Isolation and Characterization of Lecithin from Squid (Todarodes pacificus) Viscera Deoiled by Supercritical Carbon Dioxide Extraction C: Food Chemistry
Md. Salim Uddin, Hideki Kishimura, and Byung-Soo Chun
Abstract: Marine lecithin was isolated and characterized from squid (Todarodes pacificus) viscera residues deoiled by
supercritical carbon dioxide (SC-CO2 ) extraction. SC-CO2 extraction was carried out to extract the oil from squid viscera at different temperatures (35 to 45 ◦ C) and pressures (15 to 25 MPa). The extraction yield was higher at highest temperature and pressure. The major phospholipids of squid viscera lecithin were quantified by high-performance liquid chromatography (HPLC). Phosphatidylcholine (PC; 80.5% ± 0.7%) and phosphatidylethanolamine (PE; 13.2% ± 0.2%) were the main phospholipids. Thin layer chromatography (TLC) was performed to purify the individual phospholipids. The fatty acid compositions of lecithin, PC and PE were analyzed by gas chromatography (GC). A significant amount of eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA) were present in both phospholipids of PC and PE. Emulsions of lecithin in water were prepared through the use of a homogenizer. The oxidative stability of squid viscera lecithin was high in spite of its high concentration of long-chain polyunsaturated fatty acids. Keywords: marine lecithin, oxidative stability, phospholipids, squid viscera, supercritical carbon dioxide extraction
Practical Application: Squid viscera are discarded as a waste by fish processing industry. Since lecithin from squid viscera
contains higher amounts of polyunsaturated fatty acids, it may have promising effect to use in food, pharmaceutical, and cosmetic industries.
Introduction Lecithin is a sticky fatty substance composed mainly of phospholipid mixtures (especially phosphatidylcholine [PC] and phosphatidylethanolamine [PE]) with small amount of glycerides, neutral lipids, and other suspended matter. Lecithin, which occurs in egg yolk, animal and plant tissues, is used for its emulsifying properties in the food, pharmaceutical and cosmetic industries. Pharmacological use of lecithin is included in treatments for hypercholesterolemia, neurologic disorders and liver ailments. Lecithin has also been used to modify the immune system by activating specific and nonspecific defence systems (Der Marderosian 1988; Budavari 1989; Reynolds 1996). The main commercial sources of lecithin are soybeans and egg yolk (Budavari 1989; Martin-Hernandez and others 2005). To date, the soybean is the most frequently used and studied source of lecithin. However, lecithin from soybeans is rich in saturated fatty acids with few lower unsaturated fatty acids. Lecithin from soybeans does not contain important polyunsaturated fatty acids such as eicosapentaenoic acid (EPA) and docosahexaenoic acid (DHA). Egg yolk has also been used widely as a source of lecithin. Phospholipids from egg yolk are the major source used by the
MS 20101191 Submitted 10/20/2010, Accepted 12/3/2010. Authors Uddin and Chun are with Dept. of Food Science and Technology, Pukyong Natl. Univ., 599-1 Daeyeon-3dong, Nam-Gu, Busan 608-737, Republic of Korea. Author Kishimura is with Lab. of Marine Products and Food Science, Research Faculty of Fisheries Sciences, Hokkaido Univ., Hakodate, Hokkaido 041-8611, Japan. Direct inquiries to author Chun (E-mail:
[email protected]).
