Restoration composition for construction

Abstract

The present invention relates to compositions comprising at least one plant extract selected from oregano extract, thyme extract, cinnamon extract or mixtures thereof, adsorbed on an inorganic matrix, to kits of parts and detergents comprising said compositions, and to their use in cleaning surfaces, in particular in devitalization of biological patinas on the surface of stone cultural property.

Description

[0001] RESTORATION COMPOSITION FOR CONSTRUCTION

[0002] Field of the invention

[0003] The present invention relates to a composition based on plant extracts adsorbed on inorganic matrices and their use in removing biological patinas from the surface of artefacts, in particular stone artefacts.

[0004] State of the art

[0005] It is well known that essential oils (hereinafter also referred to as 'EO' for brevity) have long been used in various applications for their biocidal activity. In the last decade, numerous studies have been carried out on the use of essential oils in the restoration sector as possible substitutes for chemically synthesised biocides, especially in the devitalisation of biofilms that develop on the surface of artefacts made of stone and exposed to the external environment.

[0006] Most of these studies have shown that some EOs can be considered a viable alternative to traditional biocides, being in some cases equally effective and less environmentally impactful.

[0007] Essential oils are generally used singly / purely or in combination of two or more oils and diluted to various concentrations in a suitable solvent, usually an alcohol, such as ethanol or methanol, or a hydroalcoholic solution.

[0008] Products based on essential oils are also available on the market for the removal of biological patinas from the surface of stone cultural property. Some of these products must be diluted in water before use and generally applied as a compress to achieve the desired effectiveness. Dilution in water, as a technician in the field knows, undermines the stability and therefore the effectiveness of the product. Therefore, if the entire prepared solution is not used, it can only be used in the days immediately following dilution; thereafter, the oil separates from the water with no possibility of regaining a stable and usable emulsion. Other restorative products based on essential oils - although they do not require dilution in water - are nevertheless sold with instructions to use the product as soon as possible after opening the packaging, otherwise the effectiveness of the product will be lost.

[0009] The use of essential oils in the restoration of stone artefacts therefore has technical drawbacks, to mention a few: a) since essential oils are hydrophobic and therefore insoluble or not very soluble in water, the solvent of choice in most cases for restoration work on stone materials, the available formulations are often unstable; b) the use of alcohols or hydroalcoholic solutions as solvents precludes their use in underground contexts (where the use of short-chain alcohols is not recommended); c) essential oils are very volatile and already unstable compounds that degrade when exposed to light or air. Therefore, once diluted in the chosen solvent, they must be used quickly, otherwise their effectiveness will be diminished; d) the cost of a pure essential oil is much higher than that of chemical biocides normally used in the field of restoration. This, coupled with the product's poor efficacy after opening the packaging, makes it unattractive for consumers to purchase.

[0010] Therefore, there is still a need for a product that is effective for the removal or devitalisation of biological patinas from the surface of artefacts, in particular stone cultural artefacts, that is effective but above all stable and therefore long-lasting, even after opening.

[0011] SUMMARY OF THE INVENTION

[0012] Since many commercially available restoration products have poor stability, the present invention is based on the search for and identification of a composition based on natural substances, in particular plant extracts, more particularly essential oils, which has not only high biocidal activity but also excellent stability.

[0013] After extensive experimentation, the inventors found that emulsifying selected plant extracts, in particular essential oils, with a surfactant and then adsorbing the mixture onto an inorganic matrix, effectively solves the aforementioned technical problem.

[0014] Therefore, the present invention relates to a composition comprising at least one plant extract selected from: oregano extract, thyme extract, cinnamon extract or mixtures thereof, and an inorganic matrix. Said plant extracts are preferably essential oils. Said inorganic matrix is preferably a zeolite-based or silica-based matrix.

[0015] In the context of the experiments carried out, the inventors of the present invention have also found that the combination of at least one of the identified plant extracts, preferably at least one oregano extract, at least one thyme extract, and at least one cinnamon extract, together with propolis achieves optimal biocidal efficacy compared to combinations that do not comprise the combination of the three selected plant extracts or that do not comprise propolis. Without wishing to be bound by any theory, the increased biocidal efficacy appears to be attributable to the synergism of the components.

[0016] Therefore, the present invention also relates to compositions according to the invention further comprising propolis, in particular to a composition comprising at least one plant extract, an inorganic matrix and propolis, wherein the at least one plant extract and the inorganic matrix are as defined above. The invention further relates to the combination of at least one oregano extract, at least one thyme extract, at least one cinnamon extract and propolis. Said extracts are preferably essential oils. In the context of the experiments conducted, the inventors have also ascertained that the addition of sodium percarbonate and / or citric acid to the compositions of the invention is advantageous, in particular by allowing the amount of propolis, if present, to be reduced or eliminated. Therefore, the present invention also relates to a composition comprising at least one plant extract and at least one inorganic matrix as defined above together with, as a further additive, sodium percarbonate and / or citric acid.

[0017] A further object of the invention is also a kit of parts comprising the composition or combination according to any of the embodiments described herein for the simultaneous or sequential application of the plant extracts therein, preferably together with suitable dilution means, dosing means and / or application means.

[0018] The present invention also relates to the use of the composition or combination or kit of parts according to any of the forms of embodiment described herein for cleaning or cleansing of surfaces, in particular for removing or devitalising biological patinas from the surface of artefacts, preferably stone artefacts, in particular stone cultural heritage assets. In other words, the present invention also relates to a cleaning agent comprising the composition or combination according to any of the embodiments described herein.

[0019] The present invention also relates to a process for preparing the composition according to any of the forms of embodiment described herein comprising the following steps, not necessarily in that order: i. Mixing the essential oils and surfactant to obtain a homogeneous mixture; ii. Mixing the inorganic matrix with the mixture obtained in step i. at room temperature for about 24 h or at 60°C in a thermostat bath. iii. Optionally, add propolis, citric acid and / or sodium percarbonate to the mixture obtained in step ii. and shake to obtain a homogeneous mixture.

