`
`Copyright ©2004 by Japan Oil Chemists’ Society
`J. Oleo Sci., Vol. 53, No. 9, 417-424 (2004)
`
`JOS
`
`Extraction of Phospholipids from Salmon Roe with
`Supercritical Carbon Dioxide and an Entrainer
`
`Yukihisa TANAKA*, Ikuko SAKAKI and Takeshi OHKUBO
`Tsukuba Research Laboratory, NOF CORPORATION
`(5-10 Tokodai, Tsukuba Ibaraki, 300-2635, JAPAN)
`
`Edited by K. Takahashi, Hokkaido Univ., and accepted April 19, 2004 (received for review March 29, 2004)
`
`Abstract: Supercritical carbon dioxide (SC-C02) is a suitable substance to extract non-
`
`polar substances (triacylglycerols). However it has not proven effective in the extraction of polar
`substances. The efficient use of SC—COZ and ethanol mixture to extract and fractionate
`
`phospholipids from salmon fish roe was therefore investigated. Extraction was performed at low
`pressure and temperature (17.7 MPa and 33 °C)
`to avoid oxidation of polyunsaturated fatty acids.
`Phospholipids were not found to be extracted with 0— and 5%-ethanol in SC-COZ. However
`
`extractions with 10, 15 or 20%-ethanol in SC-COZ were effective in extracting phospholipids.
`The amount of extracted phospholipids increased with increased addition of ethanol. When the
`
`extraction was performed with SC—CO; and 20%-ethanol mixture, more than 80% of the
`
`phospholipids were recovered.
`
`Key words: supercritical carbon dioxide extraction, salmon roe, triacylglycerol,
`phospholipid, entrainer, ethanol
`
`1
`
`Introduction
`
`MPa) can be utilized to establish an energy saving pro—
`cess.
`
`Supercritical carbon dioxide (SC-C02) fluid extrac—
`
`A great deal of research has been focused on the
`
`tion is applied in the commercial production of flavor-
`ing cosmetics, pharmaceuticals and food products.
`Some examples are decaffeinating coffee (1), hop
`extraction (2), extraction of turmeric essential oils (3),
`
`and ginger flavoring (4). In the oleo-industry, a numer—
`ous of researchers have done oil-extraction from seeds
`
`and refined plant oils with SC-COz (5-11). There are
`
`several advantages in using SC-COz in industrial pro-
`duction. CD; has several desirable properties, such as, it
`is non—corrosive, non-toxic, non-flammable and non-
`
`explosive. Because C02 is stable chemically, it does not
`react with other materials in treatment. Easy separation
`and removal of CO; from the products eliminates any
`problem related to toxic residual solvents. An added
`
`bonus is, it is inexpensive and readily available. A low
`critical temperature and pressure (Tc=31.1°C, Pc=7.4
`
`intake of polyunsaturated fatty acids (PUFA), especially
`n-3 PUFA, as they have been seen to showing them to
`play a beneficial role in the prevention of cardiovascu—
`
`lar diseases (12), hypertriglyceridemia (13) and autoim-
`mune diseases (14), etc.
`
`Some works refer to the application of SC-COZ
`extraction of marine materials to obtain PUFA. Yama-
`
`guchi et al. (15) reported on the extraction of lipids
`
`from Antarctic krill. According to their report, only
`non—polar components such as cholesterol, carotenoid
`triacylglycerols and their derivatives were extracted.
`
`Phospholipids did not appear in the extracted fractions.
`
`Cheung er al. (16) tried extracting lipids from brown
`seaweed. They reported that the extracting conditions
`affected the fatty acid profiles, that is, the concentration
`of total PUFAs increased reaching a higher value than
`
`*Correspondence to: Yukihisa TANAKA, Tsukuba Research Laboratory NOF CORPORATION, 5-10 Tokodai, Tsukuba lbaraki, 300-2635,
`JAPAN
`
`E-mail: yukihisa_tanaka@nof.co.jp
`
`Journal of Oleo Science ISSN 1345—8957 print/ ISSN 1347-3352 online
`http://jos.jstage.jst.go.jp/en/
`
`417
`
`RIMFROST EXHIBIT 1015
`
`page 0001
`
`
`
`Y. Tanaka, I. Sakaki and T. Ohkubo
`
`that obtained by solvent extraction.
