WO2007048926A2

WO2007048926A2 - Marking composition based on biological vegetable materials

Info

Publication number: WO2007048926A2
Application number: PCT/FR2006/002398
Authority: WO
Inventors: Didier Tousch
Original assignee: Universite Montpellier Ii
Priority date: 2005-10-26
Filing date: 2006-10-25
Publication date: 2007-05-03
Also published as: WO2007048926A3; FR2892541B1; EP1941277A2; FR2892541A1

Abstract

The invention relates to identification and authentication marking of different products, in particular to a composition comprising a determined combination which contains from 2 to 50, preferably from 5 to 30, biological vegetable materials and is associated to a support. Said composition is suitable for marking a product. A product marking method consisting in delivering said composition to a product and a method for authenticating a product marked thereby are also disclosed.

Description

MARKING COMPOSITIONS BASED ON BIOLOGICAL MATERIALS

OF VEGETABLE ORIGIN

The present invention relates to the field of marking for the identification and authentication of various products. It is particularly concerned with the preparation and use of plant prints based on biological materials of plant origin such as pollen grains, spores, aleurone grains and secondary metabolites.

The fight against counterfeiting is a major challenge for modern societies both in terms of public health for drug monitoring and for industry in the context of manufactured products. Many solutions have been proposed, but none has yet been fully effective. Indeed, it is necessary to have a marking system fulfilling the following criteria: marking capacity of most solid or liquid products,

- resistance over time, product handling, and various environmental hazards,

- No or little visible to the tamper-proof eye, - Capable of immediate decryption with expertise of multiple confirmations, to determine the time between the marking of a product and its decryption.

The inventors have now designed a plant footprint system capable of fulfilling all the above objectives.

Pollen grains such as spores ensure the reproduction of almost all plants. Only Flowering plants, the Phanerogams, produce pollen grains or male gametophyes.

Spores are involved in the reproduction of

Cryptogams, plants without flowers such as pteridophytes and bryophytes. Spores are unicellular or multicellular corpuscles that can give birth to new individuals without fertilization.

The interest of pollen grains and plant spores lies on the one hand in their particular characteristics allowing the identification of the plant that produced them and on the other hand in their very great resistance to extreme conditions:

- the pollen grains and the plant spores have particular and specific morphological characters of a plant species allowing infinite combinations of size, shape, thickness, ornamentation of the shell, and the nature and distribution of apertures (pores, colpus). They are very small (between 5 and 500 μm) and constitute the "vegetable dust". These particular morphological characters allow the identification of pollen grains and spores by microscopic techniques. pollen grains and plant spores have cavities on their surface that contain proteins that can be very powerful and specific allergens of each plant species, which can be released in a few seconds. The peptide nature of these allergens allows the identification of pollen grains and spores by immunological methods. pollen grains and plant spores possess a very resistant envelope or exine, formed of polyterpene-type molecules, which can be preserved in sediments. Thus, pollen grains and spores have been preserved since the geological primary. The grains of aleurone constitute reserve cells that are found particularly in the albumen and cotyledons of certain mature seeds such as the seeds of castor, beans and peas. They come from the transformation, by dehydration, of a cellular vacuole during the maturation of the seeds. A grain of aleurone is in the form of globules whose diameter can vary from 0.1 to 25 microns. The interest of aleurone grains lies in the fact that they can contain a great diversity of substances varying from one plant species to another. As substances contained in the grains of aleurone, the hydrolytic enzymes, the proteins, the lipids, the sugars, the alacaloids or finally the pigments can be mentioned. These substances can be identified by techniques well known to those skilled in the art such as high performance liquid chromatography (HPLC) methods and immunological methods of the ELISA type.

The exceptional diversity of the plant world is also reflected in the great variety of secondary metabolites synthesized by plants. Secondary metabolites are compounds, usually of low molecular weight, which do not have an apparent and vital function for the organism producing it, but which seem to have a role in the defense mechanisms of plants against predatory animals or the attack of pathogens, in plant-microorganism relationships, or as a sexual attractant to pollinating insects. Secondary metabolites can be divided mainly into three major families: terpenes, phenolic compounds and nitrogenous substances.

Secondary metabolites of plant origin include alkaloids, terpenes, steroids, flavonoids, quinones, taxanes, glycosides, modified amino acids such as 4-hydroxyisoleucine and triterpenoids such as iridals.

Modified amino acids are the non-constitutive amino acids, which are most often in free form. By way of example of modified amino acids of vegetable origin, mention may be made of 4-hydroxyisoleucine, mimosine, 4-methylproline, carnavaline, albizziine, azetidine 2-carboxylic acid, γ-acid and the like. - hydroxyglutamic, hypoglycine, N-acetyl-ornithine, galégine and alliine.

Iridals are molecules produced in iris bulbs that turn into irones (odor molecules that are much sought after by perfumers) following a complex and very slow oxidation process. Today more than thirty iridals are known. Each iridal is characterized by a specific chromatographic profile detectable by high performance liquid chromatography (HPLC).

Accordingly, a first subject of the invention corresponds to a composition comprising a determined combination of 2 to 50, preferably 5 to 30 and most preferably 5 to 10 biological materials of plant origin, said combination being associated with a support .

