5 5.6 PC 16:2/18:2 PC 18:1/16:1 15.3 PC 20:3/16:0 4.1
ethanol
Soxhlet 2:1 CHL/MeOH
hexane
2.51 0.42 0.01 PCs with different fatty acid chains 25.7 26.7 34.0 15.3 19.0 4.2 1.5 20.9 15.7 3.5 8.3 7.2 9.1 11.9 8.9 14.1 1.2 5.7 4.5 8.2 2.7 7.5 14.4 13.3 4.8 3.7 8.3
Folch
SFE
ethanol
2:1 CHL/MeOH
2:1 CHL/MeOH
scCO2
scCO2/30% EtOH
0.48
0.18
0.60
