Patent/Utility Model Document Display I J-PlatPat [JPP]
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`Bibliography
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`1..
`ii,4
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`(19) [Publication country/region] JP
`
`(12) [Kind of official gazette] A
`
`(11) [Publication number] 2005065658
`
`(43) [Date of publication of application] 20050317
`
`D OR DER] VATl VE OF THE SAME
`
`(51) [International Patent Classification 7th Edition]
`
`C12P 7/40 C110 1/00 C12P 7/62
`
`(54) [Title of the invention] MEFHOD FOR PRODUCI NG UNSATURATED FA'ITY ACI
`
`https://www.j-p1atpat.inpit.go.jp/p0200
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`2019/05/28
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`

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`Patent/Utility Model Document Display I J-PlatPat [IPP]
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`2/12 &—°/°
`
`[FI]
`
`C12P 7/40
`
`0110 1/00
`C12P 7/62
`C12P 7/40
`
`C12R 1:01
`
`C12P 7/62
`
`C12R 1:01
`
`[Request for examination] Unrequested
`
`[Number of claims] 2
`
`[Format of application] OL
`
`[Total number of pages] 7
`
`(21) [Application number] 2003304141
`
`(22) [Fiiing date] 20030828
`
`(71) [Applicant]
`[Name] KAO CORP
`
`(74) [Representative]
`
`[Identification number] 110000084
`
`[Name] The Patent Oorporative Body Aruga Patent Office
`
`(74) [Representative]
`
`[Identification number] 100068700
`[Patent attorney]
`[Name] ARUGA, Mitsuyuki
`(74) [Representative]
`[Identification number] 100077562
`
`[Patent attorney]
`
`[Name] TAKANO, Toshio
`(74) [Representative]
`
`[Identification number] 100096736
`[Patent attorney]
`[Name] NAKAJI MA, Toshio
`
`(74) [Representative]
`
`[Identification number] 100101317
`[Patent attorney]
`
`[Name] MATOBA, Hiromi
`(74) [Representative]
`[Identification number] 100117156
`
`[Patent attorney]
`
`[Name] MURATA, Masaki
`
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`2019/05/28
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`Patent/Utility Model Document Display | J—PlatPat [IPP]
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`3/12 ’\°—“)
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`(74) [Representative]
`
`[Identification number] 100111028
`
`[Patent attorney]
`[Name] YAMAMOTO, Hiroto
`(72) [inventor]
`[Full name] HAGIWARA HIROSHI
`[Full name] ARAKI HIROYUKi
`
`[Theme code (reference)]
`48064
`
`4H059
`
`[F—term (reference) ]
`
`4B064AD11
`
`4BOB4AD67
`4BOG4CA03
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`480640814
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`480640009
`48064CD02
`4H059AA10
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`Overview
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`(57) [Overview]
`
`PROBLEM TO BE SOLVED: To provide a method for producing an unsaturated fatt
`
`y acid or its derivatives in a high concentration, in a high purity and under more a
`
`1
`
`i
`
`:
`
`dvantageous conditions in a medium on producing the unsaturated fatty acid or it
`
`s derivatives by a fermentation method using bacteria belonging to the genus Rh
`odococcus.
`
`
`
`
`
`
`
`
`
`SOLUTION: This method for producing the unsaturated fatty acid or its derivative
`
`s from a fatty acid or its derivatives by using unsaturated fatty acid-producing ba
`
`cteria belonging to the genus Rhodococcus is characterized by affecting seeds in t
`
`heir steady state on the fatty acid or its derivatives in the presence of phosphat
`
`https://www.j-p1atpat.inpit.go.jp/p0200
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`2019/05/28
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`Patent/Utility Model Document Display | J-PlatPat [JPP]
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`4/12 ’\°—“)
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`buffer of 20.1 M concentration exhibiting pH7.0-9.0.
`
`
`
`Scope of Claims
`;.,..,., ,.v_..,_. u.,. ,N. .,,.. .. _.. ”.mpvm ,. w ..._..._... - mm new. .«w W “M. .t . ,m... W“.-. a”-_. “ ... w.,m...w-. .. m M w A..-.,A_..,..m ”WWW WW,
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`
`[Patent Claims]
`
`[Claim 1]
`
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`
`An unsaturated fatty acid—producing bacterium belonging to the genus Rhodococc
`
`us (Rhodococcus) is used. A method for producing an unsaturated fatty acid or a
`
`derivative thereof from a fatty acid or a derivative thereof, wherein a seed of a st
`
`ationary phase is caused to act on a fatty acid or a derivative thereof in the prese
`
`nce of a phosphate buffer having a concentration of 0.1 M or more and a pH of 7.