[0020] As mentioned above, and as will be evident from the appended experimental section, thanks to the adsorption of the composition on an inorganic matrix and also thanks to the synergy of its components, the compositions according to the present invention allow obtaining an efficacy equal or superior to biocidal products, both synthetic and natural, known in the art.

[0021] Furthermore, the composition of the invention has at least the following advantages:

[0022] Adsorption on matrices stabilises the undiluted product, decreasing the volatility of the essential oils and increasing their stability (also against light or oxygen). This allows the undiluted product to be stored (even if the package is opened) for longer periods of time without compromising its efficacy (overcoming the technical drawbacks mentioned above in points c, e, f) by diluting the product in water, a stable emulsion is obtained, which remains so for long periods of time (more than two months after dilution, no traces of separated oil are visible) (overcoming the technical drawbacks mentioned under c, e, f above) water can be used as a solvent (see drawback mentioned above under a) without the stability of the product being compromised; there is no need to use alcohol as a solvent of the product, nor are alcohols used in the preparation of the product, this allows it to be used in underground environments (see drawback under b above) the stability of the product over time and the fact that it has to be diluted for use allows the purchaser to amortise the costs incurred in the purchase, in fact making the product more attractive / convenient (see inconvenience mentioned above under d) Other advantages and features of the present invention will be apparent from the following detailed description.

[0023] The terms used in this description are as generally understood by the person skilled in the art, unless otherwise indicated.

[0024] The term "extract', in the context of the present description, means any product attributable to a plant drug including all products derived from mechanical treatments (pulverisation, crushing, mixing and / or other methods) or extractive treatments (solvent extraction, distillation, and / or other specific methods) performed on a drug. The term “extrac” will therefore include alcohols, i.e. distillates resulting from the distillation of raw plant material in the presence of alcohol, e.g. ethanol, at varying concentrations; “hydrolates”, commonly known as aromatic waters, i.e. aqueous distillates obtained by steam distillation of plants (particularly aromatic plants) and, where possible, separation of the essential oils; 'concretes' or concentrates obtained by extraction of raw (fresh) plant material by means of one or more solvents which are then removed wholly or partially; “distillates’’ i.e. condensation products obtained after distillation of raw plant material; “exudate”' i.e. raw natural material excreted by the plant.

[0025] The term “essential oil” refers to complex mixtures of organic substances of various kinds, such as alcohols, aldehydes, ketones, terpenes. Once extracted, they appear as oily, liquid and volatile substances that are as fragrant as the plant from which they originate. Essential oils can be obtained by hot extraction (distillation) or cold extraction (pressing) of fresh starting material belonging to a single plant drug. Distillation is used when the starting material is not heat-sensitive, while squeezing is the preferred technique in the case of heat-sensitive components. See also the definition according to ISO 9235:2021 - Aromatic natural raw materials.

[0026] The term “biological patina" refers to a modification, especially colour change, of the surface that cannot be linked to degradation phenomena, and is mainly due to the development of biological organisms. The term film (algae, bacteria, fungi), encrustation (crusted lichens) or the more generic term film / biofilm can also be used. In the context of restoration, one can also refer to a complex biofilm composed of microorganisms belonging to different taxonomic groups, usually held together by extracellular polymeric substances (EPC) to form a more or less compact layer of varying consistency. The interaction of the micro-organisms with the substrate can vary considerably; epilithic micro-organisms live attached to the surface of stone materials, endolithic microorganisms can penetrate into the material itself. The presence of patina on the surface of an artefact can lead to chemical, physical and aesthetic damage.

[0027] The term "detergent' or "cleaning agent' in the context of this description refers to any product suitable for cleaning or cleansing surfaces, more specifically suitable for the removal of surface deposits or the removal of patinas / surface alterations. In the field of restoration, for the specific action on biological patinas, one can speak of compositions with biocidal action, compositions for disinfection, or even compositions for devitalisation. Brief description of the Figures

[0028] FIG. 1 : Graph relating to the biochemical quantification of ATP.

[0029] FIG. 2: Photographs relating to culture analysis.

[0030] FIG. 3: Photographs concerning the decrease in chlorophyll autofluorescence.

[0031] FIG. 4a: Macroscopic images of the plugs treated with the compositions of the invention Melitec and Melitec C before (left) and after (right) treatment; FIG. 4b: Graph showing the amount of ATP (pg / cm2) before and after treatment with Melitec and Melitec C; FIG. 4c: Images of the culture plates. Left: Samples taken before treatment; centre and right: samples taken after treatment with Melitec C and Melitec.

[0032] FIG. 5: Images showing a patina treated by spray application (FIG. 5a), brush application (FIG. 5b), and pack application (FIG. 5 c) of compositions according to the invention prior to treatment (left), one week after application of the product (centre), and six months after application (right).

[0033] FIG. 6: Images showing the appearance of the patina before treatment (left) after application of compositions according to the invention (centre) and after cleaning (right). FIG. 7: Graph showing the percentage decrease in ATP recorded on the patina treated with the two compositions applied by brush and pack.

[0034] DETAILED DESCRIPTION OF THE INVENTION

[0035] As mentioned above, the present invention relates to a composition comprising at least one plant extract chosen from: oregano extract, thyme extract, cinnamon extract or mixtures thereof, and an inorganic matrix said extracts being preferably essential oils.

[0036] Advantageously, the composition of the invention may also include propolis, sodium percarbonate and / or citric acid.

[0037] A brief description of the main plant extracts of the invention, as well as propolis, is provided below.

[0038] Oregano

[0039] The expression "at least one plant extract belonging to the genus Origanum" or "at least one oregano extract" in the context of this description means that the combination may comprise at least one or more plant extracts belonging to the genus Origanum. The extract(s) may be selected from, for example, extract of Origanum vulgare, Origanum vulgare subs, hirtum, Origanum compactum and mixtures thereof, and is preferably extract of Origanum vulgare subs, hirtum. The extract may be prepared according to any one of the procedures known in the art. By way of example, the oregano extract may be an essential oil of oregano having a carvacrol chemotype with a carvacrol content of min. -max. 60-80%. More preferably, it will be an essential oil of oregano as defined in ISO 13171 :2016 Essential oil of oregano [Origanum vulgare L. subsp. hirtum (Link) letsw].

