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`I7:U3
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`Frum:AKERMAN.SENTERF|TT&E|DSON
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`5616556313
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`T-802 P.06/I9
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`Job-U95
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`PATENT
`
`IN THE UNITED STATES PATENT AND TRADEMARK OFFICE
`
`Applicant: Beaudoin et al.
`
`Serial No.: 09/830,146
`
`Group Art Unit: 1651
`
`‘
`
`Filed:
`
`July 25, 2001
`
`Examiner: Witz, Jean C.
`
`TitIe: METHOD OF EXTRACTING LIPIDS FROM MARINE AND AQUATIC ANIMAL
`TISSUES
`
`AMENDMENT
`
`CERTIFICATE UNDER 37 CFR 1.8(a)
`I hereby certify that trus correspondence is being
`deposited with the U.S. Postal Service as First Class mail
`in an onvolope addresced to Commissioner for Patents, PO.
`Box 1450. Alexandria, VA 22313-1459
`
`on
`
`S "
`
`"03
`
`fifl gzgfiz Reg.No. HO, 25"’
`
`_
`FAX RECEIVED
`{OCT 2 3 2003
`
`PEI ITIONS UFHCE
`
`Mail Stop Fee Amendment
`Commissioner for Patents
`P.O. B
`1450
`Alexancdtia, VA 223134459
`
`Dear Sirs:
`
`This amendment is in response to the Office Action dated February 25, 2003.
`
`Amendments to the claims begin on page 2 of this paper.
`
`Remarks/arguments begin on page 8 of this paper.
`
`{WP146887:1)
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`Job-U95
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`Appln. No, 09/830,146
`Amendment
`Response to Office Action dated 02/25/2003
`
`Docket No. 789-47
`
`AMENDMENTS TO THE CLAIMS
`
`1-51. (Cancelled)
`
`52. (New) A method for extracting total
`
`lipid fractions from marine and aquatic animal
`
`material, said method comprising the steps of:
`
`(a)
`
`placing marine and aquatic animal material in a ketone solvent to achieve
`
`extraction of the soluble lipid fraction from said marine and aquatic animal
`
`(b)
`
`(c)
`
`material;
`
`separating the liquid and solid contents;
`
`recovering a first total
`
`lipid rich fraction from the liquid contents of b) by
`
`evaporation of the solvent present in the liquid contents;
`
`(d)
`
`placing said solid contents in an organic solvent selected from the group of
`
`solvents consisting of alcohol and esters of acetic acid to achieve extraction
`
`of the remaining soluble total
`
`lipid fraction from said marine and aquatic
`
`animal material;
`
`separating the liquid and solid contents;
`
`recovering a second total lipid rich fraction by evaporation of the solvent from
`
`(e)
`
`(f)
`
`the liquid contents of e); and
`
`(g)
`
`recovering the solid contents.
`
`53. (New) A method as in claim 52, wherein steps (b) and (d) are conducted under inert gas
`
`atmosphere.
`
`54. (New) A method as in claim 52, wherein steps (b) and (e) are effected by techniques
`
`selected from the group consisting of filtration, centrifugation and sedimentation.
`
`(WP146887;‘i)
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`2
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`T-602 P68/19 Job-085
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`Appln. No. 09/830,146
`Amendment
`Response to Office Action dated 02/25/2003
`
`Docket No. 76947
`
`55. (New) A method as in claim 52, wherein steps (c) and (f) are effected by techniques
`
`selected from the group consisting of vacuum evaporation. flash eVaP0Ta“i0“ alid SW3)’
`
`drying.
`
`56. (New) A method as in claim 52, wherein after step (b) and before step (c), the method
`
`additionally comprises the intervening step of washing the solid contents with the solvent
`
`and adding the resulting washing solution to the liquid contents of step (b).
`
`57. (New) A method as in claim 52, wherein after step (e) and before step (f). the method
`
`additionally comprises the intervening step of washing the solid contents with the organic
`
`solvent selected in step (d).
`
`58. (New) A method as in claim 52, wherein prior to step (a) the marine and aquatic animal
`
`material is finely divided.
`
`59. (New) A method as in claim 52, wherein steps (a) and (b) are conducted at solvent
`
`temperatures of not more than about 5°C.
`
`60. (New) A method as in claim 52, wherein said marine and aquatic animal is zooplankton.