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pharmaceutical industry for parenteral nutrition. In nutritional supplements, egg phospholipids do not play a large role because they have a relatively high cholesterol level and unfavourable fatty acid profiles. In the current research, it is understood that the increase level of essential fatty acids, such as EPA and DHA, leads to increased bioavailability when they are an integrated part of a phospholipid molecule. Lecithin from marine sources has several valuable nutritional benefits. Today, it is well known that the most important ω-3 fatty acids, EPA and DHA, are found in marine organisms. Marine phospholipids are valuable resources that can be applied to diverse areas such as nutrition, pharmacy, and medicine as well as basic research because they contain high levels of ω-3 fatty acids (Miniadis-Meimaroglou and others 2008). Lecithin is primarily extracted with solvents such as diethyl ether, hexane, chloroform, and ethanol. Acetone is also used to precipitate lecithin from other lipids. However, some of these solvents are considered undesirable because of environmental and health concerns (Sim 1994; Nielson 2001; Palacios and Wang 2005). To extract lecithin, the use of less hazardous solvents has a great importance to the environment. Currently, an environmentfriendly solvent, supercritical carbon dioxide (SC-CO2 ), is widely used to extract nonpolar lipids with lipid-soluble bioactive compounds from different sources (Esquivel and others 1997; Davarnejad and others 2008; Rubio-Rodriguez and others 2009; Sahena and others 2010a, 2010b). Further, SC-CO2 extraction provides some advantages over conventional extraction processes because carbon dioxide (CO2 ) is nonflammable, nontoxic, inert to most materials, inexpensive, and can be used under mild operational conditions (Ge and others 2002; Lopez and others 2004). R C 2011 Institute of Food Technologists doi: 10.1111/j.1750-3841.2010.02039.x
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Isolation of squid viscera lecithin . . .
Materials and Methods Materials Squid viscera were collected from F&F Co., Busan, Korea. The visceral waste was washed thoroughly with cold water and brought to the laboratory on ice. Pure CO2 (99.99%) was supplied by KOSEM, Korea. Standards of PC, PE, trolox, oleic, and linoleic acid were purchased from Sigma-Aldrich, St. Louis, Mo., U.S.A. All reagents used in this study were of analytical or highperformance liquid chromatography (HPLC) grade. Sample preparation Squid viscera samples were dried in a freeze-drier for about 72 h. The dried samples were crushed by a mechanical blender and sieved (700 μm) through a mesh. These samples were then stored at −60 ◦ C prior to SC-CO2 extraction. SC-CO2 extraction In this study, a laboratory-scale supercritical fluid extraction process was performed. Experiments were done in triplicate. The extraction apparatus was able to be operated at maximum pressure of 25 MPa. The flow rate of CO2 (22 g/min) was constant over the entire extraction period of 2.5 h. The process of extraction of squid viscera oil by SC-CO2 at different temperatures (35 to 45 ◦ C) and pressures (15 to 25 MPa) has been discussed in detail in our previous study (Uddin and others 2009). The extractions were performed at low temperature because fish oil is rather involatile and thermally sensitive (Singh 2004). Squid viscera providing the highest yield of oil by SC-CO2 extraction were used to isolate lecithin. Isolation of lecithin Lecithin was isolated from squid viscera according to the method of Palacios and Wang (2005) with some modifications. Briefly, 100 mL of ethanol (95%) was added to 30 g of SC-CO2 extracted squid viscera residues and stirred for almost 12 h by a magnetic stirrer. The mixture was then centrifuged at 1900 × g for 10 min. The supernatant containing mainly polar lipids with very small amounts of neutral lipids was collected in a separatory funnel. The precipitate was again extracted with 100 mL of ethanol, and, after centrifugation, the supernatant was added to the previous ethanol extract. Then twice the volume of hexane was mixed with the ethanol extract to separate the neutral lipids from the polar lipids. The ethanol phase was evaporated in a rotary vacuum evaporator at 40 ◦ C. The remaining lipid residue was dissolved in hexane. A fifth volume of chilled acetone (4 ◦ C) to hexane was added to the hexane mixture with slow stirring for precipitation of the
gummy material. The mixture was placed in an ice bath for 15 min and then centrifuged at 1500 × g for 10 min. After discarding supernatant, the collected precipitate called marine lecithin was stored at −40 ◦ C until further analysis.
