WO2015140467A1 - Procédé de perméabilisation thermique d'une biomasse de microalgues - Google Patents
Procédé de perméabilisation thermique d'une biomasse de microalgues Download PDFInfo
- Publication number
- WO2015140467A1 WO2015140467A1 PCT/FR2015/050658 FR2015050658W WO2015140467A1 WO 2015140467 A1 WO2015140467 A1 WO 2015140467A1 FR 2015050658 W FR2015050658 W FR 2015050658W WO 2015140467 A1 WO2015140467 A1 WO 2015140467A1
- Authority
- WO
- WIPO (PCT)
- Prior art keywords
- temperature
- biomass
- microalgae
- chlorella
- heat treatment
- Prior art date
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
- C12N1/066—Lysis of microorganisms by physical methods
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07H—SUGARS; DERIVATIVES THEREOF; NUCLEOSIDES; NUCLEOTIDES; NUCLEIC ACIDS
- C07H3/00—Compounds containing only hydrogen atoms and saccharide radicals having only carbon, hydrogen, and oxygen atoms
- C07H3/06—Oligosaccharides, i.e. having three to five saccharide radicals attached to each other by glycosidic linkages
-
- C—CHEMISTRY; METALLURGY
- C07—ORGANIC CHEMISTRY
- C07K—PEPTIDES
- C07K1/00—General methods for the preparation of peptides, i.e. processes for the organic chemical preparation of peptides or proteins of any length
- C07K1/14—Extraction; Separation; Purification
- C07K1/34—Extraction; Separation; Purification by filtration, ultrafiltration or reverse osmosis
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/06—Lysis of microorganisms
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12N—MICROORGANISMS OR ENZYMES; COMPOSITIONS THEREOF; PROPAGATING, PRESERVING, OR MAINTAINING MICROORGANISMS; MUTATION OR GENETIC ENGINEERING; CULTURE MEDIA
- C12N1/00—Microorganisms, e.g. protozoa; Compositions thereof; Processes of propagating, maintaining or preserving microorganisms or compositions thereof; Processes of preparing or isolating a composition containing a microorganism; Culture media therefor
- C12N1/12—Unicellular algae; Culture media therefor
Definitions
- the present invention relates to a method for thermal permeabilization of a biomass of microalgae, this treatment making it possible to release the soluble intracellular content thereof, in particular the peptides and polypeptides.
- This method of thermal permeabilization is not accompanied by cell disintegration, which also allows the easy separation of the intracellular content thus released from the residual biomass, by any solid-liquid separation method known in itself from man of the art, for example frontal or tangential filtration, centrifugation and / or flocculation.
- the method of the invention makes it possible to conserve the lipid fraction of interest in the residual biomass.
- the present invention finally relates to the recovery and fractionation of the intracellular content of microalgae in aqueous solution, intracellular content composed of soluble peptides and polypeptides, pigments, free fatty acids, oligo- and polysaccharides ... It is well known to the human Chlorella is a potential source of food because it is rich in protein and other essential nutrients.
- Chlorella biomass oil fraction which consists mainly of monounsaturated oils, provides nutritional and health benefits over saturated, hydrogenated and polyunsaturated oils often found in conventional food products.
- the protein fraction can be valued as a functional agent in the food, cosmetics and even pharmaceutical industries, for its foaming, emulsifying properties.
- Chlorellae are therefore conventionally used for human or animal nutrition, either in the form of whole biomass or in the form of flour, obtained by drying the biomass of chlorella whose cell wall has been broken by mechanical means.
- Microalgae flour also provides other benefits, such as micronutrients, dietary fiber (soluble and insoluble carbohydrates), phospholipids, glycoproteins, phytosterols, tocopherols, tocotrienols, and selenium.
- the biomass is concentrated, or harvested, from the culture medium (culture carried out by light autotrophy in photobioreactors, or in heterotrophy, in the dark in the presence of a carbon source assimilated by the Chlorella).
- the biomass At the time of harvesting the biomass of microalgae from the fermentation medium, the biomass comprises intact cells essentially suspended in an aqueous culture medium.
- a solid-liquid separation step is then carried out, by frontal and / or tangential filtration, by centrifugation, or by any other means known to those skilled in the art.
- microalgae biomass can be directly processed in order to produce vacuum-packed cakes, microalgae flakes, microalgae homogenates, intact microalgae powder, ground microalgae flour, or coconut oil. microalgae.
- microalgae biomass is also dried in order to facilitate the subsequent treatment or use of the biomass in its various applications, particularly food applications.
