JOURNAL OF OLEO SCIENCE
`
`Copyright ©2003 by Japan Oil Chemists’ Society
`J. Oleo Sci., Vol. 52, No. 6, 295-301 (2003)
`
`Extraction of Lipids from Salmon Roe with Supercritical
`Carbon Dioxide
`
`Yukihisa TANAKA* and Takeshi OnkuBo
`
`Tsukuba Research Laboratory, NOF CORPORATION
`(5-10 Tokodai, Tsukuba Ibaraki, 300-2635, JAPAN)
`
`Edited by K. Miyashita, Hokkaido Univ., and accepted February 18, 2003 (received for review December 25, 2002)
`
`Abstract: Freeze-dried salmon roe was extracted with supercritical carbon dioxide at a
`pressure range of 9.8-31.4MPa and temperature range 40-80°C. Thelipid yield andfatty acid
`profiles of the extracted lipids were affected by the extracting conditions. From the results affect
`of carbon dioxide density is estimated. At 80°C the extracts were mostly affected by the
`extracting pressure. While at 17.7MPathe extracts were affected by the extracting temperature.
`The lipids obtained, contained triacylglycerides and their derivatives while the lipids not
`extracted contained triacylglycerides and phospholipids. In other words, two groups of
`triacylglycerides extracted from the freeze-dried salmon roe were found to be present in the
`salmonroe. In the first group is triacylglyceride to be extracted with supercritical carbon dioxide.
`In the other group is triacylglyceride not to be extracted. Less than 30% of astaxanthin, a
`functional pigment in the salmon roe was extracted. The loss of astaxanthin wasless than 10% of
`the total involved in the process.
`Key words:supercritical carbon dioxide extraction, salmonroe,triacylglyceride,
`phospholipid, astaxanthin
`
`1
`
`Introduction
`
`Supercritical carbon dioxide (SC-CO,) fluid extrac-
`tion has been applied in the commercial production of
`flavoring cosmetics, pharmaceuticals and food prod-
`ucts. Examples are decaffeinated coffee (1), hop extract
`(2), extraction of turmeric essential oils (3), and ginger
`flavoring (4).
`In the oleo-industry, numerous
`researchers have tried extraction from seeds and the
`refinement of 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
`non-corrosion, non-toxicity, non-flammability and non-
`explosivity. Because CO, is stable chemically, it never
`reacts with other materials in treatment. Easy separation
`and removal of CO, from products eliminates problems
`
`related to toxic residual solvent. It is inexpensive and
`readily available. The low critical temperature and
`pressure (Tc=31.1°C, Pc=7.4MPa) can beutilized to
`establish an energy saving process.
`A great deal of research has been focused on the
`intake of polyunsaturated fatty acids (PUFA), especially
`n-3 PUFA, showing them to play a beneficial role in the
`prevention of cardiovascular diseases (12), hyper-
`triglyceridemia (13) and autoimmunediseases (14),etc.
`Some refer to the application of SC-CO, extraction
`of marine materials to obtain PUFA. Yamaguchietal.
`(15) reported on the extraction of lipids from Antarctic
`krill. According to their report, only non-polar compo-
`nents such as cholesterol, carotenoid triacylglycerides
`and their derivatives were extracted. Phospholipids did
`not appearin the extracted fractions.
`
`*Correspondence to: Yukihisa TANAKA, Tsukuba Research Laboratory NOF CORPORATION,5-10 Tokodai, Tsukuba, Ibaraki, 300-2635,
`
`
`
`
`
`AKER EXHIBIT 2018 PAGE 0001
`
`

`

`¥. Tanaka and T. Ohkubo
`
`Cheung et al. (16) tried extracting lipids from brown
`seaweed. They report that the extracting conditions
`affected the fatty acid profiles, that is, the concentration
`of total PUFAs increased reaching a higher value than
`that obtained by solvent extraction.
`Because of the high solubility in SC-CO, with EPA
`and DHAethyl esters as the extracting substrates is the
`usual approach to obtain high-purified eicosapentaenoic
`acid (20:5, EPA) and docosahexaenoic acid (22:6,
`DHA). Several researchers (17-19) have proposed con-
`tinuous processes, Mishra et al. (20) and Jaubert etal.