`
`61.
`
`(New) A method as in claim 60, wherein said zooplankton is selected from krill and
`
`Ca/anus.
`
`62.
`
`(New) A method as in claim 52, wherein said marine and aquatic animal is fish.
`
`63. (New) A method for extracting an astaxanthin-and-canthaxantin-containing total
`
`lipid
`
`fraction from a marine and aquatic animal material selected from zooplankton and fish. said
`
`method comprising the steps of:
`
`(WP146887:1)
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`3
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`T-802 P.09/I9 Job-D95
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`Appinv No, 09/830345
`Amendment
`Response to Office Action dated 02/25/2003
`
`Docket No. 789-47
`
`(a)
`
`placing said animal material in a ketone solvent to achieve an extraction of
`
`the soluble lipid fraction from said marine and aquatic animal material;
`
`separating the liquid and solid contents; and
`
`recovering a lipid rich fraction from the liquid contents by evaporation of the
`
`(b)
`
`(c)
`
`solvent present in the liquid contents.
`
`whereby an astaxanthin—and-canthaxantin—containirig total lipid fraction is obtained.
`
`64. (New) A method for extracting a total lipid fraction from a marine and aquatic animal
`
`material selected from zooplankton and fish, said method comprising the steps of:
`
`(a)
`
`placing said animal material
`
`in a solvent mixture comprising acetone and
`
`ethanol to achieve an extraction of the soluble lipid fraction from said marine
`
`and aquatic animal material;
`
`separating the liquid and solid contents; and
`
`recovering a lipid rich fraction from the liquid contents by evaporation of the
`
`(b)
`
`(c)
`
`solvents present in the liquid contents,
`
`whereby a total lipid fraction is obtained.
`
`65. (New) A method as in claim 63, wherein the animal material is selected from krill and
`
`Ca/anus.
`
`66. (New) A method as in any one of claim 63. wherein during step (a), the animal material
`
`is homogenized.
`
`67. (New) A method as in claim 63. wherein steps (b) and (d) are conducted under inert
`
`gas atmosphere.
`
`68. (New) A method as in claim 63, wherein step (b) is effected by a technique selected
`
`from the group consisting of flltratlon. centrlfugation and sedimentation.
`
`(WP146887;1)
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`4
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`T-602 P.lU/19 Job-U95
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`Appln. No. 09/830,146
`Amendment
`Response to Office Action dated 02/25/2003
`
`DOCKBI N0- 789-47
`
`69. (New) A method as in claim 63, wherein step (c) is effected by a technique selected
`
`from the group consisting of vacuum evaporation, flash evaporation and spray drying.
`
`70. (New) A method as in claim 63. wherein after step (b) and before step (c), the method
`
`additionally comprises a step of washing said solid contents with solvent and adding the
`
`resulting washing solution to the liquid contents of step (b).
`
`71. (New) A method as in claim 63, wherein prior to step (a) the marine and aquatic animal
`
`material is finely divided.
`
`72. (New) A method as in claim 63. wherein steps (a) and (b) are conducted at solvent
`
`temperatures of about 5°C or less.
`
`73. (New) A method of lipid extraction as in claim 52, wherein the solid contents of step b)
`
`is recovered and consists of a dehydrated residue containing active enzymes.
`
`74. (New) A method of lipid extraction as in claim 63, wherein the solid contents of step b)
`
`is recovered and consists of a dehydrated residue containing active enzymes.
`
`75. (New) A method for extracting total "lipid fractions from marine and aquatic animal
`
`material, said method comprising the steps of:
`
`(a)
`
`placing marine and aquatic animal material in a ketone solvent to achieve
`
`extraction of the soluble total
`
`lipid fraction from said marine and aquatic
`
`(b)
`
`(c)
`
`animal material;
`
`separating the liquid and solid contents;
`
`recovering a first total
`
`lipid—rich fraction from the liquid contents of b) by
`
`evaporation of the solvent present in the liquid contents;
`
`(d)
`
`placing said solid contents in an organic solvent selected from the group of
`
`solvents consisting of alcohol and esters of acetic acid to achieve extraction
`
`(WP14S887;1}
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`5
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`T'602 P.11/19
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`Job-095
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`Appln. No. 09/830,146
`Amendment
`Response to Office Action dated 02/25/2003
`
`Docket No. 78947
`
`of the remaining soluble total
`
`lipid fraction from said marine and aquatic
`
`animal material;
`
`«
`
`(e)
`
`(f)
`
`separating the liquid and solid contents; and
`
`recovering a second total lipid-rich fraction by evaporation or the solvent from
`
`the liquid contents of e).
