WO2003097858A1

WO2003097858A1 - Identification method for protecting products

Info

Publication number: WO2003097858A1
Application number: PCT/EP2003/004799
Authority: WO
Inventors: Christel Adomat; Tilo Weiss; Heiner Brinkmann; Ralf Reifferscheidt; Matthias Gabler
Original assignee: Henkel Kommanditgesellschaft Auf Aktien; Gabler, Julika
Priority date: 2002-05-17
Filing date: 2003-05-08
Publication date: 2003-11-27
Also published as: DE10222075A1; AU2003239846A1; AU2003239846A8

Abstract

The invention relates to an identification method for a product, comprised of the following steps: a) selecting at least one system comprising at least constituents A and B, of which one constituent is already contained in the product or is added thereto, and; b) identifying the constituent contained in or added to the product by using the other constituent, whereby constituent A is at least one enzyme and constituent B is at least one substrate.

Description

IDENTIFICATION PROCEDURE FOR PRODUCT PROTECTION

The invention relates to an identification method for a product, comprising the following steps: a) selection of at least one system comprising at least components A and B, of which a component is already contained in the product, or is added to it, b) identification of those in the product contained or added component using the other component, wherein component A is at least one enzyme and component B is at least one substrate.

The increasing imitation of branded products and the subsequent marketing of these counterfeit goods causes considerable economic damage and often affects the image of the branded product concerned due to its lower quality. For this reason, it is desirable to mark the product so that it can be clearly used for identification, but which can only be imitated with little or little effort.

Such markings of products for product protection have so far been mainly limited to marking the packaging. It is only protected as a whole, and the actual product cannot be identified in the packaging.

Biological marker systems have already been described for direct marking of the product itself.

WO 90/14441 describes a method for product protection in which a nucleic acid sequence is added to the product for identification is collected and detected. The method preferably includes the amplification of the nucleic acid sequence.

DE 199 34 573 describes a similar method for labeling and identifying solid, liquid and gaseous substances, a nucleic acid sequence being added to the product, which is then detected by hybridization with a complementary nucleic acid sequence.

In both of the cases mentioned, the identification of the added substances is associated with considerable expenditure on equipment, which is prone to errors on the one hand and time-consuming on the other hand, and therefore does not permit rapid identification of the product.

The international patent application WO 89/07272 describes a method for labeling a substance, in which the lowest concentrations of a marker substance are added to the substrate, which can then be detected by the formation of an immunologically bound pair with a complementary binding partner. For example, N-acetyl-L-lysine aflatoxin B is concentrated and detected by immunoaffinity chromatography.

The addition of fluorescent and / or luminescent compounds alone is indeed easy to detect by appropriate lighting, but is relatively unspecific as long as no wavelength-dependent identification is required in terms of device technology.

The object of the invention is therefore to provide an alternative method for identifying products which, on the one hand, is difficult or difficult to imitate, but on the other hand is simple and inexpensive to prove.

This task is solved by an identification procedure for a product, consisting of the following steps: a) selection of at least one system comprising at least components A and B, of which one component is already contained in the product or is added to it, b) identification of the component contained or added in the product with the aid of the other component, component A at least one enzyme and component B is at least one substrate.

In step a) of the identification process, a detection system must be selected which comprises at least components A and B, component A being at least one enzyme and component B being at least one substrate for at least one enzyme of component A.

One of the components A or B is already contained in the product or is added to the product. If one of the components A or B is already contained in the product, this component can also be used for identification. The other component must then be selected accordingly. This has the advantage that no further addition of component A or B has to take place to the product. This way, changes in product properties or manufacturing costs can be avoided by using a different marking method.

In step b), the presence of the component contained or added in the product is detected with the help of the other component of the system and the product is thus identified.

Another object of the present invention is an identification method for a product, consisting of the following steps: a) Selection of at least one system comprising at least components A, B and C, of which at least one component is already contained in the product, or is added to it , b) identification of the component contained or added in the product using the other component (s), where component A is at least one enzyme, component B is at least one substrate and component C is at least one substance which accelerates the enzyme reaction, in particular a cofactor, mediator and / or activator.

In the context of the invention, a substance which accelerates the enzyme reaction is understood to mean those substances which, by their presence, promote or even enable the course of the enzyme reaction.