Characterization of squid viscera lecithin Phospholipid content. The phospholipid content of lecithin from squid viscera was measured according to Stewart (1980) by a colorimetric method based on the formation of a complex between phospholipids and ammonium ferrothiocyanate. Briefly, 0.35 mg of lecithin was dissolved in 2 mL of chloroform. Then 1 mL of a solution prepared from ferric chloride (27 g/L) and ammonium ferrothiocyanate (30 g/L) was added. After vortexing, the mixture was centrifuged at 1000 × g for 15 min. The lower phase was collected, and the absorbance was recorded at 488 nm by a spectrophotometer (UVIKON 933, Kontron Instruments, Milan, Italy). The phospholipid content was calculated by constructing a calibration curve of the standard PC. Measurement of hexane insoluble matter, acid value, and peroxide value. Hexane insoluble matter of lecithin from squid viscera was determined by AOCS official method of Ja 3–87 (AOCS 1998). This method is used to determine the substances insoluble in hexane. The acid value was measured according to the AOCS official method of Ja 6–55 (AOCS 1998). The acid value was the amount of milligrams of KOH required to neutralize the acids present in 1 g of sample. The peroxide value was measured by the AOCS official method of Ja 8–87 (AOCS 1998) and expressed as milliequivalents peroxide per 1000 g sample. Free fatty acid (FFA) content. FFA content was analyzed by the method of Bernardez and others (2005). Briefly, 50 mg of lecithin was placed into Pyrex tubes with the addition of 3 mL of cyclohexane, and, then, 1 mL of cupric acetate–pyridine reagent was added. Tubes were vortexed for 30 s. After centrifugation at 2000 × g for 10 min, the upper layer was read at 710 nm by the spectrophotometer mentioned previously. The FFA content of lecithin was measured on a calibration curve constructed using oleic acid as the standard. Major phospholipids quantification by HPLC analysis. Major phospholipids of squid viscera lecithin were separated and quantified by a Jasco HPLC equipped with a controller, a 4-line degasser (DG-2080-54), a quaternary gradient unit (LG-2080-04), an intelligent HPLC pump (PU-2080 Plus), an evaporative light scattering detector (ELSD-Softa Corp., Co., U.S.A.; Model 400), and a silica column (5 μm, 4.6 × 250 mm; Waters, Mass., U.S.A.). The analysis was carried out according to the method of Letter (1992) with modification of the ELSD operation. Extracted lecithin was dissolved in chloroform and injected (20 μL) via the injector. The mobile phase was isopropyl alcohol, hexane, and water. The spray and drift tube temperature of ELSD were set to 70 and 60 ◦ C, respectively. The pressure of nitrogen gas at the nebulizer was 50 psi. The quantification of the phospholipids was performed based on the peak area of the standard phospholipids, PC and PE. Millennium software was used to analyze the data obtained by HPLC. Thin layer chromatography (TLC). The phospholipids of lecithin from squid viscera were separated by Thin layer chromatography (TLC) to determine the fatty acid profiles of PC and PE. Separation of individual lipids was performed by thinlayer chromatography using Silica plates 60 (20 cm × 20 cm, 0.2-mm thick, Macherey-Nazel, Duren, Germany). Lecithin was separated by a modified method of Miniadis-Meimaroglou and Vol. 76, Nr. 2, 2011 r Journal of Food Science C351
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Squid is a very popular food in Korea and Japan. Every year, large quantities of squid viscera are produced as a by-product in the fish processing industry. In our previous study, SC-CO2 extraction of oil from squid viscera was performed to use the extracted residue as a source of functional material (Uddin and others 2009). Although SC-CO2 is a nonpolar solvent that extracts mainly nonpolar lipids, the extracted residues may contain some polar lipids. Therefore, the aim of this research was to isolate and characterize lecithin from SC-CO2 extracted residues of squid viscera. The quality of the extraction yield was also evaluated to assess whether marine lecithin might be used as functional lipids.
Isolation of squid viscera lecithin . . .
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others (2008). The mobile phase was composed of chloroform : methanol : glacial acetic acid : water (50 : 25 : 6 : 2, v/v/v/v). Lecithin dissolved in a mixture of chloroform : methanol (2 : 1, v/v) was used for separation. Spots were visualized by iodine vapour. Spots were then scraped off in a screw cap tube separately and then extracted from the silica using the solvent system of chloroform : ethanol : water (2 : 2 : 1, v/v/v). The chloroform phase was collected by phase separation and evaporated by a vacuum rotary evaporator. The purity of the remaining residues of each phospholipid was again checked by TLC. PC and PE from the spots were again extracted as described previously. The purified PC and PE were used for fatty acid composition determinations. Determination of fatty acid composition by gas chromatography (GC). Gas chromatography (GC) analysis was carried out to determine the fatty acid compositions of lecithin and purified PC and PE. A Hewlett Packard gas chromatograph (5890 Series II GC system) with an Agilent DB-Wax capillary column (30 m length × 0.250 mm internal diameter, 0.25 μm of film) was used. The fatty acid methyl esters were prepared according to the AOCS official method of Ce 2–66 (AOCS 1998). Nitrogen was used as the carrier gas (1 mL/min) of the fatty acid methyl esters. The oven temperature was programmed starting at a constant temperature of 130 ◦ C for 3 min and then increased to 240 ◦ C at a rate of 4 ◦ C/min followed by a hold at 240 ◦ C for 10 min. Injector and detector temperatures were both 250 ◦ C. Fatty acid methyl esters were identified by comparison of retention time and standard fatty acid methyl esters mixtures (Supleco, Pa., U.S.A.).