The composition according to the invention is remarkable in that the biological materials of plant origin used make it possible to prepare a considerable number of specific combinations. Each combination associated with a support constituting a plant footprint extremely stable over time and unfalsifiable by its complexity.

Another advantage of the composition according to the invention is to offer several types of methods for identifying said composition according to the nature of the biological materials of plant origin implemented. Examples include microscopy techniques for the identification of pollen grains and spores, ELISA immunological techniques for the identification of allergens of pollen grains and spores, and identification. substances contained in the grains of aleurone and the techniques of high performance liquid chromatography allowing the identification of the secondary metabolites. Yet another advantage of the composition according to the invention is that it makes it possible to determine the time elapsed between the marking of a product and the identification of said labeled product, when at least one iridal-type biological material is used in the process. composition according to the invention. Indeed, the iridals turning into irones by a very slow oxidation process, the count of iridals during the identification of said labeled product makes it possible to evaluate the time elapsed between the marking of a product and its identification.

By definite combination is meant the result of choosing a given number of biological materials. According to the invention, the number of biological materials of plant origin chosen to form a determined combination can vary from 2 to 50, preferably from 5 to 30 and most preferably from 5 to 10.

Biological materials of plant origin are understood to mean any structure or substance present in an organism belonging to the plant kingdom. Examples of biological materials of plant origin include pollen grains, vegetable spores, corn seeds and the like. aleurones, secondary metabolites. According to a first preferred embodiment of the composition according to the invention, the biological materials of plant origin may be chosen from one or more of the following groups: pollen grains;

- vegetable spores;

- the grains of aleurones and

- secondary metabolites.

Advantageously, according to the invention, the pollen grains may be derived from a plant chosen from the class of phanerogams comprising angiosperms and gymnosperms, preferably angiosperms and particularly preferably angiosperms whose pollen grains have a size between 10 μm and 30 μm, such as the pollen grains of Corylus avellana, Pommaderis aspera, Cuscuta australis and Dendrocalamus giganteus.

Advantageously, the plant spores may be derived from a plant selected from the class of cryptogams including pteridophytes and bryophytes.

Advantageously, the grains of aleurone may be derived from a plant selected from the group comprising the

Ricinus communis, perennial Ricinus, Ricinus euphorbiaceae, Phaseolus Vulgaris, Pisum sativum and cereals such as wheat, barley,

Phaseolus Vulgaris and wheat and particularly preferably Phaseolus Vulgaris.

According to a second preferred embodiment of the composition according to the invention, the biological materials of vegetable origin may be selected from one or more of the following groups:

- pollen grains;

- vegetable spores and - grains of aleurones.

According to a third preferred embodiment of the composition according to the invention, the biological materials of plant origin can be chosen from the group of secondary metabolites comprising terpenes, steroids, triterpenoids, alkaloids, taxanes, glycosides and modified amino acids such as 4-hydroxyisoleucine, preferably triterpenoids and particularly preferably iridals.

According to a preferred embodiment of the invention, said secondary metabolites may be of natural or synthetic origin. We mean by the expression

"Secondary metabolites of natural origin" that said secondary metabolites are obtained from a plant organism such as a plant, for example by extraction methods. By the term "secondary metabolites of synthetic origin" is meant that said secondary metabolites are obtained by chemical synthesis.

According to a fourth preferred embodiment of the composition according to the invention, said composition may comprise from 1 to 10, preferably from 1 to 5 biological materials of plant origin which may be chosen from one or more of the following groups: pollen; vegetable spores and grains of aleurones, preferably from the pollen grains, in combination with 1 to 10, preferably with 1 to 5 biological materials of plant origin which may be selected from the group of secondary metabolites including terpenes, steroids, alkaloids, taxanes, glycosides, modified amino acids such as 4-hydroxyisoleucine and triterpenoxes such as iridals, preferably triterpenoids and most preferably iridals.

According to a fifth preferred embodiment of the composition according to the invention, said biological materials of plant origin come from different plant species. Examples of plant species from which biological materials of plant origin can be derived include Helianthus annuus, Ipomea purpurea, Ricinus communis, Oenothera fructicosa, Iris germanica, Iris tectorum, Iris pallida. Preferably, said biological materials of plant origin may be derived from Iris and most preferably from Iris germanica and Iris pallida.

Support means any material or substance capable of incorporating and releasing biological materials of plant origin under special conditions. Said support according to the invention must be chosen so that there is no interference between said biological material and the constituents of said support, likely to cause a change in the characteristics of said biological material such as morphological characteristics. Examples of support include capsules, glues, varnishes and paints. The term "special conditions" means the conditions allowing the release of the biological materials from the support of the composition according to the invention. These particular conditions depend on the support used and may for example be a solvent solution chosen from halogenated hydrocarbons such as dichloromethane and these derivatives, cyclohexane, ethylenebenzene, esters of dicarboxylic acids such as dimethyl adipate, dimethyl glutarate, triethylamine, acetonitrile, dimethylsulfoxide and mixtures thereof. Advantageously, when the support according to the invention is polymethyl methacrylate, the release of biological materials from said support can be done in the presence of acetone; the particular conditions may then be the presence of acetone.