`0 to 9.0.
`
`[Claim 2]
`
`The method according to claim 645, wherein the unsaturated fatty acid-producing
`
`bacterium is Rhodococcus sp. (Rhodococcus sp.) KSM—T2 strain (FERM P- 1818
`
`2).
`
`Detailed Description
`
`
`
`
`
`[Detailed description of the invention]
`
`[Technical field]
`
`[0001]
`
`The present invention relates to a method for producing unsaturated fatty acids 0
`r derivatives thereof using microorganisms.
`[Background of the Invention]
`[0002]
`Unsaturated fatty acids or derivatives thereof are widely used as perfumes, phar
`maceuticals, paints, surfactants, cosmetics, and the like, or as synthetic raw mat
`erials thereof.
`Conventionally, for preparing unsaturated fatty acids or derivatives thereof, a fer
`
`:
`l
`:
`:
`:
`l
`E
`
`mentation method using microorganisms such as filamentous fungi of * * * * * * * * *
`
`(Echinosporangium), filamentous fungi of the genus Moridoerella (Mortierella),
`
`https://www.j-p1atpat.inpit.g0.jp/p0200
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`2019/05/28
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`Patent/Utility Model Document Display I J—PlatPat [JPP]
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`5/12 4—9
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`cl bacteria of the genus Rhodococcus (Rhodococcus) has been known.
`E
`:,
`[0003]
`ii
`i
`: Among them, a fermentation method using bacteria belonging to the genus Rhod
`
`ococcus is preferred because the unsaturated fatty acids or derivatives thereof ar
`
`,
`
`I
`
`s
`
`e produced outside of the cells, and therefore, it is easy to recover (see Patent Do
`
`cument 1, see Patent Document 2), but the productivity and the thereof cannot b
`e 10 minutes. Rhodococcus.
`
`[0004]
`it
`'3
`; Accordingly, when the present inventors studied the production conditions of uns
`
`aturated fatty acids or derivatives thereof, they found that when a bacterium of R
`
`hodococcus genus is caused to act in the presence of a phosphate buffer, the bac
`
`terium can produce an unsaturated fatty acid or a derivative thereof in a high con
`
`(
`
`centration and a high purity in association with proliferation, and thus Patent appl
`ication (see Patent Document 3).
`
`However, in this method, it was necessary to use a phosphate buffer solution hav
`
`ing a concentration of 0.25 M or more.
`
`[Patent document 1]JP H 2 - 65168
`
`[Patent document 2] JP H 4 - 127188
`
`[Patent document 3] Japanese Patent Laid-Open No. 262895 2002
`
`[Disclosure of invention]
`
`[Problem to be solved by the invention]
`
`[0005]
`
`To provide a method for producing an unsaturated fatty acid or a derivative there
`
`of in a medium at a higher concentration and a high purity in a more advantageo
`
`us condition when an unsaturated fatty acid or a derivative thereof is produced b
`
`y a fermentation method using bacteria of the genus Rhodococcus (Rhodococcu
`
`s).
`
`[Means for solving the problem]
`
`[0006]
`
`To provide a method for efficiently producing an unsaturated fatty acid or a deriv
`
`ative thereof, and to provide a method for producing the same by which a seed h
`
`aving reached a stationary phase in a seed culture is used as a bacterium belongi
`
`ng to the genus Rhodococcus to be cultured. It has been found that an unsaturat
`
`ed fatty acid or a derivative thereof can be produced in a medium at high concent
`
`ration and high purity and can be efficiently produced even in the presence of a lo
`
`wer concentration of phosphate buffer,
`
`[0007]
`
`F
`
`‘;
`
`In other words, the present invention relates to a method for producing unsaturat
`
`ed fatty acids or derivatives thereof from fatty acids or derivatives thereof using
`
`5
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`https://www.j-p1atpat.inpit.g0.jp/p0200
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`2019/05/28
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`Patent/Utility Model Document Display I J—PIatPat [JPP]
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`6/12 ’Q—‘P‘
`
`n unsaturated fatty acid-producing bacterium belonging to the genus Rhodococcu
`
`s (Rhodococcus).