- microalgae have been used mainly for the production of high value-added products, but at low volume.
- an efficient cell disruption process must maximize not only the yield but also the quality of the extracted products. In other words, this optimized disintegration process must avoid chemical contamination or degradation of the targeted products.
- microalgae disintegration procedures have been studied, for example chemical, mechanical, enzymatic or even electrical (pulsed field).
- microalgae cells have very strong membrane walls, which makes the cell disintegration and the extraction of the products of interest very difficult and very expensive in energy.
- a pressure disruptor can be used to pump a suspension containing the cells through a restricted orifice to lyse the cells.
- High pressure minimum of 1500 bar
- the breaking of the cells can be achieved by three different mechanisms: encroachment on the valve, high shear of the liquid in the orifice, and sudden pressure drop at the outlet, causing an explosion of the cell.
- the method releases intracellular molecules, mixed with cell debris.
- a NIRO Homogenizer Niro homogenizer (GEA NIRO SOAVI - or any other high pressure homogenizer) can be used to treat cells with a size predominantly between 0.2 and 5 microns. This treatment of algal biomass under high pressure generally lyses more than 90% of the cells and reduces the size to less than 5 microns.
- a ball mill may also be used.
- the cells are agitated in suspension with small spherical particles.
- the breaking of the cells is caused by shear forces, grinding between the balls, and collisions with beads.
- the description of a suitable ball mill is for example made in US Patent 5,330,913. These beads break the cells to release the cell contents.
- a suspension of particles of smaller size than the original cells is then obtained in the form of an "oil-in-water" emulsion. This emulsion is then atomized and the water is removed, leaving a dry powder containing however a heterogeneous mixture composed of cellular debris, interstitial soluble compounds and oil.
- the difficulty to solve in the use of these cell disruption technologies is the isolation of the only intracellular content (excluding membrane debris and fat) and the preservation, in particular, of the quality of the protein charge.
- the energy used to break the rigidity of the microalga can indeed cause irreversible degradation or denaturation of the intracellular molecules of interest.
- This rupture of the membrane then facilitates the release of the cellular contents and, when using a complementary solvent extraction technique, also facilitates the penetration of the solvent into the cell.
- the Applicant Company has found that this need could be satisfied by a process of thermal permeabilization of the microalgae cells.
- the recovery of the molecules can be easily achieved by any solid-liquid separation technology known to those skilled in the art, since the heat treatment developed by the applicant company does not lead to the disintegration of the cell wall. Finally, the recovery of this soluble fraction opens the fractionation of its content, for example by membrane separation techniques known to those skilled in the art.
- the present invention relates to a method for thermal permeabilization of the microalgae biomass of the Chlorella genus so as to recover soluble fractions enriched in particular peptides and polypeptides and oligosaccharides.
- This process comprises the following steps:
- stepwise heat treatment at a temperature of 60 to 130 ° C, preferably 60 to 90 ° C, for 1 to 5 minutes,
- This method preferably comprises the following steps:
- microalgae of the genus Chlorella are selected from the group consisting of Chlorella vulgaris, Chlorella sorokiniana and Chlorella protothecoides, and are more particularly Chlorella protothecoides.
- the strain is Chlorella protothecoides (strain UTEX 250 - The Culture Collection of Algae at the University of Texas at Austin - USA).
- the strain is Chlorella sorokiniana (strain UTEX 1663 - The Culture Collection of Algae at the University of Texas at Austin - USA). Cultivation in heterotrophic conditions and in the absence of light conventionally leads to the production of a biomass of chlorella having a protein content (evaluated by measurement of the nitrogen content N x 6.25) of 45 to 70% by weight. dry cell weight.
- this culture is carried out in two stages:
- the biomass is then collected by solid-liquid separation, by frontal or tangential filtration, by centrifugation, or by any means known to those skilled in the art.
- the Applicant Company then recommends washing the biomass so as to eliminate the interstitial soluble compounds by a succession of concentration (by centrifugation) / dilution of the biomass.
- interstitial soluble compounds means all the soluble organic contaminants of the fermentation medium, for example the water-soluble compounds such as salts, residual glucose, oligosaccharides of degree of polymerization (or DP) 2 or 3, the peptides ...
- This biomass thus purified of its interstitial solubles is then adjusted preferably to a solids content of between 5 and 35% by weight, preferably to a solids content of between 10 and 20% with deionized water.
- the heat treatment is then carried out in stages at a temperature of between 60 ° and 130 ° C., preferably 60 ° and 90 ° C., for 1 to 5 minutes.
