Extraction of Lipid and Cholesterol from Fish Muscle with
`Supercritical Fluids
`
`INGIBJORG HARDARDOTTIR and JOHN E. KINSELLA
`
`Supercritical fluid extraction (SFE) was explored as a method for
`removing lipids and cholesterol from fish muscle. Selected conditions
`of extraction pressure, time, temperature and sample size were eval(cid:173)
`uated for effective removal of lipid and cholesterol. Increasing carbon
`dioxide (C02) pressure from 2000 to 5000 psig and the temperature
`from 40° to S0°C had little effect on the extent of lipid removal.
`Increasing the extraction time from 3 hr to 9 hr increased lipid re(cid:173)
`moval. Ethanol as an entrainer significantly enhanced lipid extraction
`at all pressures used. Supercritical carbon dioxide (SC02) and SC02
`plus ethanol removed 78% and 97% of the lipids and 97% and 99%
`of the cholesterol, respectively, from trout muscle. The solubility of
`the muscle proteins decreased following extraction.
`
`INTRODUCTION
`
`THE MARKET FOR PROTEINS as functional ingredients for
`existing foods and fabricated foods is expanding. Successful
`development of methods for the production of functional pro(cid:173)
`teins would facilitate greater use of the world's fishery re(cid:173)
`sources for human use. However, the oxidation of the highly
`unsaturated lipid components present a major problem with
`fish muscle proteins (Mackie, 1983). Organic solvent extrac(cid:173)
`tion while effective for lipid removal, results in protein den(cid:173)
`aturation and loss of functional properties (Pariser et al., 1978).
`However, the feasibility of reducing or removing lipids from
`fish muscle proteins with the recently developed techniques
`employing supercritical fluid extraction (SFE) with carbon
`dioxide (C02) warrants evaluation. The studies of Stahl et al.
`(1980, 1984), Friedrich and Pryde (1984), Snyder et al. (1984),
`Christianson et al. (1984), Bulley et al. (1984), Taniguchi et
`al. (1985) and Lee et al. (1986) showed that SFE can be used
`to extract oil from oilseeds. Stahl et al. (1984), Duwe et al.
`(1986) and Eldridge et al. (1986) reported that SFE had min(cid:173)
`imal effects on the properties of the proteins. SFE was effective
`in fractionating fish oil esters (Eisenbach, 1984; Nilsson, 1988).
`Supercritical fluids exhibit physicochemical properties in(cid:173)
`termediate between those of liquids and gases which enhance
`their efficacy as solvents. The relatively high gas density gives
`good solvent power, while the relatively low viscosity and high
`diffusivity provide appreciably higher gas permeability into the
`solute matrix. These properties impart higher rates of mass
`transfer of solutes into a supercritical fluid than into a liquid
`(Rizvi et al., 1986). In addition the inclusion of a relatively
`small amount of entrainer in the supercritical fluid (SCF) (e.g.,
`ethanol) can improve both the solubility and selectivity of the
`solvent (Brunner and Peter, 1982). C02 is a useful solvent in
`supercritical extractions of food components because it is non(cid:173)
`toxic, nonflammable, inexpensive and readily available. It has
`relatively low critical temperature (31.1°C) and pressure (1070
`psig, 7.38 MPa) (Rizvi et al., 1986).
`Although the cholesterol content of fish muscle is only 35-
`70 mg/lOOg (Anon., 1987; Nettleton, 1985), there is great
`interest in cholesterol reduction of muscle foods for nutritional
`and marketing purposes. An attempt has been made to lower
`
`Authors Hardardottir and Kinsella are with the Institute of Food
`Science, Cornell Univ., Ithaca, NY 14853.
`
`1656-JOURNAL OF FOOD SCIENCE-Volume 53, No. 6, 1988
`
`the cholesterol level of butter oil by SFE (Shishikura et al.,
`1986) but the efficacy of SFE in extracting cholesterol from
`muscle has not been reported, hence the extractability of cho(cid:173)
`lesterol from fish muscle was studied.