Advantageously, in the absence of the substance which accelerates the enzyme reaction, the conversion of the substrate by the enzyme in particular is significantly slowed down or essentially not. In the absence of component C, the corresponding components A and B can therefore be used together in the product. With the help of component C, the identification then takes place in step b). It is also possible that component C is contained or added to the product and components A and B are used in step b) to identify component C. The specificity of the detection is further increased by the three-component system.

According to a particularly preferred embodiment, in particular a cofactor, mediator and / or activator can be used as the substance accelerating the enzyme reaction.

Cofactors are substances that are necessary for the course of the enzymatic catalysis in addition to the enzyme (possibly the apoenzyme, the pure protein portion of the enzyme) and the substrate or the substrates. They are also referred to as coenzymes and differ from the substrates in that they are regenerated in a short cycle, usually in a single reaction step. Many coenzymes therefore occur in two or more different forms and are effective as electron carriers or carriers of molecular groups. As so-called cosubstrates, some cofactors, such as NAD, bring certain enzymes back into the substrate after the substrate has been converted Initial state back so that the next catalytic cycle can take place. Cofactors are often reversible and not covalently bound to the enzyme. However, there are also cofactors such as B. the heme, which can occur covalently bound to the enzyme and are often referred to as a prosthetic group. Examples of cofactors that can be used are: cations of iron, copper, zinc, calcium, manganese, cobalt, potassium or sodium, biotin, heme, FAD, FMN or thiamine pyrophosphate.

Mediators are to be understood in particular as substances which mediate the catalytic conversion of the substrate by the enzyme, in which the mediators themselves serve the enzyme as the primary substrate. The enzyme converts the mediator into an activated form. This activated form of the mediator then brings about the implementation of the substrate, the mediator generally is completely regenerated. In the net reaction, the substrate is converted by the enzyme, while the mediator remains unchanged.

Such a reaction chain can often be observed with redox enzymes. The reaction with the enzyme puts the mediator in an activated state with a high oxidation or reduction potential. In the subsequent reaction of the mediator with the substrate, the substrate is oxidized or reduced.

In the absence of the mediator, the reaction between enzyme and substrate advantageously takes place only to a small extent or in particular not.

For example, the mediation of the enzyme reaction via a wide variety of mediators is known for polyphenol oxidases. These include 1-hydroxybenzotriazole (HBT), violuonic acid, 2,2'-azino-bis- (3-ethylbenzthiazoline-6-sulfonic acid (ABTS), 2-nitroso-1-naphthol-4-sulfonic acid (HNNS) or methylsyringate. Polyphenol oxidases advantageously show color changes during the reaction with a suitable substrate and mediator, which can often be observed with the naked eye and therefore simply and inexpensively. In addition to the cofactors already mentioned, activators in the context of the invention are understood to mean those substances which, by their presence, accelerate the catalytic conversion. Such activators can be, for example, metal ions, for example calcium, magnesium, copper, nickel or iron, or else complexing agents.

Glutathione (L-γ-glutamyl-L-cysteinylglycine, glutathione-SH) is a tripeptide that is converted into the disulfide by reversible oxidation and is therefore a buffer system for the redox state. For example, peroxides and radicals are reduced under the catalysis of selenium-containing glutathione peroxidase. Glutathione is also a cofactor of glutathione S-transferase.

Ubiquinones (Coenzyme Q) are also cofactors that can serve as electron and proton carriers. A distinction is made between the ubiquinones depending on the number of isoprene units linked in the side chain or on the number of carbon atoms. Coenzyme Q10, which carries 10 isoprene units, is particularly well known.

NAD (nicotinamide adenine dinucleotide) is involved in many enzymatic redox reactions and serves, especially in connection with dehydrogenases, as a hydrogen transfer agent. The oxidized form is called NAD ⁺ , while the reduced form is known as NADH. NAD ⁺ can be used, for example, as a free acid, lithium or sodium salt. NADH is often used as dipotassium, disodium, cyclohexylammonium and tris (hydroxymethyl) methylammonium salt.