Oxidative stability To measure the oxidative stability, emulsions of lecithin in water were oxidized at 37 ◦ C. Total of 3 emulsions of lecithin in water (w/w) (linoleic acid 4%, lecithin 1%, water 95%; lecithin 5%, water 95%; trolox 1%, lecithin 4%, water 95%) were prepared. Deionized and degassed water were used for emulsion preparation. Linoleic acid and standard trolox were used to measure the oxidative stability of marine lecithin. The mixture was properly homogenized by a homogenizer. Oxidative stabilities were checked by the thiocyanate (TC) (Mitsuda and others 1966) and thiobarbituric acid (TBA) (Ottolenghi 1959) methods, which were used to measure the antioxidant activity. In this study, these 2 methods were conducted to measure the quality of the extracted material in terms of its oxidative stability.
mixture was centrifuged at 3000 rpm for 20 min. Absorbance of the supernatant containing TBARS was measured at 532 nm.
Results and Discussion SC-CO2 extraction SC-CO2 extraction curves of squid viscera oil at different temperatures (35 to 45 ◦ C) and pressures (15 to 25 MPa) are shown in Figure 1. The highest oil yield obtained by SC-CO2 extraction was 0.34 g/g squid viscera at 45 ◦ C and 25 MPa. The variation of applied pressures and temperatures greatly affected the oil solvating power of SC-CO2 and, hence, the amount of yield. The SC-CO2 extraction curves of oil yield at different temperatures and pressures have been described in detail in our previous study (Uddin and others 2009). Characterization of squid viscera lecithin In this study, 4.25% (w/w) of lecithin was isolated from squid viscera residues. The phospholipid content of squid viscera lecithin was 91.56% (w/w). Therefore, 3.89% of phospholipids (w/w) was found in squid viscera residues. Cho and others (2001) reported that the phospholipid content of squid viscera extracted by the chloroform–methanol was 3.8% of the total lipid content. The value of phospholipid content was higher than that obtained in this study. Phospholipid content in squid viscera may vary depending on sample age, habitat, fishing time, extraction process and time, extracting solvents, and so on. Hexane insoluble matter, FFA content, acid value, and peroxide value Hexane insoluble matter, FFA content, acid value, and peroxide value of squid viscera lecithin are given in Table 1. These parameters provide the quality index of the marine lecithin. Hexane insoluble matter was less than 1%. However, this value was within the range of commercially-available soy lecithin. The FFA content, peroxide value, and acid value are dependent on the nature of processing and the factors such as the surrounding moisture, air, temperature, and so on. The FFA content of isolated lecithin
TC method The peroxide formed by lipid peroxidation reacted with ferrous chloride and formed ferric ions. Ferric ions then combined with ammonium thiocyanate and produced ferric thiocyanate. Briefly, 0.1 mL of emulsion solution was added to 4.7 mL of 75% ethanol and 0.1 mL of 30% ammonium thiocyanate. Precisely 3 min after the addition of 0.1 mL of 0.02 M ferrous chloride in 3.5% HCl to the reaction mixture, the absorbance of a red color was measured at 500 nm. The absorbance was recorded at 24-h intervals during the incubation. TBA method The TBA method was used to evaluate the extent of lipid peroxidation. Malonadehyde, the product of lipid breakdown caused by oxidative stress, binds with TBA to form a red complex of thiobarbituric acid reactive substance (TBARS). Briefly, 2 mL of 20% trichloroacetic acid and 2 mL of 0.67% 2-thiobarbituric acid were added to 1 mL of emulsion solution. The mixture was placed Figure 1–SC-CO2 extraction of oil from squid viscera at different temperain a boiling water bath (100 ◦ C) for 10 min. After cooling, the tures and pressures. C352 Journal of Food Science r Vol. 76, Nr. 2, 2011
was 1.1% ± 0.1%. Due to presence of moisture in lecithin, FFA may be liberated by its hydrolytic rancidity. Determination of FFA content therefore provided an index of the quality of the marine lecithin. The peroxide value provides the oxidation state of the lipids. This value was used to measure the rancidity that occurred by auto-oxidation. The peroxide value of isolated lecithin was 3.8 ± 0.3 milliequivalent/1000 g. In contrast, the acid value of squid viscera lecithin was 33.1 ± 1.8 mg KOH/g of lecithin. The acid value was used to determine the acidity of the lecithin. The peroxide value and the acid value of food grade lecithin recommended by FAO/WHO are found to be up to 10 milliequivalent/1000 g and 36 mg KOH/g of lecithin, respectively (Nieuwenhuyzen and Tomas 2008).
