JOURNAL OF OLEO SCIENCE
`
`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 OukUBO
`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-CO,) is a suitable substance to extract non-
`polar substances(triacylglycerols). Howeverit has not proven effective in the extraction of polar
`substances. The efficient use of SC-CO, 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-CO;. However
`extractions with 10, 15 or 20%-ethanol in SC-CO, 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
`
`Supercritical carbon dioxide (SC-CO,) fluid extrac-
`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-CO, (5-11). There are
`several advantages in using SC-CO, in industrial pro-
`duction. CO, has several desirable properties, such as,it
`is non-corrosive, non-toxic, non-flammable and non-
`explosive. Because COQ,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
`bonusis, it is inexpensive and readily available. A low
`critical temperature and pressure (Tc=31.1°C, Pc=7.4
`
`MPa) can be utilized to establish an energy saving pro-
`cess.
`
`A great deal of research has been focused on the
`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-
`munediseases (14), etc.
`Some worksrefer to the application of SC-CO,
`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 appearin the extracted fractions.
`Cheungetal. (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 Ibaraki, 300-2635,
`JAPAN
`
`E-matl: yukihisa_tanaka@nof.co.jp
`
`Journal of Oleo Science ISSN 1345-8957 print / ISSN 1347-3352 online
`http://jos jstage.jst.go.jp/en/
`RIMFROST EXHIBIT 1015
`
`417
`
`page 0001
`
`

`

`Y. Tanaka, I. Sakaki and T. Ohkube
`
`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 9.8-31.4 MPa,
`temperature 40-80°C) and the behaviorsoflipids in SC-
`CO). Triacylglycerols (TGs) were not extracted com-
`pletely. Phospholipids (PLs) were not extracted with
`SC-CO,atall.
`SC-CO, 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-CQ,. The presence
`of an entrainer enhances the solubility of SC-CO,. 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-CO,. Temelli (20) extracted PLs
`from canola flakes and presscake with SC-CO, 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-CO, and 10 wt% ethanol.
`They reported phosphatidylcholine (PC) enrichment at
`low pressures although the total yields increased with
`increasing pressure. Teberikler ef al. (23) used SC-CO,
`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-CO, 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 powderis 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, monoacy]-
`glycerols and lysophospholipids, etc.).
`
`2°2 SC-CO, Extraction
`The extraction vessel used in this work was of 10.0-
`mm interior diameter and 129 mm length (model EV-4,
`
`418
`
`JASCO, Hachioj1, 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. CO, and ethanol were delivered by two sep-
`arate pumps, mixed and passed through a preheating
`coil.
`
`2:3 Analysis
`In this work, the lipids extracted with SC-CO, are
`referred to as the extracted lipids (EL). After the SC-
`CO, 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 EL, RL and TL were analyzed by means of
`silica gel thin layer chromatography (TLC, plate 5721,
`Merck, Darmstadt, Germany) with hexane-diethy]l
`ether-acetic acid (80:20:1 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 td. X 200 mm height) withsilica 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
`TGsand the original PLs.
`The fatty acid profiles were analyzed by gas chro-
`matography of the methyl esters prepared by trans-
`methylation with BF;/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 i.d.) (J & W Scientific, Folsom, CA)
`was used. The column temperature wasraised 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 I HPLC sys-
`tem (Toso, Tokyo, Japan) equipped with DEVELOSIL
`
`J. Oleo Sci., Vol. 53, No. 9, 417-424 (2004)
`RIMFROST EXHIBIT 1015
`
`page 0002
`
`