According to a fifth preferred embodiment of the composition according to the invention, the support may be a capsule. By capsule is meant any material consisting of hollow globules, of variable shape, for example, cylindrical, spherical or ovoid. Advantageously, said capsule may be composed of at least one polymer which may be chosen from the group comprising polyprolactone, polymethyl methacrylate, Eudragit or Pluronic® such as Pluronic®P6100 or L92, preferably polymethyl methacrylate. methyl, a mixture of polymethyl methacrylate and polyprolactone, a mixture of polymethyl methacrylate and Pluronic and even more preferably polymethyl methacrylate.

Such supports are well known to those skilled in the art and can be obtained using solvent evaporation encapsulation processes (emulsion / precipitation), by coacervation, by interfacial polymerization, by nebulization, by spray drying, by coating in a fluidized bed, by gelling by freezing drops, preferably by solvent evaporation. These processes are described in particular in the following articles: Abrant A et al., Eur.polym. J., 37, 955-963, (2001) "Microencapsulation by Evaporation of Solvent"; Taverdet JL et al., Ann. Do. Exp. Chim. , 94, 955, 103-113 (2001) "Microencapsulation of active ingredient by complex coacervation: influence of certain factors on the release rate"; Fugit JL et al., Polymer Int., 52, 670-675, (2003) "Treatment of a plasticized PVC to reduce plasticizer / solvent migration: optimization with an experiment design"; Ponsart S. et al. Eur.J.Sci. 4 Suppl. S75-S76, 996 "microencapsulation of β-glucuronidase by spray-drying". These methods are also described in the following publications: Lech Pawlowski's The Science and Engineering of Thermal Spray Coatings and Journal of Thermal Spray Technology, ASM International, vol. 13, No. 1, March 2004).

According to a sixth preferred embodiment of the composition according to the invention, the support may be an adhesive. By glue is meant any adhesive type fastening material. According to the invention, the glues may be of mineral, vegetable, animal or synthetic origin. Advantageously, the adhesive according to the invention may be chosen from the group comprising neoprene, polyurethane, cyanoacrylate or a mixture thereof, preferably neoprene.

According to a seventh preferred embodiment of the composition according to the invention, the support may be a varnish. The term "varnish" means any substance which, by application, is likely to form a hard, translucent, shiny and resistant layer. Advantageously, the varnish may be chosen from the group comprising natural and derived resins, polymethacrylate of methyl, Pluronic® such as Pluronic® P6100 or L92, polyethylene, polystyrene, polypropylene or a mixture thereof.

According to a preferred embodiment of the invention, each secondary metabolite is present in the composition according to the invention at a content of at least 0.1 nmol, in particular 1 nmol and more particularly 10 nmol per liter of support, particular per liter of polymer composing said support.

According to a preferred embodiment of the invention, each pollen grain or spore is present in the composition according to the invention at a content of at least 10, in particular 20 and more particularly 40 per nanolitre of support, in particular by nanolitre of polymer component said support.

Advantageously, the composition according to the invention may further contain at least one element selected from the group comprising unmodified amino acids, markers capable of producing a detectable signal. By unmodified amino acid is meant the constituent amino acids of the proteins. For example, the composition according to the invention may further contain at least one unmodified amino acid such as isoleucine, glycine, lysine. By way of example again, the composition according to the invention may further contain at least one marker capable of producing a detectable signal such as fluorescent compounds, bioluminescent compounds, radioisotopes and dyes.

According to a preferred embodiment of the composition according to the invention, the composition may comprise acids modified amino acids and unmodified amino acids associated with a capsule, said amino acids being identified by high performance liquid chromatography techniques.

A second subject of the invention corresponds to a method of marking a product comprising supplying a product with a composition according to the invention.

Examples of products include perfume bottles, bottles such as wine bottles, packaging, banknotes, clothing, watches, electrical products, books, passports, works of art, drugs, chemical formulations, food products such as meats, perfumes, wines, articles made from canvas, paper, plastic, inks, paint.

By supply means the delivery to a product of a composition according to the invention. The provision to a product of a composition according to the invention can be done in any appropriate manner that does not cause modification of the characteristics of said biological materials such as morphological characteristics.

For example, the supply to a product can be done by gluing or varnishing if the support of the composition used is an adhesive or a varnish respectively.

When the support of the composition is a capsule and the product is a fluid, the composition according to the invention can be supplied to the product by a mixing operation. When the support of the composition is a capsule and the product is a paste or a solid, the composition according to the invention can be supplied to the product during the manufacture of the paste or the solid. By way of examples, when the support of the composition is a capsule, the supply to a product of a composition according to the invention can be carried out by:

mixing a composition according to the invention with an ink or ink vehicle composition or with water or a combination of water and organic solvent and printing directly on a product, or

- Immersing at least a portion of the surface of a product in a solution comprising a composition according to the invention for fixing, for example adsorbing said composition on the product, or

the mixture of at least one composition according to the invention with a dyestuff or a paint intended to be used with a product, or the addition of a composition according to the invention directly to solutions or formulations of chemical products such as medicines or food products.

Advantageously, the composition according to the invention can be supplied to the product by spraying. Spraying can be done for example using a nozzle apparatus for marking many products or using an apparatus of the "aerosol" type.

A third subject of the invention corresponds to a labeled product that can be obtained by supplying said product with a composition according to the invention.