`
`whereby total lipid fractions are obtained.
`
`76. (New) A method of total lipid extraction as in claim 75. wherein the solid contents of
`
`step b) is recovered and consists of a dehydrated residue containing active enzymes.
`
`77. (New) A method as in claim 52, wherein the ketone solvent is acetone.
`
`78.
`
`(New) A method as in claim 52. wherein the alcohol
`
`is selected from the group
`
`consisting of ethanol. isopropanol and t-butanol.
`
`79. (New) A method as in claim 52, wherein the ester of acetic acid is ethyl acetate.
`
`80. (New) A method as in claim 58, wherein the marine and aquatic animal material is finely
`
`divided to an average particle size of not more than 5mm.
`
`81. (New) A method as in claim 63, wherein said marine and aquatic animal material
`
`is
`
`viscera.
`
`82 (New) A method as in claim 63. wherein the ketone solvent is acetone.
`
`83 (New) A method as in claim 64, wherein said marine and aquatic animal material
`
`is
`
`viscera.
`
`(WP146B87'.1}
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`T-802 P.l2/19 Job-U95
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`Appln. No. 09/830,146
`Amendment
`Response to Office Action dated 02/25/2003
`
`Docket No. 759-47
`
`84. (New) A method as in claim 64, wherein the animal material is selected from krill and
`
`Calanus.
`
`«
`
`85. (New) A method as in claim 71. wherein the animal material is finely divided to an
`
`average particle size of not more than 5 mm.
`
`86. (New) A method as in claim 52. wherein the solid contents of step (e) is recovered and
`
`consists of a dehydrated residue containing active enzymes.
`
`87 (New) A method as in claim 75. wherein the ketone solvent is acetone.
`
`88. (New) A method as in claim 75, wherein the alcohol
`
`is selected from the group or
`
`ethanol. isopropanol and t-butanol.
`
`89. (New) A method as in claim 75, wherein the ester of acetic acid is ethyl acetate.
`
`90. (New) A method of lipid extraction as in claim 75, wherein the solid contents of step (3)
`
`is recovered and consists of a dehydrated residue containing active enzymes.
`
`(WP146887:1)
`
`7
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`5615596313
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`T-B02 P.l3/l9 Job-U55
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`Appln. No. 09/830,146
`Amendment
`Response to Office Action dated 02/25/2003
`
`Docket No. 769-47
`
`REMARKS
`
`The foregoing amendments and these remarks are in response to the Office Action
`
`dated February 25, 2003. This amendment is filed with a request for a three month
`
`extension of
`
`time and authorization to charge Deposit Account No. 50-0961 for
`
`the
`
`extension of time. At the time of the Office Action, claims 1, 4-12, 14-17, 19-27, 30-33 and
`
`37-51 were pending in the application.
`
`I.
`
`Claim rengmbering and amendments
`
`Reconsideration of this Application and entry of the foregoing amendments are
`
`requested. Claims 1, 4-12, 14-17, 19-25. 27, 30-32, 36-44 and 47-51 have been cancelled
`
`and new claims 52-90 have been entered.
`
`Independent claims 52, 63, 64 and 75 recite a
`
`method to extract a gal lipid fraction. Support for the amendments to these claims can be
`
`found, for example. at page 5, lines 5 to 7 and at page 8, lines 15 to 19. Further support for
`
`this terminology may be found throughout the disclosure where it is shown that the lipid
`
`fraction extracted from marine and aquatic animal is the total lipid fraction as may be seen
`
`more particularly in Table 13. Table 13 describes the various components of the lipids
`
`extracted according to the present claims which include triglycerides, free fatty acids,
`
`monogiycerides and phospholipids.
`
`Claims 52, 63 and 64 do not recite “filletlng-by-products". Support for this deletion
`
`may be found in the claims 15 and 16 as originally filed.
`
`ll.
