EP3365419A1 - Kit de bionettoyage et procédé d'élimination de films biologiques de substrats - Google Patents
Kit de bionettoyage et procédé d'élimination de films biologiques de substratsInfo
- Publication number
- EP3365419A1 EP3365419A1 EP16779177.1A EP16779177A EP3365419A1 EP 3365419 A1 EP3365419 A1 EP 3365419A1 EP 16779177 A EP16779177 A EP 16779177A EP 3365419 A1 EP3365419 A1 EP 3365419A1
- Authority
- EP
- European Patent Office
- Prior art keywords
- bio
- cleansing
- formulation
- kit
- substrate
- Prior art date
- Legal status (The legal status is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the status listed.)
- Granted
Links
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- XLYOFNOQVPJJNP-UHFFFAOYSA-N water Substances O XLYOFNOQVPJJNP-UHFFFAOYSA-N 0.000 claims description 12
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- KCXVZYZYPLLWCC-UHFFFAOYSA-N EDTA Chemical compound OC(=O)CN(CC(O)=O)CCN(CC(O)=O)CC(O)=O KCXVZYZYPLLWCC-UHFFFAOYSA-N 0.000 claims description 6
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- 239000001509 sodium citrate Substances 0.000 claims 2
- NLJMYIDDQXHKNR-UHFFFAOYSA-K sodium citrate Chemical compound O.O.[Na+].[Na+].[Na+].[O-]C(=O)CC(O)(CC([O-])=O)C([O-])=O NLJMYIDDQXHKNR-UHFFFAOYSA-K 0.000 claims 2
- VDDCANDLLVELCJ-UHFFFAOYSA-N 4-(3-carboxypropanoyloxyamino)oxy-4-oxobutanoic acid Chemical compound C(CC(=O)ONOC(=O)CCC(=O)O)C(=O)O VDDCANDLLVELCJ-UHFFFAOYSA-N 0.000 claims 1
- 108090000270 Ficain Proteins 0.000 claims 1
- 229910019142 PO4 Inorganic materials 0.000 claims 1
- ABLZXFCXXLZCGV-UHFFFAOYSA-N Phosphorous acid Chemical class OP(O)=O ABLZXFCXXLZCGV-UHFFFAOYSA-N 0.000 claims 1
- 229920000388 Polyphosphate Polymers 0.000 claims 1
- 108010077895 Sarcosine Proteins 0.000 claims 1
- YDONNITUKPKTIG-UHFFFAOYSA-N [Nitrilotris(methylene)]trisphosphonic acid Chemical compound OP(O)(=O)CN(CP(O)(O)=O)CP(O)(O)=O YDONNITUKPKTIG-UHFFFAOYSA-N 0.000 claims 1
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- DUYCTCQXNHFCSJ-UHFFFAOYSA-N dtpmp Chemical compound OP(=O)(O)CN(CP(O)(O)=O)CCN(CP(O)(=O)O)CCN(CP(O)(O)=O)CP(O)(O)=O DUYCTCQXNHFCSJ-UHFFFAOYSA-N 0.000 claims 1
- 235000019836 ficin Nutrition 0.000 claims 1
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- WYTZZXDRDKSJID-UHFFFAOYSA-N (3-aminopropyl)triethoxysilane Chemical compound CCO[Si](OCC)(OCC)CCCN WYTZZXDRDKSJID-UHFFFAOYSA-N 0.000 description 2
- 229910002013 Aerosil® 90 Inorganic materials 0.000 description 2
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- 239000004475 Arginine Substances 0.000 description 1
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- ZAMOUSCENKQFHK-UHFFFAOYSA-N Chlorine atom Chemical compound [Cl] ZAMOUSCENKQFHK-UHFFFAOYSA-N 0.000 description 1
- 241000173697 Euchloe naina Species 0.000 description 1
- YTRPTVLTUWVLKO-ZDUSSCGKSA-N Ficine Natural products CN1CCC[C@H]1C1=C(O)C=C(O)C2=C1OC(C=1C=CC=CC=1)=CC2=O YTRPTVLTUWVLKO-ZDUSSCGKSA-N 0.000 description 1
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- 239000004472 Lysine Substances 0.000 description 1
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- YTRPTVLTUWVLKO-UHFFFAOYSA-N ficine Chemical compound CN1CCCC1C1=C(O)C=C(O)C2=C1OC(C=1C=CC=CC=1)=CC2=O YTRPTVLTUWVLKO-UHFFFAOYSA-N 0.000 description 1
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- SWQJXJOGLNCZEY-UHFFFAOYSA-N helium atom Chemical compound [He] SWQJXJOGLNCZEY-UHFFFAOYSA-N 0.000 description 1
- 229910002011 hydrophilic fumed silica Inorganic materials 0.000 description 1
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- 230000002093 peripheral effect Effects 0.000 description 1
- 239000002985 plastic film Substances 0.000 description 1
- 229920006255 plastic film Polymers 0.000 description 1
- 231100000719 pollutant Toxicity 0.000 description 1
- 229920000728 polyester Polymers 0.000 description 1
- 229920001223 polyethylene glycol Polymers 0.000 description 1
- 229920001184 polypeptide Polymers 0.000 description 1
- 238000004321 preservation Methods 0.000 description 1
- 238000007639 printing Methods 0.000 description 1
- 102000004196 processed proteins & peptides Human genes 0.000 description 1
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- 231100000370 skin sensitisation Toxicity 0.000 description 1
- AJPJDKMHJJGVTQ-UHFFFAOYSA-M sodium dihydrogen phosphate Chemical compound [Na+].OP(O)([O-])=O AJPJDKMHJJGVTQ-UHFFFAOYSA-M 0.000 description 1
- SUKJFIGYRHOWBL-UHFFFAOYSA-N sodium hypochlorite Chemical compound [Na+].Cl[O-] SUKJFIGYRHOWBL-UHFFFAOYSA-N 0.000 description 1
- 229910000162 sodium phosphate Inorganic materials 0.000 description 1
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Classifications
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/16—Organic compounds
- C11D3/38—Products with no well-defined composition, e.g. natural products
- C11D3/386—Preparations containing enzymes, e.g. protease or amylase
- C11D3/38681—Chemically modified or immobilised enzymes
-
- B—PERFORMING OPERATIONS; TRANSPORTING
- B08—CLEANING
- B08B—CLEANING IN GENERAL; PREVENTION OF FOULING IN GENERAL
- B08B7/00—Cleaning by methods not provided for in a single other subclass or a single group in this subclass
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
- C11D17/04—Detergent materials or soaps characterised by their shape or physical properties combined with or containing other objects
- C11D17/041—Compositions releasably affixed on a substrate or incorporated into a dispensing means
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D17/00—Detergent materials or soaps characterised by their shape or physical properties
- C11D17/04—Detergent materials or soaps characterised by their shape or physical properties combined with or containing other objects