`The objectives of this study were to explore the use of su(cid:173)
`percritical carbon dioxide and carbon dioxide/ethanol for the
`lipid and cholesterol extraction of fish muscle and to assess
`the effects of the extraction on the solubility of the muscle
`proteins.
`
`MATERIALS & METHODS
`
`Samples
`Samples of post-rigor muscle form rainbow trout (Sa/mo gairdneri)
`were cut into small pieces (1 x 1 cm), frozen in liquid nitrogen and
`ground into particles (1-2 mm diameter) to increase the surface area.
`After SFE, the samples were freeze-dried to <2% water (Labconco
`Corp. Kansas City, MS) and stored in airtight bottles under nitrogen
`at 4°C for subsequent analyses.
`
`Extraction
`
`A laboratory scale supercritical extraction system, Model X-10
`(Milton Roy, Rivera Beach, FL) was used to extract the lipids from
`the trout muscle. The C02 (a liquid at - 5°C) was pumped at a flow
`rate of 460 mL/hr and pressurized above its critical pressure (1070
`psig) in a 50 mL extraction vessel as it emerged from the metering
`pump. The temperature inside the vessel was maintained at a selected
`temperature above the critical temperature (31.1°C). The SC02 was
`passed through the muscle particles at 40-50°C and 2000-5000 psig
`(13.8-34.5 MPa). The discharged C0 2 containing the extracted lipids
`was pumped into a separator vessel where the C02 was deprcssurized
`the dissolved lipids separated from the C02 •
`For determination of the effects of extraction on the composition
`and solubility, proteins were prepared using the following conditions:
`SC02 extraction. 460 mL C02/hr were pumped through muscle
`samples (5 g) for 9 hr (total C02:3900 g).
`SCO:Jethanol extraction. 460 mL C02 + 60 mL ethanol/hr were
`pumped through (10 g) muscle samples for 6 hr (total C02:2600 g;
`total ethanol: 280 g, i.e., about 10% by weight). Extractions were
`performed at 4000 psig and 40°C.
`The extracted protein preparations from each treatment were then
`analyzed.
`
`Composition
`
`The efficiency of the lipid extraction of the fish muscle was mea(cid:173)
`sured by the residual lipids in the SC02 extracted proteins using chlo(cid:173)
`roform/methanol extraction as in the method of Bligh and Dyer (1959).
`The total phospholipid content of the lipids in the protein preparations
`were estimated by the method of Stewart (1980). The cholesterol
`content of the lipids in the muscle proteins was measured by the
`method of Rudel and Morris (1973). The fatty acid composition of
`the lipids in the muscle proteins was quantitated by gas chromatog(cid:173)
`raphy using the methods of Swanson et al. (1987).
`The protein content of the fish protein preparations was determined
`by a micro-Kjeldahl procedure (% nitrogen x 6.25). The moisture
`content of the extracted fish proteins was determined after drying to
`constant weight at 110°C for 18 hr.
`.
`Solubility of the protein was analyzed by the procedure of Morr et
`al. (1985). The composition of soluble muscle proteins following SFE
`was determined by electrophoresis using an SE-200 miniature slab gel
`electrophoresis unit (Hoefer Scientific Instruments, San Fransisco,
`
`RIMFROST EXHIBIT 1164 Page 0001
`
`

`

`f===l
`
`~
`.SP
`~
`i:'
`-0
`"'
`8
`'§
`g
`Ji
`~
`1
`
`·~
`
`l ~c l
`
`50'C
`
`0
`
`.E
`"' ·~
`~
`"' 0
`'§
`'O g
`"£
`"'
`·o.