[0040] Thyme

[0041] The expression "at least one plant extract belonging to the genus Thymus" or "at least one thyme extract" in the context of the present description means that the composition may comprise at least one or more plant extracts belonging to the genus Thymus. According to an embodiment, the extract(s) may be selected from extract of Thymus vulgaris, extract of Thymus serpillum, extract of Thymus capitatus, Thymus zygis and preferably extract of Thymus vulgaris.

[0042] The extract may be prepared according to procedures known in the art, for example, it may be an essential oil of red thyme and / or white thyme. The thyme essential oil is preferably a thyme essential oil having a thymol chemotype, in particular 35-50%. By way of example, the essential oil of thyme will be as defined in ISO 19817:2017 Essential oil of thyme [Thymus vulgaris L. and Thymus zygis L.J, thymol type.

[0043] Thyme extracts prepared according to the methods described above are particularly suitable for the purposes of the present invention, but other commercially available thyme extracts are still suitable.

[0044] Cinnamon

[0045] The expression "at least one plant extract belonging to the genus Cinnamomum" or "at least one cinnamon extract" in the context of the present description means that the composition may comprise at least one or more plant extracts belonging to the genus Cinnamomum. According to a form of embodiment, the extract(s) may be chosen from extracts of Cinnamomum zeylanicum and Cinnamomum verum.

[0046] The extract may be prepared according to procedures known in the state of the art, for example, it may be an essential oil of cinnamon (from bark).

[0047] By way of example, but in no way limiting, the plant extract belonging to the genus Cinnamomum will be an essential cinnamon oil extracted from bark having a chemotype in cinnamaldehyde, preferably, 50-70%, more preferably 54-68%. Cinnamon extracts prepared according to the methods described above are particularly suitable for the purposes of the present invention, but other commercially available cinnamon extracts are also suitable.

[0048] Propolis

[0049] “Propolis" in the context of the present description refers to that resinous substance, generally brown in colour, produced by tree buds which bees rework and use, for example, to close holes and crevices in the hive, to attack honeycombs and paint them, and to make them immune to attack by bacteria and fungi.

[0050] Although for optimum efficacy the concomitant presence of the above-mentioned active components is preferred, the present invention also relates to a composition comprising one, two or more of the following: oregano extract, thyme extract, cinnamon extract adsorbed on an inorganic matrix, with and without propolis, variously combined with each other. By way of illustration, but by no means limitation, the invention relates to compositions comprising oregano extract and propolis; or oregano extract and thyme extract; or cinnamon extract and propolis; or thyme extract, cinnamon extract and propolis; or thyme extract and propolis; or oregano extract and sodium percarbonate; cinnamon extract and citric acid, etc.

[0051] As will be evident from the appended examples, in the context of the experiments conducted to fine-tune the compositions of the invention, the inventors surprisingly found the combination of at least one oregano extract, at least one thyme extract, at least one cinnamon extract and propolis to have optimal efficacy.

[0052] Therefore, further subject matter of the invention is a combination of at least one oregano extract, at least one thyme extract, at least one cinnamon extract and propolis.

[0053] Preferably, said plant extracts are essential oils. Surprising efficacy has also been obtained for compositions comprising in addition to the adsorbed matrix of at least one of the plant extracts as defined herein, also sodium percarbonate and / or citric acid. Indeed, as shown in the appended experimental part, the addition of percarbonate and / or citric acid enabled the desired efficacy to be obtained even in the absence of propolis. In other words, the present invention also relates to a composition according to any of the embodiments described herein in which percarbonate and / or citric acid are further added or to compositions according to any of the embodiments described herein in which percarbonate and / or citric acid replace propolis.

[0054] Notwithstanding the foregoing, the present invention relates to a composition comprising at least one plant extract selected from: oregano extract, thyme extract, cinnamon extract or mixtures thereof, an inorganic matrix and - optionally - also propolis, sodium percarbonate and / or citric acid, wherein

[0055] - the oregano extract is a plant extract belonging to the genus Origanum selected from extracts of Origanum vulgare, Origanum vulgare subs, hirtum, Origanum compactum and mixtures thereof, and is preferably essential oil of Origanum vulgare subs, hirtum’, and / or wherein

[0056] - the thyme extract is a plant extract belonging to the genus Thymus selected from extracts of Thymus vulgaris, Thymus serpillum, Thymus zygis and mixtures thereof, and is preferably essential oil of Thymus vulgaris L; and / or wherein

[0057] -the cinnamon extract is a plant extract belonging to the genus Cinnamomum chosen from extract of Cinnamomum verum, Cinnamomum zeylanicum or mixtures thereof, and is preferably essential oil of Cinnamomum zeylanicum.

[0058] According to one embodiment, the composition comprises at least one plant extract selected from: oregano extract, thyme extract, cinnamon extract, propolis, sodium percarbonate and / or citric acid as the only biocidal ingredients.

[0059] According to another embodiment, the composition comprises other components, regardless of their biocidal power, for example one or more (two, three, four) additional plant extracts, preferably selected from: clove extract, rosemary, mint and / or lavender. In other words, the composition according to any one of the embodiments described herein may further comprise clove extract, rosemary extract, mint extract and / or lavender extract, preferably in the form of essential oils.

[0060] According to a further embodiment, the composition may comprise other ingredients or active ingredients or excipients.

[0061] During the studies carried out, the inventors of the present invention were able to ascertain that, by adding a surfactant to the composition, a stable emulsion is obtained which guarantees the reproducibility of the biocidal efficacy of the composition (see also the comparative tests included in the appended experimental section). The composition according to any of the embodiments described herein may thus further comprise a surfactant, preferably a non-ionic surfactant, more preferably chosen from: polysorbate 20, polysorbate 80 or mixtures thereof.

[0062] As mentioned above, the inventors of the present invention have found that, by adsorbing the combination described herein onto an inorganic matrix, preferably a zeolite or silica-based matrix, the stability of the combination increases (also with respect to light or oxygen) and the volatility of the plant extracts, in particular essential oils, contained therein decreases. This makes it possible to store the undiluted biocide product (even if the package is opened) for longer periods of time without compromising its efficacy, and to dilute it in water before use, obtaining - in contrast to commercially available products - a stable emulsion that remains so for more than two months after dilution, without having to use alcohols as solvents.

