`
`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
`
`Yukilu'sa TANAKA* and Takeshi OHKUBo
`
`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. The lipid yield and fatty 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 17.7MPa the extracts were affected by the extractingtemperature.
`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
`salmon roe. 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 was less than 10% of
`
`the total involved in the process.
`
`Key words: supercritical carbon dioxide extraction, salmon roe, triacylglyceride,
`
`phospholipid, astaxanthin
`
`1
`
`Introduction
`
`Supercritical carbon dioxide (SC-C02) 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-COZ (5-11). There are
`
`several advantages in using SC—COZ in industrial pro-
`
`related to toxic residual solvent. It is inexpensive and
`
`readily available. The low critical temperature and
`pressure (Tc=3l.1°C, Pc=7.4MPa) can be utilized 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 (l3) and autoimmune diseases (14), etc.
`Some refer to the application of SC—COz extraction
`of marine materials to obtain PUFA. Yamaguchi et al.
`
`duction. C02 has several desirable properties, such as
`
`(15) reported on the extraction of lipids from Antarctic
`
`non-corrosion, non-toxicity, non-flammability and non—
`explosivity. Because C02 is stable chemically, it never
`
`krill. According to their report, only non-polar compo-
`nents such as cholesterol, carotenoid triacylglycerides
`
`reacts with other materials in treatment. Easy separation
`
`and their derivatives were extracted. Phospholipids did
`
`and removal of C02 from products eliminates problems
`
`not appear in the extracted fractions.
`
`*Correspondence to: Yukihisa TANAKA, Tsukuba Research Laboratory NOF CORPORATION, 5-10 Tokodai, Tsukuba, Ibaraki, 300-2635,
`
`
`
`
`
`AKER EXHIBIT 2018 PAGE 0001
`
`
`
`Y. Tanaka and T Ohkubo
`
`Cheung er al. (16) tried extracting lipids from brown
`
`2-3 Analysis
`
`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
`
`In this work, the lipids extracted with SC—C02 are
`
`referred to as the extracted lipids. After the SC-COz
`
`extraction, the lipids retained in the spent FD-sample
`
`that obtained by solvent extraction.
`Because of the high solubility in SC—C02 with EPA
`
`and DHA ethyl esters as the extracting substrates is the
`
`were extracted by Folch’s method. This is referred to as
`
`the residual lipids, in this work. The lipids content of
`the extracted lipids, residual lipids and total lipids were
`
`usual approach to obtain high—purified eicosapentaenoic
`acid (2025, EPA) and docosahexaenoic acid (22:6,
`
`analyzed by means of silica gel thin layer chromatogra—
`
`phy (TLC, plate 5721, Merck, Darmstadt, Germany)
`
`DHA). Several researchers (17-19) have proposed con~
`
`with hexane-diethyl ether-acetic acid (802011 v/v/v) or
`
`tinuous processes. Mishra et al. (20) and Jaubert et al.
`
`chloroform—methanol—water (65 :25 :4 v/v/v).
`
`(21) reported phase behavior with ethyl ester in SC-
`C02. Yu et al. (22) compared the solubilities of fatty
`
`The residual lipids and total lipids (150mg) were run
`through the column (20 mm id. X 200 mm height)
`
`acids, esters, triacylglycerides, fats and oils in SC-CO2.
`
`The authors report herein on extracting lipids fi'om
`
`with silica gel 60 (mesh 70-230, Merck) to fractionate
`the TG and FL. The TG and PL were eluted with
`
`salmon roe with SC—COz to clarify the extraction condi-
`
`300mL of chloroform and 200mL of methanol, respec—
`
`tions and behavior of the lipids in SC-COZ. The extract—
`
`ed lipids and residual lipids remaining in the FD-sam—
`
`ple alter the extraction were characterized (yield, lipid
`contents, fatty acid profiles, etc.).
`
`2 Experimental
`
`21 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 (23) is defined as the total lipid (TL). It con-
`
`tively. The TG and PL fractionated from the total lipids
`are referred to as the original TG and original PL.
`
`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
`
`(30M >< 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 SOmL/min, and hydrogen and air were sup—
`
`plied to the FID. The fatty acids were identified by
`
`tains triacylglycerides (TG), phospholipids (PL) and
`
`comparison of retention times with lipid standards
`
`their derivatives (diacylglycerides, monoacylglycerides
`
`and lysophospholipids, etc.).