NADP (nicotinamide adenine dinucleotide phosphate) carries an additional phosphoric acid group on the 2'-hydroxy group of the NAD. This cofactor, also known as coenzyme 2, is also involved in many enzymatic redox reactions and often acts as a hydrogen carrier. The oxidized form NADP ⁺ is commercially available as the potassium, sodium, disodium and tris salt. The reduced form NADPH is often commercially available as tetrapotassium, tetrasodium and tris salt. FAD (flavin-adenine dinucleotide) plays a particular role as a prosthetic group of numerous flavoenzymes, in particular dehydrogenases, oxidases or reductases, as hydrogen and electron carriers. The reduced form of FAD is called FADH ₂ . FAD often occurs in connection with trace elements such as copper, iron or manganese.

FMN (flavin mononucleotide or riboflavin 5'-phosphate), for example, is contained in some flavoenzymes instead of FAD as a prosthetic group and, like this, acts as a cofactor of hydrogen transfer.

Methylcobalamin is the vitamin B12-derived coenzyme of methionine synthase that catalyzes the transfer of methyl groups.

Coenzyme A (CoA) plays an important role in the metabolism as a carrier of acyl groups, especially acetyl groups. An important methyl group donor is S-adenosylmethionine (S-adenosin-5'-yl-methioninium, ademethionine).

Tetrahydrofolic acid (N - [(6S) -5,6,7,8-tetrahydropteroyl] -L-glutamic acid) is also known as coenzyme F and transfers one-carbon body (C-i).

Lipoic acid ([(R) -5- (1,2-dithiolan-3-yl) valeric acid also called thioctanoic acid) is used as a coenzyme z. B. the pyruvate dehydrogenase complex is known. Furthermore, lipoic acid with dihydrolipoic acid and correspondingly liponamide with dihydroliponamide (R-6,8-dimercaptooctanoic acid) form important redox pairs, which can also act as biological antioxidants.

Pyridoxal-5'-phosphate plays in transamination and

Decarboxylation reactions and other important metabolic pathways play an important role. As further cofactors, nucleotide phosphates, such as. B. nucleotide triphosphates can be used. Adenosine, cytosine, uridine and guanidine are to be mentioned as nucleotides. Adenosine 5'-triphosphate (ATP) and the corresponding di- and monophosphate are particularly preferred. The nucleotide phosphates often act as phosphogroup transfer agents and as energy suppliers for a large number of enzymatically catalyzed reactions.

According to a particularly preferred embodiment, the cofactor is selected from the group consisting of glutathione, ubiquinones, NAD, NADP, nucleotide phosphates, FAD and FMN.

According to a further particularly preferred embodiment, the cofactor is selected from the group of non-covalently bound cofactors. This has among other things the advantage that the cofactor does not have to be added to the product (together with the enzyme). A covalently bound cofactor could be released from the enzyme under appropriate conditions. Especially with colored cofactors, such as. B Heme, the presence of the cofactor in the product is not always desirable.

According to a preferred embodiment, the enzyme is genetically modified so that it has an altered enzymatic activity compared to the wild type.

In this context, a genetically modified enzyme which has a modified enzymatic activity is to be understood in particular as an enzyme which is modified by genetic engineering measures or with the aid of genetic engineering methods in such a way that its enzymatic activity, in contrast to the enzyme to be designated as a wild type is changed.

The enzymatic activity is the catalytic effect of the enzyme, ie the measurable increase in the rate of the reaction under defined conditions due to the presence of the enzyme as the difference in the conversion of the catalyzed and the uncatalyzed reaction understood in a certain period. A catal (kat), the substrate turnover in mol per second, serves as the measured variable. A change in the activity of the enzyme, for example an activation by an activator, an inhibition by an inhibitor or a change in the reaction rate as a function of cofactors or a mutant compared to the wild type, can be determined using this measured variable.

In this sense, both the naturally occurring native enzyme, which is produced, for example, by bacteria in the course of their metabolism, and an enzyme which has the same enzymatic activity and an essentially the same amino acid sequence as the enzyme occurring in the original organism is considered to be wild type , Among them, e.g. B. those enzymes are to be understood, which for easier purification, e.g. are modified at the C- or N-terminus without the activity being changed in comparison to the enzyme which can be isolated from the original organism.

In particular, enzymes in which a mutation, in particular a point mutation (variation of an amino acid), leads to an increase or decrease in the enzymatic activity should not be regarded as a wild type.

A genetic modification or modification of an enzyme in the sense of the invention is an induced (random or targeted) mutation, i.e. understand the change in at least one amino acid within the amino acid sequence of the enzyme. A random mutation can be induced, for example, by selection pressure or a specific mutagen.