[0063] Thus, the present invention also relates to a composition according to any of the embodiments described herein in which the biocidal agents are adsorbed on an inorganic matrix, preferably based on zeolites or silica. In other words, the present invention relates to an inorganic matrix, preferably based on zeolites or silica, comprising the composition according to any one of the embodiments described herein.

[0064] The zeolites may in particular be selected from: clinoptilolite fauiasite, paulingite, stilbite or mixtures thereof. Zeolites, as known to a technician in the field, are hydrated techtoaluminosilicates of alkali and alkaline earth metals with a microcrystalline tetrahedron structure; the more regular their structure, the greater their adsorption capacity. A key feature is the vast presence of empty spaces within their structure, which allows zeolites to adsorb, for example, essential oils and release them without altering or destroying their structure. In particular, clinoptilolite is particularly suitable for the purpose of the invention due to its grain size of 1- 2.5 pm and cavity size of 2.6 to 7.4 A, i.e. a very large internal surface area.

[0065] Other matrices suitable for the purpose of the invention are in general silica- based matrices, consisting of an ordered porosity and a large surface area, such as mesoporous matrices belonging to the MCM-41 family or related. MCM-41 silicas have an ordered hexagonal structure, with an available surface area of 800 to 1000 m2 / g. Although not an essential feature of the invention, the inventors have identified optimal amounts of the components of the composition which are preferably as follows:

[0066] - the at least one plant extract or mixture of plant extracts is present in an amount between 5 and 40, preferably 24 % w / w;

[0067] - propolis, where present, is present in an amount between 0.5 and 5, preferably 2 % w / w;

[0068] - sodium percarbonate and / or citric acid, if present, is present in an amount between 5 and 40 % w / w.

[0069] - the inorganic matrix is present in an amount between 20 and 80, preferably 50 % w / w;

[0070] - the at least one surfactant, if present, is present in an amount between 10 and 40, preferably 24 % w / w; on the total weight of the dry mixture.

[0071] The composition may thus comprise a combination of essential oils according to any of the embodiments described herein in a weight ratio of 1 :1 to the surfactant. The weight ratio of the mixture of plant extracts with surfactant and propolis is generally 2:0.2.

[0072] In embodiments comprising additional ingredients to those enumerated herein, the additional ingredients are generally added in an amount between 5 and 15, preferably 10 % w / w.

[0073] The composition should be diluted in a suitable solvent, preferably water, prior to use. The composition may be diluted in suitable solvent in a concentration of between 0.5 and 5, preferably at 2 % w / w, on the volume of the final suspension.

[0074] The pH of the diluted product is between 6.5 and 7.

[0075] Thus, further subject matter of the invention is a detergent comprising the composition or combination according to any of the embodiments described herein and at least one suitable solvent, preferably water.

[0076] The composition of the invention can be prepared as generally known in the art. In order to obtain a product with optimal efficacy, the inventors of the present invention have also devised an ad hoc process in which the combination of plant extracts is adsorbed onto the matrix itself by contacting the mixture with the inorganic matrix at room temperature in a closed container or at 60 °C in a thermostat bath. Propolis - as well as sodium percarbonate and / or citric acid - can be added indifferently either before or after loading the mixture of essential oils and surfactant(s) onto the inorganic matrix.

[0077] The present invention thus also relates to a process for preparing the composition according to any of the described embodiments comprising the following steps, not necessarily in that order: i. Mixing the essential oils and surfactant to obtain a homogeneous mixture; ii. Charging or mixing the inorganic matrix with the mixture obtained in step i. at room temperature for about 24 h or at 60°C in a thermostat bath. iii. Optionally, adding propolis, sodium percarbonate and / or citric acid to the mixture obtained in step i. or ii. and shaking to obtain a homogeneous mixture.

[0078] Preferably, the essential oils and surfactant are in a 1 :1 ratio while propolis is preferably added in an amount of 2% w / w to the final mixture (essential oils adsorbed on inorganic matrix). Adsorption of the essential oils onto the matrix is preferably conducted in a closed container.

[0079] As known to a technician in the field, the composition may be applied to the surface to be treated by spray, brush and / or pack.

[0080] The aforementioned combination of active ingredients may be used formulated in a single composition according to the various embodiments described above or in a kit of parts comprising the different ingredients separately, for example, in single compositions suitably formulated by adsorption onto inorganic matrix, for sequential or simultaneous "administration" I application of the different active ingredients onto the surface to be treated. Thus, the present invention also relates to a kit of parts comprising the composition or combination according to any one of the embodiments described herein for the simultaneous or sequential application of the plant extracts and biocidal actives therein, preferably together with suitable dilution means, dosing means and / or application means.

[0081] Suitable dilution means, dosage means and / or means of application are those generally known to a technician in the field, particularly in the field of restoration of stone artefacts. By way of example, suitable dilution media may be, water, hydroalcoholic mixtures, alcohol, hydrocarbons, etc. The composition according to any one of the embodiments described herein before use is preferably diluted in a suitable solvent, usually water. The resulting suspension preferably has a concentration of 0.5% to 5% and more preferably 2% w / v.

[0082] Suitable means of dosage may be pipettes, Erlenmeyer flasks, syringes, dispensers or dispensers in general, etc. Suitable means of application may be, for example, brushes, spray dispensers, compresses e.g. with cellulose pulp or other supporting agents, etc.

[0083] As will be apparent to the person skilled in the art from the examples provided below, the present invention constitutes a viable alternative to biological film removal products known in the art. The action of the compositions subject matter of the present invention has been evaluated by means of in vitro tests which, moreover, have shown that the composition of the invention is particularly effective in the treatment of biological film also due to the reinforcing (synergistic) action of its components.

[0084] Therefore, the present invention also relates to the use of the composition, combination, detergent and kit of parts, according to any one of the embodiments described herein, for cleaning or cleansing of surfaces, in particular for removing or devitalising biological patinas from the surface of artefacts, preferably wherein said artefacts are stone artefacts, in particular stone cultural heritage. The compositions, combinations, cleaners and kits of parts of the invention appear to be particularly suitable for the restoration of stone artefacts, in particular stone cultural heritage.