A fourth subject of the invention corresponds to a method for identifying a labeled product according to the invention, characterized in that it comprises the following steps: (i) extracting a composition according to the invention of said labeled product, and

(iii) identifying said biological materials of said composition.

By extraction of a composition according to the invention of said labeled product is meant the recovery of the composition according to the invention of said labeled product. The extraction of the composition according to the invention from the labeled product can be carried out in any appropriate manner which does not cause modification of the characteristics of said biological materials such as morphological characteristics.

For example, when the composition is extracted from a product in a liquid form, the liquid can be evaporated using an apparatus such as a vacuum or filtered centrifuge to recover the composition in the form of filtration residues. and to allow the identification of biological materials of said composition. The filtration of said product can be carried out, for example, using ultrafiltration unit of Amicon type (Millipore) with "cellulose YM 1 Dia 44.5 μm" type filters (Millipore).

By way of example again, when a composition is extracted from a product in a solid form, the product can be dissolved or appropriately treated with for example a solvent. Halogenated hydrocarbons, such as dichloromethane and these derivatives, cyclohexane, ethylenebenzene, dicarboxylic acid esters such as dimethyl adipate, dimethyl glutarate, triethylamine, acetonitrile, cyclohexane and the like may be mentioned as solvents. dimethyl sulfoxide and mixtures thereof.

Identification means the characterization of the biological materials of said composition. Several techniques can be used according to the invention for allow the identification of biological materials of plant origin. Examples include microscopy techniques for the identification of pollen grains and spores, ELISA immunological techniques for the identification of allergens of pollen grains and spores, and identification. substances contained in the grains of aleurone and the techniques of high performance liquid chromatography allowing the identification of the secondary metabolites.

As an example of a microscopy technique allowing the identification of the pollen grains and spores of a composition according to the invention, the following technique may be mentioned:

Pollen grains and plant spores after being fixed on slides and treated with safranin-type dye formolated with Sémichon or phloxine B alcoholic or red congo SDS or iodine green, can be photographed and digitized under optical microscope (with a magnification which may be between 6Ox and 100Ox in immersion), in sequences of 100 to 1000 images (with a step which may be between 0.5 and 1 μm, preferably 0.5 μm) taken at different focal lengths to represent grains in three dimensions. Measurements on the central section of pollen grains or plant spores can then be made to analyze some of their characteristics such as color, size, shape, pores, nucleation of the cytoplasm, convexity of the pollen grain. . The characteristics, in particular the morphological characteristics of pollen grains or plant spores can also be analyzed from their three-dimensional representation. The different measures and characteristics of pollen grains or spores then can be compared with databases of different characteristics of pollen grains and known plant spores. These databases are available on http sites; // perso .wanadoo. en / pollen / library / sought after. htm and http; //apimo.dk/pollen.htm. By this comparison, the pollen grains and plant spores of a composition according to the invention can thus be easily identified.

The identification of the pollen grains and spores of a composition according to the invention can also be carried out by analyzing the measurements performed in a single focal plane (with a magnification that may be 10Ox in immersion).

The secondary metabolites of the composition according to the invention can be easily identified by techniques well known to those skilled in the art, such as by the analysis of their elution profile after migration on a C8 or C18 type column (phase reverse) by high performance liquid chromatography (HPLC). Each secondary metabolite has a particular elution profile by its molecular weight, its hydrophobicity properties and its charge.

According to a preferred embodiment of the method for identifying a labeled product according to the invention, the method further comprises a step of extracting (ii) said biological materials from said support. The extraction of said biological materials from said support is understood to mean the release and recovery of said biological materials from said support. The extraction of the composition according to the invention from the support can be carried out in any appropriate manner which does not cause modification of the characteristics of said biological materials such as morphological characteristics.

When the support is an adhesive, a varnish or a capsule, the extraction of said biological materials from said support can be carried out using solvents such as, for example, halogenated hydrocarbons such as dichloromethane and these derivatives, cyclohexane, ethylenebenzene dicarboxylic acid esters such as dimethyl adipate, dimethyl glutarate, triethylamine, acetonitrile, dimethylsulfoxide and mixtures thereof.

Advantageously, when the support according to the invention is composed of polymethyl methacrylate or Pluronic® or a mixture thereof, the extraction of said biological materials from said support can be carried out using acetone.

According to another preferred embodiment of the method for identifying a labeled product according to the invention, the composition comprises at least one pollen grain and the step

(iii) corresponds to the identification of said pollen grain by its morphological characteristics.

Advantageously, the identification of said pollen grain and / or said spore can be carried out directly in the support without any prior manipulation.

According to another preferred embodiment of the method for identifying a labeled product according to the invention, the step of identifying (iii) said biological materials may comprise an immunological method, for example of the type

ELISA. According to yet another preferred embodiment of the method for identifying a labeled product according to the invention, the step of identifying (iii) said biological materials may comprise a high performance liquid chromatography method, for example using a column type C8 or cl8 (reverse phase).

A fifth object of the invention corresponds to the use of a composition according to the invention as a product marking agent.

Other advantages and features of the invention will appear with reference to the figures and examples which follow.