`

`Extraction of Phospholipids from Salmon Roe with SC-CO, and Ethanol
`
`60
`
`40
`
`20
`
`0
`
`30
`
`50
`
`70
`
`90
`
`es
`
`=>
`
`Pp
`
`g =+
`
`)o
`
`£Ea
`
`fal
`
`Temp (C)
`Fig. 1 Effect of Extraction Temperature on Extraction
`Yield.
`
`SC-CO, flow was 3 mL/min, Extraction time was 10
`hours.
`
`The extraction pressure was 17.7 MPa.
`Eachresult represents the mean + 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-CO,. 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-CO, 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-CO, because: (i) It is suitable for
`food use; and (1i) the phase behavior of CO,/ethanol
`mixes at high pressure is available (26, 27).
`CO, 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-CO,/etanol mixture
`have been reported (21). In our study ethanol was used
`as the entrainer to extract PLs from salmonroe.
`
`3:2 Effect of Ethanol on the Lipid Extrac-
`tion
`The extraction was performed at 17.7 MPa and 33°C
`with 5, 10, 15 or 20%-ethanol in SC-CO,. The ethanol
`
`419
`
`model 60-5 HPLC-column (259 mm X 4.6 mmi.d.)
`(Nomura-chemicals, Tokyo, Japan) was used. The
`mobile phase was acetonitrile/methanol/phosphoric
`acid (780:50:9, 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 5C-CO,. 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; 44.18+0.21, 46.714
`0.83, and 51.030.71% (average +SD), 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; C20:5, n-3) and docosahexaenoic
`acid (DHA; C22:6, 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
`consumea great deal of energy to produce and maintain
`them.
`
`Since the authors had already shownthat 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-COQ, at
`17.7 MPa and 33°C.
`
`J. Oleo Sci., Vol. 53, No. 9, 417-424 (2004)
`RIMFROST EXHIBIT 1015
`
`page 0003
`
`

`

`Y. Tanaka, I. Sakaki and T. Ohkubo
`
`100
`
`80
`
`60
`
`20
`
`ExtractionYield(%)
`
`
`
`
`
`
`
`
`
`
`
`
`
`0
`
`5
`
`16
`
`15
`
`20
`
`Entrainer (%)
`
`Fig. 2 Effect of Entrainer on Extraction Yield.
`The extraction conditions were 17.7 MPa, 33°C.
`Eachresult represents the mean = S.D.
`
`flows were 5, 10, 15 or 20% of CO, flow by volume
`(CO, 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-CO, and 5%-ethanol mixture, the extraction yield
`rose above 50%, while an addition of 10-, 15-, or 20%-
`ethanol in SC-CO, achieved an extraction yield of more
`than 75%.
`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-CO,
`and 5%-cthanol mixture. When extraction was per-
`formed with SC-CO, and 10- or 15%-ethanol mixture,
`PLs were observed in the EL fractions. In all the
`
`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-CO,and 20%-
`ethanol mixture within an initial 30 min extraction
`almost all the TGs were extracted. In the following
`fractions (1 h-4 h) and RL fraction, slight TGs and
`some amount of PLs were observed.
`
`The PL concentrationsin all the fractions were quan-
`tified by HPLC. The results are shown in Table 1.
`Extraction with SC-CO, 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-
`CO, and ethanol mixtures increased with increase in the
`ethanol amount in SC-CO, (Fig. 3). When extraction
`was performed with SC-CO, 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 TGsto the original TGs
`are indicated as the TG recovery rate. When extraction
`was performed with SC-CO, 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-CO, and remained present
`in the RL fraction. When the lipids were extracted with
`SC-CO, and 10-, 15-, or 20%-ethanol mixtures more
`than 90% of the TGs were recovered to the EL fraction
`
`(Fig. 4).
`
`3°3 Effect of Ethanol on Fatty Acid Pro-
`files of Lipids
`The fatty acid profiles of the TGsin all the fractions
`were analyzed. The concentrations of oleic acid (OA;
`C18:1, n-9) and DHA of the EL are shown in Figs. 5
`and 6. The addition of the entrainer and extracting peri-
`
`
`
`Table 1 Phospholipid Content (wt%) in the Extracted Lipid Fractions.
`
`
`Extraction time (h)
`Ethanol
`
`(*)
`0.5
`1
`5
`3
`4
`
`14.2647.36
`10654561
`12.5543.10
`8.30L141
`2.444 2.96
`10
`39.3144.25
`64.1347.70
`67.37+6.71
`52.61+8.43
`8.80+4.21
`15
`
`20 97,204 1.33 7.88 £4.25 94.70+8.45 84.9028.41 92.9944.02
`
`
`
`
`
`
`
`
`
`Phospholipid content was analyzed by HPLC.
`Each result represents the mean + S.D.
`
`420
`
`J. Oleo Sci., Vol. 53, No. 9, 417-424 (2004)
`RIMFROST EXHIBIT 1015
`
`page 0004
`
`