- C11D17/049—Cleaning or scouring pads; Wipes
-
- C—CHEMISTRY; METALLURGY
- C11—ANIMAL OR VEGETABLE OILS, FATS, FATTY SUBSTANCES OR WAXES; FATTY ACIDS THEREFROM; DETERGENTS; CANDLES
- C11D—DETERGENT COMPOSITIONS; USE OF SINGLE SUBSTANCES AS DETERGENTS; SOAP OR SOAP-MAKING; RESIN SOAPS; RECOVERY OF GLYCEROL
- C11D3/00—Other compounding ingredients of detergent compositions covered in group C11D1/00
- C11D3/02—Inorganic compounds ; Elemental compounds
- C11D3/12—Water-insoluble compounds
- C11D3/124—Silicon containing, e.g. silica, silex, quartz or glass beads
Definitions
- the present invention relates to a kit for the enzymatic bio-cleansing and a method based on said kit to remove biofilms from surfaces made of stone, wood, ceramic or other materials and preferably from surfaces of delicate and unique works of art e.g. an ancient mosaics or stone works.
- the present invention provides a new and inventive solution to the problem of removing biofilms comprising microorganisms such as molds, lichens, cyanobacteria, microalgae, etc. without affecting the integrity of a delicate and valuable surface.
- Microorganisms such as molds, cyanobacteria, microalgae, lichens, etc., often in combination with organic and inorganic substances, gradually adhere to floors, walls or stone-works in particular when they are exposed to atmospheric agents.
- These organic deposits technically defined as 'biofilms', deteriorate the substrate they adhere thereto by altering, often irreparably, firstly the surface aesthetics and then the surface structure of the substrate.
- the kinetics of the surface deterioration induced by biofilms depends on the nature of the substrate and therefore on the substrate material and porosity.
- formulations containing enzymes have been commercially introduced.
- formulations containing enzymes present a selective cleaning action.
- biofilm removal from the surface occurs as the result of the enzyme attack on a selective bond recognized by the enzyme chemical structure.
- enzymes act on the chemical mechanism of biofilm removal.
- a list of enzyme-based cleaning compositions can be found, for example, in the book ' L'uso degli enzimi nella pulitura di opere policrome ' by P. Cremonesi (Il Prato ed. 2002), or in the text-book ' La Chimica nel Restauro ' of M. Matteini and A. Moles (Nardini ed. 2007).
- the use of such compositions is particularly suitable, and sometimes it is the only choice when the use of traditional cleaning systems would result in damage or destruction of the work to be restored.
- WO2014006424A1 discloses a novel cleaning material that finds particular application in cleaning processes, preferably of fabrics.
- the novel cleaning material operates in the presence of limited quantities of water and use a cleaning formulation which comprises solid polymeric cleaning particles in combination with suitable detergent component.
- the system utilizes a cleaning formulation wherein the detergent component is an enzyme immobilized on the solid polymeric cleaning particles.
- Enzyme immobilization improves cleaning performance, as well as the ability to re-use these detergent components over multiple washes, thereby providing environmental and economic benefits.
- WO2014006424A1 the use of the novel cleaning material to stone-like substrates is not provided, and it appears clearly out of the scope of this patent.
- the pores of the stone-like substrate can trap the solid polymeric cleaning particles, which degrade and yellow under the effect of solar radiation. For this reason, they are unsuitable in the field of historic heritage where restoration must not alter the original substrate nor leave residues thereon.
- This patent claims a single-use-composition based on an insoluble enzyme preparation comprising an enzyme immobilized on a solid carrier (preferably fumed silica), wherein the insoluble enzyme preparation is dispersed in the single-use-composition.
- a solid carrier preferably fumed silica
- the single-use insoluble enzyme preparations of WO2014067933A1 are particularly useful in care compositions and food applications, while washing or cleaning applications are only described generally in a useless way for the skilled in the art.
- no useful cleaning compositions is disclosed, although the inventors declare that they can be used for providing enzyme activity to single-use hard surface (e.g. surface of equipment used in the food industry) preparations.
- removal of biofilms is cited only with reference to biofilms formed in lens cases, which are very different from biofilms formed on stone-like substrate. In this case, the inventors do not disclose any useful composition also.
- embedding enzymes in coatings, paint and varnish yields a protection against the settlement of algae and mold. Therefore, such compositions prevent biofilm formation and they are not useful to remove biofilms already adhered to the substrate.
- Example 4 the only cleaning composition of WO2014067933A1 is presented in the Example 4 and provides lipase activity to a commercial household cleaner. It is relevant to point out that first, the formulation of Example 4 is an aerosol (and not a liquid or gel), and then that lipase is immobilized on Aerosil® 90 an hydrophilic fumed silica manufactured by Evonik Industries AG, which is characterized by a non-porous structure (see for instance data-sheet of Aerosil® 90). In addition, the results of the performance testing of Example 4 are obscure and are not useful to the skilled in the art wishing to develop a liquid or gel formulation useful to remove biofilms from stone-like substrates.
- WO2014067933A1 relates to the use of insoluble enzyme preparations, having reduced safety concerns compared to the free enzyme.