`(21) reported phase behavior with ethyl ester in SC-
`CO,. Yu et al. (22) compared the solubilities of fatty
`acids, esters, triacylglycerides, fats and oils in SC-CO,.
`The authors report herein on extracting lipids from
`salmon roe with SC-CO,to clarify the extraction condi-
`tions and behaviorofthe lipids in SC-CO,. The extract-
`ed lipids and residual lipids remaining in the FD-sam-
`ple after the extraction were characterized (yield, lipid
`contents,fatty acid profiles, etc.).
`
`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-sampie by Folch’s
`method (23) is defined as the total lipid (TL). It con-
`tains triacylglycerides (TG), phospholipids (PL) and
`their derivatives (diacylglycerides, monoacylglycerides
`and lysophospholipids,etc.).
`
`2:2 SCF-CO, Extraction
`The extraction vessel used in this work was of 10.0-
`mm interior diameter and 129mm length (model EV-4,
`JASCO, Hachioji, Japan) with a volume of 10mL. 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).
`2.5g of FD-sample were applied in the vessel.
`Extraction trials were performed at temperatures
`between 40 and 80°C and pressures between 9.8 and
`31.4MPa. SC-CO, was introduced into the vessel at a
`
`2:3 Analysis
`In this work, the lipids extracted with SC-CO, are
`referred to as the extracted lipids. After the SC-CO,
`extraction, the lipids retained in the spent FD-sample
`were extracted by Folch’s method. Thisis referred to as
`the residual lipids, in this work. The lipids content of
`the extracted lipids, residual lipids and total lipids were
`analyzed by meansofsilica gel thin layer chromatogra-
`phy (TLC, plate 5721, Merck, Darmstadt, Germany)
`with hexane-diethyl ether-acetic acid (80:20:1 v/v/v) or
`chloroform-methanol-water (65:25:4 v/v/v).
`The residual lipids and total lipids (150mg) were run
`through the column (20 mm i.d. X 200 mm height)
`with silica gel 60 (mesh 70-230, Merck) to fractionate
`the TG and PL. The TG and PL were eluted with
`300mL of chloroform and 200mL of methanol, respec-
`tively. The TG and PL fractionated from the total lipids
`are referred to as the original TG andoriginal PL.
`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-WAXcapillary column
`(30M X 0.25mm 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 at
`a flow of 80mL/min, and hydrogen and air were sup-
`plied to the FID. The fatty acids were identified by
`comparison of retention times with lipid standards
`(Sigma, Saint Louis, MA).
`The concentration of astaxanthin (AX), the function-
`al red pigment contained in the salmon roe wasestimat-
`ed at 488nm in benzene by using an absorbance coeffi-
`cient, A j, of 1,990.
`
`3 Results and Discussion
`
`3:‘1 Effect of Extracting Temperature and
`Pressure on the Lipid Yield
`The SC-CO, extractions were treated for 6 hours at
`24.5MPa and 60°C to determine the CO, flow rate and
`the extracting time (data not shown). The CO,flow rate
`and extracting time were determined as 3 mL/min and 2
`hours, respectively. The FD-sample was extracted with
`SC-CO, for 2 hours at 9.8-31.4MPa and 40-80.
`
`oproon
`preppeep
`
`AKER EXHIBIT 2018 PAGE 0002
`
`

`

`Extraction of Salmon Roe Lipids with SCF-CO,
`
`temperature conditions, in this work, only the results
`from the pressure range 17.7MPa to 31.4MPaare dis-
`cussed herein.
`Every sample was fluid and bright red because of the
`AX contained in it. PL was not found in any extraction.
`Neutral lipids (TG and derivatives) and carotenoid were
`extracted. According to previous reports (24, 25) the
`SC-CO),extraction is suitable for the separation of polar
`and non-polar lipids. Although the non-polar lipids are
`extracted, the polar lipids are not.