Figure 1 illustrates the capsules 50 microns in diameter, obtained according to the manufacturing method of Example 1 using polymethyl methacrylate as polymer. This is a photograph of a scanning electron microscope (Hitachi S-2600) view of the capsules obtained according to the manufacturing method of Example 1. The white arrow indicates the capsules of the order of 50 nm. of diameter existing at the periphery of the capsules of 50 microns in diameter. The white line indicates the length of 20 μm.

Figure 2 shows chromatograms of an amino acid solution isoleucine, before (Figure 2A) and after (Figure 2B) encapsulation obtained by passage over column Alltima C8. (Figure 2A): 25 μL of a solution containing 150 μM isoleucine were mixed with 25 μL of a solution of polymethyl methacrylate and Pluronic®P6100 50/50 by weight. This mixture was then analyzed in High Performance Liquid Chromatography on a C8 column. (Figure 2B): The content of a polymethyl methacrylate and Pluronic® P6100 capsule, comprising isoleucine, was extracted and analyzed in High Performance Liquid Chromatography on a C8 column.

Figure 3 shows chromatograms of a solution of amino acids, glycine and 4-hydroxyisoleucine, before (Figure 3A) and after (Figure 3B) encapsulation obtained by passage over column Alltima C8. 25 μl of a solution containing 150 μM of glycine and 4-hydroxyisoleucine were mixed with 25 μl of a solution of polymethyl methacrylate and 50/50 by weight Pluronic®P6100. This mixture was then analyzed in High Performance Liquid Chromatography on a C8 column. (Figure 3B): The content of a polymethyl methacrylate and Pluronic® P6100 capsule, comprising glycine and 4-hydroxyisoleucine, was extracted and analyzed in High Performance Liquid Chromatography on a C8 column.

FIG. 4 shows chromatograms obtained by high performance liquid chromatography analysis of a solution of iridals (16-hydroxyiridal and 10-deoxy-17-hydroxyiridal) (FIG. 4A) before inclusion in a carrier based on polymethyl methacrylate, (Figure 4B) after extraction of the polymethyl methacrylate support.

Figure 5 illustrates the principle of marking an ink using a determined combination of biological materials of plant origin incorporated into capsules and identifying the combination determined by the High Performance Liquid Chromatography technique.

Figure 6 illustrates glass slides coated with a layer of polymethyl methacrylate. 50 μl of a solution of polymethyl methacrylate / dichloromethane were deposited on a glass slide (FIG. 6A) in the form of a thick layer (FIG. 6B) in the form of a drop, (FIG. 6C) in the form of a spread drop. with a spatula.

Figure 7 illustrates glass slides coated with a layer of pollen grains incorporated into a support based on polymethyl methacrylate and dichloromethane. A mixture of althea and ephemeral pollen grains was incorporated into a solution of polymethyl methacrylate and dichloromethane and then deposited on a glass slide.

Figure 8 illustrates the optical microscopic observation of pollen grains from althea (Hibiscus syriacus) and ephemeral (Tradescantia flumineusis) stained with methyl green before (Figure 8A and Figure 8B) and after

(Figure 8C) their incorporation into a thin layer of

Polymethacrylate methyl / dichloromethane. Photograph of pollen grains from althea (Figure 8A) and ephemeral

(FIG. 8B) stained with methyl green before being incorporated in a support based on polymethyl methacrylate / dichloromethane. Photograph of pollen grains from althea (Figure 8C) after their extraction from a support based on polymethyl methacrylate / dichloromethane. EXAMPLE 1 Method for Producing Capsules Comprising an Amino Acid Modified as a Biological Material of Plant Origin Associated With an Unmodified Amino Acid

1) Preparation of the polymer solution constituting the capsules

Polymethylmethacrylate (PMMA) or a mixture of PMMA powder and 50/50 (w / w) Pluronic®P6100 was poured into a solution of volatile organic solvent, ethyl acetate or dichloromethane in proportions of 25 g. for 500 ml and was solubilized by stirring to obtain a solution A.

This solution A was mixed with an aqueous solution B whose preparation protocol is as follows: 0.002% (weight / volume) of amino acid, ie an unmodified amino acid, isoleucine alone (MW = 131.17 g) ), or an unmodified amino acid mixture, glycine (MW = 75.07 g) and a modified amino acid, 4-hydroxyisoleucine (MW = 147.5 g), which corresponds to 0.1 mg d amino acid dissolved in 4.9 mL of distilled water, then adjusted to 5 mL. The final solution B thus comprises approximately 200 to 270 μM of each amino acid. This aqueous solution B enriched with biological materials of plant origin must be immiscible with the solvents of the polymer. The mixture of the 2 immiscible solutions A and B was treated for 1 minute by an ultrasound sonicator (Bioblock Scientific Ultrasonic processor) set to 100% observing pulses of 5 seconds alternated by 5 seconds poses in order to obtain a fine emulsion C. This operation made it possible to obtain droplets of very fine water in the solvent.

The emulsion C thus obtained was then mixed with a solution of polyvinyl alcohol (PVA) used as a surfactant, which makes it possible to prevent any agglomeration of the polymer or capsules if they have formed. This emulsion process allows the solvent polymers to precipitate around the droplets of the aqueous solution B as the solvent evaporates. This emulsion process lasted 13 hours (with a minimum of 12 hours) while stirring with a mixer (Rayneri from VMI) set at a speed of 2500 rpm to obtain an emulsion D.