The genetic modification or modification of an enzyme often takes place by exchanging or deleting at least one nucleotide in the gene sequence which codes for the corresponding enzyme. The enzyme is then expressed in a suitable expression system. In addition to the introduction of targeted or spontaneous mutations, other methods for generating genetically modified enzymes are error-prone PCR or gene shuffling.

According to a special embodiment of the invention, the enzyme is genetically modified in such a way that it has an altered specific activity compared to a substrate. The specific activity relates directly to the binding or reaction of a substrate with an enzyme and is calculated as substrate conversion in mol per second and mass of the enzyme (cat / kg). The wild type already described serves as a reference for determining the changed specific activity.

The genetically modified enzyme particularly preferably has a higher specific activity with respect to the substrate than the wild type. This has the advantage that a smaller amount of the enzyme is sufficient to achieve a reliable identification. This enables proof to be carried out more cost-effectively and efficiently.

According to a further particular embodiment of the method according to the invention, the enzyme is genetically modified in such a way that, in the presence of an inhibitor and / or an activator, it exhibits a modified enzymatic activity compared to a non-genetically modified enzyme (wild type). An independent change in the specific activity of the enzyme is also possible.

The property that the genetically modified enzyme can be regulated by an inhibitor or an activator in a way that is different from that of the wild type has the advantage that the authenticity of the enzyme can be demonstrated even more easily. For example, an enzyme contained in or added to the product may be less inhibited by an inhibitor. In such a case, despite the addition of an inhibitor during identification, the substrate is bound and possibly converted. Comparable or counterfeit products without the enzyme with these specific changes could then do not bind or reposition the substrate. This difference contributes in particular to a more efficient and clear identification. An exact determination of the specific activity is then not necessary, since the observation that the catalyzed reaction takes place despite the inhibitor is completely sufficient for an inexpensive rapid test.

According to a further embodiment of the method according to the invention, the component contained in or added to the product is component A. Advantages of adding one or more enzymes to the product is that they are both more difficult to fully identify in their amino acid sequence and more difficult to imitate than a substrate , It is particularly difficult to completely mimic genetically modified enzymes, since the corresponding methods for producing an enzyme defined by its amino acid sequence are generally very time-consuming and costly.

In detergents and cosmetic compositions, for example, enzymes are often added, which can be used as component A in the identification method according to the invention. Most of these are enzymes that have been modified and optimized for their special tasks using genetic engineering methods. For example, proteases, amylases, oxidases and lipases are mentioned here for detergents and cleaning agents.

According to a further embodiment, the component contained in or added to the product is component B. In particular, it may be advantageous for cosmetics to use the substrate in the product, since on the one hand the substrates often have a lower sensitizing potential than the corresponding enzyme (for example saccharides) and on the other hand, certain enzymes cannot be used without further undesirable side effects. It is often not favorable to use proteases in cosmetics, for example in skin creams. For a corresponding identification of the product, it may therefore make more sense to add the substrate to the product, for example a peptide, which is identified using a protease. According to a further particularly preferred embodiment, at least two enzymes are used as component A. This has the advantage that the identification of the product can be carried out even more reliably by using two different enzymes for identification. This has the advantage that several parameters can be checked simultaneously during the identification, so that the branded product can be identified particularly precisely and efficiently.

According to a further embodiment of the invention, at least two substrates are used as component B. This has the advantage that higher authenticity can also be guaranteed by varying the substrate combination. For example, several substrates can be bound and converted by an enzyme at different speeds, so that the comparison between the conversion rates ensures a particularly high degree of authenticity. The ratio of the implementation rates to each other then gives a very reliable criterion for checking the product identity. Likewise, there can be different substrates that are implemented by several different enzymes. This can be particularly advantageous if it is possible to demonstrate both implementations by performing a single identification step b).

According to a particularly preferred embodiment, component A is selected from the group of proteases, cellulases, lipases, oxidases, reductases, amylases and transferases. These enzymes are used, for example, in detergents and cleaning agents or in cosmetics to improve product properties. This has a double benefit. On the one hand, the added enzymes improve the product properties, on the other hand, they serve to identify the product properly. The amylases and the proteases are particularly preferred in detergents and cleaning agents. Proteases often have different enzymatic activity in relation to differently constructed oligopeptides. A different one Reaction, for example, can then simply be used to check the authenticity of the product.