Quantification of major phospholipids The major phospholipids of lecithin obtained from squid viscera are shown in Table 2. The main phospholipids of squid viscera were PC and PE. PC and PE comprised 80.5% ± 0.7% and 13.2% ± 0.2% of the total phospholipids, respectively. Cho and others (2001) reported that phospholipids from squid viscera contained 79.2% PE and 12.7% PC. The composition of phospholipids (PC and PE) from squid viscera obtained by Cho and others (2001) were almost opposite in trend to those values obtained in this study. However, Cho and others (2001) and Igarashi and others (2001) also reported 3 to 5 times higher PC than PE content in phospholipids from muscle, mantle, fin, arm, and integument of squid, all of which were similar to the values found in this study. The phospholipid compositions may differ due to the habitat, intake of food varieties, variation of isolation and quantification process, variation of seasons for harvesting, and more. Fatty acid compositions of lecithin, PC, and PE For fatty acid analysis, the individual phospholipids were separated by TLC. The fatty acid compositions of lecithin, PC and PE from squid viscera obtained by GC are shown in Table 3. In Table 3, for lecithin fatty acids showed which was present more than 1% of total fatty acids. The percentages of the total polyunsaturated fatty acid were higher in all lipid fractions (lecithin, PC, and PE). The important polyunsaturated fatty acids were EPA (ranging from 10.4% to 16.4% of total fatty acids) and DHA (ranging from 14.5% to 22.7% of total fatty acids), which were found in significantly large amount in all lipid fractions. Among the mo-
nounsaturated fatty acids, C18:1 was present in higher amounts. The most significant saturated fatty acids was C16:0 (ranging from 24.3% to 32.5% of total fatty acids) in all lipid fractions. The DHA/EPA ratios for lecithin, PC, and PE were 1.4, 1.7, and 1.1, respectively. The DHA/EPA ratio for squid viscera phospholipids and salmon head lecithin were 0.90 and 1.65, respectively (Cho and others 2001; Belhaj and others 2010).
Oxidative stability The oxidative stabilities of marine lecithin are shown in Figure 2A and B. In this study, the oxidation trend was evaluated instead of determining the absolute state of oxidation of the incubated sample. Lecithin with linoleic acid emulsions showed the increase in absorbance value from the first day. The increase in absorbance value was an indicator of auto-oxidation by formation of peroxides during incubation. Only the marine lecithin emulsion showed low absorbance values indicating low levels of lipid peroxidation until 15 d. The marine lecithin showed significantly increased oxidation after 20 d. In contrast, marine lecithin emulsions with trolox showed high oxidative stability. Trolox inhibited the peroxide formation of the lipids by peroxidation over a certain period. Initially, squid viscera lecithin emulsion showed slightly higher absorbance’s as compared to lecithin within the linoleic acid emulsion. This might be due to the presence of peroxide from the oxidation of neutral lipids of marine lecithin. When using the thiobarbituric acid method, the absorbance measured on the 15th day was also similar to the lecithin and lecithin with trolox emulsions. However, this value was also high in the lecithin with linoleic acid emulsion indicating a low oxidative stability. On the other hand, a significant increase in absorbance was found on the 20th day of the lecithin emulsion sample. EPA and DHA in lecithin (the major component of the unsaturated fatty acids) were the most susceptible to oxidation. However, marine lecithin showed high oxidative stability. In our previous study, it was found that squid viscera contained a natural antioxidant, astaxanthin. Lecithin from squid viscera may contain small amounts of natural antioxidants that might be one of the causes of its higher oxidative stability. Gogolewski and others (2000) also reported that long chain polyunsaturated fatty acids which esterified with polar lipids had synergistic effects with antioxidants. High oxidative stabilities of lecithin from animal and plant sources were also reported by using different methods (Palacios and Wang 2005; Wang and Wang 2008; Belhaj and others 2010).