`
`The method for producing the unsaturated fatty acid or its derivative is characteri
`
`zed by allowing a seed of a stationary phase to act on a fatty acid or a derivative
`
`1
`
`thereof in the presence of a phosphate buffer having a concentration of 0.1 M or
`
`more and a pH of 7.0 to 9.0.
`
`[Effect of the Invention]
`
`[0008]
`
`According to the present invention, an unsaturated fatty acid or a derivative there
`
`of can be highly efficiently and highly efficiently produced outside of microbial cell
`
`3 under more favorable conditions.
`
`[Best mode for carrying out the invention]
`
`[0009]
`
`The unsaturated fatty acid of the present invention or the manufacturing method
`
`of the derivative makes seed of the stationary phase of the unsaturated fatty acid
`
`production bacillus belonging to a Rhodococcus (Rhodococcus) group act on fatty
`
`acid or its derivative under existence of pH 7.0 to 9.0 phosphate buffer solution b
`
`y more than concentration 0.1M.
`
`The unsaturated fatty acid-producing bacteria belonging to the genus Rhodococcu
`
`8 may be any one having an ability to produce unsaturated from saturated fatty a
`
`cids. RhodococcusExamples thereof include (Rhodococcus sp.) KSIVI—B—Z. 3 m stra
`
`in (FERM BP1531) and Rhodococcus sp. (Rhodococcus sp.) KSIVI-T 645 (FERM P-
`
`18182). Of these, a Rhodococcus SP. KSM—T 645 strain is particularly preferred fr
`
`cm the viewpoint of the production of unsaturated fatty acids or derivatives there
`
`of. The Rhodococcus sp. KSIVI—T 645 strain is a mutant strain obtained by irradiati
`
`ng a Rhodococcus sp. KSM-B—2 M strain with ultraviolet rays. 8.
`
`[0010]
`
`A seed in a stationary phase means a bacterial cell or a bacterial cell culture in w
`
`hich proliferation and death are in equilibrium in seed culture. In other words, wh
`
`en a log of a viable cell count is plotted against time, a state (a stationary phase)
`
`in which a viable cell count is kept constant (a stationary phase) (a closed phase,
`
`a "Microbiology", a 11 revised version, a Hirokawaen Shoten, Feb. 15, 1975, p. 6
`
`9). Alternatively, it means a bacterial cell or a bacterial cell culture liquid in a stat
`
`e (Example 1) in which the bacterial cell growth degree in which the viable cell cc
`
`4
`
`;
`
`unt is expressed by the turbidity cf the cell is constant.
`
`[0011]
`
`In general, in fermentative production using bacteria, seed culture is performed i
`
`
`
`n order to prepare a seed which shortens an induction period in main fermentat
`
`
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`https://www.j —p1atpat.inpit.gc.jp/pOZOO
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`7/12 “5-“;
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`n (main culture), but in the present invention, a seed in a stationary phase of see
`d culture is used. By using seeds of stationary phase, unsaturated fatty acids or d
`
`‘
`
`'
`
`erivatives thereof can be produced efficiently and highly efficiently compared with
`
`the case of using seeds of exponential phase (logarithmic growth phase), and goo
`
`d production can be achieved even in a low concentration of phosphate buffer (Ex
`
`ample 2, Example 3).
`
`[0012]
`.
`I 1, Usually,
`
`in an industrial microbial reaction, since bacteria in the exponential phas
`
`e are most actively divided, their physiological activities are considered to be stro
`ng, and thus seeds in the exponential phase are used for transplantation and inoc
`
`ulation. Thus, it is considered that growth of bacteria is rapidly continued even in
`
`a large culture tank, and the reaction can be started quickly (Yamayama et aI., "B
`
`iological Reaction Engineering", First Edition, Industrial Book 00., Ltd., Feb. 29, 1
`
`M
`
`;
`
`980, p. 155).
`
`Thus, it is quite surprising that unsaturated fatty acids or derivatives thereof coul
`
`d be successfully produced when a stationary phase is used as a seed rather than
`
`an exponential phase.
`
`[0013]
`
`Here, the seed culture is an unsaturated fatty acid of the genus Rhodococcus (Rh
`
`odococcus) or a derivative thereof. Any condition (medium, pH, temperature, cult
`
`ure time, and the like) may be used as long as it is sufficiently grown, and the cul
`
`ture method may be carried out by a conventional microorganism culture metho
`d, but preferably is aerobically cultured at 30 ° C. for 1 to 2 days.