`
`Rejections Under 35 U.S.C. § 193
`
`The Office Action rejected claims 1, 4-12, 14-17, 19-25, 27. 30-32, 36-44, 47-51
`
`under 35 U.S.C. § 103(a) as being unpatentable over the disclosures of CA 2,155,571, WO
`
`8401715, JP 360035057A and US 6,055,936 ("Collins") taken as a whole.
`
`Prior to discussing the rejections. a brief presentation on the nature and constituents
`
`of lipids including those of marine and aquatic animal lipids is provided.
`
`{WP146887:1}
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`T-802 P.I4/19
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`Job-095
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`Appln. No. 09/830,146
`Amendment
`Response to Office Action dated 02/25/2003
`
`Lipids from animal and plant tissues
`
`Docket No. 78947
`
`Within its generally recognized meaning and within the meaning of that which is
`
`recited by the claims, the term “lipid” refers to naturally occurring substances soluble in
`
`organic solvents, but insoluble in water. The diverse groups of substances encompassed by
`
`this definition can be divided into two broad classes: the acyl
`
`lipids and the terpenoids
`
`(minor components). The acyl lipids may then be subdivided into further subclasses: neutral
`
`acyl
`
`lipids (glycerides. free fatty acids and cholesterol esters), and polar lipids including
`
`glycerophospholipids, glyceroglycolipids and sphingolipids. Terpenoids are comprised of
`two subclasses of minor components: the sterols and the chlorophylls and carotenoids.
`
`Carotenoids constitute a very minor constituent of lipids in marine animals. The Examiner is
`
`referred to Table 17 on page 29 of the present application providing the content
`
`in
`
`astaxanthin and canthaxanthin of fractions obtained and to page 10, lines 26 to 31 where It
`
`is indicated that
`
`the content of astaxanthin is about 75-124pg/g and the cont nt of
`
`canthaxanthin is about 250-700 pg/g.
`
`it is apparent throughout the present application that
`
`the term “total lipid" as used therein is meant to encompass all these various types of lipids
`
`and that the method of the present invention is able to extract all these various types of
`
`lipids.
`
`Marine oils
`
`The oils extracted from marine and aquatic materials can be separated into two
`
`types, according to their composition. The oils extracted from most fishes constitute the
`
`first oil type.
`
`It is composed of more than 90% of neutral acyl lipids, the major part of
`
`which are glycerides. Glycerides are fat reserves of marine animals. Cod liver oil for
`
`instance, is mostly constituted of glycerides.
`
`The second oil type is composed of a lesser proportion of neutral acyl lipids and of
`
`glycerophospholipids, and sphingollpids. Krill
`
`lipids for instance may contain about 44%
`
`neutral acyl
`
`lipids (triglycerides, monoglycerides and free fatty acids) and about 54% of
`
`phospholipids (Phosphatidylcholines. Phosphatidylethanolamines, lysophosphatidylcholines
`
`(WP146887;1)
`
`'
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`9
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`Appln. No. 09/830,148
`Amendment
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`Response to Office Action dated 02/25/2003
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`DOCKGI N0. 73947
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`and phosphatidylserines) and sphingolipids (sphingomyelin) (see table 13 of the present
`
`application).
`
`~»
`
`Prior to the present invention,
`
`the only solvents that appeared to produce good
`
`results to extract total
`
`lipids from Krill was a combination of chloroform and methanol.
`
`Notably, however, these solvents are unacceptable for the food industry, because after their
`
`evaporation. some toxic residues could remain in the lipids. There is no reported success of
`
`total
`
`lipids extraction with non-toxic solvents generally recognized as safe (GRAS).
`
`Furthermore, although acetone was used to extract certain components of lipids,
`
`it was
`
`never disclosed to extract total lipids from marine and aquatic animals.
`
`CA 2,155,571, QP 360035057/X and Collin
`
`It is respectfully submitted that JP 36003505‘/‘A, Collin and CA 2,155,571, do not
`
`describe the use of acetone or ethanol for extracting "total lipids" within the meaning of the
`
`present application but to extract only a small fraction of total lipids, namely carotenoids for
`
`JP 360035057A and Collin, and polyunsaturated fatty acids for CA 2,155,571.
`
`JP 360035057A and Collin teach that acetone is a conventional solvent for the
`
`recovery of fatty acids and lipid pigments (astaxanthin and canthaxanthin) of marine
`
`animals. Both polyunsaturated fatty acids and pigments constitute minor components of
`
`marine animal lipids.