`
`In our previous report (17) we extracted lipids from
`freeze-dried salmon roe to obtain an information on the
`
`effects of extracting conditions (pressure 9.8-3l.4 MPa,
`temperature 40-80°C) and the behaviors of lipids in SC—
`C02. Triacylglycerols (TGs) were not extracted com—
`
`pletely. Phospholipids (PLs) were not extracted with
`SC—COZ at all.
`
`SC-COZ does not provide a means to dissolve PLs
`effectively, but recovery of PLs can be achieved by the
`addition of a polar entrainer to SC-COz. The presence
`of an entrainer enhances the solubility of SC-COz. The
`choice of a suitable entrainer must be based on thermo-
`
`dynamic considerations and with regard to food safety,
`that is, it should be “Generally recognized as safe”
`(GRAS)(18). Prosise (19) noted that ethanol was an
`excellent solvent for isolating PLs for food use. Some
`researchers have already studied its role as an entrainer
`to extract PLs in SC—COz. Temelli (20) extracted PLs
`
`from canola flakes and presscake with SC-COZ and
`
`ethanol. Dunford et al. (21) reported a positive effect of
`ethanol on the extraction of PLs from canola meal.
`
`Montanari et al. (22) observed the extraction of PLs
`
`from soybean flakes with SC-COZ and 10 wt% ethanol.
`They reported phosphatidylcholine (PC) enrichment at
`low pressures although the total yields increased with
`increasing pressure. Teberikler er al. (23) used SC—COZ
`
`to produce a 95% purified PC from soybean lecithin
`containing a low percentage of PC.
`The authors report herein, the extraction of Docosa—
`hexaenoic acid (DHA)—rich PLs from freeze-dried
`salmon roe with SC-COZ and ethanol as the entrainer.
`
`2 Experimental
`
`2-1 Materials
`
`Frozen salmon roe was obtained from Nippon Kaken
`
`(Tokyo, Japan) and stored at —20°C before use. It was
`thawed and freeze-dried. In this report freeze-dried
`
`salmon roe powder is referred to as the FD—sample. The
`lipid extracted from the FD-sample by Folch’s method
`(24) is defined as the total lipid (TL). It contains TGs,
`
`PLs and their derivatives (diacylglycerols, monoacyl-
`glycerols and lysophospholipids, etc.).
`
`2-2 80—002 Extraction
`The extraction vessel used in this work was of 10.0-
`
`mm interior diameter and 129 mm length (model EV—4,
`
`418
`
`JASCO, Hachioji, Japan) with a volume of 10 mL. The
`equipment used for the work consisted of a high-pres—
`
`sure liquid chromatograph system (pump, JASCO PU-
`1586, column oven, JASCO 865-CO) and back pressure
`regulator (JASCO 880-81).
`
`4.0 g of the FD~sample was applied in the vessel.
`Extraction trials were performed at 33°C and 17.7 MPa.
`The extracted lipid was collected several times during
`extraction. C02 and ethanol were delivered by two sep-
`
`arate pumps, mixed and passed through a preheating
`coil.
`
`23 Analysis
`
`In this work, the lipids extracted with SC-COz are
`
`referred to as the extracted lipids (EL). After the SC—
`C02 extraction, the lipids retained in the spent FD—sam—
`ple were extracted by Folch’s method. This is referred
`to as the residual lipids (RL),
`in this work. The lipids
`content of BL, RL and TL were analyzed by means of
`
`silica gel thin layer chromatography (TLC, plate 572l,
`Merck, Darmstadt, Germany) with hexane-diethyl
`ether-acetic acid (80:20zl v/v/v) or chloroform-
`methanol-water (65:25:4 v/v/v).
`
`The extracting yield is referred to as the ratio of the
`weight of EL to the weight of TL.
`TL and RL (150 mg) were run through the column
`
`(20 mm id. X 200 mm height) with silica gel 60 (mesh
`70-230, Merck) to fractionate the TGs and PLs. The
`TGs and PLs were eluted with 300 mL of chloroform
`
`and 200 mL of methanol, respectively. The TGs and
`PLs fractionated from TL are referred to as the original
`TGs and the original PLs.