`'O
`:J
`
`5000psig
`
`4000psig
`
`0 11---~~-~-~--r----.----,
`4000
`5000
`2000
`3000
`
`2-l--~~~~r---~~~--,,-~~~--,.~~~~--,
`12
`
`0
`
`Pressure (psig)
`
`Fig. 1-Effect of extraction pressure and temperature on the
`amount of lipids remaining in the protein preparations after SC02
`extraction of TOg of muscle for 6 hr at 2000 to 5000 psig and 40
`and 50°C.
`
`Time (hr)
`
`Fig. 2-Effect of extraction time on the amount of lipids re(cid:173)
`maining in the protein preparations after SC02 extraction of 5g
`of muscle at 4000 and 5000 psig and 40°C.
`
`CA) and standard electrophoresis procedures (Laemmli, 1970). The
`gels were scanned by a densitometer (7003, E-C Apparatus Corp. St.
`Petersburg, FL) and the curves integrated and printed out by a Hewlett
`Packard 3390 A Integrator (Hewlett Packard, Avondale, PA).
`
`Statistical analysis
`
`The supercritical extractions were done in duplicate under each set
`of conditions and the mean of the two values are reported. For each
`graph showing the extent of lipid removal the data were pooled and
`the error mean square (EMS), calculated from the One Way Analysis
`of Variance (ANOVA), was used to calculate 95% confidence inter(cid:173)
`vals around the mean. ·
`
`RESULTS & DISCUSSION
`
`Extraction
`There was no significant difference in the amount of total
`lipids removed from the fish muscle by extraction with SC02
`at pressures between 2000 and 5000 psig (Fig. 1). The pres(cid:173)
`sures tested were limited by the critical pressure of carbon
`dioxide at 1070 psig, and the maximum working pressure of
`the extraction unit, 5000 psig. Extraction at 50°C removed
`slightly more lipids from the trout muscle than extraction at
`40°C (Fig. 1). Increasing the extraction time from 3 hr to 9 hr
`increased the lipid removal from the muscle (Fig. 2).The sol(cid:173)
`vent power of SC02 for organic substances is highly dependent
`on its pressure and temperature. The solvent power increases
`with temperature at given density (Brogle, 1982), but at lower
`pressures (below 6000 psig) the increased solubility effect due
`to temperature may be counteracted by the decrease in density
`and related decrease in solute holding power (Friedrich and
`Pryde, 1984). The amount of lipids removed from the muscle
`was almost constant regardless of pressures and temperatures
`examined. This is in accord with the findings of Yamaguchi
`et al. (1986) for SC02 extraction of lipids from antarctic krill.
`The inclusion of ethanol as an entrainer increased the lipid
`removal from the fish muscle (Fig 3). Extractions at 4000 psig
`using SC02 with and without ethanol resulted in muscle protein
`preparations containing 0.43 and 5.86g lipids/lOOg dry weight,
`respectively. Varying extraction pressures, times, temperature,
`and sample size did not significantly affect lipid removal by
`extraction with SCO)ethanol. The addition of ethanol to the
`SCF improves the solubility of lipids in the solvent (Brunner
`and Peter, 1982). Ethanol also increases the polarity of the
`solvent and may have helped dissociate phospholipid protein
`
`0%EtOH
`
`4000
`
`5000
`
`0 ;;·~b:+=:=-.----+----~=:==I :=='.o%EtoH ~
`
`2000
`
`3000
`
`Pressure (psig)
`
`Fig. 3-Effect of using an entrainer (10 weight % ethanol) with
`the SC02 on the amount of lipids remaining in the protein prep(cid:173)
`aration after extraction of TOg of muscle for 6 hr at 2000 to 5000
`psig and 40°C.
`
`complexes and thereby, improved extraction. This was con(cid:173)
`sistent with the composition of the extracted lipids.