Table 1– Hexane insoluble matter, free fatty acid, acid value, and peroxide value of lecithin as quality index. Quality index
Table 3–Fatty acid compositions (percent) of squid viscera lecithin, PC, and PE.
0.9 ± 0.1 a 0.1 ± 0.1 Fatty acids 33.1 ± 1.8 (% of total fatty acids) 3.8 ± 0.3 C14:0 a C16:0 Mean value of 2 replicates ± S.E. C16:1 Table 2– Major phospholipid compositions of squid viscera C18:0 C18:1n-9 lecithin. C18:2n-6 Cho and others (2001) C20:0 Squid viscera Squid viscera Squid muscle C20:1 phospholipid C20:3n-6 Phospholipidsa (%) (in this study) phospholipid C20:4n-6 C20:5n-3 (EPA) PC 80.5 ± 0.7 12.7 71.7 C22:6n-3 (DHA) PE 13.2 ± 0.2 79.2 24.7 Others 6.3 ± 0.9 8.1 3.6 DHA/EPA Hexane insoluble mattera (%) Free fatty acida (%) Acid valuea (mg KOH/g) Peroxide valuea (milliequivalent/1000 g)
a
Mean value of 2 replicates ± S.E.
a
Lecithin
PC
PE
3.1 ± 0.1 32.5 ± 0.9 2.6 ± 0.1 6.8 ± 0.2 10.6 ± 0.2 5.2 ± 0.1 1.9 ± 0.1 2.7 ± 0.1 3.3 ± 0.1 2.1 ± 0.1 10.4 ± 0.3 14.5 ± 0.3 1.4
2.4 ± 0.1 25.5 ± 0.5 ND 7.1 ± 0.2 11.5 ± 0.3 3.2 ± 0.1 ND 6.7 ± 0.3 3.6 ± 0.1 4.3 ± 0.1 13.1 ± 0.3 22.7 ± 0.6 1.7
3.8 ± 0.1 24.3 ± 0.6 ND 4.7 ± 0.1 9.2 ± 0.3 8.9 ± 0.3 1.4 ± 0.1 5.8 ± 0.2 4.3 ± 0.1 3.5 ± 0.1 16.4 ± 0.5 17.6 ± 0.4 1.1
Results are the mean value of 2 replicates. ND = not detected.
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Isolation of squid viscera lecithin . . .
Isolation of squid viscera lecithin . . .
Acknowledgments This research was supported by a grant from the Marine Bioprocess Research Centre of the Marine Biotechnology Program funded by the Ministry of Land, Transport and Maritime, Republic of Korea.
C: Food Chemistry
References
Figure 2–A and B: Oxidative stability of squid viscera lecithin: (A) TC method and (B) TBA method. Results are the mean value of 3 replicates ± S.E.
Conclusions In this study, marine lecithin was isolated from SC-CO2 extracted squid viscera residues and characterized by measuring the FFA content, acid value, and peroxide value. The major phospholipids of squid viscera lecithin were PC and PE. The phospholipids contained higher amounts of polyunsaturated fatty acids (especially EPA and DHA). The oxidative stability of marine lecithin was also high. Therefore, it can be concluded that nonpolar lipids obtained by SC-CO2 extraction may have different purposes and that the marine lecithin isolated from extracted samples provide polyunsaturated fatty acids, which may be useful in the food industry as well as in the pharmaceutical industry.
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