`The lipid yield is reported as a percentage of the
`original TG (the TG fraction separated from the TL by
`silica gel 60). The effect of the extracting temperature
`and pressure on lipid yield is shown in Fig. 1. The
`extracting conditions affected the lipid yield. At 80°C
`the lipid yield increased drastically from 17.7 to
`31.4MPa (p<0.01). At 60°C the lipid yield increased
`significantly from 17.7 to 24.5MPa. At 40°C the lipid
`yield tended to increase with the extracting pressure.
`From the point of extracting pressure, 17.7MPa, the
`lipid yield decreased with significantly the extracting
`temperature (p<0.01). At 24.5MPathe lipid yield at
`80°C reached its highest point. At 31.4MPathelipid
`yield increased with the extracting temperature
`(p<0.05). Bhupsesh et al. (26) reported that the extract-
`ing conditions have an effect on the lipid yield in toma-
`to seed oil extraction. The oil yield increases with pres-
`sure. Shen et al. (27) observed similar behavior in the
`case of application to rice bran oil extraction. Gomez et
`al. (28) proposed that the change was due to variations
`in the physical properties of CO», particularly the densi-
`ty which is closely related to the solvent capacity.
`The effect of the extracting pressure was that at con-
`stant temperature the lipid yield increased with the
`extracting pressure. While the effect of the extracting
`temperature was at constant pressure the lipid yield
`decreased with the extracting temperature. This
`decreasing tendency in the lipid yield from temperature
`was significant at low pressure and decreased with
`increasing pressure. These results coincide with several
`previous reports. The results suggested close participa-
`tion of the CO, density in the extraction of salmonroe.
`The sum ofthe extracted lipid and residuallipid yield
`resulted in the sameyield asthetotal lipid.
`
`80
`
`60
`
`40
`
`20
`
`Yield(%)
`
`17.7
`
`31.4
`
`24.5
`Pressure (MPa)
`Fig. 1 Effects of SC-CO, Extract Conditions on Yield of
`Extracted Lipids.
`SC-CO, flow was 3mL/min, Extract time was 2
`hours. Extractions were treated at 40 (MD, 60(C),
`and 80 (&)°C. Each result represents the meant
`S.D.
`
`3:2 Effect of Extracting Temperature and
`Pressure on Fatty Acid Profiles of the
`Extracted Lipid
`After a 2-hour extraction, the fractions of the extract-
`ed lipid were analyzed for their fatty acid profiles
`(Table 1). The extracting conditions affected the DHA
`concentration of the extracted lipid. This increased with
`the extracting pressure. A significant increase in the
`DHAconcentration was observed at 80°C. At 60 and
`40°C a DHAconcentration of 17.7MPa was significant-
`ly lower than those of 24.5 and 31.4MPa (p<0.01). At
`17.7MPa the DHA concentration increased significantly
`with extracting temperature (p<0.01). At 24.5MPa the
`DHAconcentration at 80°C wassignificantly lower
`than those at 40 and 60°C (p<0.05).
`The DHAconcentrations at 80°C/17.7MPa and 60°C
`/17.7MPa were lower than that of the original TG
`(19.75%). This result suggested that the solubility of
`TG containing DHA changed drastically with the
`extracting conditions. The extracting conditions, except
`for the two conditions above, were more suitable for
`dissolving TG containing DHA than any of the other
`TG molecules.
`
`oar
`aeppapeen
`
`AKER EXHIBIT 2018 PAGE 0003
`
`

`

`Y, Tanaka and T. Ohkubo
`
`
`
`Table 1 Fatty Acid Compositions of Extracted Lipids.