The implementation, binding, inhibition or activation of the reaction of suitable substrates (e.g. catechol, resorcinol, 2,3,4-trihydroxybenzaldehyde, 3,4-dihydroxy-benzoic acid, 3- (3,4-dihydroxyphenyl) -L-alanine or 2 , 3,4-trihydroxy-benzoic acid) by the polyphenol oxidase gives particularly visible color reactions, for example can be easily detected using an appropriate spot test and a color scale or with a spectrophotometer.

According to a further particular embodiment, component B is selected from the group of peptides, multiple sugars and fatty acids. Multiple sugars or fatty acids are advantageously relatively inexpensive to produce, so that a corresponding identification method can be carried out inexpensively. In this context, multiple sugars are in particular oligosaccharides and polysaccharides. Certain multiple sugars are converted, for example, by cellulases or amylases, while proteases bind peptides and lipases and catalyze their conversion. The substrates mentioned are used in particular in cosmetics because of their further positive properties.

According to a further particularly preferred embodiment, component B is at least one peptide, in particular an oligopeptide. Oligopeptides are the combination of several amino acids, usually from two to about ten, for example four amino acids. In particular in the case of detergents which already contain proteases, the identification can take place simply by selecting the corresponding oligopeptide (s), since the rate of degradation and / or probabilities between the oligopeptides often strongly depend on the protease used.

According to a further embodiment of the identification method according to the invention, at least one can additionally be used as a further component Inhibitor can be used together with one of the components. The use of an inhibitor together with one of the components A, B or possibly C advantageously leads to the authenticity verification being able to be carried out even more clearly.

If, for example, component A is used in the product, it is advantageous to use the inhibitor together with component B in the detection reaction, since otherwise the activity of the enzyme, for example in detergents and cleaning agents, is reduced. The genetically modified enzymes that are used as component A can, for example, be modified so that they have an increased resistance to an otherwise specific inhibitor. For example, after a certain genetic modification, protoporphyrinogen oxidase shows increased resistance to the inhibitor oxifluorofen (2-chloro-4-trifluoromethylphenyl-3-ethoxy-4-nitrophenyl ether). The advantage of such a change is that the genetically modified enzyme can be distinguished very well from other enzymes with the same effect.

According to a further embodiment of the present invention, component B is identified in step b) by reacting it, binding, inhibiting the reaction and / or activating the reaction with component A, if appropriate in the presence of at least one component C. For example, component B can also be identified by inhibiting the reaction catalyzed by component A. Furthermore, absorption maxima of the substrates can be shifted by the reaction of substrates with the enzyme, so that the reaction can be easily monitored with the naked eye or a spectrophotometer. The binding of component A to an immobilized component B can also be detectable by changing the refractive index.

According to a particular embodiment of the identification method according to the invention, the product in step b) is identified by identifying at least two different components B by their implementation, Binding, inhibition of the reaction and / or activation of the reaction with at least one component A optionally in the presence of at least one component C instead. For example, a protease can have different activities against different oligopeptides. The ratio of the substrates converted per unit of time to each other allows a much more accurate and at the same time less expensive authenticity check than the analysis of only one component B. Advantageously, the determination of the ratio of the activities makes the determination of the specific activity of the enzyme (cat / kg) superfluous, which otherwise ia with the isolation of the enzyme (to determine its mass) from the product. This method has the further advantage that the identification of the branded product is improved by the greater certainty of the test result by determining the ratio of at least two implementation reactions.