- enzyme activity enhancement and cost optimization is out of the scope of this invention.
- it refers to an insoluble enzyme 'single-use-composition', i.e. a composition, which after its primary use is either discarded, used up or rendered in a form unsuitable for subsequent reuse.
- the teachings provided by C-Lecta GmbH in the international application WO2014067933A1 are not useful to remove biofilms from stone-like substrates, by means of an enzyme composition wherein the enzyme can be reused. Therefore, the compositions disclosed are definitely not a viable alternative to known biocides or enzyme formulations currently used in the restoration and maintenance of historic heritage or in fields having similar problems with regard to biofilm removal from stone-like substrates.
- bio-cleansing formulation including a stable enzyme system that is useful to remove biofilms from stone-like substrates, and that is simple to synthesize and conserve, and compared to prior art solutions, fulfill the requirements of demanding fields such as historic heritage or fine-arts.
- Such requirements covers improved cleaning efficiency and flexibility in use, low cost per unit surface treated, preservation of substrate integrity (i.e. no substrate damage nor alteration, no residues on substrate after treatment) and finally negligible risks for users and for the environment.
- the present inventors intend to overcome the limitations existing in the state of the art related to enzyme cleaning systems useful for removing biofilm from surfaces, particularly of natural stone, stone-like materials, ceramic and wood.
- bio-cleansing kit including an enzymatic system as set forth in the appended independent claim.
- Said bio-cleansing kit is useful to remove pre-existing biofilms from various kinds of substrates, preferably stone-like substrates, more effectively and less expansively than known solutions.
- an important task of the present invention is to provide a stable bio-cleansing kit having an improved enzyme cleansing activity and which is storable and reusable several times.
- another important task of the present invention is to provide a bio-cleansing kit, which can be customized according to the needs of different fields, particularly of cultural heritage.
- a second important object of the present invention is a method for making said bio-cleansing kit , as set forth in the appended independent claim, and particularly a scalable manufacturing method that is easily achievable with known technologies at competitive costs compared to the enzyme-based cleaning solutions already in the market.
- a third object of the present invention is to provide a method for removing biofilms from substrates , preferably stone-like substrates, which allows cost-cutting and a simplification of restoration plans, particularly in the field of cultural heritage.
- a further object of the present invention is to provide a bio-cleansing kit and to develop a method for removing biofilm from substrates, based on said kit, which do not damage or alter the substrate, and do not present chemical hazard for users as well for the environment.
- the present inventors have made many studies related to enzyme systems consisting of enzymes immobilized on inorganic particles. These studies were mainly directed to address optimization of the enzyme system, enhancement of enzyme cleansing activity, and optimization of a formulation comprising such enzyme systems compatible with the working conditions of restorers or other professionals involved in restoration interventions.
- an enzymatic bio-cleansing kit suitable to remove biofilms from substrates, preferably stone-like substrates, comprising: an enzyme system consisting of immobilized enzymes on inorganic particles, preferably silica nanoparticles; a formulation comprising said enzyme system; a support for said formulation.
- said enzyme is trypsin, but it can be selected from those known in the art according to the characteristics of the biofilm to be removed from the substrate.
- said formulation impregnates the support consisting of a hydrophilic fabric but in another embodiment, the formulation is coated on a flexible or rigid support, such as a plastic film, so that the formulation forms a substantially distinct phase from such support. Still in another embodiment, the kit does not have a support (i.e. a 'support-less' kit configuration) and said formulation is dispersed in a means being a component of said formulation. Said means is in form of a liquid, or a gel, or a solid means suitable to deliver and to put in contact the enzyme system with the substrate to be cleaned.
- additional components are included in the bio-cleansing kit such as means to control the cleaning process parameters (i.e. temperature, moisture content and pH), in order to optimize the enzyme action and thus substrate cleaning, according to the characteristics of the enzyme system and the external climatic conditions.
- Additional components includes also means to facilitate adhesion to the substrates, particularly on vertical substrates, and protective films to prevent contamination of the formulation by external agents, as well as to maintain the proper moisture content and temperature in the impregnated support to enable an effective cleaning action.
- the present invention also claim a method for removing biofilms comprising microorganisms such as molds, cyanobacteria, microalgae, lichens, etc. without affecting the integrity of a delicate and valuable surface, in particular works of art.
- said method starts with an analysis of the substrate and the environmental conditions which help to select the proper enzymatic system and bio-cleansing kit configuration. Said enzyme formulation and the bio-cleansing kit are then freshly prepared. The method proceeds with kit application on the biofilm-contaminated substrate. After the enzymatic cleansing process has concluded, the kit is removed from the substrate and stored for future use.
- the enzyme formulation is not freshly prepared, and a pre-stored formulation or kit is used.
- the method includes activation of the enzymatic system.
- the enzyme formulation is in form of a gel, which is activated before application on the work of art ('wet form'). In still another embodiment, the activation is performed after the application on the work of art ('dry form').
- the enzymatic bio-cleansing kit and the related method for removing biofilms from substrates are characterized in that the formulation comprising the enzyme system can be easily removed from the substrate by water washing so that no formulation residues are left on the substrate after the treatment.
- the term ' particle ' as used in the description and in the claims of the present invention shall designate an aggregate of atoms, molecules or other fundamental constituents, such aggregate having sub-micrometric size or super-micrometric and a substantially spherical shape but also a non-symmetrical shape.
- the terms 'nanoparticle' or 'nanostructure', singular or plural, shall indicate exclusively a particle of size less than about 1 micrometer.
- biofilm ' it is meant any association, simple or complex, of organic and/or inorganic substances with microorganisms such as molds, algae, bacteria, lichens and the like, wherein such association adhere to a substrate, for instance a rock substrate.
- bio-cleansing ' shall refer to the removal of a pre-existing (i.e. a biofilm covering the substrate before removal) biofilm from a substrate taking advantage of the bio-cleansing system according to the present invention.