`bility of TG. The vapor pressure of TG is due to the
`
` Temp. . Pressure (MPa)
`fatty acid combination. The combination of the other
`
`(cy Fatty acid
`75
`24.5
`31.4
`two fatty acids in TG affects the extract efficiency of
`16:0
`12.29+034 11934010 11.93 +057
`each fatty acid. Therefore not only the carbon number
`
`16:1=8254084 74240.09 7.464 0.19
`but also the double bonds depend on the solubility. In
`18:0
`4.00 0.06
`3.62 £0.25
`4.06 + 0.56
`general a low molecular weight TG is extracted easily
`18.70 £ 0.56 19.31 + 0.06 19.37 + 0.58
`18:1
`at low pressure and a high molecular weight TG is
`4394016 4.16 +015 4.21 + 0.08
`18:2
`extracted easily at high pressure. Because the fatty acid
`
`20:4=2.1L £0.03 2.124001 2.08 + 0.02
`
`profiles result in the combination of three fatty acids
`20:5
`11.06 + 0.14 11.16 £0.07 11.38 + 0.38
`our result does not reflect any specific fatty acid proper-
`22:6
`19.72 + 1.42 21.544 0.73 21.46 + 0.52
`ty.
`In the case of marine oils, as the numberof fatty
`acids is large, combination is innumerable and com-
`13.37£0.72 12414018 12.02 +0.13
`16:0
`plex. All the fatty acids were separated to extract or
`9.13040 7.614033
`7.45 + 0.36
`16:1
`remain due to the difference in combinations of fatty
`«3.08 £0.22
`3.614035
`3.414037
`18:0
`acids in TG. Furthermore, analysis of fatty acid behav-
`21.01 £0.84 20.45 +011 19.71 £0.75
`18:1
`ior in SC-CO, from the point of view of the TG molec-
`4.454005 4.23007 4.07 + 0.05
`18:2
`ular unit might be necessary. A combination of thermal
`«2.09 0.04 2184010 215+ 0.02
`20:4
`gradient fractionation could be applied to separate TG
`20:5=10.91 + 0.30 11.474£0.27 11.55 +037
`motestrictly.
`22:6
`1732 $1.21 21284043 21.84 + 0.48
`Cheunget al. (16) reported on the extraction of lipids
`from brown seaweed. They showed the oil yield and
`17.02 £ 0.94 12.76 £0.15 11.92 + 0.22
`16:0
`10.40 £0.66 8574029 7.66 +0.35
`16:1
`extracting conditions affected the DHA concentration
`3.424023 2874008 3.46+0.52
`18:0
`of the extracted lipid. The DHA concentration increased
`«21.53 £0.45 20.32 + 0.22 19.25 + 0.70
`«SL
`but the saturated fatty acid decreased with extracting
`439 £0.21 4374012 4324 116
`18:2
`pressure.
`20:4=2.10 £0.12 2144002 2144002
`Extraction of tomato seed oil resulted in a change in
`20:5
`11.14+0.63 11424021 11.50 +031
`the fatty acid profile (26), because of the difference in
`12.07 £ 0.91 1927+ 0.80 20.97 + 0.52
`the fatty acid solubility in SC-CO,. Soluble components
`can be extracted first in SC-CO,. Linolenic acid (LNA,
`18:3) and LA decreased but OA, SA and PA increased.
`Fatty acids with shorter chain length and a higher
`degree of double bondedness have higher solubilities
`(22). Snyder et al. (29) reported the same behavior for
`soybeanoil extraction.
`In plant oil extraction such as with wheat germ oil
`(28) and rice bran oil (27), the fatty acid profiles were
`not significantly affected by the operating conditions.
`
`donic. acid (AA, 20:4), linoleic acid (LA, 18:2) or
`stearic acid (SA, 18:0). Palmitic acid (PA, 16:0), palmi-
`toleic acid (POA, 16:1) and oleic acid (OA, 18:1)
`decreased with the extracting pressure. The fatty acid
`concentrations of SA, OA and AA in the extracted
`lipids were lower than those in the original TG, whereas
`PA, POA and EPA were higher than in the original TG.