[0085] Without wishing to be bound by any theory, the inventors hypothesise that the product acts as a biocide on microbial patinas due to the presence of the selected essential oils and propolis which interact with the plasma membrane of the microorganisms by inducing cell lysis or, by penetrating the cell, interfere with certain metabolic pathways and thus cause the death of the microorganism. The use of selected essential oils, as is known from tests carried out, gives good devitalisation results against different types of autotrophic and heterotrophic microorganisms. In addition, the addition of propolis improves efficacy against Gram-negative bacteria, on which essential oils tend to have little action.

[0086] As will be evident from the experimental section, the composition of the invention was tested in vitro against isolated microorganisms (Gram+ and Gram- bacteria; algae of the genera Chlorella and Desmodesmus; cyanobacteria of the genera Leptolyngbya and Nostoc; fungal strains of the genera Aspergillus, Penicillium, Fusarium, Aerobasidium, Alternaria, Cladosporiuni). Cultivation, ATP quantification and evaluation of residual chlorophyll autofluorescence were carried out to assess efficacy.

[0087] EXAMPLES

[0088] The following are non-limiting examples of compositions according to the present invention. Modifications or variations of the embodiments exemplified herein, obvious to one skilled in the art, are covered by the appended claims.

[0089] First, the efficacy of a composition comprising an inorganic matrix based on zeolites and a plant extract selected from thyme extract, oregano extract and cinnamon extract has been tested. For example, compositions comprising thyme extract adsorbed on zeolite and cinnamon extract adsorbed on zeolite were tested. The compositions were prepared by adding: essential oil 25 % w / w, surfactant 25 % w / w, zeolites 50 % w / w. The effectiveness of an inorganic matrix loaded with all three plant extracts (thyme, oregano, cinnamon) was then tested. This composition comprised

[0090] - essential oil of plant belonging to the genus Origanum 10 % w / w; - essential oil of plant belonging to the genus Thymus 10 % w / w;

[0091] - essential oil of plant belonging to the genus Cinnamomum 5% w / w;

[0092] - surfactant 25 % w / w;

[0093] - zeolites 50 % w / w.

[0094] In all these cases, the efficacy was assessed as suitable for use in restoration or more generally in surface cleaning / cleaning.

[0095] Subsequently, it was decided to test more complex compositions containing e.g. propolis, sodium percarbonate and / or citric acid.

[0096] The quali-quantitative formulations of the tested compositions are reported below together with the relevant experimental evidence.

[0097] EXAMPLE 1: composition according to an embodiment of the invention

[0098] - essential oil of plant belonging to the genus Origanum 10 % w / w

[0099] - essential oil of plant belonging to the genus Thymus 4 % w / w

[0100] - essential oil of plant belonging to the genus Cinnamomum 10 % w / w

[0101] - propolis 2 % w / w

[0102] - surfactant 24 % w / w

[0103] - zeolites 50 % w / w

[0104] EXAMPLE 1a: Composition according to an embodiment of the invention essential oil of plant belonging to the genus Origanum 15% w / w

[0105] - essential oil of plant belonging to the genus Thymus 9.5% w / w

[0106] - essential oil of plant belonging to the genus Cinnamomum 15% w / w

[0107] - propolis 0.5 % w / w

[0108] - surfactant 40 % w / w

[0109] - zeolite 20 % w / w

[0110] EXAMPLE 2: Composition according to an embodiment of the invention

[0111] - essential oil of plant belonging to the genus Thymus 15% w / w

[0112] - essential oil of plant belonging to the genus Cinnamomum 5 % w / w

[0113] - propolis 5 % w / w

[0114] - surfactant 20 % w / w

[0115] - zeolites 55 % w / w

[0116] EXAMPLE 3: Composition according to an embodiment of the invention

[0117] - essential oil of plant belonging to the genus Cinnamomum 20% w / w

[0118] - propolis 0.5% w / w

[0119] - surfactant 19.5% w / w

[0120] - zeolites 60% w / w EXAMPLE 4: to an embodiment of the invention

[0121] - essential oil of plant belonging to the genus Origanum 10 % w / w

[0122] - essential oil of plant belonging to the genus Thymus 4% w / w

[0123] - essential oil of lavender 5% w / w

[0124] - essential oil of cloves 5 % w / w

[0125] - propolis 2 % w / w

[0126] - surfactant 24 % w / w

[0127] - zeolites 50 % w / w

[0128] EXAMPLE 5: to an embodiment of the invention

[0129] - essential oil of plant belonging to the genus Origanum 20% w / w

[0130] - propolis 0.5% w / w

[0131] - surfactant 19.5% w / w

[0132] - zeolites 60% w / w to an embodiment of the invention essential oil of plant belonging to the genus Origanum 5% w / w zeolites 34% w / w sodium percarbonate 33% w / w

[0133] EXAMPLE 7: Composition according to an embodiment of the invention

[0134] - essential oil of plant belonging to the genus Thymus 20% w / w

[0135] - propolis 0.5% w / w

[0136] - surfactant 19.5% w / w

[0137] - zeolites 60% w / w

[0138] The composition set forth in Example 1 comprising at least one oregano extract, at least one thyme extract and at least one cinnamon extract together with propolis achieved optimal results in the efficacy tests below. Highly effective biocidal activity was also recorded for the compositions in EXAMPLES 2 - 7.

[0139] Tests for evaluating biocidal efficacy in vitro

[0140] The product was tested in vitro on the following micro-organisms: Gram+ and Grambacteria; algae of the genera Chlorella and Desmodesmus; cyanobacteria of the genera Leptolyngbya and Nostoc; fungal strains of the genera Aspergillus, Penicillium, Fusarium, Aerobasidium, Alternaria, Cladosporium.