`
`2-2 SCF-COZ 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 lOmL. The
`equipment used for the work consisted of a high-pres-
`
`sure liquid chromatograph system (pump, JASCO PU-
`
`15 86, 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—COZ was introduced into the vessel at a
`
`(Sigma, Saint Louis, MA).
`The concentration of astaxanthin (AX), the function-
`
`al red pigment contained in the salmon roe was estimat—
`
`ed at 488nm in benzene by using an absorbance coefi‘i—
`cient, A 1%, of 1,990.
`
`3 Results and Discussion
`
`3-1 Effect of Extracting Temperature and
`Pressure on the Lipid Yield
`The SC-COZ extractions were treated for 6 hours at
`
`24.5MPa and 60°C to determine the C02 flow rate and
`
`the extracting time (data not shown). The C02 flow rate
`
`and extracting time were determined as 3 mL/min and 2
`
`hours, respectively. The FD-sample was extracted with
`
`.n
`““3:1
`
`.mmmmpg...,.._
`
`
`Wanna,,.
`
`AKER EXHIBIT 2018 PAGE 0002
`
`
`
`,1..§
`flit?
`
`"""llllEllIW'ls"
`il
`,.
`
`‘ M
`
`Extraction of Salmon Roe Lipids with SCF-CO;
`
`temperature conditions, in this work, only the results
`
`from the pressure range 17.7MPa to 31.4MPa are 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—COZ 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 afi‘ected 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 24.5MPa the lipid yield at
`80°C reached its highest point. At 31.4MPa the lipid
`yield increased with the extracting temperature
`(p<0.05). Bhupsesh et al. (26) reported that the extract-
`
`80
`
`Yield(%)
`
`AO
`
`NG
`
`17 .7
`
`24.5
`Pressure (MPa)
`
`31.4
`
`Fig. 1 Effects of SC—COz Extract Conditions on Yield of
`
`Extracted Lipids.
`SC-COZ flow was 3mL/min, Extract time was 2
`
`hours. Extractions were treated at 40 (I), 60(0),
`and 80 (A)°C. Each result represents the meani
`SD.
`
`3 - 2 Effect of Extracting Temperature and
`Pressure on Fatty Acid Profiles of the
`
`ing conditions have an effect on the lipid yield in toma-
`
`Extracted Lipid
`
`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
`a1. (28) proposed that the change was due to variations
`
`in the physical properties of C02, particularly the densi-
`
`ty which is closely related to the solvent capacity.
`The effect of the extracting pressure was that at con-
`
`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
`DHA concentration was observed at 80°C. At 60 and
`
`40°C a DHA concentration of 17.7MPa was significant—
`
`stant temperature the lipid yield increased with the
`extracting pressure. While the effect of the extracting
`
`ly lower than those of 24.5 and 31.4MPa (p<0.01). At
`17.7MPa the DHA concentration increased significantly
`
`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
`
`with extracting temperature (p<0.01). At 24.5MPa the
`DHA concentration at 80°C was significantly lower
`than those at 40 and 60°C (p<0.05).
`The DHA concentrations at 80°C/17.7MPa and 60°C
`
`increasing pressure. These results coincide with several
`
`/l7.7MPa were lower than that of the original TG
`
`previous reports. The results suggested close participa—
`tion of the C02 density in the extraction of salmon roe.
`
`(19.75%). This result suggested that the solubility of
`TG containing DHA changed drastically with the
`
`The sum of the extracted lipid and residual lipid yield
`
`resulted in the same yield as the total lipid.
`
`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.
`
`AKER EXHIBIT 2018 PAGE 0003
`
`
`
`Y. Tanaka and T. Ohkubo
`
`Table 1 Fatty Acid Compositions of Extracted Lipids.
`
`Pressure (MPa)
`24.5
`17.7
`31.4
`12.29 i 0.34 11.93 i 0.10 11.93 i 0.57
`8.25 i 0.84
`7.42 i 0.09
`7.46 i 0.19
`4.00 i- 0.06
`3.62 i 0.25
`4.06 i- 0.56
`
`.