`LA was found to be almost the sameas in the original
`
`22:6
`
`The solubility depends on the vapor pressure and the
`density of the solvent. The extraction of free fatty acids
`or ethylesters was affected by the singular property of
`each fatty acid. The results of TG extraction were dif-
`ferent from the extraction of free fatty acids or ethyl
`
`SC-CO,extraction has not proved suitable for good
`
`3:3 Effect of Extracting Temperature and
`Pressure on Fatty Acid Profiles of the
`Residual Lipid
`After the SC-CO,extraction, the spent FD-sample
`was extracted with solvent and fractionated thorough a
`silica gel column to obtain the TG fraction of the resid-
`ual lipids not extracted with SC-CO,. Its fatty acid pro-
`file was analyzed and compared with those ofthe origi-
`
`
`
`AKER EXHIBIT 2018 PAGE 0004
`
`

`

`Extraction of Salmon Roe Lipids with SCF-CO,
`
`shown in Table 2. Naturally the DHA concentration
`decreased with the extracting pressure. At 40°C the
`DHA concentration decreased significantly from 24.5
`to 31.4MPa. At 60°C it decreased significantly from
`17.7 to 24.5MPa. At 80°C it decreased significantly
`with the extracting pressure (p<0.01), DHA concentra-
`tions under all the extracting conditions were lower
`than that of the original TG.
`The extracting conditions did not affect the extraction
`of SA nor LA. EPA and AA decreased with the extract-
`ing pressure. PA, POA and OA increased with the
`extracting pressure. The concentrations of POA and OA
`of the extracted lipids were higher than those of the
`original TG, whereas in PA, AA and EPA, they were
`lower than those of the original TG. In LA they were
`almost the same as in the original TG. These results
`support the fatty acid profiles of the extracted lipid
`
`Table 2 Fatty Acid Compositions of Residual Lipids (TG
`
`fraction).
`
`.
`Pressure (MPa)
`(Cy Fatty acid
`17.7
`24.5
`31.4
`
`16:0
`9.59 + 0.43
`11.01 £0.23 10.36 + 0.32
`16:1
`7.254054 7.964029 9.074 0.13
`18:0
`3.014009 2954014 2.63 + 0.44
`18:1
`27.87 + 0.62 27.11 £0.11 27.36 + 0.23
`18:2
`3.862014 4104021 4374 0.13
`20:4
`129+0.03
`13940.03
`1.21 + 0.02
`20:5
`8.56 £0.13 10.05 + 0.24 10.26 + 0.17
`22:6
`17.21 £1.12 18.02 £1.98 10.25 + 1.82
`
`16:0
`16:1
`
`18:0
`18:1
`18:2
`
`20:4
`20:5
`22:6
`
`16:0
`16:1
`18:0
`18:1
`18:2
`20:4
`
`20:5
`
`10.20+0.22
`6.274 0.36
`
`84940.21
`7.374053
`
`8.97 + 0.22
`7.79 + 0.41
`
`3.01 + 0.27
`2.904£0.21
`2.98£041
`21.80 + 0.49 22.35 + 0.41 22.26 + 0.62
`3.674012 3474009 3.61 40.12
`
`1.59 + 0.02
`161 40.02
`1.70 £0.03
`10.26+0.15 6264017 5.81 +022
`19.77+0.39
`8.88 +1.59
`7.31 + 1.38
`
`11444017 9104028 83724011
`7124023 637+£031
`8.55 + 0.28
`3154015 2.674019 2.35 + 0.47
`22.60 + 0.39 22.21 £0.51 25.81 + 0.69
`407 £0.11
`3.3940.20
`3.72 + 0.19
`1.9140.08
`1444£0.07
`0.73 + 0.10
`
`11.01 40.71
`
`9.394043
`
`7.24 + 0.55
`
`(Chapter 3:2).
`The fatty acid profiles of the PL fraction for all the
`extracting conditions were almost the same as that of
`the original PL. The results suggest that no PL was
`extracted with SC-CO,.
`
`3:4 Effect of Extracting Temperature and
`Pressure on Astaxanthin Behavior
`The AX concentrations of the extracted lipids and the
`residual lipids are shown in Fig. 2. All the AX concen-
`trations in the extracted lipids were lower than those in
`all the residual lipids and total lipids. The AX extract
`yield of the extracted lipids is shown in Fig. 3. The AX
`extract yield increased with pressure and temperature.
`The maximum yield was about 30%. Less than 30% of
`AX was extracted with SC-CO, and more than 70% of
`AX was found to have remained in the spent FD-sample
`after the extraction.