`
`Composition
`
`The proximate composition and the fatty acid content of
`fresh muscle and protein preparations after extraction with SC02
`with and without ethanol are shown in Table 1 and 2, respec(cid:173)
`tively. Extraction with SC02 arid SC02/ethanol at 4000 psig
`and 40°C removed 78% and 97% of the lipids from the trout
`muscle, respectively. SC02 preferably extracted the triglyc(cid:173)
`erides, whereas SCOzf ethanol extracted both triglycerides and
`phospholipids.
`SC02 extraction of krill yielded oils that were composed
`solely of nonpolar lipids, largely triglycerides without phos(cid:173)
`pholipids (Yamaguchi et al., 1986), because the phospholipids
`were less soluble in the SC02 than the triglycerides. The ad(cid:173)
`dition of ethanol to the SCF also increased the polarity of the
`solvent and may have disrupted the noncovalent bonds between
`the phospholipids and the proteins making the phospholipids
`available for extraction.
`Cholesterol was effectively removed by both solvents, i.e.,
`
`Volume 53, No. 6, 1988-JOURNAL OF FOOD SCIENCE-1657
`RIMFROST EXHIBIT 1164 Page 0002
`
`

`

`LIPID EXTRACTION FROM MUSCLE ...
`
`Table 1- Composition of the original muscle and the lipid extracted mus(cid:173)
`cle protein preparations after extraction with SCOl and SCOi/ethanolb
`Original
`SC02
`SC021ethanol
`fish muscle extracted proteins extracted proteins
`Component
`22.5d
`97.19d
`99.19d
`Protein (%N x 6.25)c
`74.90
`1.71•
`0.68•
`Moisturec
`10.0
`2.18
`0.29
`Lipids'
`Phospholipids1
`3.69
`1.35
`0.04
`0.31
`0.01
`<0.001
`Cholesterol1
`•Conditions: 5g muscle extracted for 9 hr at 4000 psig 40'C.
`b Conditions: 10g muscle extracted for 6 hr at 4000 psig 40'C.
`c Reported as g/100g wet weight
`d Values not corrected for nonprote1n nitrogen
`• Measured after freeze-drying (see text)
`1 Reported as g/100 g dry weight
`
`Table 2-Fatty acid content of trout muscle and protein preparations after
`lipid extraction with SC02 and SCO,/ethanol (g/IDOg dry weight of ex(cid:173)
`tracted protein and % of original content in fresh muscle}
`Fatty
`Fresh SC02 extracted
`SC02/ethanol
`Name
`acid
`muscle
`proteins
`extracted proteins
`0.41" 0.06 (14.6%)b
`0.002 (0.5%)
`14:0 myristic acid
`16:0
`palmitic acid
`1.80
`0.69 (38.3%)
`0.05 (2.8%)
`16:1
`palmitoleic acid
`0.56
`0.06 (10.7%)
`0.005 (0.9%)
`0.36
`0.14 (38.9%)
`0.05 (13.9%)
`18:0
`stearic acid
`18:1
`oleic acid
`1.50
`0.26 (17.3%)
`0.03 (2.0%)
`18:2n-6 linoleic acid
`0.23
`0.03 (13.0%)
`0.004 (1.7%)
`18:3n-3 linolenic acid
`0.94
`0.11 (10.6%)
`0.009 (1.0%)
`0.07
`0.02 (28.6%)
`0.007 (10.0%)
`20:4n-6 arachidonic acid
`0.49
`0.11 (22.4%)
`0.02
`(4.1 %)
`20:5n-3 eicosopentaenoic acid
`22: 1
`erucic acid
`1.17
`0.09 (7.7%)
`0.005 (0.4%)
`0.05
`0.01 (20.0%)
`0.001 (2.0%)
`22 :4n-6 docosatetraenoic acid
`22: 5n-3 docosapentaenoic acid
`0.15
`0.04 (26. 7%)
`0.009 (6.0%)
`1.67
`0.45 (26.9%)
`0.10 (6.0%)
`22:6
`docosahexaenoic acid
`• g/100 g dry muscle protein
`b Percentage of original amount in fresh muscle
`
`97% and 99.5% of the cholesterol was removed, with the lip(cid:173)
`ids, by extraction with SC0 2 and SC02/ethanol, respectively.