2) Purification of the Capsules In order to purify the capsules by eliminating the surplus of unencapsulated amino acids and polymers, the emulsion D containing the capsules was passed through a 0.25 μm porosity filter with the aid of a Millipore ™ filtration unit. The filter on which the capsules were retained was rinsed with E buffer (composed of 20mM Tris HCl and 5mM EDTA) to remove any traces of aqueous solution. This operation made it possible to obtain on the filter a large number of capsules with a diameter of 50 μm.

These capsules were taken up in 5 ml of buffer E and then filtered again on a membrane of 0.45 μm porosity and the filtrate was recovered. This operation made it possible to obtain a solution rich in capsules of diameter 50 nm; these capsules existing on the periphery of the capsules of 50 microns in diameter.

3) Tests of the encapsulation efficiency of amino acids

The amino acids were determined according to the protocol described in Example 2 before and after encapsulation. By difference, a percentage of encapsulation of the aqueous solution B enriched in amino acids was obtained; averaging the results of three manipulations independent, an encapsulation rate of 70% to 75% was obtained.

The 50 μm diameter capsules obtained with polymethyl methacrylate are shown in FIG.

The level of impermeability of the capsules has been evaluated. For this, they were left in the recovery buffer A filter at room temperature. Each day a 200 μL sample was taken to measure the amino acids. This experiment was conducted on three independent capsule syntheses. The longest experience spanned a month. No significant amino acid value in the samples taken was obtained, which shows that the capsules obtained do not secrete very few amino acids.

EXAMPLE 2 Analysis of the Content of a Capsule

The contents of the capsules obtained by the manufacturing method illustrated in Example 1 were analyzed by the following method:

1) Extraction of the Content of the Capsules After estimating the density of the capsules by electron microscopy, the F solutions were diluted in order to obtain solutions containing a single capsule in 100 μl. A 100 .mu.l sample was evaporated using a vacuum centrifuge to recover a capsule. The recovered capsule was then incubated for 2 minutes in 5 .mu.l of acetone and then 20 .mu.l of a water / ethanol mixture (1/70, volume / volume) was added. 2) Identification of the amino acids contained in the capsules

The 25 μl of the solution containing a capsule with water and ethanol were incubated for 1 minute at room temperature with 25 μl of a solution based on ortho-phthalic acid (OPA) prepared according to the following protocol :

A 38.2 mM OPA solution in methanol was mixed with a 0.2M borate solution (prepared in 0.2M KCl and adjusted to pH 9.9 with sodium hydroxide) and an acid solution. mercaptopropionic in proportions 5/20 / 0,05 (volume / volume / volume); The pH of the solution thus obtained was then adjusted to 9.1.

The 50 μl thus obtained were then injected onto an Alltima C8 High Performance Liquid Chromatography column (250 × 4.6 mm). The apparatus used is a Varian 5000 Liquid Chromatograph coupled to a Shimadzu RF-530 fluorimeter (sensitivity: high, gain: infinity, excitation and emission wavelength: 337/454). Data acquisition was performed using the Millenium ³² ® Chromatography Manager software.

The eluents used are the eluent G of 8mM sodium acetate at pH 5.7 and the eluent H composed of a methanol / tetrahydrofuran (THF) mixture (93.1 / 6.9 v / v). The eluent flow rate was 1.5 mL / min.

The gradient that was applied to the column was 91% eluent G sodium acetate and 9% eluent H for 10 minutes, 66% eluent G and 34% eluent H for 5 minutes. minutes then 38% eluent G and 62% eluent H for 10 minutes and finally 100% eluent H for 2 minutes. Data collection was stopped 30 minutes after the sample was injected onto the column. The results obtained are shown in FIGS. 2 and 3. FIG. 2 represents chromatograms of an isoleucine amino acid solution, before (FIG. 2A) and after (FIG. 2B) encapsulation obtained by passage over the Alltima C8 column. Figure 3 shows chromatograms of a solution of amino acids, glycine and 4-hydroxyisoleucine, before (Figure 3A) and after (Figure 3B) encapsulation obtained by passage over column Alltima C8. An identical chromatographic profile in each experiment before (Figure 3A) encapsulation and after (Figure 3B) extraction of amino acids from the capsule was observed.

These experiments therefore demonstrate that it is possible to identify a product labeled with a composition comprising a specific combination of amino acids encapsulated by the extraction of a single capsule of said product.

In theory each capsule regardless of size contains the amino acids at a concentration of approximately 150 μM each. The experiments were carried out with a capsule 50 μm in diameter; its volume is therefore 65.5 pico liter. We can therefore conclude that the 50 μm diameter capsule contains about 10 pmoles. l / ^10th of the solution was loaded on the chromatographic column is approximately 1 pmol of each amino acid.

In view of these results and knowing that the detection sensitivity of this method is a femtomole, we can therefore consider using in the compositions according to the invention capsules of about 50 nm in diameter. By encapsulating an amino acid solution 10 times more concentrated, it is possible to reduce the diameter of the capsules to 5 nm.

4) Quantification of the amino acids contained in the capsules

From the obtained chromatogram, the peak area of the standard amino acid is calculated using the Millennium ³² Chromatography Manager ® software. The area of the peak is directly correlated to the amount of amino acid deposited.