`Zosel (1) suggested that SC-CO, is suitable for the
`isolation of thermally labile substances because of the
`low critical temperature. In this work, the authors tried
`high temperature conditions. To obtain information on
`the effect of extracting temperature on the decomposi-
`tion of AX during the extraction the authors compared
`the AX concentration in the TL with those of both the
`extracted lipids and the residual lipids. The results are
`shown in Table 3. The loss of AX was independent of
`the extracting conditions. The loss of AX during extrac-
`tion was less than 10% for all conditions in this work.
`Almost no AX had decomposed in the high pressure
`and high temperature conditions. Miki (30) showed AX
`is stable at 100°C at atmospheric pressure, whereas
`Yamaguchi et al. (15) reported that AX in Antarctic
`krill is decomposed by high temperature and high pres-
`sure. In particular, all or most of the AX had disap-
`peared after the extraction at 80°C. Our results show
`that most of the AX had remained after extraction with
`SC-CO,. It was resistant to the high pressure and tem-
`perature involved in the process,
`
`Acknowledgments
`
`This work was performed for “The Japanese
`Research and Development Association for New Func-
`tional Foods” and presented at the 41st JOCS annual
`meeting (Musashino, Japan, Sep. 2002).
`
`
`
`
`
`AKER EXHIBIT 2018 PAGE 0005
`
`

`

`Y. Tanaka and T. Ohkubo
`
`40
`
`30
`
`20
`
`10
`
`
`
`AXconcentration(mg/100g)
`
`Pressure
`
`(MPa)
`2. P. HUBERT and 0.G. VITZTHUM,Fluid Extraction of Hops, 10
`
`Fig. 2 Effects of SC-CO, Extract Conditions on AX delivery to Extracted and Residual Lipids.
`EL: Extracted Lipid, RL: Residual Lipid, TL Total Lipid (extracts by organic solvent)
`Eachresult represents the mean S.D.
`
`Table 3 Effect of Extraction Conditions on Astaxanthin.
`
`Temp.
`(C)
`40
`60
`80
`
`Pressure (MPa)
`31.4
`24.5
`17.7
`98.31 + 8.62
`98.6447.49 102.644 7.11
`95.22 + 9.65
`91.564 8.16
`97.30 + 8.68
`90.45 £9.22
`90.88 £9.99 101.36 + 7.89
`
`Spices, and Tobacco with Supercritical Gases, Angew. Chem.
`Ind. Ed. Engl., Vol. 17, 710-715 (1978).
`3. B. GOPALAN, M. GOTO, A. KODAMAand 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-
`
`31.4
`
`17.7
`
`24.5
`Pressure (MPa)
`Fig. 3 Effects of SC-CO, Extract Conditions on AX
`Yield of Extracted Lipids.
`Symbols are the same as in Fig. 1. Eachresult
`represents the mean+8.D.
`
`References
`
`1. K. ZOSEL, Separation with Supercritical Gases: Practical
`Application, Angew. Chem. Ind. Ed. Engl., Vol. 17, 702-709
`
`
`
`:
`L
`
`
`
`AKER EXHIBIT 2018 PAGE 0006
`
`

`

`Extraction of Salmon Roe Lipids with SCF-CO,
`
`. G.R. ZIEGLER, and Y.-J. LIAW Deodorization and Deacidifi-
`cation of Edibles Oils with Dense Dioxide, J. Am. Oil Chem.
`Soc., Vol. 70, 947-953 (1993).
`. S. LI, 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).
`M.S. KUK and M.K. DOWD,Supercritical CO, Extraction of
`Rice Bran, J. Am. Oil Chem. Soc., Vol. 75, 623-628 (1998).
`I, PAPAMICHAIL, V. LOULI and K. MAGOULAS,Supercriti-
`cal Fluid Extraction of Celery Seed Oil, J. Supercritical Fluids,
`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. ILLINGWORTHand 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
`
`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).