`

`Extraction of Phospholipids from Salmon Roe with SC-CO, and Ethanol
`
`100
`
`80
`
`60
`
`40
`
`26
`
`PLRecoveryRate(%)
`
`
`
`OAconcentration(%)
`
`
`
`30
`
`
`25
`
`20
`
`15
`
`
`
`0
`
`S
`
`10
`
`15
`
`20
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`Entrainer (%)
`Fig. 3 Effect of Entrainer on Phospholipids Recovery Rate.
`The extraction conditions were 17.7 MPa, 33°C.
`Each result represents the mean + $.D.
`
`Time(h)
`Fig.5 Effect of Entrainer on Extracted Oleic Acid
`Concentration in TGs.
`
`The extraction conditions were 17.7 MPa, 33°C.
`Entrainer flows were 5 (LJ), 10 (A), 15 (>) and 20
`(C)%.
`no----- designates the oleic acid concentration in the
`original TG.
`Each result represents the mean + S.D.
`
`
`
`DHAconcentration(%)
`
`
`
`
`20
`
`30
`
`25
`
`15
`
`10
`
`a
`
`
`
`0
`
`1
`
`2
`
`3
`
`4
`
`5
`
`Time (h)
`Fig. 6 Effect of Entrainer on Extracted Docosahexaenoic
`Acid Concentration in TGs.
`
`The extraction conditions were 17.7 MPa, 33°C.
`Entrainer flows were 5 (L)), 10 (A), 15 (>) and 20
`(Cy%.
`n------ designates the oleic acid concentration in the
`original TG.
`Eachresult represents the mean + S.D.
`
`
`
`
`
`
`
` 0
`
`10
`
`15
`
`20
`
`5
`
`
`
`TGRecoveryRate(%)
`
`120
`
`100
`
`wooS
`
`60 |
`
`40
`
`20
`
`
`
`Fig. 4
`
`Entrainer (%)
`Effect of Entrainer on Triacylglycerols Recovery
`Rate.
`
`The extraction conditions were 17.7 MPa, 33°C.
`Eachresult represents the mean + S.D.
`
`J. Oleo Sci., Vol. 53, No. 9, 417-424 (2004)
`RIMFROST EXHIBIT 1015
`
`page 0005
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`421
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`

`

`Y. Tanaka, I. Sakaki and T. Ohkubo
`
`od did not affect the fatty acid profiles only the OA and
`DHA compositions. The OA 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 ofthe extrac-
`tions with SC-CO,and less than 15%-ethanol. The OA
`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-
`CO, and ethanol mixture occurs. The polarity of SC-
`CO, added with ethanol accelerated the DHAextraction
`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 andlinolenic acid (C18:3, n-3) decreased.
`
`and 20%-ethanol mixture. During the initial Lh extrac-
`tion almost all the TGs and 75% of the PLs were recoy-
`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 im thefirst frac-
`tion was approximately the sameas that for the TL such
`as 75:25 wiw.
`
`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 TGs and
`PLs using their retention time lag. Instead, the authors
`investigated a three-step extraction. In the first step, a
`SC-CO, 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 | h. In the third step, the extrac-
`tion was performed for 3 h with SC-CO, 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-
`
`=2
`
`bo
`
`6&
`
`oaf
`
`u %
`
`fa
`
`0
`
`0
`
`2
`
`4
`
`6
`
`8
`
`10
`
`According to the present results almost all lipids
`were extracted from the salmon roe FD with SC-CO,
`
`422
`
`‘Time(h)
`Fig. 7 Extraction of Lipid from Salmon Roe FD bythree-
`step Extraction.
`The extraction conditions were 17.7 MPa, 33°C.
`Eachresult represents the mean + S.D.
`
`J. Olea Sci., Vol. 53, No. 9, 417-424 (2004)
`RIMFROST EXHIBIT 1015
`
`page 0006
`
`3:4 Separation of TGs and PLs with SC-
`CO, and Ethanol
`Some researches have reported a two-step process for
`the extraction of the PLs with SC-CO, 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 ef a/. (22) reported on the
`extraction of PLs from canola and soybean, respective-
`ly. According to their reports the oil was removed at
`2 80|1st fraction
`high pressure (60-70 MPa) and high temperature (70-
`80°C). Since Montanari ef 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-CO, 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.
`
` 100
`
`60
`
`40
`
`20
`
`