`The fatty acid profiles were analyzed by gas chro—
`matography of the methyl esters prepared by trans-
`
`methylation with BFg/methanol. An Agilent 6890A
`series gas chromatograph (Yokogawa Analytical Sys—
`
`tems, Musashino, Japan) equipped with a flame ioniza—
`tion detector (FID) and DB-WAX capillary column (30
`M X 0.25 mm id.) (J & W Scientific, Folsom, CA)
`
`was used. The column temperature was raised from 150
`
`to 210°C at 5°C/min. Both the injector and detector
`temperatures were 250°C. The carrier gas was helium
`with hydrogen and air supplied to the FID. The fatty
`acids were identified by comparison of the retention
`times with lipid standards (Sigma, Saint Louis, MA).
`PLs analyses were performed by high pressured liq—
`uid chromatography (HPLC). A LC Model 1 HPLC sys-
`tem (Toso, Tokyo, Japan) equipped with DEVELOSIL
`
`J. 0160 Sci., Vol. 53, No. 9, 417—424 (2004)
`
`RIMFROST EXHIBIT 1015
`
`page 0002
`
`
`
`Extraction of Phospholipids from Salmon Roe with SC—C02 and Ethanol
`
`model 60—5 HPLC-column (259 mm X 4.6 mmi.d.)
`
`60
`
`is
`
`E,
`
`2 40
`>1
`E
`.2H
`U
`EX
`W
`
`20
`
`0
`
`30
`
`50
`
`70
`
`90
`
`Temp (C)
`
`Fig. 1 Effect of Extraction Temperature on Extraction
`Yield.
`
`SC—COZ flow was 3 mL/min, Extraction time was 10
`hours.
`
`The extraction pressure was 17.7 MPa.
`Each result represents the mean i S.D.
`
`Many researchers have already reported since a pure
`
`carbon dioxide does not dissolve PLs effectively,
`extraction of PLs might be achieved by the addition of a
`polar entrainer to SC—COZ. An entrainer is a substance
`
`of medium volatility added to a mixture of compressed
`gas and a low volatility substance (20). As the solubility
`
`in SC—COZ at the same extracting conditions (tempera-
`ture and pressure) is drastically enhanced, extraction
`can be conducted at a lower pressure (25). The logical
`choice for a co-solvent in the food industry would be
`ethanol. The authors used ethanol as the entrainer to
`
`extract PLs in SC-CO2 because: (i) It is suitable for
`
`food use; and (ii) the phase behavior of COz/ethanol
`mixes at high pressure is available (26, 27).
`
`C02 and ethanol were mixed and passed through the
`preheating coil, and delivered to the vessel in the oven
`to extract the lipids. Extractions of PLs from canola,
`soybean and cottonseed with SC—COz/etanol mixture
`have been reported (21). In our study ethanol was used
`as the entrainer to extract PLs from salmon roe.
`
`3 ' 2 Effect of Ethanol on the Lipid Extrac-
`tion
`
`(Nomura-chemicals, Tokyo, Japan) was used. The
`
`mobile phase was acetonitrile/methanol/phosphoric
`acid (780:50z9, v/v/v). All solvents were HPLC grade
`(Wako Pure Chemical Industries, Ltd., Osaka, Japan).
`
`The flow of mobile phase was 1.5 ml/min. The column
`oven temperature was 45 °C. Detective absorbance was
`at 220 nm. Zephiramine (Wako Pure Chemical Indus-
`tries, Ltd.,) was used as the inner standard to analyze
`quantitatively.
`
`3 Results and Discussion
`
`3 ' 1 Effect of Extracting Temperature on
`the Lipid Yield at 17.7 MPa
`
`The authors investigated the most suitable conditions
`to extract lipids from salmon roe with SC—COZ. The
`results in our previous report (17) suggested that the
`extraction at higher pressure gained a higher extraction
`yield. Extractions performed at 50 MPa and 40, 60 and
`
`80°C gave high extraction yields; 44.18i0.21, 46.71 i
`0.83, and 51.03 i0.71% (averageiSD), respectively.
`The extraction yields were significantly higher than
`those found at 31.4 MPa and 40—80°C, mentioned in the
`
`previous report. No significant amount of PLs was
`extracted at these conditions.
`
`The authors further investigated improved the extrac—
`tion conditions from the following standpoints. The
`authors wanted to avoid PUFAs such as eicosapen-
`
`taenoic acid (EPA; C2025, n-3) and docosahexaenoic
`acid (DHA; C2226, n-3) being oxidized through being
`
`exposed to high temperatures during extraction. Extrac—
`tion at higher pressure also increases the risks of acci-
`dents in the handling of equipment. The authors further-
`more tried to establish an energy saving protocol.