`
`In the present application, the fraction 1
`
`is a lipid fraction extracted
`
`with acetone according to a specific embodiment of the present invention. Analysis of lipid
`
`classes shows that free fatty acids constitute about 23.7% (See table 13) of total lipids of
`
`that fraction. The pigments, found in the hydrocarbon group (see Table 13) are only present
`
`as trace. As may further be seen from Table 17 of the present application, pigments
`
`constitute 0.0364% of the total
`
`lipid extracts of krill obtained according to the present
`
`invention (i.e. 93.1+270.4 =363.5pg/g oil).
`
`it is therefore submitted that techniques such as
`
`those described in JP 360035057A and Collin, which are able to extract less than 25% of
`
`total lipids, cannot be considered to be methods for tciai lipid extraction.
`
`it is also respectfully submitted that it was not predictable from references such as
`
`those describing the extraction of minor components of marine animal
`
`lipids, namely
`
`(WP146887;i)
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`T-802 P.16/19
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`Job-U95
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`Appln. No. 09/830,146
`Amendment
`
`Response to Office Action dated 02/25/2003
`
`Docket No. 789-47
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`polyunsaturated fatty acids and lipid pigments with acetone,
`
`that the remaining major
`
`components of lipids could also be extracted from marine animal with acetone. None of the
`
`cited references JP 360035057A, Collin or CA 2,115,571 disclose or suggest that acetone
`
`can efficiently extract glycerides, glycerophosphollplds and sphlngollplds from marine
`
`animals.
`
`Notably.
`
`the cited prior art
`
`teaches away from using acetone to extract
`
`glycerophospholipids, sphingolipids and olycerides. Dawson et aI., Textbook of RMC, Third
`
`Ed. “Data for Biochemical Research" and The Merck Index. 12th edition, Budavari et al.,
`
`Ed. Merck Research Laboratories,
`
`1996
`
`teach
`
`that
`
`phosphatidylethanolamines,
`
`sphingomyelin and
`
`phosphatidylcholine are practically insoluble in acetone and that
`
`phosphatidylserine and lysophosphatidylcholine are only slightly soluble in acetone. The
`
`Table below presents the solubility of various glycerophospholipids and sphingolipids
`
`in
`
`acetone as disclosed in these references. Please find enclosed copies of the rel vant
`
`extracts of these references.
`
`(WP148887',1)
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`1 1
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`Appln. No. 09/830,146
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`Amendment
`Response to Office Action dated 02/25/2003
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`DOCKBI N0- 739-47
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`soluble in acetone
`
`I
`
`
`
`
`U)2L:1.7
`tfififérstfiét
`
` Phosphatidylcholine
`Dawson p.181
`
`insoluble in acetone partially soluble in ethanol
`
`Merck #5452. p.925
`
`Phosphatidylethanolamines Dawson p.181
`
`insoluble in acetone
`
`
`
`Merck #2022, p.328
`
`Phosphatidylserines
`
`Dawson p.183
`
`Phosphatidylinositol
`
`Dawson p.183
`
`
`
`
`
`
`
`insoluble in acetonej
`insoluble in acetonej
`insoluble in acetonej
`insoluble in ethanol
`insoluble in acetone_
`
`Sphingomyelin
`
`
`
`Merck #8899, p.1495
`
`Dawson p.184
`
`insoluble in acetone
`
`it
`
`is also noteworthy that acetone is not mentioned as a potential solvent
`
`for
`
`triglycerides and that many of these neutral lipids are indicated to be only slightly soluble in
`
`ethanol.
`
`It is therefore respectfully submitted that at the time of the Applicants‘ invention,
`
`th
`
`prior art taught away from using acetone as a solvent for extracting lipids from marine
`
`animal. The Applicants themselves fortuitously discovered acetone‘s potential as a solvent
`
`for the extraction of krill lipids while they were investigating for a means to dehydrate krill
`
`tissues.
`
`In summary. the pnor art does not disclose or suggest that the main classes of
`
`lipids (glycerophospholipids. sphingolipids and glycerides) can be efficiently extracted with
`
`acetone or ethanol.