`This indicated that the supercritical solvent removed choles(cid:173)
`terol more effectively than the lipids and hence might be of
`value in reducing cholesterol in foods.
`The fresh fish muscle contained 74.9% moisture, but after
`extraction, prior to freeze-drying, the moisture content of the
`protein preparations had decreased to 13.1% and 5.9% for
`SC02 and SCOzfethanol, respectively. Fresh fish muscle and
`protein preparations made by extraction with SC02 and SC02/
`ethanol contained 22.5%, 97.2% and 99.2% protein
`(%N x 6.25), respectively (Table 1). The increase in proteins
`resulted from the removal of both fat and moisture, thus con(cid:173)
`centrating the proteins in the preparations.
`The solubility of muscle proteins was reduced as a result of
`extraction. This suggested that some of the proteins might have
`been denatured during the extraction, probably reflecting in(cid:173)
`creased intermolecular protein-protein interactions. Studies by
`Weder (1980) showed that SC02 had some denaturing effects
`on ribonuclease and Christianson et al. (1984) showed that the
`SFE denatured the proteins· in the extracted corn germ flour.
`Analyses by gel electrophoresis revealed that myosin became
`insoluble during extraction (Fig. 4). The dehydration of the
`fish muscle during SFE might be responsible for the observed
`loss in solubility. Addition of dithiothreitol (DTI, lOmM) to
`the extracted protein preparation solutions did not increase the
`solubility of the proteins, whereas addition of 1 % sodium do(cid:173)
`decyl sulfate (SDS) to the protein increased protein solubility
`to that observed in fresh fish. These results indicated that for(cid:173)
`mation of intermolecular disulfide bonds was not responsible
`for the insolubility of the proteins but that secondary forces,
`i.e., hydrogen bonds and hydrophobic interactions, were re(cid:173)
`sponsible for the insolubilization of the myosin components
`during SFE. The protein preparations showed poor emulsifying
`properties and did not form gels following heating probably
`because of the limited solubility of the myosin component,
`
`048
`
`1.58
`
`1.66
`
`1.72
`
`c
`
`B
`
`0.44
`
`1.56
`
`1.6 1.76
`
`Fig. 4-Densitometric scans of the protein bands obtained fol(cid:173)
`lowing e/ectrophoretic separation of soluble proteins from fresh
`fish and protein preparations made following SC02 extraction:
`(A) Soluble proteins from fresh fish. (30 Mg protein) (0.44:myosin,
`1.56:actin, 1.62:troponin, 1.76:tropomyosin); (BJ Soluble pro(cid:173)
`teins from fresh fish after extraction with SCO:z- (30 Mg proteins)
`(1.58:actin, 1.66:troponin, 1.72:tropomyosin); (C) Soluble pro(cid:173)
`teins from fish after extraction with SC02 stirred with 1 % SDS.
`(15 Mg protein} (0.48:myosin, 1.59:actin, 1.64:troponin,
`1. 77.;tropomyosin).
`
`which is considered the most important of the fish proteins for
`both gel and emulsion formation (Asghar et al., 1985).
`
`CONCLUSION
`SUPERCRITICAL C02 extraction with and without ethanol
`removed 97% and 78% of the lipids and 99.5% and 97% of
`cholesterol from trout muscle, respectively. The solubility of
`the proteins decreased following extraction apparently because
`of myosin aggregation.
`
`REFERENCES
`Anon. 1987. Composition of seafoods. Section 8.15. In "USDA Handbook.
`The Composition of Foods." US Dept. of Agriculture, Washington, DC.
`Asghar, A., Samejima, K., and Yasui, T. 1985. CRC Crit. Rev. Food Sci.
`Nutr. 22:27.