[0141] The microorganisms were grown on the following agar media:

[0142] Bacteria: Plate count Agar (PCA) Oxoid

[0143] Fungi: Malt Extract Agar (MEA) PanReac AppliChem Cyanobacteria: BG11o

[0144] Algae: BG11

[0145] Once the microorganisms had grown, the plates were sprayed with the product (i.e. the composition of EXAMPLE 1) (approx. 600 pl per plate) and sealed with parafilm. To verify the biocidal efficacy, the following analyses were carried out:

[0146] 1. ATP quantification: all microorganisms tested.

[0147] 2. Culture analysis: fungi and bacteria.

[0148] 3. Estimation of the decrease in chlorophyll autofluorescence: cyanobacteria and algae.

[0149] Analyses in points 1. and 3. were carried out immediately before application and after four days of incubation; analyses in point 2. were carried out after four days of incubation. The protocols followed are given below.

[0150] 1. ATP quantification

[0151] A known quantity of micro-organisms was taken from each plate and suspended in 1 ml of sterile water; for fungi, the taken mycelium was fragmented by vortexing in the presence of sterile glass beads;

[0152] 20 pl suspension of microorganisms transferred into 1.5 ml eppendorf with 80 pl Water-Gio Lysis Reagent (Promega);

[0153] Lysis time 5 minutes at room temperature;

[0154] 100 pl of Water-Gio detection reagent (Promega) added;

[0155] Reading of Relative Light Units (RLU) by bench-top luminometer LB 9526 Sirius 2 PC-1 (Berthold) combined with FB12 / Sirius Software V2.0 (Berthold); Parameter setting: waiting time 2 seconds, integration time 2 seconds;

[0156] For measurement of free ATP: 20 pl of un-lysed suspension in 80 pl of ATP-free water;

[0157] The possible interference of the product on ATP quantification was assessed by adding 1 pl of the product to 99 pl of standard ATP; the RLUs recorded were compared to those obtained by replacing the product with 1 pl of sterile deionised water.

[0158] The RLUs recorded were converted to pg of ATP per mg of microorganisms taken.

[0159] All tests were performed in triplicate; the average values obtained are shown in the 'Results' section below.

[0160] The efficacy of the treatment was assessed by comparing the amount of ATP present before and after treatment and calculating the percentage decrease, indicating a reduction in the metabolic activity of the micro-organisms. The protocol was set up according to the literature

[0001] and based on previous laboratory experience, which allowed the WaterGlo kit to be adapted to different types of microorganisms frequently found on historic-artistic artefacts preserved outdoors.

[0161] 2. Culture analyses

[0162] The cultural analyses are aimed at assessing any residual viability and clarifying whether the metabolic decrease revealed by the biochemical investigations is actually related to a microbicidal and not a microbistatic effect.

[0163] After 4 days of incubation, the treated microorganisms were harvested with a sterile loop and transferred onto sterile, biocide-free culture medium. The same media were used as at the beginning of the text. The inoculated plates were incubated at 27 °C ± 1 °C and checked for 14 days at regular intervals. The efficacy of the product was assessed in terms of absence / presence of growth. [1]

[0164] 3. Estimation of the decrease in chlorophyll autofluorescence

[0165] Efficacy on photosynthetic microorganisms was tested by assessing the physiological state of the cells via chlorophyll autofluorescence. When excited with blue or ultraviolet light, healthy photosynthetic cells show a characteristic primary red / orange fluorescence caused by the presence of chlorophyll a. After biocide treatment, the cells undergo progressive depigmentation and the red colour tends to change, turning brown to greenish-white, depending on the level of degradation of the cell. [2]

[0166] The test was carried out on fresh preparations obtained from suspensions prepared as reported in step 1. For each microorganism, three slides were analysed (25 pl of suspension per slide). The evaluation of viable cells was conducted by observing 10 visual fields along the diagonals of each slide[2] . The level of viability was expressed as the percentage decrease of viable cells present in the treated plates compared to those present before treatment. Observations were carried out with a Leica DM750 microscope equipped with a solid-state fluorescent light source (Leica SFL100), 450 / 490 nm bandpass excitation filters and a 515 nm barrier filter.

[0167] Test Results

[0168] 1. ATP quantification

[0169] Biochemical ATP quantification tests showed a marked decrease in metabolic activity in all microorganisms after treatment with the product of the invention. The percentage decrease in the amount of ATP was over 97% for all microorganisms, with the exception of Penicillium, which retained a residual amount of ATP of just over 5%. Table 1 below and the graph in FIG. 1 summarise the results obtained.

[0170] Table 1 : Residual ATP (%) Decrease (%)

[0171] Penicillium 5,55 94,45

[0172] Aspergillus 0,07 99,93 Alternaria 0,46 99,54 Aureobasidium 0,00 100,00

[0173] Fusarium 0,05 99,95

[0174] Cladosporium 0,02 99,98 G+ 2,43 97,57

[0175] G- 2,11 97,89

[0176] Nostoc 0,36 99,64

[0177] Leptolyngbya 0,34 99,66 Chlorella 0,14 99,86

[0178] Desmodesmus 0,94 99,06

[0179] The test conducted to assess the possible influence of the product of the invention in the quantification of ATP showed no detectable effect. The standard ATP values recorded with and without the product were comparable, with a percentage variation of less than 1 % compared to the values of the pure standard. This variation is usually observed in a set of measurements made on the pure standard, so it can be considered insignificant.

[0180] 2. Culture analyses

[0181] The culture analyses showed that none of the treated microorganisms (both fungi and bacteria) were viable after treatment. Mycelium fragments and bacteria inoculated on sterile medium were in fact unable to grow (see FIG. 2).

[0182] In tests with the different formulations, an ATP decrease of less than 90% was obtained for the product without propolis, and residual viability of the Gram- bacteria was recorded in culture tests.

[0183] 3. Estimation of the decrease in chlorophyll autofluorescence

[0184] Microscopic investigations showed that there was a clear decrease in fluorescence for all microorganisms investigated. Residual fluorescence was estimated to be less than 10% of the fluorescence present before treatment.

[0185] The observation of the micro-organisms in visible light also revealed that the filaments of the cyanobacteria had undergone a strong disintegration, as they appeared as small fragments or as single cells detached from the others. In some cases, it was no longer possible to identify individual cells; an amorphous mass of degraded biological material was present in the slide. In the case of the green algae, the phenomena of cell lysis were less evident; however, it was possible to show that for many cells only the cell wall remained intact, which appeared empty as a result of the lysis phenomena. In the case of the Chlorella algae, similarly to the cyanobacteria, clusters of shapeless material were visible in which few residual cells were still discernible.