`
`3f
`
`I
`
`For example, a Rhodococcus sp. KSM — T 645 strain is MSG 225 9.
`
`Yeast extract 309,K2HPC>4 309, glucose 2259,MgSO4 7H20 7.5g,FeSO4 7H2
`
`O 225mg,CuSO4 5H20 18mg,MnSO4, 5 h 2 O, 9 mg
`
`Medium in which 30 g of silicon-based antifoaming agent (manufactured by Shin—
`
`Etsu Chemical Co., Ltd.) is dissolved in 15 L of ion-exchanged water
`
`f;
`
`i VWen inoculated into a 30 Ljar fermenter containing the same and subjected to s
`
`;
`
`eed culture at 30 ° C. under a 350rpm,0.5vvm condition, it shifts into a stationar
`
`y phase from about 30 hours of culture (FIG. 1).
`
`In addition, in the separation of seeds, the obtained culture solution may be reco
`
`vered as it is or from the culture solution by a method such as centrifugation.
`
`[0014]
`
`r
`I
`
`t
`
`As a fatty acid used as a raw material, a saturated or unsaturated fatty acid havin
`g 1 to 22, preferably 14 to 20 carbon atoms may be mentioned, and as a derivati
`
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`ve thereof, an alkyl (having 1 to 10 carbon atoms, preferably 2 to 4 carbon atom
`
`
`
`s) or * may be exemplified by an aryl ester, a secondary or *, or an alkali. 2 1.
`
`II
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`8/12 N—‘J
`
`3l
`
`il.l lri
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`,
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`mong these, n-hexadecanoic acid(palmitic acid), n-tetradecanoic acid(myristic aci
`
`d), Preferred are the esters, ammonium salts, salts, and the like of the methyl, et
`
`hyl, n—propyl, isopropyl, n—butyl,
`aric Acid.
`
`[0015]
`
`iso—butyl, and tert-butyl of these fatty acids. Ste
`
`According to the reaction of the present invention, such fatty acids or derivatives
`
`thereof are converted into unsaturated fatty acids or derivatives thereof in which
`
`4
`
`i
`
`,y
`
`an unsaturated bond is introduced at a specific position of a hydrocarbon chain of
`
`a fatty acid. For example, when palmitic acid or an ester thereof is used as a raw
`
`material, a cis—6-hexadecenoic acid or an ester thereof can be obtained as a main
`
`product.
`
`[0016]
`
`Main culture is performed by making seed (fungus body culture medium or fungu
`
`3 body) of the stationary phase mentioned above act on fatty acid or its derivativ
`
`e under existence of pH 7.0 to 9.0 phosphate buffer solution by more than conce
`
`ntration 0.1M.
`
`Here,
`
`it is preferable to use a bacterial cell culture solution or a bacterial cell in an
`
`amount of 0.1 to 10%, particularly 0.5 to 5%, and a fatty acid or a derivative the
`
`é
`
`;
`
`reof is preferably in a range of 1 to 30%, particularly 10 to 25%, with respect tot
`he reaction solution.
`
`[0017]
`
`In the present invention, it is intended that the phosphate buffer solution has a p
`
`H of 7.0 or more or a concentration of 0.1 M or more because the phosphate buff
`
`I
`
`er solution is efficiently desaturated, and that the pH is 9.0 or less for ensuring th
`
`e growth of microorganisms. In view of such effect, the concentration of the phos
`
`phate buffer is preferably 0.1 M or more, particularly preferably 015 M or more,
`
`and the pH is preferably 7.0 or more and 9.0 or less. Particularly, those having a
`
`concentration of 0.1 M to 0.5 M and a pH of 7.0 to 9.0 are preferred, and those h
`
`aving a pH of 7.0 to 8.0 are more preferred in a concentration 0.15M~0.4M.
`
`[0018]
`
`Further, as the phosphate buffer, any counter ion may be used, but a phosphoric
`
`acid 1 * * * * * -phosphoric acid2* * * * or a phosphoric acid1* * * * — phosphoric acid2* *
`
`** is preferred.