`Bligh, E.G. and Dyer, W.J. 1959. A rapid method of total lipid extraction
`and purification. Can. J. Biochem. Phys. 37(8):911.
`Brogle, H. 1982. C02 as a solvent: Its properties and applications. Chem.
`and Ind. 19:385.
`Brunner, G. And Peter, S. 1982. On the solubility of !flycerides and fatty
`acids in compressed gases in the presence of an entramer. Sep. Sci. Tech(cid:173)
`nol. 17(1):199.
`Bulley, N.R., Fattori, M., Meisen, A., and Moyls, L. 1984. Supercritical
`fluid extraction of ve~etable oilseeds. J. Am. Oil Chem. Soc. 61:1362.
`Christianson, D.D., Fnedrich, J.P., List, G.R., Warner, K., Bagley, E.B.,
`Stringfellow, A.C., and Inf?lett, G.E. 1984. Supercritical fluid extraction
`of dry-mi!led corn germ with carbon dioxide. J. Food Sci. 49:229.
`-Continued on page 1661
`
`1658-JOURNAL OF FOOD SCIENCE-Volume 53, No. 6, 1988
`
`RIMFROST EXHIBIT 1164 Page 0003
`
`

`

`Table 3-Percentage compositions of fatty acids in the food material, dregs and drip obtained by extrusion cooking and its pretreatment of chum salmon
`at the spawning migration stage
`
`14:0
`4.20
`±0.45
`
`16:0
`15.81
`± 0.80
`
`16:1
`6.98
`±0.78
`
`18:0
`3.71
`±0.17
`
`18:1
`20.28
`± 1.13
`
`Sample
`Dregs through
`a screw
`press
`Oils in the drip
`
`8.70
`±0.51
`4.29
`±1.24
`
`10.32
`± 0.29
`15.41
`± 0.28
`
`Dregs + wheat
`flour (10%) through
`an extruder
`•Each value represents the mean ± standard deviation for triplicate analyses.
`
`9.55
`±0.51
`7.04
`±0.72
`
`2.77
`±0.08
`3.53
`±0.03
`
`26.60
`± 0.55
`20.74
`± 0.08
`
`Fatty acids•
`18:3
`0.60
`±0.02
`
`20:0
`0.75
`±0.06
`
`0.38
`±0.04
`0.57
`±0.06
`
`0.61
`±0.02
`0.74
`±0.04
`
`18:2
`1.76
`±0.26
`
`1.58
`±0.13
`3.68
`±2.24
`
`20:1
`7.77
`± 0.72
`
`16.75
`± 0.35
`7.83
`± 1.07
`
`20:4
`0.57
`±0.05
`
`0.18
`±0.01
`0.65
`±0.15
`
`20:5
`8.48
`±0.35
`
`3.13
`±0.14
`7.74
`±0.57
`
`22:1
`5.01
`± 0.62
`
`11.38
`± 0.18
`5.42
`± 0.53
`
`22:6
`19.29
`± 2.08
`
`3.73
`± 0.24
`17.39
`± 0.93
`
`REFERENCES
`Ando, S., Hatano, M., and Zama, K. 1985. A consumption of muscle lipid
`during spawning migration of chum salmon Oncorhynchus keta. Bull.
`Jpn. Soc. Sci. Fish. 51: 1817.
`Hatano, M., Takama, K., Kojima, H., and Zama, K. 1983. Proximate com(cid:173)
`position of fall chum salmon. Bull. Jpn. Soc. Sci. Fish. 49: 213.
`Isobe, S. and Noguchi, A. 1987. High moisture extrusion with twin screw
`extruder-Fate of soy protein during the repetition of extrusion cooking.
`J. Jpn. Soc. Food Sci. Technol. 34: 456.
`Jacquot, R. 1961. Organic constituents of fish and other aquatic animal
`foods. "Fish as Food," Vol. 1. Borgstrom G. (Ed.) Academic Press Inc.,
`New York.