[0186] The images shown in FIG. 3 are for illustration purposes only; please note that the assessment of the percentage decrease was performed by averaging what was observed in a large number of fields of view. The photos in UV light were acquired using the same exposure to make the fluorescence levels comparable.

[0187] Comparative tests a. Alternative compositions to that of the invention

[0188] Various compositions were tested during the development of the composition of the invention; the main tests performed and results obtained are given below:

[0189] 1 . Use of a hydroalcoholic solution or ethanol or hydrolates instead of the surfactant: when the product was diluted in water, a homogeneous and stable emulsion of the essential oils was not obtained, this compromised the biocidal efficacy of the product.

[0190] 2. Use of soft soap instead of surfactant: the final product obtained has too low a pH (not compatible for use on stone materials of a carbonate nature). In fact, the pH of the product of the diluted invention is between 6.5 and 7, while with the use of soft soap it was below 5.

[0191] 3. Use of propolis mixed with essential oil, without surfactant: when the product was diluted in water, a homogeneous and stable emulsion of the essential oils was not obtained, which compromised the biocidal effectiveness of the product.

[0192] 4. Use of the mixture of surfactant and essential oil not loaded on matrix: an opalescent product is obtained, which is difficult to dilute in water; moreover, the aqueous emulsion is not stable over time (limit of usability of the final product).

[0193] All the above formulations were discarded as they do not give satisfactory results.

[0194] The results of efficacy tests of certain compositions according to the invention are shown in more detail below.

[0195] Efficacy evaluation tests

[0196] 1- Formulation based on three essential oils (oregano, thyme and cinnamon) according to EXAMPLE 1

[0197] Materials and methods

[0198] The composition is applied to the area to be treated with a brush twice at three-day intervals (treatment method usually used by restorers). The area to be treated presents a black patina, composed mainly of cyanobacteria of the genus Chroococcus.

[0199] Efficacy is assessed by measuring cellular ATP according to the following procedure. Extraction of the patina using a sterile scalpel, suspension of 10 mg of patina in 1 ml of sterile saline;

[0200] 20 pl suspension of microorganisms are transferred into 1.5 ml eppendorf with 80 pl Water-Gio Lysis Reagent (Promega) Lysis time 5 minutes at room temperature

[0201] 100 pl of Water-Gio detection reagent (Promega) added

[0202] Reading of Relative Light Units (RLU) by bench-top luminometer LB 9526 Sirius 2 PC-1 (Berthold) combined with FB12 / Sirius Software V2.0 (Berthold); parameter setting: waiting time 2 seconds, integration time 2 seconds

[0203] For measurement of free ATP: 20 pl of un-lysed suspension in 80 pl of ATP free water

[0204] The detected RLUs were converted to pg of ATP per mg of patina taken.

[0205] All tests were performed in triplicate, the average values obtained are reported in the results section.

[0206] The efficacy of the treatment was evaluated by comparing the amount of ATP present before and after treatment and calculating the percentage decrease, indicating a reduction in the metabolic activity of the microorganisms.

[0207] The protocol was set up according to the literature .[1]

[0208] Results

[0209] ATP assay analyses showed a decrease in viability of the treated micro-organisms of 93.52% ± 2.80%.

[0210] 2- Comparison of two single-oil formulations (oregano and cinnamon)

[0211] Materials and methods

[0212] The two formulations, hereafter referred to as Melitec and Melitec C, contain essential oil of oregano (composition of EXAMPLE 5) and cinnamon (composition of EXAMPLE 3), respectively.

[0213] The products were applied by brush twice three days apart on a patina composed of actinomycetes in a hypogeal environment. Efficacy evaluation was performed by:

[0214] - ATP assay with the Water-Gio™ Microbial Water Testing Kit (Promega): sampling performed with a sterile swab soaked in lysis reagent and rubbed onto a 1 cm2surface; in the laboratory, the swab was immersed in 500 pl of lysis reagent; the continuation of the procedure is as reported in step 1. The detected RLU were converted to pg of ATP per cm2of sampled surface.

[0215] Culture analysis: micro-organisms taken with sterile buffer and transferred onto Actinomycetes isolation agar culture medium; the test was performed before and after treatment, efficacy was assessed by observing the presence / absence of colonies on the culture medium.

[0216] Results

[0217] Figure 4a shows the appearance of the plugs treated with the two compositions before and after treatment. The ATP assay showed a decrease in ATP levels on the treated surfaces of 96.03% ± 1.65% for Melitec and 98.82% ± 0.37% for Melitec C (see Figure 4b). Culture surveys showed the absence of actinomycete colonies on the culture medium for both products (Fig. 4c).

[0218] 3- Oregano essential oil formulation according to EXAMPLE 5

[0219] Materials and methods

[0220] The composition was applied to a green patina composed of cyanobacteria and green algae on the pavement of a semi-hypogean archaeological context.

[0221] The product was applied to three test tiles:

[0222] By brush, two applications three days apart (FIGURE 5a);

[0223] By spraying, two applications three days apart (FIG. 5b);

[0224] - With a cellulose pulp pack (FIG. 5c), interposing a sheet of Japanese paper between the treated surface and the pack; the system was kept humid by covering it with a sheet of transparent plastic film; the application was carried out only once and lasted about a week, at the end of which it was still possible to perceive the odour of the essential oil present in the product.

[0225] The evaluation of efficacy was carried out by monitoring the macroscopic appearance of the patina over time, also in order to identify a residual effect that would limit surface recolonisation.

[0226] Results

[0227] Observations showed a clear decrease in the pigmentation of the treated patina (Figs. 5a, 5b, 5c), which was particularly evident in the case of the treatment performed with brush application. Six months after application, no signs of recolonisation were present on the surfaces except for small portions.

[0228] 4- Comparison of two single-oil compositions (oregano and thyme)

[0229] Materials and methods

[0230] The two compositions tested, hereafter referred to as Melitec (composition in EXAMPLE 5) and Melitec T (composition in EXAMPLE 7), contain oregano essential oil and thyme essential oil, respectively.