- ' immobilized ' it is meant any chemical bound (covalent bond, ionic, hydrogen bond or Van der Waals interaction), physical bound (e.g. electrostatic attraction) or physio-chemical bond that binds or associates permanently or temporarily an enzyme, or more in general a polypeptide, to at least one particle or a nanoparticle.
- the same reference sign will be used to indicate the ' support ' (or, more generally, the 'means') and the 'impregnated support' (or more generally the 'impregnated means'), i.e. the support where the formulation containing the enzyme system has been applied thereto.
- the same reference sign will also be used to identify the corresponding dry or dehydrated form of the 'impregnated support'.
- ' preserving substrate integrity ' and similar wordings (e.g. 'to preserve substrate integrity'), as used in the description and in the claims, will be referred to a cleansing treatment of a substrate that does not damage nor affect the original (i.e. before biofilm contamination) aesthetic, colors and surface structure of the substrate, according to the best practices of works of art restorations.
- the kit for enzymatic bio-cleansing according the present invention and the related methods based on said kit have a number of remarkable advantages over prior-art solutions. These benefits and advantages are described in the following.
- the kit is stable and presents an improved enzyme cleansing capability, compared to known gel formulations having a greater enzyme content.
- the inventors experimentally demonstrated that removal of the same biofilm can be achieved with up to 20 times less enzyme, thus reducing enzyme cost and allergenic potential and immunogenicity.
- the kit for bio-cleansing according the present invention is storable and reusable up to 8 times (single-use is also possible at competitive costs), which represents an outstanding achievement in stone-like biofilm cleaning.
- a storable and multiple-use kit allow to both lowering the product cost (due to economies of scale in manufacturing) and the cost for unit of cleaned surface.
- this features allow to improve flexibility in management and scheduling of extensive restoration and maintenance programs, particularly in the field of cultural heritage.
- the kit according to the present invention is non-toxic for users and the environment because the silica nanoparticles are bound in a gel or in a liquid formulation, no aerosol containing free nanoparticles and/or enzyme is formed and little enzyme is used.
- formulations based on micro-scale particles can also prepared.
- bio-cleansing kit according to the present invention and the related bio-cleansing method do not damage the substrate (during use, salts formation is minimized on the surface which may affect the art-work) as traditional biocide-based cleansing composition, or known composition based on enzymes bound to polymeric particle.
- the formulation residues remained on the substrate after the treatment, can be easily removed from the substrates using water washing.
- particles trapped in the pores of the substrate do not affect the surface integrity because, being metal oxide (e.g. silica), they are stable and chemically compatible with many rock compositions.
- bio-cleansing kit according to the present invention is simple to prepare in various configurations, as the examples herein provided demonstrate.
- the advantages of the bio-cleansing kit according to the present invention allow to remove biofilms from various kinds of substrates, particularly stone-like substrates, more effectively, economically and safely than known solutions. It will apparent to those skilled in the art that the advantages of the present invention cannot be achieved by prior-art cleaning system based on enzymatic systems.
- the enzymatic bio-cleansing kit is indicated with the number (1) and comprises a formulation (10) containing an enzymatic system (20); a means (30) above which said formulation (10) is applied, or in which said formulation is dispersed; a protective film (40) and temperature control means (50).
- Said enzyme system (20) includes trypsin immobilized to mesoporous silica nanoparticles by means of a process that will be described later.
- the inventors have found that an effective bio-cleansing action can be achieved by immobilizing a quantity of trypsin between about 3 and about 9 mg/g onto mesoporous silica nanoparticles having diameter between about 100 and about 300 nm and a pore diameter between about 2.1 and about 2.3 nm.
- different values are possible depending on the morphology and chemical composition of the nanoparticles, on the enzyme and the biofilm compositions.
- the bio-cleansing treatment by means of the kit (1) requires that the support (30) is placed over the substrate (L) and requires that the formulation (10) comes effectively in contact with this substrate.
- said formulation (10) is in the form of an enzyme gel that is applied on a rigid or flexible support (30) so that the gel impregnate effectively this support.
- a hydrophilic non-woven sterile gauze made of a viscose/polyester (67%/33%) fabric e.g. produced by Dermatess Mater Aid.
- other kind of fabrics can be used, but also hydrophilic or superabsorbent polymers, provided they have suitable wetting properties to ensure a good impregnation of the substrate with the gel (10) containing the enzyme system (20).
- the inventors have experimentally found that maintaining the right moisture content of the impregnated support (30) constitutes a fundamental requirement to ensure an effective bio-cleansing action.
- said protective film (40) may be an extensible film such as Parafilm® or Domopak® which is placed over the surface (S) of the bio-cleansing kit (1) opposite to the substrate (L) to be cleaned, as illustrated by way of example but not limitation in the Figure 1.
- the protective film (40) may be shaped to protect also the active surface (S') of the kit that will be placed in direct contact of the substrate (L) during the bio-cleansing treatment. This configuration is particularly useful when the bio-cleansing kit is not immediately used and must be therefore stored for future use.
- the protection of the kit active surface (S') can be simply achieved by including an additional protective film (40').
- said bio-cleansing (1) kits comprises temperature control means (50), external to said kit and to the impregnated support (30).
- Said temperature control means (50) can be a temperature-controlled hair-dryer, an aquarium heater or other equivalent devices, but other advantageous solutions useful to control and maintain the temperature within a predefined range during the bio-cleansing treatment have been identified.
- bio-cleansing kit described herein in the best mode is suitable to numerous variations, all falling within the more general scope of the present invention.
- the bio-cleansing kit can be optimized for removing biofilms from vertical walls, or from uneven or non-uniform surfaces or from large floorings.
- Some components of the kit, as the temperature control means, may advantageously be integrated in the support of the formulation to further increase the application flexibility of the bio-cleansing kit according to the present invention.
- Such alternative solutions will be later described with reference to other embodiments.
- kit configuration ensure optimal hydration of the formulation without altering the pH value. It also maintain a constant contact between the active surface (S') and the substrate by increasing the cleaning activity of the enzyme.