`
`Extraction at higher pressures and higher temperatures
`consume a great deal of energy to produce and maintain
`them.
`
`Since the authors had already shown that reducing
`the temperature lead to an increase in the extraction
`yield at 17.7 MPa (17), to extract lipids from salmon
`roe FD at lower temperatures than 40°C suggested a
`higher extraction yield. The extraction yield at 33°C
`was significantly higher than that at 40°C (p<0.05, Fig.
`1). This was comparable to that at 50 MPa and 60°C.
`No PLs were extracted under these conditions. The
`
`authors investigated the behavior of lipids in SC-COZ at
`17.7 MPa and 33°C.
`
`The extraction was performed at 17.7 MPa and 33°C
`with 5, 10, 15 or 20%-ethanol in SC—COZ. The ethanol
`
`J. Oleo Sci., Vol. 53, No. 9, 417—424 (2004)
`
`RIMFROST EXHIBIT 1015
`
`page 0003
`
`419
`
`
`
`Y. Tanaka, I. Sakaki and T. Ohkubo
`
`100
`
`ccO
`
`aO
`
`NG
`
`ExtractionYield(%)
`
`
`
`
`
`
`
`
`
`
`
`
`
`0
`
`5
`
`10
`
`15
`
`20
`
`Entrainer (%)
`
`Fig. 2 Effect of Entrainer on Extraction Yield.
`The extraction conditions were 17.7 MPa, 33°C.
`Each result represents the mean i S.D.
`
`flows were 5, 10, 15 or 20% of C02 flow by volume
`
`(C02 flow : 3.0 ml/min, Ethanol flow = 0.15, 0.30,
`0.45 or 0.60 ml/min). The effect of the entrainer on the
`extraction yield is shown in Fig. 2. The extraction yield
`increased with increase in the ethanol percentage. 39%
`of the total lipids were extracted without the entrainer
`after 4 hours. When the extraction was performed with
`SC—COZ and 5%—ethanol mixture, the extraction yield
`rose above 50%, while an addition of 10—, 15—, or 20%—
`
`extracted fractions both TGs and PLs were observed
`
`after a 4-hour extraction, and present in the RL fraction.
`When extraction was performed with SC-COZ and 20%—
`ethanol mixture within an initial 30 min extraction
`
`almost all the T65 were extracted. In the following
`fractions (1 h-4 h) and RL fraction, slight TGs and
`some amount of PLs were observed.
`
`The PL concentrations in all the fractions were quan—
`tified by HPLC. The results are shown in Table 1.
`
`Extraction with SC-COZ and 20%-ethanol mixture pro-
`duced fractions containing PLs without TGs. The rate
`
`of extracted PLs to the original PLs are indicated as the
`PL recovery rate. The rate of extracted PLs with SC—
`C02 and ethanol mixtures increased with increase in the
`
`ethanol amount in SC-COZ (Fig. 3). When extraction
`was performed with SC-COZ and 10%—ethanol mixture
`30% of the PLs were recovered in the EL fractions.
`
`When the ethanol percentage was increased up to 20%
`more than 80% of the PLs were recovered in the EL
`
`fractions. The rate of extracted TGs to the original TGs
`are indicated as the TG recovery rate. When extraction
`
`was performed with SC-COZ and 5%-ethanol mixture,
`nearly 80% of the TGs were recovered during the four-
`hour extraction. On the other hand, 20% of the TGs
`
`were not extracted with SC-COZ and remained present
`in the RL fraction. When the lipids were extracted with
`SC-COZ and 10—, 15-, or 20%-ethanol mixtures more
`than 90% of the TGs were recovered to the EL fraction
`
`ethanol in SC—COZ achieved an extraction yield of more
`than 75%.
`
`(Fig. 4).
`
`The lipid compositions in all the fractions were
`observed using TLC. No PLs were observed in the EL
`fractions when extraction was performed with SC-COZ
`and 5%~ethanol mixture. When extraction was per~
`formed with SC-COZ and 10- or 15%—ethanol mixture,
`PLs were observed in the EL fractions. In all the
`
`3 - 3 Effect of Ethanol on Fatty Acid Pro—
`files of Lipids
`
`The fatty acid profiles of the TGs in all the fractions
`were analyzed. The concentrations of oleic acid (0A;
`
`C18zl, n-9) and DHA of the EL are shown in Figs. 5
`and 6. The addition of the entrainer and extracting peri~
`
`
`
`Table l Phospholipid Content (wt%) in the Extracted Lipid Fractions.