[0231] The compositions were applied to a green patina consisting of green algae, mosses and filamentous fungi in the following manner:

[0232] By brush twice at three-day intervals By pulp wrap, the system was kept moist by covering it with a sheet of transparent plastic film, the application was performed only once and lasted about a week at the end of which it was still possible to perceive the smell of the essential oil in the product.

[0233] The evaluation was carried out by means of the ATP assay, following the procedure in section 1.

[0234] Results

[0235] Figure 6 shows the appearance of the patina before and after treatment with the two compositions. The ATP assay showed a decrease in ATP levels on the treated surfaces of over 82% for Melitec and over 91% for Melitec T (FIG. 7). The latter achieved devitalisation levels of 97% with the pack application.

[0236] 5- Effectiveness of the sodium percarbonate composition

[0237] Materials and methods

[0238] The composition of EXAMPLE 6 was applied by brush twice three days apart on an artefact coated with a black patina, composed mainly of cyanobacteria of the genus Chroococcus.

[0239] Evaluation of efficacy by cellular ATP assay, procedure as in point 1 , performed after both the first and second application.

[0240] Results

[0241] ATP assay analyses showed a decrease in viability of the treated microorganisms of 90.5% ± 1.17% after the first application and 97.51% ± 0.05% after the second application.

[0242] BIBLIOGRAPHY

[0243] [1] F. Antonelli, M. Bartolini, M.-L. Plissonnier, A. Esposito, G. Galotta, S. Ricci, B. Davidde Petriaggi, C. Pedone, A. Di Giovanni, S. Piazza, F. Guerrieri, M. Romagnoli, Essential Oils as Alternative Biocides for the Preservation of Waterlogged Archaeological Wood, Microorganisms. 8 (2020) 2015. https: / / doi. org / 10.3390 / microorganisms8122015.

[0244] [2] M. Bartolini, A.M. Pietrini, S. Ricci, Application of fluorescence microscopy to evaluate the viability of photoautotrophic biodeteriogens, in: B. Georgali, E.E. Toumbakari (Eds.), Proceeding 8th Euroseminar Microsc. Appl. to Build. Mater, Athens, 2001: pp. 511-516.

Claims

CLAIMS1. A composition comprising at least one plant extract and an inorganic matrix, wherein said plant extract is selected from oregano extract, thyme extract, cinnamon extract or mixtures thereof, and wherein said inorganic matrix is preferably a zeolitebased or silica-based matrix.

2. Composition according to claim 1 , wherein said silica-based matrix is a clinoptilolite, fauiasite, paulingite, stilbite or mixtures thereof, and preferably is a clinoptilolite-based matrix.

3. Composition according to claim 1 or 2 further comprising clove extract, rosemary extract, mint extract and / or lavender extract.

4. Composition according to any one of the preceding claims further comprising a surfactant, preferably selected from polysorbate 20 and polysorbate 80.

5. Composition according to any one of the preceding claims further comprising propolis.

6. Composition according to any one of the preceding claims further comprising sodium percarbonate and / or citric acid.

7. Composition according to claim 5 or 6 wherein:- the at least one plant extract or mixture of plant extracts is present in an amount between 5 and 40 % w / w, preferably 24 % w / w;- propolis, where present, is present in an amount between 0.5 and 5 % w / w, preferably 2 % w / w;- sodium percarbonate and / or citric acid, if present, is present in an amount between 5 and 40 % w / w;- the inorganic matrix is present in an amount between 20 and 80 % w / w, preferably 50 % w / w;- at least one surfactant is present in an amount between 10 and 40 % w / w, preferably 24 % w / w; on the total weight of the dry mixture.

8. Composition according to the preceding claim comprising:- at least one plant extract chosen from oregano extract, thyme extract, cinnamon extract or mixtures thereof,- sodium percarbonate and / or citric acid,- one or more surfactants, and- an inorganic matrix based on zeolites or silica.

9. Combination of at least one oregano extract, at least one thyme extract, at least one cinnamon extract and propolis.

10. Composition according to any one of claims 1 to 8 or combination according to claim 9, wherein:- the oregano extract is a plant extract belonging to the genus Origanum selected from extracts of Origanum vulgare, Origanum vulgare subs, hirtum, Origanum compactum and mixtures thereof, and is preferably essential oil of Origanum vulgare subs, hirtum’, and / or wherein- the thyme extract is a plant extract belonging to the genus Thymus selected from extracts of Thymus vulgaris, Thymus serpillum, Thymus zygis and mixtures thereof, and is preferably essential oil of Thymus vulgaris L; and / or wherein the cinnamon extract is a plant extract belonging to the genus Cinnamomum selected from extract of Cinnamomum verum, Cinnamomum zeylanicum or mixtures thereof, and is preferably essential oil of Cinnamomum zeylanicum.

11. Kit of parts comprising the composition according to any one of claims 1 to 8 and 10 or the combination according to claim 9 or 10 for the simultaneous or sequential application of the plant extracts contained therein, preferably together with suitable dilution means, dosing means and / or application means.

12. Detergent comprising the composition according to any one of claims 1 to 8 or the combination according to claim 9 or 10 and a suitable solvent, preferably water.

13. Use of the composition according to any one of claims 1 to 8, the combination according to claim 9 or 10, the kit of parts according to claim 11 or the detergent according to claim 12 for cleaning or cleansing of surfaces, in particular for removing or devitalising biological patinas from the surface of artefacts, preferably wherein said artefacts are stone artefacts, in particular stone cultural heritage assets.

14. A process for preparing the composition according to any one of claims 1 to 8 or the combination according to claim 9 or 10 comprising the following steps, not necessarily in that order: i. Mixing the essential oils and surfactant to obtain a homogeneous mixture; ii. Mixing the inorganic matrix with the mixture obtained in step i. at room temperature for about 24 h or at 60°C in a thermostat bath. iii. Optionally, adding propolis, sodium percarbonate and / or citric acid to the mixture obtained in step ii. and shake to obtain a homogeneous mixture.

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