`Kinsella, J. E. 1987. "Seafoods and Fish Oils in Human Health and Dis(cid:173)
`ease," p. 1. Marcel Dekker, Inc., New York.
`Snedecor, G. W. and Cochran, W. G. 1967. "Statistical Methods," 6th ed.
`Iowa State University Press, Ames, IA.
`Suzuki, H., Wada, S., Hayakawa, S., and Tamura, S. 1985. Effects of oxy(cid:173)
`gen absorber and temperature on w3 polyunsaturated fatty acids of sar(cid:173)
`dine oil during storage. J. Food Sci. 50: 358.
`Suzuki, H., Hayakawa, S., Wada, 0., Yamamoto, A., and Ono, T. 1986a.
`Effect of age on the fatty acid composition of the lipids of rat brain. Jpn.
`J. Hyg. 41: 410.
`Suzuki, H., Okazaki, K., Hayakawa, S., Wada, S., and Tamura, S. 1986b.
`Influence of commercial dietary fatty acids on polyunsaturated fatty acids
`of cultured freshwater fish and comparison with those of wild fish of the
`same species. J. ~ic. Food Chem. 34: 58.
`Tsutsumi, C. 1984. 'Methods of Food Analysis," 2nd ed., Jpn. Soc. Food
`Sci. Technol., Korin, Tokyo, Japan.
`Wada, S., Suzuki, H., and Hayakawa, S. 1987. Effect offish oil diet on the
`brain polyunsaturated fatty acids, phospholipids and enzyme activities
`in aged rats. "Polyunsaturated Fatty Acids and Eicosanoids." Am. Oil
`Chem. Soc., Champaign, IL.
`Ms received 12/4/87; revised 6/20/88; accepted 717/88.
`
`Table 4-Effects of extrusion cooking and its pretreatment on w-3 poly(cid:173)
`unsaturated fatty acid content in chum salmon muscle during spawning
`migration
`
`w-3 Polyunsaturated fatty acids•
`20:5
`22:6
`
`Whole muscle
`0.46
`0.18
`Raw material
`0.90
`0.40
`Through a screw press
`0.82
`0.36
`Through an extruder
`•Values represent the mean calculated numerically from total lipid content in the
`muscles and percentages of the acids in the lipids.
`
`g/1 OOg wet wt
`
`lipid content is equal to the total fatty acid content. The con(cid:173)
`centrations of 20:5 and 22:6 in the food material were about
`twice those in the raw materials. This is due to the increase of
`lipid content by dehydration in screw pressing.
`The present study indicates that the muscle lipids, especially
`in the outer layer, of chum salmon during spawning migration
`contain a higher percentages of w-3 PUFAs than those in the
`fish during feeding migration_ Also, w-3 PUFAs were not lost
`by extrusion cooking and its pretreatment. The production of
`foods containing a latge concentration of PUF As may be pos(cid:173)
`sible by processing chum salmon muscle during spawning mi(cid:173)
`gration.
`
`LIPID EXTRACT/ON FROM MUSCLE. .. From page 1658 - - - - - - - - - - - - - - - - -
`
`Duwe, H., Sonnenkalb, W., Roethe, K.-P., and Rosahl, B. 1986. Destraktion
`von lipiden aus mikrobiellen eiweissisolaten. Die Nahrung 30:667.
`Eisenbach, W. 1984. Supercritical fluid extraction. A film demonstration.
`Ber. Bunsenges. Phys. Chem. 88:882.
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`Ms received 2/12/88; revised 4/29/88; accepted 6/13/88.
`
`Supported in part by grant from the New York Sea Grant program. The help and
`advice of Prof. Syed S.H. Rizvi is gratefully acknowledged.
`
`Volume 53, No. 6, 1988-JOURNAL OF FOOD SCIENCE-1661
`RIMFROST EXHIBIT 1164 Page 0004
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