`
`
`Ethanol
`Extraction time (h)
`
`(%)
`0.5
`l
`2
`3
`4
`
`14.26i7.36
`10.65:5.61
`12.55i3.10
`8.30:1.41
`2.44i2.96
`10
`39.31i4.25
`64.13i7.70
`67.37i6.7l
`52.61i8.43
`8.80i4.21
`15
`
`
`
`
`
`84.90i8.41 92.99 24.0294.70:8.457.88i42520 97.20i 1.33
`
`
`
`
`
`Phospholipid content was analyzed by HPLC.
`Each result represents the mean i SD.
`
`420
`
`J. 0160 Sci., Vol. 53, No. 9, 417-424 (2004)
`
`RIMFROST EXHIBIT 1015
`
`page 0004
`
`
`
`Extraction of Phospholipids from Salmon Roe with SC-CO; and Ethanol
`
`b) O
`
`0Aconcentration(%)
`
`
`
`
`N U]
`
`NC
`
`p—i U]
`
`
`
`0
`
`5
`
`10
`
`15
`
`20
`
`0
`
`l
`
`2
`
`3
`
`4
`
`5
`
`100
`
`80
`
`60
`
`40
`
`20
`
`0
`
`A 5
`
`V a
`
`°
`.)
`
`*5
`m
`
`2‘
`
`3O
`
`UG
`
`.)
`:4
`b]
`D:
`
`Entrainer (%)
`
`Fig. 3 Effect of Entrainer on Phospholipids Recovery Rate.
`The extraction conditions were 17.7 MPa, 33°C.
`Each result represents the mean i SD.
`
`Fig. 5
`
`Time (h)
`Effect of Entrainer on Extracted Oleic Acid
`Concentration in TGS.
`
`The extraction conditions were 17.7 MPa, 33°C.
`Entrainer flows were 5 (D), 10 (A), 15 (O) and 20
`(O)%.
`------- designates the oleic acid concentration in the
`original TG.
`Each result represents the mean i S.D.
`
`
`
`
`
`
`
`
`
`
`
`DHAconcentration(%)
`
`Nc
`
`H U]
`
`H e
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`Fig. 6
`
`Time (h)
`Effect of Entrainer 0n Extracted Docosahexaenoic
`Acid Concentration in TGs.
`
`The extraction conditions were 17.7 MPa, 33°C.
`Entrainer flows were 5 (D), 10 (/_\), 15 (O) and 20
`(O)%.
`------- designates the oleic acid concentration in the
`original TG.
`Each result represents the mean i SD.
`
`421
`
`
`
`
`
`
`
`20
`
` 0
`
`5
`
`10
`
`15
`
`
`
`TGRecoveryRate(%)
`
`120
`
`100
`
`NO
`
`Entrainer (%)
`
`Fig. 4 Effect of Entrainer on Triacylglycerols Recovery
`Rate.
`
`The extraction conditions were 17.7 MP3, 33°C.
`Each result represents the mean i S.D.
`
`J. 0130 Sci., Vol. 53, N0. 9, 417-424 (2004)
`
`RIMFROST EXHIBIT 1015
`
`page 0005
`
`
`
`Y. Tanaka, I. Sakakz' and T. Ohkubo
`
`and 20%-ethanol mixture. During the initial 1h extrac—
`tion almost all the TGs and 75% of the PLs were recov-
`ered at the same time. As a result both the TGs and PLs
`
`were observed in the same fraction. A further 22% of
`
`the PLs were extracted in the following 3 h. When
`
`lipids were extracted from the spent FD powder with an
`organic solvent. 3% of the PL was observed as the RL
`fraction. In the second the EL fraction and RL fraction,
`the PLs were isolated without TGs. With these extrac—
`
`tion conditions, most of the PLs were obtained along
`with the TGs. The ratio of TGs and PLs in the first frac—
`
`tion was approximately the same as that for the TL such
`as 75:25 w/w.