According to a further particular embodiment, the conversion, binding, inhibition of the reaction and / or activation of the reaction of component B by component A, if appropriate in the presence of at least one component C in step b), causes a color reaction, a fluorescence signal or its change, another spectroscopically measurable effect, an electrochemical potential change, a detectable change in the size of one of the components (e.g. the cleavage of a peptide into smaller components) or an odor effect. The advantage of a color reaction is that a positive result can be recognized with the naked eye, for example by a clearly visible color change. A fluorescence signal can be easily detected using a UV lamp. Processes such as NMR, IR, MS or GC-MS spectroscopy, among other things, are to be understood as additional effects which can be measured spectroscopically. Electrochemical potential changes can easily be detected by simple sensors without any equipment. Electronic noses, which can detect changes in odors, are also suitable for detecting an odor effect, for example the release of a fragrance. According to a further preferred embodiment of the invention, component B carries a marker group. This marker group serves to make the implementation of component B by component A or its binding easier or easier to detect. Dyes, chromophoric groups, chromogens, fluorescent dyes or isotopes (in particular radioisotopes) can be used as markers, for example. Within the scope of the invention, chromogens are understood to be those groups which contain chromophoric groups but do not absorb in the visible wave range. The enzyme reaction can result in the introduction of auxochrome dyes from these groups. Suitable auxochromes are, for example, substituents with free electron pairs such as - NR ₂ , -OR, -COOH or -SO ₃ H are suitable. The radioactivity is particularly suitable as a marker when component A is used in the product and in specific cases other means of identification are not sufficient. Within the meaning of the invention, markers are to be understood in particular as groups whose presence can be demonstrated in a simplified manner by their property or properties or the change in these properties. For example, p-nitroanilide or 5-bromo-4-chloro-3-indolyl phosphate nitro blue tetrazoleum (BCIP / NBT) is also suitable as a substrate of a genetically modified alkaline phosphatase.

According to a preferred embodiment, at least one property of the marker group in step b) is changed by the reaction, binding, inhibition of the reaction and / or activation of the reaction of component A with component B, if appropriate in the presence of component C. This includes, for example, a shift in the absorption maximum, in particular in the visible or ultraviolet range, an increase (or decrease) or shift in fluorescence, an increase (or decrease) or shift in the extinction coefficient or another detectable property.

According to a particularly preferred embodiment, the change in the property of the marker group consists in a color reaction, a fluorescence signal, another spectroscopic effect, an electrochemical potential change or an odor effect. The identification by means of a color change as a result of such a change in properties leads to the corresponding identification particularly quickly and easily. The component contained in the product can be mixed with the other component (s), a color reaction making the identity of the product visible as a color change. If the color changes are not obvious, a corresponding shift in the extinction coefficient can also be detected by means of an analysis device. Such devices, especially those for the UV / VIS range, can be used regardless of location.

Another possibility for product labeling is the enzymatically controlled odor release through the implementation of the marker group or substrate. Electronic scanners that can detect such odor effects can be manufactured and optimized for the reaction to be observed.

In addition, the individuality of the combination of component A and B and possibly component C gives the possibility of coding further information about the product to be protected. For example, different batches of a product can be equipped with different components, for example different enzymes or enzyme mixtures. When checking for authenticity, not only the manufacturer but also the batch must be determined directly on the product.

The following chromogenic substances, for example, can be bound to the substrates as fluorescent dyes: 2,2'-azino- (3-ethyl-benzothiazoline-6-sulfonic acid) (ABTS), o-phenylenediamine (OPD), 3,3'-5, 5'-tetramethylbenzidine (TMB), naphthyl isocyanate, 1-OPTA (o-phthalic anhydride), N-4- (2-benzimidazolyl) phenylmaleimide (BIPM), 2-hydroxy-3-naphtholic acid, 4-methyl-7-hydroxycoumarin, o -Dianesidine, 5-aminosalicylic acid (5AS) or, for example, fluorescein, monobromobiman (especially for thiol groups) or rhodamine 640. Terminal amino groups can be, for example, with fluorescamine, 2-methoxy-2,4-diphenyl-3 (2H) -furanone (MPDF) or dansyl chloride. carboxylic acids can, for example, be reacted with 4-bromomethyl-7-methoxycoumarin (BrMmC) or 4-bromomethyl-7-acetoxycoumarin (BrMaC) and thus labeled with fluorescent probes.

Fluorescent groups, for example Cy-2, Cy3 (Amersham Life Sciences, Inc., Arlington Heights, USA) or Cy-5 (Amersham Life Sciences, see above), FITC (fluorescein isothiocyanate), CT (5, (6) - Carboxytetramethylrhodamine-N-hydroxysuccinimide ester (Molecular Probes Inc., Eugene, USA)), TRITC (Tetramethylrhodamine-5,6-isothiocyanate (Molecular Probes Inc., see above) or FLUOS (5, (6) -carboxyfluorescein-N -hydroxysuccinimide ester (Boehringer Mannheim, Mannheim, Germany)) Alternatively, chemiluminescent groups or radioactive labels, for example ³⁵ S, ³² P, ³³ P, ¹²⁵ J, are used. If component A is an enzymatically active molecule, for example an alkaline phosphatase, acid Phosphatase, peroxidase, horseradish peroxidase, β-D-galactosidase or glucose oxidase, a number of chromogens are known for each of these enzymes, which can be converted into colored or fluorescent products instead of the natural substrate n. Examples of such chromogens are given in Table 1 below.