- Step 1 Preparation and functionalization of the particles
- Suitable mesoporous silica nanoparticles can be easily synthesized by known procedures; alternatively, various types are available commercially, for example from Sigma-Aldrich SBA-15 (777242), or MCM-41 (643 645).
- the silica nanoparticles may be functionalized using various known techniques, for example making them react at room temperature for about 2 hours in a solution of cyclohexane with 3-aminopropyltriethoxysilane (APTES) and n-propylamine, both in the proportion of between about 1.5 and about 3.0% v/v, so as to obtain a first precipitate.
- APTES 3-aminopropyltriethoxysilane
- said first precipitate is filtered and washed with cyclohexane, and finally dried in an oven at about 40 °C for a sufficient time to remove solvent residues in order to obtain functionalized silica particles.
- the functionalized silica particles are then dispersed in a buffer solution containing a predefined amount of trypsin.
- a good enzyme immobilization is achieved with an amount of trypsin comprised between about 3 and about 9 mg per gram of nanoparticles and a phosphate buffer having a pH of about 6 in a concentration of between about 10 and about 20 microMol.
- This dispersion is continuously stirred for about 2 hours at room temperature to obtain a second precipitate. After filtering and drying said second precipitate, for example in air within a protected environment, it is obtained the enzyme system in the form of a white powder composed of silica nanoparticles where trypsin molecules are immobilized thereto.
- compositions that remove specific biofilms from specific substrates by a proper selection of the particles and of the enzyme.
- customization of the enzyme system can be achieved by acting on one or more of the following parameters: particles composition, size or surface morphology; enzyme type or amount in the formulation.
- silica particles with dimensions greater than about 1000 nm can be usefully used, as well as particles of other metal oxides such as zirconia (ZrO 2 ), titania (TiO 2 ), or mixed oxides such as zirconia/titania.
- spherical nanoparticles characterized by a uniform distribution of immobilized enzyme are particularly useful for removing biofilms from macroporous or non-homogeneous substrates.
- particles having an oblong shape it is preferable using particles having an oblong shape to improve the contact surface of the immobilized enzyme system with the biofilm on the substrate.
- hydrolytic and non-hydrolytic enzymes can be used, e.g. pepsin, lipase, papain, amylase, chymotrypsin, elastase, xylanase, cellulase, ligninase, ficine, bromelain.
- the immobilization process may not require nanoparticle functionalization, but only the creation of a non-covalent bound between the enzyme and the substrate.
- the process parameters e.g. the pH of the precursor solution, the temperature, concentration, dripping speed of the precursor
- the process parameters can be altered to obtain changes to the morphology, shape or average size of the particles.
- Step 3 Bio-cleansing kit preparation and conservation
- the enzyme system (20) is dispersed in a buffer solution, preferably having a pH between about 7.2 and about 8.0.
- This solution may be, for example, a phosphate buffer NaH 2 PO 4 • H 2 O, but other equivalent solutions may be used.
- a gelling agent is then added so as to obtain the formulation (10) in the form of a homogeneous gel of proper consistency that can be coated on the support (30).
- said gelling agent is methylcellulose (e.g.Tylose® MH300P), added in a ratio of about 1:10 w/w, or another compound equivalent conveniently chosen to obtain a suitable viscosity and to maintain the most suitable pH value for an effective trypsin (or other enzyme) action.
- methylcellulose e.g.Tylose® MH300P
- a uniform layer of gel formulation (10) is formed on the support (30) by dip-coating a pre-cut fabric into a vessel containing the formulation (10) for a time long enough to ensure a good impregnation (e.g. 5 minutes or more).
- other known coating techniques e.g. roll-coating or spraying
- these known techniques allow the formulation (10) deposition on specific areas of the support (30) according to a predefined pattern.
- the protective film (40) preferably a stretchable film (e.g. Parafilm®, Domopak® or other extensible films), concludes the preparation of the bio-cleansing kit according to the best mode of the present invention.
- the protective film (40) can be shaped to cover also the active surface (S').
- a second protective film (40') can be applied on the active surface (S') in direct contact with the biofilm-contaminated substrate (L).
- one or more protective films can be shaped and applied to protect the surfaces S, and/or S' of the kit according to the various conditions of use and conservation of the kit itself.
- the bio-cleansing kits thus prepared is ready to be applied to the substrate (L) according to a procedure that will be described below.
- the bio-cleansing kit can be stored and used several days or months after its preparation.
- drying of the impregnated support (30) must be included in the kit preparation procedure.
- This additional step can be performed by placing the kit in a protected environment such as a vented thermostat dryer.
- the inventors have surprisingly found that after drying the support (30) impregnated with the gel formulation (10) can be stored even for months without losing its bio-cleansing action properties.
- the impregnated support (30) is kept in a plastic container, or wrapped in the protective films (40,40'), and stored in a standard refrigerator at temperature of about 4 ° C degrees.
- the enzymatic system (20) is trapped in the dried support and is not dispersed.
- the enzyme autolysis is prevented or minimized.
- Restoration of the cleaning properties before use requires bringing the kit to room temperature and dripping a few drops of buffer solution on the support to re-activate the formulation.
- the inventors have found that the formulation (10) in the form of a gel should not be dried and preserve well for a long time without losing its cleaning properties.
- the bio-cleansing kit (1) thus prepared is useful for removing pre-existing biofilms from substrates, preferably stone-like substrates by means of a method comprising the following steps. Said method is described hereinafter with reference to the best mode of the present invention.
- Step 1 Analysis of the substrate and the environmental conditions
- the method for removing biofilm starts with a preliminary step which includes an analysis of the characteristics of the surface to be treated (material, substrate porosity, biofilm type and severity of attack, etc.) as well as the local environmental conditions (temperature, humidity, presence of wind and pollutants, etc.) which may affect the bio-cleansing treatment. Care must be taken, in case the substrate is a valuable work of art such as an ancient mosaic.
- This preliminary step allows the selection of the most suitable bio-cleansing kit (particularly the enzyme system) according to the working conditions and other requirements.
- Step 2 Bio-cleansing kit pre-application preparation
- the method for removing biofilms proceeds with the preparation of the bio-cleansing kit.