`
`The authors tried to obtain fractions containing high
`concentrations of the PLs in the short time available
`
`10min. taken for the TGs to be already extracted. The
`authors judged that it was difficult to separate T65 and
`PLs using their retention time lag. Instead, the authors
`
`investigated a three—step extraction. In the first step, a
`SC-COZ and 5%—ethanol mixture was used for 4 h to
`
`extract as much of the TGs as possible. In the second
`step, the amount of ethanol was increased from 5% to
`20% in as short a time as possible, and the extraction
`
`progressed for a further I h. In the third step, the extrac-
`tion was performed for 3 h with SC—COz and 20%-
`ethanol. In the first step, approximately 80% of the TGs
`and none of the PLs were recovered. But thereafter, all
`
`of the TGs and part of PLs were recovered in the sec-
`
`
`
`100
`
`53
`
`80
`
`60
`
`40
`
`20
`
`0.
`
`E.
`
`2
`>1
`
`5
`.5
`1
`
`UNL
`
`32'
`III
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`
`Time (h)
`
`Fig. 7 Extraction of Lipid from Salmon Roe FD by three-
`step Extraction.
`The extraction conditions were 17.7 MPa, 33°C.
`Each result represents the mean i S.D.
`
`0d did not affect the fatty acid profiles only the 0A and
`
`DHA compositions. The DA concentrations in all the
`extracted fractions were lower than that in the original
`TGs. They increased with extracting time. The DHA
`concentrations in most of the extracted fractions were
`
`higher than that in the original TGs. After a four-hour
`extraction, TGs were observed in the RL of the extrac-
`tions with SC-COz and less than 15%-ethanol. The CA
`
`concentration in the RL was higher than in the original
`TGs. While the DHA concentration was lower than in
`
`the original TG. The results suggest a polarity of SC-
`C02 and ethanol mixture occurs. The polarity of SC-
`CO; added with ethanol accelerated the DHA extraction
`in the TGs.
`
`While the entrainer did not affect the fatty acid pro-
`files of the extracted PLs, significantly, the fatty acid
`
`profiles of the PLs in the EL and RL fractions were the
`same as those in the original PLs. Temelli (20) in his
`report on the fatty acid profiles of canola oil extracted
`with ethanol, remarked that the relative concentrations
`of OA and linolenic acid (C18z3, n-3) decreased.
`
`34 Separation of TGs and PLs with SC-
`002 and Ethanol
`
`Some researches have reported a two‘step process for
`the extraction of the PLs with SC—COZ to obtain high
`
`concentrated PLs. The first extraction is performed
`without ethanol to remove the oil. Subsequent extrac—
`
`tions are performed with ethanol to extract the PLs.
`Temelli (20) and Montanari et al. (22) reported on the
`extraction of PLs from canola and soybean, respective-
`
`ly. According to their reports the oil was removed at
`high pressure (60—70 MPa) and high temperature (70-
`80°C). Since Montanari et al. (22) performed the
`
`extraction at high temperature and high pressure, almost
`of all soybean TGs were obtained. Both high tempera-
`ture and pressure was necessary to extract the PLs from
`
`soybean. However when extraction temperature was
`
`high (80°C), the extracted amount of the PLs was
`reduced when extraction was performed at low pressure
`(23.9 MPa). Therefore a significant amount of the PLs
`was lost at 16.6 MPa. The extraction rate does not
`
`depend on SC-COZ and ethanol mixture density.
`In the present report, our choice of mild extraction
`conditions (33°C, 17.7 MPa) resulted in an incomplete
`extraction of TGs.
`
`According to the present results almost all lipids
`were extracted from the salmon roe FD with SC—COZ
`
`422
`
`J. 0160 Sci., Vol. 53, N0. 9, 417—424 (2004)
`
`RIMFROST EXHIBIT 1015
`
`page 0006
`
`
`
`Extraction of Phospholipids from Salmon Roe with SC—COZ and Ethanol
`
`ond step. In the third step, almost all of the PLs were
`recovered. The authors applied this three-step extraction
`to obtain high concentrated PLs from 4 g of salmon roe
`FD. The results are shown Fig. 7. The lipid delivery in
`the first, second, third and the RL fractions were 59.9i
`
`6.9, l7.1i5.6, 12.4i0.6, and 10.5: 1.7% (averagei
`
`SD), respectively. In the third and the RL fractions the
`PLs but none of the TGs were observed. The ratio of
`
`the TGs and PLs was 2:1 (w/w) in the second fraction.
`
`Acknowledgments
`
`This work was performed for “The Japanese
`Research and Development Association for New Func-
`tional Foods”.
`
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