Example 3: Marking of a glass plate with a film of polymethyl methacrylate containing a determined combination of iridals.

1) Purification of iridals from iris rhizomes

The extraction and analysis of iridals from iris rhizomes was performed according to the following protocol:

Rhizomes of various genotypes and varieties of iris; Iris germanica L. var. Rococo, Iris tectorum Maxim., Iris pallida Lam. , were washed with distilled water and then cut into very small pieces before being crushed into fine powder by the grinder (Janke & Kunkel, model AlO). The juice was deposited on a cellulose cartridge (whatman, 41 x 123 mm), then the latter was rinsed 3 times with EtOH-H ₂ O (7/3, v / v) solution at room temperature in a cooling device. Tecator type. The eluents were pooled and the solution was filtered through an Analypore EC membrane (0.45 μm, Nalgene Filtration System) and the solvent was evaporated under vacuum on a rotary evaporator (Rota Vapor, Bioblock). Distilled water (100 mL) was added and the resuspension solution was washed 4 times (4 x 100 mL) with Et ₂ O. The organic solution was concentrated at 40 ^° C. under vacuum on a rotary evaporator (Rota Vapor, Bioblock). At the end of this treatment, there remains a residue of about 5g which was passed on two columns HPLC (Liquid Chromatography High Performance) successive according to the following protocol:

High Performance Liquid Chromatography (HPLC) Protocol: The HPLC instrument consists of two Gilson pumps, a 305 pump and a 302 pump (25 SC head), a Gilson UV-Vis 112 detector set at 254 nm. The first semi-preparative column is Hibar Lichrospher RP-18, 100 Å, 10 μm, 250 × 25 mm (Merck, Germany). The second preparative column is a Kromasil analytical, C18, 5 μm, 100 Å, 250 x 4.6 mm (Touzart & Matignon, France). For 2 minutes, a mixture of MeOH / H ₂ O (4/1, v / v) was passed and a gradient was then programmed in order to pass to a 100% MeOH eluting solvent in 20 min, then the MeOH was again been spent on the column for 30 min.

2) Incorporation of iridals in polymethyl methacrylate and then deposit on a glass plate

A mixture of two iridals: 16-hydroxyiridal and 10-deoxy-17-hydroxyiridal, each at 500 μM was carried out. This solution was then mixed with a solution of polymethyl methacrylate / dichloromethane (2.5 mg / 50 mL). The fluid mixture was poured and then spread on a glass slide to cover the blade. The slide was placed at 50 ^° C. in order to evaporate the solvent and generate a film of uniform thickness. Within a few minutes, the highly volatile solvent was removed and the polymethyl methacrylate formed a thin, solid, transparent film. 3) HPLC Analysis of Iridals Before and After Their Inclusion in Polymethylmethacrylate

The next day, on this film a volume of 500 .mu.l of acetone was spread then a volume of 400 .mu.l was recovered which was then filtered on a 0.25 .mu.m filter (Millipore) fitted on a 1 ml syringe.

50 .mu.l of this solution were then injected onto the pre-equilibrated C18 analytical column. The results are shown in FIG. 4. FIG. 4 shows chromatograms obtained by high performance liquid chromatographic analysis of a solution of iridals (16-hydroxyiridal and 10-deoxy-17-hydroxyiridal) (FIG. 4A) before inclusion in a polymethylmethacrylate-based support (FIG. 4B) after extraction of the polymethylmethacrylate-based support.

This experience demonstrates the possibility:

- Incorporate a specific combination of biological materials of plant origin such as iridals in a support such as polymethyl methacrylate to mark a product such as a glass plate.

extracting the determined combination of the support by solubilization of polymethyl methacrylate. to identify the determined combination of biological materials previously supplied to the product by the High Performance Liquid Chromatography technique.

Example 4 Observation Using a Light Microscope of a Specific Combination of Pollen Beads Before and After Its Extraction from a Support Based on Polymethylmethacrylate 1) Marking of a glass plate with transparent layers of polymethyl methacrylate containing a determined combination of pollen grains

A solution of polymethyl methacrylate was prepared by solubilizing 250 mg of polymethyl methacrylate in 5 ml of dichloromethane. This solution was stored at room temperature in a perfectly sealed bottle.

Using a micropipette, 50 μl of the solution of polymethyl methacrylate / dichloromethane was deposited in the center of the glass slide in the form of a large droplet. This drop was either dried or spread in a very thin layer using a glass spatula. In a few minutes, a rigid and strong and perfectly transparent, sometimes slightly opalescent film was obtained as shown in Figure 6.

A mixture of pollen grains from althea and mayfly was then incorporated in the solution of polymethyl methacrylate / dichloromethane according to the following protocol:

Pollen grains from althea (Hibiscus syriacus) and mayfly (Tradescantia virginiana) were harvested and stored in the dry and dark. They come in the form of a fine yellow powder.

A volume of 50 μl of the solution of polymethyl methacrylate / dichloromethane was poured into an eppendorf tube (specific PCR 75 μL) and quickly closed to prevent evaporation. Using a plastic spatula, a mixture of pollen grains from althea and ephemeral (the pollen grains are fixed by electrostatic effect with the spatula) was immersed some seconds in the 50 μL of the solution of polymethyl methacrylate / Dichloromethane

The mixture was then deposited and spread on a glass slide using a micropipette. Once the solvent was completely evaporated (after 2 minutes), a thin transparent layer was obtained.