`

`Extraction of Phospholipids from Salmon Roe with SC-CO, 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. Theresults are shown Fig. 7. The lipid delivery in
`the first, second, third and the RL fractions were 59.9
`6.9, 17.145.6, 12.4£0.6, and 10.5+1.7% (average =
`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 secondfraction.
`
`Acknowledgments
`
`This work was performed for “The Japanese
`Research and Development Association for New Func-
`tional Foods”.
`
`References
`
`1. K. ZOSEL, Separation with Supercritical Gases: Practical
`Application, Angew, Chem. Ind. Ed. Engl., Vol. 17, 702-709
`(1978).
`2, P. HUBERT and O.G. VITZTHUM, Fluid Extraction of Hops,
`Spices, and Tobacco with Supercritical Gases, Angew. Chem.
`Ind. Ed. Engl., Vol. 17, 710-715 (1978).
`3. B. GOPALAN, M. GOTO, A. KODAMA and T. HIROSE,
`Supercritical Carbon Dioxide Extraction of Turmeric (Curcuma
`longa), J. Agric. Food Chem., Vol. 48, 2189-2192 (2000).
`4. Y. YONEI, H. OHINATA, R. YOSHIDA, Y. SHIMIZU and C.
`YOKOYAMA, Extraction of Ginger Flavor with Liquid or
`Supercritical Carbon Dioxide, J. Supercritical Fluids., Vol. 8,
`156-161 (1995).
`5. P. BONDIOLI, C. MARIANI, A. LANZANI, E. FEDLI, A.
`MOSSA and A. MULLER, Lampante Olive Oil Refining with
`Supercritical Carbon Dioxide, J, Am. Oil Chem. Soc., Vol. 69,
`477-480 (1992).
`6. A. BIRTIGH, M. JOHANNSEN, G. BRUNNERand N. NAIR,
`Supercritical-fluid Extraction of Oil-palm Components, J.
`Supercritical Fluids, Vol. 8, 46-50 (1995).
`7. GR. LIST, J.W. KING, J.H. JOHNSON, K. WARNERand T.L.
`MOUNTG, Supercritical CO, Degumming and Physical Refin-
`ing of Soybean Oil, J. Am. Oil Chem. Soc., Vol. 70, 473-476
`(1993).
`8. G.R. ZIEGLER and Y.-J. LIAW, Deodorization and Deacidifica-
`tion of Edibles Oils with Dense Dioxide, J. Am. Oi! Chem. Soc.,
`Vol. 70, 947-953 (1993),
`9. §. LE and S. HARTLAND, A New Industrial Process for
`Extracting Cocoa Butter and Xanthines with Supercritical Car-
`bon Dioxide, J. Am. Oil Chem. Soc., Vol. 73, 423-429 (1996).
`10. M.S. KUK and M.K. DOWD, Supercritical CO, Extraction of
`Rice Bran, J. Am. Oil Chem. Soc., Vol. 75, 623-628 (1998).
`1. PAPAMICHAIL, V. LOULI and K. MAGOULAS, Supereriti-
`cal Fluid Extraction of Celery Seed Oil, J. Supercritical Fluids,
`
`11.
`
`Vol. 18, 213-226 (2000).
`. S.H. GOODNIGHT, The Effects of n-3 Fatty Acids on Arte-
`riosclerosis and the Vascular Response to Injury, Arch. Pathol.
`Lab. Med., Vol. 117, 102-106 (1993).
`. W.C. HARRIS, D.W, ROTHROCK, A. FANNING, S.B. INKE-
`LES, S.H. GOODNIGHT, D.W. ILLINGWORTH and W.E.
`CONNOR,Fish Oil in Hypertriglyceridemia, A Dose Response
`Study, Am. J. Clin. Nutr., Vol. 51, 399-406 (1990).
`. J.M. KREMER, D.A. LAWRENCE, W.JUBIZ, R. Di GIACO-
`MA,K. RYNES, L.E. BARTHOLOMEW and M. SHERMAN,