- This treatment can include 2 to 6 temperature steps. For example, it may comprise several incremental temperature steps and then, optionally, several decreasing temperature steps.
- the temperature steps may be 10 to 40 ° C, for example about 10, 20, 30 or 40 ° C.
- a first step may allow the biomass to be brought to a temperature of about 60-70 ° C. By “about” is meant + or - 10%, preferably + or - 5%.
- Intermediate bearings can be made between 60 ° C and the maximum applied temperature, for example between about 90 and 130 ° C.
- Each step can last between about 10 seconds and 4 minutes, preferably between 30 seconds and 3 minutes.
- the treatment may comprise a first stage allowing the biomass to be brought to a temperature of approximately 60-70 ° C., one or more bearings allowing achieve a maximum applied temperature of about 90 to 130 ° C, and optionally one or more bearings to reduce the temperature.
- the treatment can be carried out in three phases: raising the temperature of the ambient temperature to 60 ° C. in 30 seconds; - temperature rise from 60 ° to 90 ° C for another 30 seconds;
- the method comprises the following steps: raising the temperature from 28 ° C. to 60 ° C. for 30 seconds,
- This treatment makes it possible to allow the intracellular components to diffuse into the reaction medium.
- the Applicant Company has thus found that the heat treatment, performed under these operating conditions, thus acts as a membrane embrittlement process which allows the spontaneous release of the soluble components of the intracellular compartment.
- organic substances such as carbohydrates (predominantly DP1 and DP2), peptides and polypeptides are drained out of the cell.
- the process according to the invention therefore does not lead to the formation of an emulsion, but to an aqueous suspension.
- a lag time may be necessary to allow sufficient diffusion after the heat treatment which permeabilizes the membrane.
- reaction time between 30 minutes and 3 hours additional can be implemented to optimize the diffusion of soluble compounds of the cell compartment.
- the residual biomass is then removed by a solid-liquid separation technique by frontal or tangential filtration, by flocculation, by centrifugation, or by any other means known to those skilled in the art, which makes it possible to easily recover the freed soluble fraction. microalgae cells.
- This soluble fraction consists essentially of proteins (50 - 80% w / w) and carbohydrates (5 - 15% w / w).
- the conventional processes for recovering soluble proteins generally rely on a step of precipitating said proteins with trichloroacetic acid (10% w / v) or with ammonium sulphate.
- the method according to the invention makes it possible, on the contrary, to release native, intact peptides and polypeptides, all of whose functionalities can still be expressed.
- the Applicant Company found that the size of the soluble peptides and polypeptides released varied in proportion to the processing temperature used. It is also considered that the processing time can have an impact.
- the applicant company recommends proceeding in two stages:
- compositions enriched in soluble proteins and in oligosaccharides from soluble fractions (freed from heat-treated microalgae) filtered on a membrane system chosen from the group consisting of microfiltration, ultrafiltration, nanofiltration and diafiltration, taken alone or in combination,
- compositions subjecting said compositions to additional membrane fractionation treatments of reverse osmosis type in order to separate peptides and polypeptides on the one hand, and oligosaccharides on the other hand.
- additional membrane fractionation treatments of reverse osmosis type in order to separate peptides and polypeptides on the one hand, and oligosaccharides on the other hand.
- the strain used is Chlorella protothecoides UTEX 250
- composition of the medium (in g / L):
- the incubation takes place under the following conditions: duration: 72 h; temperature: 28 ° C; agitation: 1 10 rpm (Infors Multitron Incubator).
- the middle is the following: Table 2.
- the initial volume (Vi) of the fermenter is adjusted to 17 L after seeding. It is brought to 20 to 25 L in final.
- the driving parameters of fermentation are as follows:
- a glucose supply in the form of a concentrated solution at approximately 800 g / l is carried out so as to maintain the glucose content between 0 and 20 g / l in the fermenter.
- the biomass obtained according to Example 1 is:
- the biomass thus obtained is separated from the soluble fraction by centrifugal separation. Said soluble fraction is then microfiltered on a 0.14 m ceramic membrane at 60 ° C.
- the transmembrane pressure is set at a value between 0.2 and 0.6 bar and the microfiltration is conducted to obtain a volume concentration factor of 2.5 (100 liters of this microfiltered soluble fraction thus generate 40 liters of retentate "R1 And 60 liters of permeate "P1").
- the microfiltration permeate "P1" obtained at the end of Example 2 at 4% of dry matter is in particular ultrafiltered on a membrane with a cut-off threshold of 10 kDa, so as to obtain:
- a retentate "R2" at 10% dry matter containing peptides having a molecular weight greater than or equal to 5 kDa and oligosaccharides of high DP;
- P2 permeate at 1% dry matter, containing peptides having a molecular weight of less than 5 kDa and oligosaccharides DP less than or equal to 2.