`(°C) Fatty and
`16:0
`16:1
`1820
`
`18:1
`18:2
`20:4
`20:5
`226
`
`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
`
`22:6
`
`18.70 i 0.56 19.31 i 0.06 19.37 i 0.58
`4.39 3: 0.16
`4.16 i 0.15
`4.21 :1: 0.08
`2.11 i 0.03
`2.12 3: 0.01
`2.08 :1: 0.02
`11.06 i 0.14 11.16 i 0.07 11.38 i- 0.38
`19.72 i 1.42 21.54 i 0.73 21.46 3: 0.52
`
`13.37 i 0.72 12.41 i 0.18 12.02 i 0.13
`9.13 :t 0.40
`7.61 i 0.33
`7.45 :t 0.36
`3.08 i- 0.22
`3.61 i 0.35
`3.41 :l: 0.37
`21.01 i 0.84 20.45 i 0.11
`19.71 :t 0.75
`4.45 i- 005
`4.23 i 0.07
`4.07 i 0.05
`2.09 i 0.04
`2.18 i- 0.10
`2.15 : 0.02
`10.91 i 0.30 11.47 i 0.27 11.55 i 0.37
`17.32 i 1.21 21.28 i 0.43 21.84 i- 0.48
`
`1702 i 0.94 12.76 :L- 0.15 11.92 i 0.22
`10.40 i- 0.66
`8.57 i 0.29
`7.66 i 0.35
`342 :1: 0.23
`2.87 i 0.08
`3.46 :r 0.52
`
`2153 :1: 0.45 20.32 i 0.22 19.25 i 0.70
`4.39 i 0.21
`4.37 3: 0.12
`4.32 3: 1.16
`2.10 i 0.12
`2.14 i 0.02
`2.14 :1: 0.02
`ll.l4i0.63 11.42i 0.21 11.50i0.31
`12.07 'J: 0.91 19.27 i 0.80 20.97 :t: 0.52
`
`
`
`bility of TG. The vapor pressure of TG is due to the
`
`fatty acid combination. The combination of the other
`
`two fatty acids in TG affects the extract efficiency of
`
`each fatty acid. Therefore not only the carbon number
`
`but also the double bonds depend on the solubility. In
`general a low molecular weight TG is extracted easily
`
`at low pressure and a high molecular weight TG is
`
`extracted easily at high pressure. Because the fatty acid
`profiles result in the combination of three fatty acids
`
`our result does not reflect any specific fatty acid proper-
`ty.
`In the case of marine oils, as the number of fatty
`
`acids is large, combination is innumerable and com—
`
`plex. All the fatty acids were separated to extract or
`
`remain due to the difference in combinations of fatty
`acids in TG. Furthermore, analysis of fatty acid behav-
`
`ior in SC~C02 from the point of View of the TG molec—
`
`ular unit might be necessary. A combination of thermal
`
`gradient fractionation could be applied to separate TG
`
`more strictly.
`
`Cheung et al. (16) reported on the extraction of lipids
`from brown seaweed. They showed the oil yield and
`
`extracting conditions affected the DHA concentration
`
`of the extracted lipid. The DHA concentration increased
`but the saturated fatty acid decreased with extracting
`pressure.
`
`Extraction of tomato seed oil resulted in a change in
`
`the fatty acid profile (26), because of the difference in
`
`the fatty acid solubility in SC-COz. Soluble components
`
`can be extracted first in SC-COz. Linolenic acid (LNA,
`18:3) and LA decreased but 0A, SA and PA increased.
`
`donic. acid (AA, 2024), linoleic acid (LA, 18:2) or
`
`Fatty acids with shorter chain length and a higher
`
`stearic acid (SA, 18:0). Palmitic acid (PA, 16:0), palmi-
`toleic acid (POA, 16:1) and oleic acid (0A, 18:1)
`
`degree of double bondedness have higher solubilities
`
`(22). Snyder et al. (29) reported the same behavior for
`
`decreased with the extracting pressure. The fatty acid
`concentrations of SA, OA and AA in the extracted
`
`soybean oil extraction.
`In plant oil extraction such as with wheat germ oil
`
`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 same as in the original
`
`(28) and rice bran oil (27), the fatty acid profiles were
`
`not significantly affected by the operating conditions.
`
`3 -3 Effect of Extracting Temperature and
`
`The solubility depends on the vapor pressure and the
`
`Pressure on Fatty Acid Profiles of the
`
`density of the solvent. The extraction of free fatty acids
`or ethylesters was affected by the singular property of
`
`Residual Lipid
`
`After the SC-COz extraction, the spent FD-sample
`
`each fatty acid. The results of TG extraction were dif-
`
`was extracted with solvent and fractionated thorough a
`
`ferent from the extraction of free fatty acids or ethyl
`
`silica gel column to obtain the TG fraction of the resid—
`
`SC-COZ extraction has not proved suitable for good
`
`file was analyzed and compared with those of the origi-
`
`ual lipids not extracted with SC-COZ. Its fatty acid pro-
`
`
`
`
`
`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-
`
`(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—COZ.