`Dietary Fish Oil and Olive Oil Supplementation in Patients with
`Rheumatoid Arthritis, Arthritis Rheum., Vol. 33, 810-820
`(1990).
`. K. YAMAGICHI, M. MURAKAMI, H. NAKANO, S. KONO-
`SU, T. KOKURA, H. YAMAMOTO, M. KOSAKAand K.
`HATA, Supercritical Carbon Dioxide Extraction of Oils from
`Antarctic Krill, J. Agric. Food Chem., Vol. 34, 904-907 (1986).
`P.C.K. CHEUNG, A.Y.H. LEUNG and P.O. ANG,Jr., Compari-
`son of Supercritical Carbon Dioxide and Soxhlet Extraction of
`Lipid from a Brown Seaweed, Sargassum hemiphyllum (Turn.)
`C. Ag., J. Agric. Food Chem., Vol. 46, 4228-4232 (1998).
`. Y. TANAKA and T. OHKUBO, Extraction of Lipids from
`Salmon Roe with Supercritical Carbon Dioxide, J. Oleo Sci.,
`Vol. 52, 295-301 (2003).
`. L. MONTANARY, J.W. KING, G.R. LIST and K.A. RENNICK,
`Selective Extraction of Phospholipid Mixtures by Supercritical
`Carbon Dioxide and Cosolvents, /. Food Sci., Vol. 61, 1230-
`1233, 1253 (1996),
`. W.E. PROSISE, Commercial Lecithin Products: Food Use of
`Soybean Lecithin,
`in Lecithins, (B.F. SZUHAJ and G.R. LIST,
`ed), American Oil Chemists’ Society, Champaign IL, pp.163-
`182 (1985).
`F. TEMELLI, Extraction of Triglycerides and Phospholipids
`from Canola with Supercritical Carbon Dioxide and Ethanol, J.
`FoodSci., Vol. 57, 440-442, 457 (1992).
`N.T. DUNFORD and F. TEMELLI, Extraction of Phospholipids
`from Canola with Supercritical Carbon Dioxide and Ethanol, J.
`Am. Oil Chem. Sac., Vol. 72, 1009-1015 (1995).
`L. MONTANARI, P. FANTOZZI, J.M. SNYDER and J.W.
`KING, Selective Extraction of Phospholipids from Soybeans
`with Supercritical Carbon Dioxide and Ethanol, J. Supercrit.
`Fluids, Vol. 14, 87-93 (1999).
`L. TEBERIKLER,
`8S. KOSEOGLU and A. AKGERMAN,
`Selective Extraction of Phosphatidylcholine from Lecithin by
`Supercritical Carbon Dioxide/Ethanol Mixture, J. Am, Qil
`Chem. Soc., Vol. 78, 115-119 (2001).
`J. FOLCH, M. LEE and 8.G.H. SLOANE, A Simple Method for
`the Isolation and Purification of Total Lipids from Animal Tis-
`sues, J. Biol, Chem., Vol. 226, 497-509 (1957).
`G. BRUNNER and §. PETER, On the Solubility of Glycerides
`and Fatty Acids in Compressed Grases in the Presence of an
`Entrainer, Sep. Sci. Technol, Vol. 17, 199-214 (1982).
`H. POHLER and E. KIRAN, Volumetric Properties of Carbon
`Dioxide + Ethanol at High Pressure, J. Chem. Eng. Data, Vol.
`
`os
`
`24,
`
`25.
`
`26,
`
`16.
`
`20.
`
`Bde:
`
`22.
`
`J. Oleo Sci., Vol. 83, No. 9, 417-424 (2004)
`RIMFROST EXHIBIT 1015
`
`page 0007
`
`423
`
`

`

`¥. Tanaka, I. Sakaki and T. Ohkubo
`
`42, 384-388 (1997).
`27. C.Y. DAY, C.J. CHANG and C.Y. CHEN, Phase Equilibrium of
`
`Ethanol + CO, and Acetone + CO, at Elevated Pressures, J.
`Chem. Eng. Data, Vol. 41, 839-843 (1996).
`
`424
`
`J. Oleo Sci., Vol. 53, No. 9, 417-424 (2004)
`RIMFROST EXHIBIT 1015
`
`page 0008
`
`