2) Marking of a glass plate using transparent layers of polymethyl methacrylate in which are incorporated pollen grains stained with methyl green

Pollen grains from althea (Hibiscus syriacus) and mayfly (Tradescantia virginiana) were harvested and stored in the dry and dark. A coloration of the pollen grains with methyl green was carried out according to the following protocol:

A drop of water was deposited in the middle of a glass slide. Using a plastic spatula, pollen was collected and dispersed in the drop of water. The blade was then placed at 70 ⁰ C in order to evaporate all the water. A drop of ethanol was deposited on the dry residue to dehydrate the biological tissues. After a few minutes, the ethanol is removed. This treatment was repeated 2 times). A drop of 5% methyl green was then deposited on the slide and left for 5 minutes before being dispersed with ethanol. It is also possible to remove excess dye with a paper towel by placing on the edge of. the drop of ethanol.

The colored pollen was then covered with a thin layer of 50 μl of the solution of polymethyl methacrylate / dichloromethane. The thin transparent layer formed on the slide was photographed (Figure 7) and observed in optical microcopy (Figures 8A and 8B). A tiny portion of the clear film was recovered by resolubilization with dichloromethane. Resolubilization can also be carried out with a mixture of chloroform / dichloromethane / isoamyl alcohol, 12/12/1, v / v / v). The recovered product was deposited on a glass slide in order to identify by optical microscopy the pollen grains (FIG. 8C).

3) Observation of pollen grains by optical microscopy

Photographs of the pollen grains (Figure 8) were made using a Leitz-Diaver microscope with 1000X magnification under immersion oil. FIG. 8 illustrates the optical microscopic observation of pollen grains from althea (Hibiscus syriacus) and ephemeral (Tradescantia flumineusis) stained with methyl green before (A and B) and after (C) their incorporation into a thin layer of polymethylmethacrylate / dichloromethane. Photograph of pollen grains from althea (A) and ephemeral (B) stained with methyl green prior to incorporation into a polymethyl methacrylate / dichloromethane support. Photograph of pollen grains from althea (C) after their extraction from a polymethylmethacrylate / dichloromethane support.

Pollen grains from althea and mayfly were perfectly identified by their morphological characteristics. The coloration of the pollen grains by methyl green, their incorporation into a support based on polymethyl methacrylate and dichloromethane as well as their release by solubilization of the support did not alter the morphology of the pollen grains, which allows their identification.

Claims

A composition characterized in that it comprises a determined combination of 2 to 50, preferably 5 to 30 biological materials of vegetable origin, said biological materials of plant origin being selected from one or more of the following groups:

- pollen grains;

- vegetable spores; - the grains of aleurones and

- secondary metabolites; and in that said combination is associated with a support adapted to incorporate and release under particular conditions for their identification, said materials and in that said support is selected from the group comprising glues, varnishes and capsules.

2. Composition according to claim 1, characterized in that said biological materials of plant origin are selected from the group of secondary metabolites.

3. Composition according to claim 1, characterized in that it comprises from 1 to 10, preferably from 1 to 5 biological materials of vegetable origin chosen from one or more of the following groups:

- pollen grains; plant spores and aleurone grains; in combination with 1 to 10, preferably with 1 to 5 biological materials of plant origin selected from the group of secondary metabolites.

4. Composition according to any one of claims 1 to 3, characterized in that said Secondary metabolites are selected from the group consisting of terpenes, steroids, triterpenoids, alkaloids, t.axanes, glycosides and modified amino acids, preferably from triterpenoids.

5. Composition according to claim 4, characterized in that said secondary metabolites are iridals.

6. Composition according to any one of claims 1 to 5, characterized in that said biological materials of plant origin are from different plant species.

7. Composition according to any one of claims 1 to 6, characterized in that said support is a capsule.

8. Composition according to claim 7, characterized in that said capsule is composed of at least one polymer selected from the group comprising polycaprolactone and polymethyl methacrylate.

9. Composition according to any one of claims 1 to 6, characterized in that said support is an adhesive selected from the group comprising neoprene, polyurethane, cyanoacrylate and a mixture thereof.

10. Composition according to any one of claims 1 to 6, characterized in that said support is a varnish selected from the group comprising natural and derived resins such as casein, polymethyl methacrylate, polyethylene, polystyrene polypropylene and a mixture thereof.

11. A method of marking a product characterized in that it comprises the supply to a product of a composition according to any one of claims 1 to 10.

12. A method of marking according to claim 11, characterized in that said composition is supplied to said product by spraying.

13. Marked product obtainable by the process according to any one of claims 11 or 12.

14. A method of identifying a labeled product according to claim 13 characterized in that it comprises the following steps: (i) extracting a composition according to any one of claims 1 to 10 of said labeled product, and

(iii) identifying said biological materials of said composition.

15. The method of identification according to claim 14 of a labeled product, characterized in that it further comprises a step of extracting (ii) said biological materials from said support.

16. The identification method as claimed in claim 14, wherein the composition comprises at least one pollen grain and in that step pollen grain by its morphological characteristics.

17. Use of a composition according to any one of claims 1 to 10 as a product marking agent.