`[0019]
`To the reaction solution, a magnesium sulfate salt, an iron sulfate salt, a copper s
`
`EE
`I
`
`ulfate salt, a manganese sulfate salt, a nitrogen source, a vitamin, and the like m
`
`ay be added as long as the growth of the cell is not prevented. In this case, it is p
`
`referable to add the magnesium sulfate salt in an amount of 0.005 to 1%, prefera
`
`bly 0.01 to 0.5%, and the iron sulfate salt in an amount of 1 to 100 ppm, prefe ‘
`
`l
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`https://www.j -p1atpat.inpit.go.jp/p0200
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`Patent/Utility Model Document Display I J-PlatPat [JPP]
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`9/12 /\°—“/°
`
`bly 5 to 50 ppm.
`[00201
`A reaction is temperature conditions (20-37 degrees C, preferably 25—30 degrees
`
`;
`,
`i}
`I
`
`C), and it is preferable under aerobic conditions to carry out especially for 60 to 9
`6 hours for 48 to 120 hours.
`
`[0021]
`
`Thus, an unsaturated fatty acid or a derivative thereof formed in a reaction soluti
`
`on can be easily recovered and separated by an extraction and isolation method 0
`
`ommonly used in natural organic compounds and the like. For example, a reactio
`
`n solution is separated into an oil phase, an oil phase and an aqueous phase by c
`
`entrifugation, and an oil phase is separated, or a reaction liquid is directly extract
`ed by an organic solvent, so that an object can be recovered very easily. In additi
`
`H
`
`on, a high—purity target substance can be recovered by this reaction, but when fu
`
`rther purification is necessary, purification can be carried out by usual column chr
`
`omatography, distribution extraction, solvent crystallization, and the like.
`
`[Examples]
`
`[0022]
`
`g Hereinafter, the present invention will be described in more detail by way of Exa
`
`mples.
`
`Example 1 Measurement of fungus body growth degree aging in seed culture
`
`The Rhodococcus sp. KSM — T 645 strain contains 5 g of sodium glutamate (here
`
`inafter abbreviated as MSG), a yeast extract (manufactured by Deutsche Hefewer
`ke GmbH & Co.oHG ; hereinafter simply abbreviated as yeast extract) Zg,K2HPO
`
`4 29, 10 g of glucose, and 4 g of MgSO. sub. 7H20 O.59,FeSO4To a 500 mL shir
`
`red Erlenmeyer flask containing 30 mL of a medium dissolved in 1 L of ion—excha
`
`.,
`
`nge, 1 platinum ears were inoculated and subjected to shaking culture at 30 ° C.
`
`for 1 days at 210 rpm for days, followed by shaking at rpm. 7H20 15mg,CuSO4
`
`5H20 1.2mg,MnSO4 5H20 0.6mg.
`
`[0023]
`
`All of this culture broth is 225 g MSG, a yeast extract 309,K2HPO4 309, a glucos
`
`e 2259,MgSO4 7H20 7.5g,FeSO4 7H20 225mg,CuSO4 5H20 18mg,MnSO4, 5
`
`i.:1i.
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`h 2 O 9 mg. A 30 L volumejar fermenter containing a medium prepared by dissol
`V;
`ving 30 g of a silicon—based antifoaming agent (manufactured by Shin-Etsu Chem
`
`ical Co., Ltd.) in 15 L of ion-exchange water was inoculated and subjected to aera
`
`t
`
`t
`:
`
`2,
`
`:
`
`I
`
`tion stirring culture (seed culture) at 30 ° C. for 2 days in a 350rpm,0.5vvm.
`
`[0024]
`
`l During culture, the seed broth was withdrawn in small portions at an appropriate
`time, and the absorbance (A 0) of the solution appropriately diluted with physio
`
`
`
`
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`

`

`Patent/Utility Model Document Display I J—PlatPat [JPP]
`
`10/12 ’\°—“)
`
`ng the cell growth degree on a logarithmic basis with respect to the culture time,
`
`it was found that, under the above seed culture conditions, the present strain und
`
`erwent a logarithmic growth phase and then shifted from about 30 hours to a stat
`
`
`
`ionary phase (FIG. 1).
`
`[0025]
`
`gical saline was measured to determine the cell viability (OD). As a result of plotti
`
`
`
`
`
`Drawings
`
`[FIG.1]‘
`mt}
`
`"V's.1:25;}e'x
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`{no.2}
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`https://www.j-p1atpat.inpit.go.jp/p0200
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`2019/05/28
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`Patent/Utility Model Document Display I J—PlatPat [JPP]
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`11/12 ’\°—‘>¢
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