- the support (30) which in the best mode is a hydrophilic fabric (e.g. Dermatess Mater Aid), is first cut to size according to the shape and surface extension of the stone-work contaminated by the biofilm.
- the bio-cleansing kit is ready for application on the surface (L) of the stone-work to be cleaned.
- the bio-cleansing kit was previously made and stored (see the kit producing method in the best mode described hereinabove)
- the bio-cleansing method requires the enzymatic system (20) activation, i.e. the restoration of the proper formulation moisture content, temperature and pH.
- the activation of the enzymatic formulation (10) can occur both before and after the application of the bio-cleansing kits on the surface (L) of the stone-work.
- a buffer solution (preferably a phosphate buffer) is sprayed to properly wet the impregnated hydrophilic fabric support (30).
- the bio-cleansing kit is thus ready for the next application step.
- the enzyme formulation (10) is activated after the application of the bio-cleansing kits on the surface (L) of the object to be treated.
- the buffer solution is sprayed directly on the impregnated support (30).
- Step 3 Application of the bio-cleansing kit on the surface
- the restorer or other professionals, applies the bio-cleansing kit on the substrate (L), taking care that the entire surface (L), flat or curved, is in contact with the formulation (10) containing the enzyme system (20), which in the best mode is in the form of a gel.
- enzyme system activation can also be made in the present step 3.
- an optimal bio-cleansing kit activation can achieved by, first, applying a phosphate buffer in form of a gel (and not in liquid form) directly on the substrate (L), and then applying the impregnated fabric (30) with the enzyme gel (10) over the phosphate buffer layer.
- this arrangement limits salts formation which may deteriorate the stone surface and must be avoided (especially in treatment of historic art-works).
- it prevents migration of particles-immobilized enzyme from the support, and finally, it limits the use of buffer solution to humidify the kit, further simplifying the bio-cleansing process and reducing costs.
- Domopak® or other extensible films such as Parafilm®
- this component is not already part of the of bio-cleansing kit.
- the method according to the present invention requires waiting the necessary time to allow a selective and satisfactory cleaning of the substrate by the enzyme included in the kit.
- cleaning times between about 30 minutes to about 6 hours are sufficient for effective bio-cleansing of stone-works, provided that the suitable temperature, pH and moisture content are maintained.
- the temperature control means (50) it is possible to adjust the temperature control means (50), according to the external environmental conditions.
- the temperature control unit (50) is an aquarium heater which is a component external to the support (20). Nevertheless, the inventors have identified other advantageous solutions, which will be described with reference to other embodiments.
- Step 4 Removal of the kit from the substrate
- the kit is removed from the substrate.
- the excess formulation (10) is removed by rinsing the cleaned surface with demineralized water or by dabbing the surface with a sponge or tissue soaked with demineralized water.
- the biofilm removing method according to the present invention did not alter the original colors nor the substrate of the stone works, as will described in the following examples.
- the bio-cleansing method according to the present invention concludes with the storage of the bio-cleansing kit, in a home or laboratory refrigerator at a temperature of about 4 °C.
- This step includes de-hydration of the impregnated support (30) (as described previously) to de-activate the enzyme system, and finally the protection of the kit (1) by means of a protective film (40) or other technically equivalent means.
- FIG. 2 shows stone substrates that were used for testing the bio-cleansing kit and the biofilm removal process herein disclosed.
- the inventors have experimentally verified the efficacy of the kit and the cleaning process on two works of art from the Baths of Caracalla in Rome: a marble capital and a black and white mosaic floor tiles.
- both works were contaminated by a biofilm consisting of a heterogeneous microbial/fungal consortium comprising, inter alia, invertebrates ( Macrobiotus Tardigrado ), protozoa ( Aspidisia ) microalgae, fungi ( Zygomycetes and Ascomycetes ) and by a multitude of bacteria.
- the kit was prepared according to the teachings of the best mode of the present invention.
- Supports made of hydrophilic gauze (from Dermatess) 5x5 cm 2 in size were dip-coated with a gel formulation of methylcellulose (Tylose MH300P) comprising trypsin immobilized on mesoporous silica nanoparticles (synthetized by the inventors via standard techniques) in a percentage comprised between about 5 and about 10%.
- the temperature was maintained within a range between about 30 °C and about 40 °C using an aquarium heater as temperature control means.
- the proper moisture content of the formulation and a pH between about 7.2 and about 8.0 was maintained by gently wetting the hydrophilic fabric with buffer solution.
- the kit was protected with a Parafilm® film.
- the impregnated fabric with the bio-cleansing dehydrated gel was stored for 5 months in a home refrigerator. Once reactivated with a phosphate buffer solution, it was possible to reuse the same kit up to 8 times thus demonstrating that bio-cleansing efficacy is maintained over time according to the teachings of the present invention.
- the inventors found that the combination of trypsin and silica mesoporous nanoparticles enhanced the biofilm removal capability and reusability compared to prior-art cleansing compositions (having the same enzyme content), particularly formulations based on enzyme immobilized on fumed silica or formulations based on free enzyme. According to the best knowledge of the present inventors, this achievement is not obvious and although further research and experiments are required to understand better this phenomenon, it may depend on the peculiar enzyme distribution or charge distribution around the nanoparticle induced by the mesoporous structure.
- Bio-cleansing kits substantially similar to that of Example 1 were applied on different substrates: rock (basalt, marble, colored marble), terracotta, plastered and painted walls (with tempera paint or washable paint). The surfaces of the samples suffered a biofilm attack consisting principally of mold.
- kits were applied on the substrates both in wet form (i.e. gel enzyme activation before application) and in dry form (i.e. gel enzyme activation after application).
- the combination of the hydrophilic fabric support and the gel formulation showed an optimal adaptation to the uneven surface profile of the different sample materials.
- the bio-cleansing treatment conducted for about 90 minutes was effective and easy to perform.
- the same results were achieved on a wooden surface (natural, polished oak) and a plastic surface (transparent methacrylate) both suffering a mold attack.