- This "P2" permeate can then be filtered in particular on a reverse osmosis membrane (having a NaCl rejection rate of 93%), so as to obtain:
Abstract
Description
Claims
Priority Applications (7)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
MX2016011970A MX2016011970A (es) | 2014-03-18 | 2015-03-18 | Metodo para la permeabilizacion termica de una biomasa de microalgas. |
JP2016557884A JP2017507662A (ja) | 2014-03-18 | 2015-03-18 | 微細藻バイオマスの熱透過処理のための方法 |
CN201580014474.4A CN106103693A (zh) | 2014-03-18 | 2015-03-18 | 用于微藻生物质的热透化的方法 |
EP15718966.3A EP3119872A1 (fr) | 2014-03-18 | 2015-03-18 | Procédé de perméabilisation thermique d'une biomasse de microalgues |
US15/126,370 US20170081630A1 (en) | 2014-03-18 | 2015-03-18 | Method for thermal permeabilization of a microalgae biomass |
KR1020167022948A KR20160134657A (ko) | 2014-03-18 | 2015-03-18 | 미세조류 바이오매스의 열 투과화를 위한 방법 |
US15/943,665 US20180223245A1 (en) | 2014-03-18 | 2018-04-02 | Method for thermal permeabilization of a microalgae biomass |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
FR1452219 | 2014-03-18 | ||
FR1452219 | 2014-03-18 |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
US15/126,370 A-371-Of-International US20170081630A1 (en) | 2014-03-18 | 2015-03-18 | Method for thermal permeabilization of a microalgae biomass |
US15/943,665 Continuation US20180223245A1 (en) | 2014-03-18 | 2018-04-02 | Method for thermal permeabilization of a microalgae biomass |
Publications (1)
Publication Number | Publication Date |
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WO2015140467A1 true WO2015140467A1 (fr) | 2015-09-24 |
Family
ID=53008802
Family Applications (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
PCT/FR2015/050658 WO2015140467A1 (fr) | 2014-03-18 | 2015-03-18 | Procédé de perméabilisation thermique d'une biomasse de microalgues |
Country Status (7)
Country | Link |
---|---|
US (2) | US20170081630A1 (fr) |
EP (1) | EP3119872A1 (fr) |
JP (1) | JP2017507662A (fr) |
KR (1) | KR20160134657A (fr) |
CN (1) | CN106103693A (fr) |
MX (1) | MX2016011970A (fr) |
WO (1) | WO2015140467A1 (fr) |
Cited By (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR3056225A1 (fr) * | 2016-09-21 | 2018-03-23 | Inria Institut National De Recherche En Informatique Et En Automatique | Bioreacteur pour la selection de microalgues |
US10519204B2 (en) | 2014-07-18 | 2019-12-31 | Corbion Biotech, Inc. | Method for extracting soluble proteins from microalgal biomass |
Families Citing this family (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
FR3031987B1 (fr) * | 2015-01-26 | 2019-05-24 | Corbion Biotech, Inc. | Procede de fractionnement des composants d'une biomasse de microalgues riches en proteines |
CA3068983A1 (fr) * | 2017-07-05 | 2019-01-10 | Inventprise, Llc | Purification de polysaccharides pour la production de vaccins a l'aide d'enzymes lytiques, d'une filtration tangentielle et d'une chromatographie multimodale |
CN109734264A (zh) * | 2018-11-20 | 2019-05-10 | 江南大学 | 一种促进水华蓝藻内容物释放的方法 |
BR112022008618A2 (pt) * | 2019-11-08 | 2022-07-19 | Affibody Ab | Método e sistema para extração de uma proteína citoplasmática ou periplasmática |
Citations (7)
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US5330913A (en) | 1991-09-11 | 1994-07-19 | Hideo Nakayama | Method of disrupting the chlorella cell wall by cell rupture |
JPH0975094A (ja) * | 1995-09-14 | 1997-03-25 | Japan Kurorera Konsaruteeshiyon:Kk | クロレラ藻体由来の緑色抽出液とその製造法 |
WO2006095964A1 (fr) * | 2005-03-08 | 2006-09-14 | Hyun Jin Jin | Procede d'extraction d'un extrait liquide a partir d'une chlorelle |
WO2010104922A1 (fr) * | 2009-03-10 | 2010-09-16 | Srs Energy | Fractionnement d'une biomasse d'algues |