`VJ. KRUKONIS, J-E. VIVIAN, C.J. BAMBARA, W.B. NILS-
`SON, and R.E. MARTIN, Concentration of Eicosapentaenoic
`Acid by Supercritical Fluid Extraction: A Design Study of a
`Continuous Production Process, in Seafood Biochemistry: Com-
`position and Quality,
`(G. FLICK and R.E. MAETIN, ed.),
`Technometric Publishing Co., Lancaster PA, pp.169-179 (1992),
`H. SUZUKI, J. CHEN, T. ADSCHTRI, H. INOMATAand K.
`ARAJIPurification of EPA Ethyl Esters from Fish Oil with a
`Supercritical CO, Temperature Gradient Rectification Column,
`
`19.
`
`20.
`
`21.
`
`22.
`
`23.
`
`24,
`
`25,
`
`26.
`
`27.
`
`28.
`
`29,
`
`J. Jpn Oil Chem, Soc., Vol. 46, 331-340 (1997).
`W.B. NILSSON, E.J. GAUGLITZ Jr., JAK. HUDSO, V.F.
`STOUTand J. SPINELLI, Fractionation of Menhaden Oil Ethyl!
`Esters Using Supercritical Fluid CO,, J. Am. Oil Chem. Soc.,
`Vol. 65, 109-117 (1988).
`J.-N. JAUBERT, P. BORG, L. CONIGLIO and D. BARTH,
`Phase Equilibria Measurements and Modeling of EPA and DHA
`Ethyl Esters in Supercritical Carbon Dioxide, J. Supercritical
`Fluids, Vol. 20, 145-155 (2001).
`Z.-R.YU, B. SINGH and S.8.H. RIZVI, Solubilities of Fatty
`Acid, Fatty Acid Esters, Triglycerides, and Fats and Oils in
`Supercritical Carbon Dioxide, J. Supercritical Fluids, Vol. 7, 51-
`59 (1994).
`J. FOLCH, M. LEE, and S.G.H. SLOANE, A Simple Method
`for the Isolation and Purification of Total Lipids from Animal
`Tissues, J. Biol. Chem., Vol. 226, 497-509 (1957).
`C.R. BHUPESH, M. GOTO and T. HIROSE, Temperature and
`Pressure Effects on Supercritical CO, Extraction of Tomato Seed
`Oil, Int. J. Food Sci. Tech., Vol. 31, 137-141 (1996).
`L. CALVE, M.J. COCERO and J.M. DIEZ, Oxidative Stability
`of Sunflower Oil Extracted with Supercritical Carbon Dioxide,
`J. Am. Oil Chem. Soc., Vol. 71, 1251-1254 (1994),
`F.P. CUPERUS, G. BOSWINKEL, B.G. MUUSE,and J.T.P.
`DERKSEN,Supercritical Carbon Dioxide Extraction of Dimor-
`photheca pluvialis Oil Seeds, J. Am. Oil Chem. Soc., Vol. 73,
`1675-1679 (1996).
`Z. SHEN, M.V. PALMER, S.S.T. TING and R.J. FAIR-
`CLOUGH,Pilot Scale Extraction of Rice Bran Oil with Dense
`Carbon Dioxide, J. Agric. Food Chem., Vol. 44, 3033-3039
`(1996).
`A.M. GOMEZ and E. MARTINEZ de la OSSA, Quality of
`Wheat Germ Oil Extracted by Liquid and Supercritical Carbon
`Dioxide, J. Am. Oil Chem. Soc., Vol. 77, 969-974 (2000).
`JM. SYNDER,J.P. FRIEDRICH and D.D. CHRISTINSON,
`Effect of Moisture and Partical Size on the Extractability of Oils
`from Seeds with Supercritical CO,, J. Am. Oil Chem. Soc., Vol.
`61, 1851-1856 (1984),
`W. MIKI, N. TORIU, Y. KONDO, K. MURAKAMI, S. YAM-
`AGUCHI, 8. KONOSU, M. SATAKE and T. FUJITA, The Sta-
`bility of Carotenoid Pigmants in the Antarctic Krill, Euphausia
`superba, Bull. Japan. Soc. Sci. Fish., Vol. 49, 1417-1420 (1983).
`
`
`
`
`
`AKER EXHIBIT 2018 PAGE 0007
`
`