- bio-cleansing kit herein disclosed can effectively remove biofilms from substrates different than stone-like materials, or from substrates characterized by a non-uniform surface morphology, thus achieving a further object of the present invention.
- Example 3 Treatment of substrates having complex shape
- bio-cleansing kit facilitate removal of biofilm from the surface of stone-work having complex shape.
- a suitable elastic pre-shaped hydrophilic fabric (30) with the enzyme formulation (10), it is advantageously possible to remove biofilm from curved or complex surfaces such as columns or statues heads.
- the inventors have found useful the impregnation of standard hydrophilic pre-shaped elastic gauzes and tubular nets used in wound treatment (e.g. produced by 3M®).
- the formulation (10) can be deposited on specific areas of the support, in this case using standard printing techniques such as ink-jet, or screen printing frames, depending on the viscosity of the formulation.
- the bio-cleansing kit is 'supportless' i.e. it present a configuration which does not comprise any support (30).
- the formulation (10) is directly applied on the biofilm-contaminated substrate (L) and includes a dispersion of the enzymatic system (20) in a suitable medium (30') which is part of the formulation (10) itself.
- Said means (30') can be a gel (e.g. a suitable gel buffer solution); in this case, formulation application is performed with a spatula or brush, a technique, which is also suitable for application on vertical substrates.
- the formulation and the detached biofilm can be removed by simply washing the substrate (L) at the end of the bio-cleansing treatment.
- the alleged Figure 5 shows the results of the bio-cleansing treatment on two stone works located in Palazzo Marchesale in the town of Arnesano, Lecce (Italy). This historic site was selected for testing the bio-cleansing kit in the 'supportless' configuration according to the second embodiment of the present invention. In fact, the stone works were heavily contaminated by a variegated association of lichens and other microorganism which other cleansing technique failed to remove.
- the same formulation of Example 1 was freshly prepared, applied to the substrates and maintained in contact of the substrates for about 30 40 minutes. The residues of the gel formulation on the substrates were completely removed with ease by using demineralized water. As the Figure 5 clearly demonstrate, the bio-cleansing kit and the related method removed completely lichens, thus revealing the original substrate without affecting its integrity and without leaving residues of the formulation after the treatment.
- the enzymatic bio-cleansing kit comprehends fixing means (70) to promote adhesion between the substrate and the active surface (S') of the kit and thus enzyme activity especially when removing biofilms from vertical surfaces such as a wall.
- fixing means (70) to promote adhesion between the substrate and the active surface (S') of the kit and thus enzyme activity especially when removing biofilms from vertical surfaces such as a wall.
- said fixing means (70) is a component integrated to the bio-cleansing kit, as schematically depicted in the attached Figure 3, and it is preferably a reversible adhesive system applied along the peripheral edges of the support (30).
- a reversible adhesive system applied along the peripheral edges of the support (30).
- adhesive systems can be advantageously used: double-sided adhesive tape for moist and delicate surfaces (e.g. 3MTM ActiveTM Strips or other tapes for medical tape), velcro-based fastening products, micro-suction systems, but also water glue or other physical or chemical reversible adhesives.
- said fixing means (70) is an external non-integrated component of the bio-cleansing kit, which can retain the bio-cleansing kit in contact with the wall or floor to be cleaned, such as a frame fixed to a scaffold or a temporary platform, or other technically equivalent known solution.
- Bio-cleansing kits advantageously allows an effective biofilm removal from vertical walls (L), thus achieving a further object of the present invention.
- the inventors have found useful impregnating an hydrophilic fabric (30) with the same gel formulation (10) of the best mode.
- the gel presents a good tackiness which limits fall by gravity and the fabric structure has proved a good wettability, because dripping by gravity of the buffer solution is effectively balanced by the capillary rise of the same solution. Nevertheless, if needed, the correct impregnation of the upper portions of the impregnated fabric (30) can be adjusted by providing further buffer solution.
- the temperature control unit (50) may be an external component, such as an aquarium heater or a standard IR lamp, or it can be integrated to the bio-cleansing kit as in the second embodiment.
- the bio-cleansing kit includes two additional elements consisting of temperature control means (50) integrated to the bio-cleansing of enzymatic kits (which is not an external component as in the best mode), and a humidifier (80).
- the temperature control unit e.g. printed resistive heating elements manufactured by GSI
- the temperature control unit e.g. heating microwires such as Gerbing MICROWIRE® Heat Technology
- a humidifier for home use or a precision humidifier for lab are both suitable.
- the process conditions are managed by a control system (90).
- the formulation and the support can be the same by the preferred embodiment.
- the formulation containing the enzymatic system (20) comprises a complexing agent capable of forming a complex with the substances of the substrate (or biofilm) inhibiting the enzymatic activity of trypsin.
- a complexing agent capable of forming a complex with the substances of the substrate (or biofilm) inhibiting the enzymatic activity of trypsin.
- the inventors have found useful EDTA (ethylenediaminetetraacetic acid), a chelating agent inhibiting the action of divalent ions.
- EDTA ethylenediaminetetraacetic acid
- other known chelating agents can be advantageosuly used.
- the hydrophilic fabric was maintained in position on the wall by fixing the edges with a scotch paper.
- the temperature on the wall was constantly maintained at about 37 °C (using a standard IR lamp), while the fabric was periodically sprayed with buffer solution for maintaining the proper hydration and a pH value between about 7.2 and about 8.
- the kit for enzymatic bio-cleansing and the related cleaning method according the present invention find application in many professional and consumer fields where biofilm removal from a substrate is required, or desired, for aesthetic, structural, sanitary or conservative purposes.
- the advantages exhibited by the present invention make it most suitable in the field of cultural heritage, as an alternative to traditional biocides an enzymatic gel formulations, particularly in the restoration of delicate and valuable works of art e.g. archeologic sites, mosaics, ceramics and ancient frescoes.
- the bio-cleansing kit has an excellent ability, not found in the known solutions, to remove biofilm from stone-like surfaces without altering colors or damaging the substrate.