WO2015001261A1 (fr) * | 2013-07-04 | 2015-01-08 | Roquette Freres | Procede optimise de rupture des parois de chlorelles par broyage mecanique |
WO2015007997A1 (fr) * | 2013-07-19 | 2015-01-22 | Roquette Freres | Procédé optimise de rupture des parois de chlorelles par homogénéisation a très haute pression |
WO2015079169A1 (fr) * | 2013-11-29 | 2015-06-04 | Roquette Freres | Granules de farine de biomasse de microalgues riches en protéines et leur procédé de préparation |
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JPH01180832A (ja) * | 1988-01-05 | 1989-07-18 | Sanwa Kagaku Kenkyusho Co Ltd | 高ヨウ素含有淡水クロレラからの熱水抽出物、その製法、およびその用途 |
JP3430176B2 (ja) * | 1993-04-16 | 2003-07-28 | 日本合成化学工業株式会社 | アンギオテンシン変換酵素阻害ペプチドの精製方法 |
JPH08275793A (ja) * | 1995-04-06 | 1996-10-22 | Ishikawajima Harima Heavy Ind Co Ltd | 微細藻類を用いた有用高分子の製造方法並びにその有用高分子を用いた製紙方法と生分解性プラスチックの製造方法 |
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JPH09239275A (ja) * | 1996-03-10 | 1997-09-16 | Daicel Chem Ind Ltd | イミン化合物合成用触媒、及びこれを用いたイミン化合物の製造法 |
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-
2015
- 2015-03-18 MX MX2016011970A patent/MX2016011970A/es unknown
- 2015-03-18 JP JP2016557884A patent/JP2017507662A/ja active Pending
- 2015-03-18 EP EP15718966.3A patent/EP3119872A1/fr not_active Withdrawn
- 2015-03-18 KR KR1020167022948A patent/KR20160134657A/ko unknown
- 2015-03-18 CN CN201580014474.4A patent/CN106103693A/zh active Pending
- 2015-03-18 US US15/126,370 patent/US20170081630A1/en not_active Abandoned
- 2015-03-18 WO PCT/FR2015/050658 patent/WO2015140467A1/fr active Application Filing
-
2018
- 2018-04-02 US US15/943,665 patent/US20180223245A1/en not_active Abandoned
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WO2006095964A1 (fr) * | 2005-03-08 | 2006-09-14 | Hyun Jin Jin | Procede d'extraction d'un extrait liquide a partir d'une chlorelle |
WO2010104922A1 (fr) * | 2009-03-10 | 2010-09-16 | Srs Energy | Fractionnement d'une biomasse d'algues |
WO2015001261A1 (fr) * | 2013-07-04 | 2015-01-08 | Roquette Freres | Procede optimise de rupture des parois de chlorelles par broyage mecanique |
WO2015007997A1 (fr) * | 2013-07-19 | 2015-01-22 | Roquette Freres | Procédé optimise de rupture des parois de chlorelles par homogénéisation a très haute pression |
WO2015079169A1 (fr) * | 2013-11-29 | 2015-06-04 | Roquette Freres | Granules de farine de biomasse de microalgues riches en protéines et leur procédé de préparation |
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Cited By (5)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
US10519204B2 (en) | 2014-07-18 | 2019-12-31 | Corbion Biotech, Inc. | Method for extracting soluble proteins from microalgal biomass |
US10815281B2 (en) | 2014-07-18 | 2020-10-27 | Corbion Biotech, Inc. | Method for extracting soluble proteins from microalgal biomass |
FR3056225A1 (fr) * | 2016-09-21 | 2018-03-23 | Inria Institut National De Recherche En Informatique Et En Automatique | Bioreacteur pour la selection de microalgues |
WO2018055282A1 (fr) * | 2016-09-21 | 2018-03-29 | Inria Institut National De Recherche En Informatique Et En Automatique | Bioreacteur pour la selection de microalgues |
US11427796B2 (en) | 2016-09-21 | 2022-08-30 | Inria Institut National De Recherche En Informatique Et En Automatique | Bioreactor for the selection of microalgae |
Also Published As
Publication number | Publication date |
---|---|
JP2017507662A (ja) | 2017-03-23 |
US20180223245A1 (en) | 2018-08-09 |
MX2016011970A (es) | 2016-12-05 |
US20170081630A1 (en) | 2017-03-23 |
KR20160134657A (ko) | 2016-11-23 |
CN106103693A (zh) | 2016-11-09 |
EP3119872A1 (fr) | 2017-01-25 |
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