`
`{WP146887:1)
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`p_]g/1g
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`J°b-ng5
`
`Appln. No. 09/830,146
`Amendment
`Response to Office Action dated 02/25/2003
`
`Destruction of main components of krill lipids
`
`DOCKGI NO. 759-47
`
`JP 36003505‘/A discloses an process by which krill
`
`is combined with an organic
`
`solvent such as acetone or hexane and a lipase. The main process seeks to decompose
`
`glycerldes and phosphollpids lnto fatty acids in order to achieve the Concentration of
`
`carotenoids which can then be utilized as a dye. Now, as indicated earlier, marin
`
`and
`
`aquatic animals lipids contain glycerides, free fatty acids, phospholiplds, glyceroglycolipids
`
`and sphingolipids. Each of these components are constituted in part of fatty acids. Thus,
`
`the method of JP 360035057A would damage the major components of marine and aquatic
`
`animals lipids.
`
`For medical. cosmetic and nutritional applications.
`
`it
`
`is advantageous to preserve
`
`marine lipid integrity because they are then more easily assimilated in the organism. Hence,
`
`phospholiplds for instance contribute to the formation of an emulsion with triglycerides
`
`during digestion. thereby facilitating lipase action which in turn may progressively release
`
`fatty acids and enable their good absorption by the organism. Furthermore, many if not all
`
`components of the total lipid extracts have nutritional values. A method able to extract the
`
`total lipids is therefore advantageous.
`
`yyo 8401715
`
`WO 8401715 describes a carbon tetrachloride (CCI4) extraction of enzymes from
`
`water-extracted lipids (see page 10'. lines 4 to 10 of WO 8401715). CCI4 is toxic according to
`
`the Merck index (#1864. page 297 Merck index, 12"‘ edition): it may cause liver and kidney
`
`damage and may be a carcinogen.
`
`in stark contrast. the method of the present invention is directed to an extraction of
`
`total lipids of marine and aquatic animals with a ketone solvent or a mixture of a ketone and
`
`an ethanol solvent or ethyl acetate. According to a specific embodiment of the present
`
`invention, active enzymes may be recovered from the solid residues produced. WO
`
`8401715 does not disclose or suggest a method for extracting total lipids from marin
`
`and
`
`aquatic animals with a ketone solvent, the solvent used in W0 8401715 for extracting lipids
`
`is CCI4.
`
`(WP1-$6887;1)
`
`13
`
`NEPN 2015
`
`NEPN 2015
`
`
`
`OCT-23-U3
`
`17:06
`
`From:AKERMAN.SENTERFiTT&EiD$0N
`
`5515535313
`
`T-auz mg/lg Job..gg5
`
`Appln. No. 09/830,146
`Amendment
`Response to Office Action dated 02/25/2003
`
`Docket No. 75947
`
`in view of the above and foregoing, withdrawal of the rejection under 35 U.S.C. §103,
`
`first paragraph is respectfully requested. The rejections of the original claims arebelieved
`
`to have been overcome by the present remarks and the introduction of new claims. From
`
`the foregoing, further and favorable action in the form of a Notice of Allowance is believed to
`
`be next in order. and such action is earnestly solicited.
`
`ill.
`
`Qouciuflm
`
`Applicant has made every effort to present claims which distinguish over the prior art,
`
`and it
`
`is believed that all claims are in condition for allowance. Nevertheless, Applicant
`
`invites the Examiner to call the undersigned if it is believed that a telephonic interview would
`
`expedite the prosecution of the application to an allowance.
`
`in view of the foregoing
`
`remarks, Applicant respectfully requests reconsideration and prompt allowance of
`
`the
`
`p nding claims.
`
`Date:
`
`?" 25 '0: 3
`
`Docket No. 789-47
`
`Respectfully submitted,
`
` .¢/‘2-d.—--
`J. Rodrnan Steele, Jr.
`Registration No. 25,931
`Mark D. Passler
`
`Registration No. 40,764
`AKERMAN SENTERFITT
`Post Office Box 3188
`
`West Palm Beach, FL 33402-3188
`Telephone: (551) 653-5000
`
`(WP146887;1)
`
`14
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`NEPN 2015
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`OCT-23-U3
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`17:40
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`OCT-23-U3
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`5616596313
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`OCT-23-03
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`17:40
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`From:AKERMAN.SENTERF|TT G EIDSON
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`OCT-23-U3
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