Table 1

Enzymes chromogens

1. Alkaline phosphatase and 4-methylumbelliferyl phosphate (*) acid phosphatase bis (4-methylumbelliferyl phosphate), (*)

3-O-methylfluorescein, flavon-3-disphosphate triammonium salt (*), p-nitrophenylphosphate disodium salt

2. peroxidase tyramine hydrochloride (*),

3- (p-hydroxyphenyl) propionic acid (*), p-hydroxyphenethyl alcohol (*), 2,2'- Azino-bis- (3-ethylbenzothiazoline sulfonic acid) (ABTS), ortho-phenylenediamine dihydrochloride, o-dianisidine, 5-aminosalicylic acid, p-urcresol (*), 3,3'-dimethyloxybenzidine, 3-methyl l-2-benzothiazole in hyd razon, tetramethylbenzidine

3. Horseradish peroxidase H ₂ O ₂ + diammonium benzidine H ₂ O ₂ + tetramethylbenzidine

4. β-D-galactosidase o-nitrophenyl-β-D-galactopyranoside, 4-methylumbelliferyl-β-D-galactoside

5. Glucose oxidase ABTS, glucose and thiazolyl blue

* Fluorescence

According to a further preferred embodiment, the product to be protected is a washing and / or cleaning agent, cosmetic agent or adhesive. Not only luxury goods, but also consumer goods are increasingly affected by product piracy. It is therefore necessary to use simple and inexpensive identification procedures to prove the authenticity of the corresponding product. Enzymes, in particular those which are genetically modified and which can therefore be used without extensive modifications or directly as components of the detection system, are advantageously already present in cosmetics or washing and / or cleaning agents. This results in a correspondingly inexpensive way to protect the product without adding further ingredients to the product.

Another object of the invention is a test kit for identifying the labeled substance according to the identification method according to the invention, the contains the component (s) not contained in the product and optionally further substances which are required in step b) for identification of the component contained or added to the product. For example, it can be one or more substrates (component B) and possibly component C, the addition of which identifies component A contained in the product. The substances optionally used in this kit can be, for example, inhibitors or buffer systems for pH regulation, salts or solutions for adjusting the ionic strength or also markers which indicate a reaction of component B with component A, if appropriate in the presence of component C. ,

Another object of the invention is a test kit for identifying a product which contains components A and B and possibly component C. In addition, other substances that are necessary for the identification of the component to be used in the product can optionally be contained. This kit can then be used to mix a product with one of the components contained in the kit and then to use the other component (s) to demonstrate the presence of the added component in the product.

The following example is intended to illustrate the invention without restricting it thereto.

Example 1 :

Determination of substrate-specific activities of proteases:

A stock solution of 1 mg / ml in 50% (w / v) 1, 2-propanediol / distilled water is prepared from the proteases to be tested (Savinase, Alkalase; EC 3.4.21.62). The samples are further diluted with Tris / HCl buffer (Sigma). Substrate solutions of two different substrates (tetrapeptide A: alanine-alanine-proline-phenylalanine-p-nitroanilide and tetrapeptide B: alanine-alanine-proline-leucine-p-nitroanilide) are prepared by dissolving 70 mg of tetrapeptide in 1 ml of DMSO. The 0.1 M Tris / HCl buffer pH 8.6 is preheated to 25 ° C. The following solutions are pipetted into various semi-micro cuvettes and then mixed:

Sample batch A: Sample batch B: Blank value:

10 ul substrate solution A 10 ul substrate solution B 10 ul substrate solution 10 ul enzyme solution 10 ul enzyme solution 1000 ul O, 1M Tris pH 8.6 1000 ul O, 1M Tris pH 8.6 1000 ul 0.1M Tris pH 8.6

After mixing, the absorption of the reaction mixture is monitored on a photospectrometer at 410 nm. The protease activity in the solution (C = cat / l) is calculated using the following formula: C = ΔE / s * VKÜ * Fy [mol / (sx I)] ε ₄₁₀ * ^V PiP * ^" with ΔE / s: Absorbance change per second

VK _ÜV : Volume of the solution in the cuvette

Fy: dilution factor of the enzyme solution ε ₄₁₀ : molar decadal extinction coefficient at 41 Onm

d: light path through the cuvette = 1 cm

V _P j _P : pipetted volume of the enzyme solution The specific activity (cat / kg) is based on the mass of the enzyme used.