- the present invention has demonstrate to be useful and effective in the removal of treatment of molds from domestic walls or floors.
- applications in other fields are also possible, e.g. industrial plant cleaning in the food industry.
Landscapes
- Chemical & Material Sciences (AREA)
- Organic Chemistry (AREA)
- Engineering & Computer Science (AREA)
- Chemical Kinetics & Catalysis (AREA)
- Oil, Petroleum & Natural Gas (AREA)
- Wood Science & Technology (AREA)
- Life Sciences & Earth Sciences (AREA)
- Inorganic Chemistry (AREA)
- Detergent Compositions (AREA)
- Cosmetics (AREA)
- Immobilizing And Processing Of Enzymes And Microorganisms (AREA)
- Enzymes And Modification Thereof (AREA)
- Chemical Or Physical Treatment Of Fibers (AREA)
- Cleaning By Liquid Or Steam (AREA)
Abstract
Priority Applications (1)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
EP23205328.0A EP4317390A3 (fr) | 2015-08-28 | 2016-08-27 | Kit de bionettoyage et procédé d'élimination de biofilms de substrats |
Applications Claiming Priority (2)
Application Number | Priority Date | Filing Date | Title |
---|---|---|---|
ITUB2015A003557A ITUB20153557A1 (it) | 2015-08-28 | 2015-08-28 | Kit e metodo per biopulitura enzimatica di superfici |
PCT/IB2016/055131 WO2017037602A1 (fr) | 2015-08-28 | 2016-08-27 | Kit de bionettoyage et procédé d'élimination de films biologiques de substrats |
Related Child Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP23205328.0A Division EP4317390A3 (fr) | 2015-08-28 | 2016-08-27 | Kit de bionettoyage et procédé d'élimination de biofilms de substrats |
EP23205328.0A Division-Into EP4317390A3 (fr) | 2015-08-28 | 2016-08-27 | Kit de bionettoyage et procédé d'élimination de biofilms de substrats |
Publications (3)
Publication Number | Publication Date |
---|---|
EP3365419A1 true EP3365419A1 (fr) | 2018-08-29 |
EP3365419B1 EP3365419B1 (fr) | 2023-10-25 |
EP3365419B8 EP3365419B8 (fr) | 2023-11-29 |
Family
ID=54884190
Family Applications (2)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP23205328.0A Pending EP4317390A3 (fr) | 2015-08-28 | 2016-08-27 | Kit de bionettoyage et procédé d'élimination de biofilms de substrats |
EP16779177.1A Active EP3365419B8 (fr) | 2015-08-28 | 2016-08-27 | Kit de bionettoyage et procédé d'élimination de films biologiques de substrats |
Family Applications Before (1)
Application Number | Title | Priority Date | Filing Date |
---|---|---|---|
EP23205328.0A Pending EP4317390A3 (fr) | 2015-08-28 | 2016-08-27 | Kit de bionettoyage et procédé d'élimination de biofilms de substrats |
Country Status (7)
Country | Link |
---|---|
EP (2) | EP4317390A3 (fr) |
JP (1) | JP2018529012A (fr) |
CN (1) | CN108473916A (fr) |
BR (1) | BR112018003641A2 (fr) |
IT (1) | ITUB20153557A1 (fr) |
RU (1) | RU2018110873A (fr) |
WO (1) | WO2017037602A1 (fr) |
Families Citing this family (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
BE1023885B1 (fr) * | 2016-06-29 | 2017-09-04 | Realco | Support imprégné d'au moins une composition pour le prélèvement de microorganismes présents sur une surface |
MX2021006072A (es) * | 2018-11-28 | 2021-07-06 | Univ Pennsylvania | Robots de pequeña escala para la erradicación de biopelículas. |
Family Cites Families (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
KR100981519B1 (ko) * | 2007-12-24 | 2010-09-10 | 재단법인서울대학교산학협력재단 | 생물막 형성 억제 효소를 이용한 분리막 생물반응조 수처리 공정 |
US8911986B2 (en) * | 2011-04-29 | 2014-12-16 | Toyota Motor Engineering & Manufacturing North America, Inc. | Coatings containing polymer modified enzyme for stable self-cleaning of organic stains |
GB201212098D0 (en) * | 2012-07-06 | 2012-08-22 | Xeros Ltd | New cleaning material |
WO2014067933A1 (fr) * | 2012-10-31 | 2014-05-08 | C-Lecta Gmbh | Préparation de support bioactif pour une sécurité améliorée dans les produits de soin et les aliments |
-
2015
- 2015-08-28 IT ITUB2015A003557A patent/ITUB20153557A1/it unknown
-
2016
- 2016-08-27 RU RU2018110873A patent/RU2018110873A/ru not_active Application Discontinuation
- 2016-08-27 CN CN201680059669.5A patent/CN108473916A/zh active Pending
- 2016-08-27 EP EP23205328.0A patent/EP4317390A3/fr active Pending
- 2016-08-27 JP JP2018530193A patent/JP2018529012A/ja active Pending
- 2016-08-27 BR BR112018003641A patent/BR112018003641A2/pt not_active Application Discontinuation
- 2016-08-27 EP EP16779177.1A patent/EP3365419B8/fr active Active
- 2016-08-27 WO PCT/IB2016/055131 patent/WO2017037602A1/fr active Application Filing
Also Published As
Publication number | Publication date |
---|---|
BR112018003641A2 (pt) | 2019-03-06 |
EP4317390A2 (fr) | 2024-02-07 |
EP4317390A3 (fr) | 2024-04-24 |
WO2017037602A9 (fr) | 2017-06-08 |
WO2017037602A1 (fr) | 2017-03-09 |
CN108473916A (zh) | 2018-08-31 |
EP3365419B1 (fr) | 2023-10-25 |
JP2018529012A (ja) | 2018-10-04 |
ITUB20153557A1 (it) | 2017-02-28 |
RU2018110873A (ru) | 2019-09-27 |
EP3365419B8 (fr) | 2023-11-29 |
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