`
`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-
`
`ing pressure. PA, FDA and 0A increased with the
`
`trations in the extracted lipids were lower than those in
`
`extracting pressure. The concentrations of FDA and 0A
`
`all the residual lipids and total lipids. The AX extract
`
`of the extracted lipids were higher than those of the
`
`yield of the extracted lipids is shown in Fig. 3. The AX
`
`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
`
`extract yield increased with pressure and temperature.
`
`The maximum yield was about 30%. Less than 30% of
`AX was extracted with SC-C02 and more than 70% of
`
`support the fatty acid profiles of the extracted lipid
`
`Table 2 Fatty Acid Compositions of Residual Lipids (TG
`
`fraction).
`
`_
`Pressure (MPa)
`
`(*0) Fatty ““1 T T T
`16:0
`9.59 i 0.43
`11.01 i 0.23 10.36 i 0.32
`16:1
`7.25 i 0.54
`7.96 i: 0.29
`9.07 i- 0.13
`18:0
`301 i 0.09
`2.95 i 0.14
`2.63 i 0.44
`18:1
`27.87 i 0.62 27.11 i 0.11 27.36 i: 0.23
`18:2
`3.86 i 0.14
`4.10 :1: 0.21
`4.37 :1: 0.13
`20:4
`1.29 :t 0.03
`1.39 i 0.03
`1.21 i 0.02
`20:5
`8.56 i 0.13
`10.05 :1: 0.24 10.26 i 0.17
`22:6
`17.21 :1: 1.12 18.02 :1: 1.98 10.25 i 1.82
`
`16:0
`16:1
`
`18:0
`18:1
`1822
`
`20:4
`20:5
`22:6
`
`16:0
`16:1
`18:0
`
`18:1
`18:2
`20:4
`
`20:5
`
`10.20 i 0.22
`6.27 :1: 0.36
`
`8.49 i 0.21
`7.37 i 0.53
`
`8.97 i 0.22
`7.79 i 0.41
`
`3.01 i 0.27
`2.90 i 0.21
`2.98 i 0.41
`21.80 :1: 0.49 22.35 :1: 0.41 22.26 i 0.62
`3.67 i 0.12
`3.47 :1: 0.09
`3.61 i 0.12
`
`1.70 i 0.03
`10.26 i 0.15
`19.77 i 0.39
`
`1.61 :1: 0.02
`6.26 i 0.17
`8.88 i 1.59
`
`1.59 i 0.02
`5.81 :1: 0.22
`7.31 i 1.38
`
`11.44 i 0.17
`7.12 i 0.23
`3.15 i: 0.15
`
`9.10 i 0.28
`6.37 :1: 0.31
`2.67 i 0.19
`
`8.37 i 0.11
`8.55 i 0.28
`2.35 i 0.47
`
`22.60 i 0.39 22.21 :1: 0.51 25.81 i 0.69
`4.07 :1: 0.11
`3.39 d: 0.20
`3.72 :1: 0.19
`1.91 1‘: 0.08
`1.44 i 0.07
`0.73 :1: 0.10
`
`11.01 :1: 0.71
`
`9.39 i 0.43
`
`7.24 i 0.55
`
`AX was found to have remained in the spent FD-sample
`afier the extraction.
`
`Zosel (1) suggested that SC-C02 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—COz. 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)
`
`
`
`Fig. 2 Effects of SC—COZ Extract Conditions on AX delivery to Extracted and Residual Lipids.
`EL: Extracted Lipid, RL: Residual Lipid, TL Total Lipid (extracts by organic solvent)
`Each result represents the meani SD.
`
`Table 3 Effect of Extraction Conditions on Astaxanthin.
`
`Temp.
`
`(°C)
`40
`60
`80
`
`Pressure (MPa)
`
`31.4
`24.5
`17.7
`98.31 i 8.62
`98.64 i‘ 7.49 102.64 i 7.11
`95.22 i 9.65
`91.56 i 8.16
`97.30 :l: 8.68
`90.45 i 9.22
`90.88 i 9.99 101.36 i 7.89
`
`2. P. HUBERT and O.G. VITZTHUM, Fluid Extraction of Hops, 10
`
`V
`17.7
`
`24.5
`Pressure (MPa)
`Fig. 3 Effects of SC-C02 Extract Conditions on AX
`Yield of Extracted Lipids.
`Symbols are the same as in Fig. 1. Each result
`
`31.4
`
`represents the meani S.D.
`
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