The following specific protease activities of the various proteases were determined for the various substrates, tetrapeptide A and tetrapeptide B, using the method indicated.

Savinase 12.0 T (Novozyme, granules): tetrapeptide A: 0.12 cat / kg; Tetrapeptide B: 0.018 cat / kg

Alkalase 2.0 T (Novozyme, granules): tetrapeptide A: 0.65 cat / kg; Tetrapeptide B: 0.48 cat / kg

Example 2:

Evidence of a color change:

When adding 0.1% by weight of a mediator (methylsyringate or 1-hydroxybenzotriazole) to a 2% by weight solution of 3,4-dihydroxybenzoic acid in water with polyphenol oxidase (0.3 ml / kg, concentrate, denilite, Novozyme) a color change of the reaction solution from colorless to red-brown could be observed.

Claims

claims

1. Identification method for a product, consisting of the following steps: a) selection of at least one system comprising at least components A and B, of which a component is already contained in the product or is added to it, b) identification of the components contained in or added component with the aid of the other component, component A being at least one enzyme and component B being at least one substrate.

2. Identification method for a product, consisting of the following steps: a) selection of at least one system comprising at least components A, B and C, of which at least one component is already contained in the product, or is added to it, b) identification of the im Component contained or added to the product with the aid of the other component (s), component A being at least one enzyme, component B being at least one substrate and component C being at least one substance which accelerates the enzyme reaction, in particular a mediator, cofactor and / or activator ,

3. Identification method according to claim 2, characterized in that component C is at least one cofactor, in particular selected from the group glutathione, nucleotide phosphates, ubiquinones, NAD, NADP, FAD and FMN.

4. Identification method according to one of claims 2 or 3, characterized in that the cofactor is selected as component C from the group of non-covalently bound cofactors.

5. Identification method according to one of claims 1 to 4, characterized in that the enzyme is genetically modified so that it has a modified enzymatic activity.

6. Identification method according to claim 5, characterized in that the enzyme is genetically modified so that it has a modified, in particular a higher, specific activity compared to the substrate.

7. Identification method according to one of claims 5 or 6, characterized in that it has a modified enzymatic activity in the presence of an inhibitor and / or an activator compared to a non-genetically modified enzyme.

8. Identification method according to one of the preceding claims, characterized in that the component contained in or added to the product is component A.

9. Identification method according to one of claims 1 to 8, characterized in that the component contained in or added to the product is component B.

10. Identification method according to one of the preceding claims, characterized in that at least two enzymes are used as component A.

11. Identification method according to one of the preceding claims, characterized in that at least two substrates are used as component B.

12. Identification method according to one of the preceding claims, characterized in that the enzyme is selected from the group of proteases, cellulases, lipases, oxidases, reductases, amylases and transferases, in particular the proteases and peroxidases.

13. Identification method according to one of the preceding claims, characterized in that the substrate is selected from the group of fatty acids, multiple sugars or peptides, in particular the oligopeptides.

14. Identification method according to one of the preceding claims, characterized in that at least one inhibitor is additionally used as a further component together with one of the components.

15. Identification method according to one of the preceding claims, characterized in that the identification in step b) by the reaction, binding, reaction, inhibition of the reaction and / or activation of the reaction of component B with component A, if appropriate in the presence of component C. takes place.

16. Identification method according to claim 15, characterized in that the identification in step b) by the implementation, binding, inhibition of the reaction and / or activation of the reaction of at least two different components B with at least one component A, if appropriate in the presence of component C. takes place.

17. Identification method according to one of claims 15 or 16, characterized in that the reaction, binding, inhibition of the reaction and / or activation of the reaction of component B with component A, if appropriate in the presence of component C, a color reaction, a fluorescence signal, a ( other) effect that can be measured spectroscopically, an electrochemical potential change or an odor effect.

18. Identification method according to one of the preceding claims, characterized in that component B carries a marker group.

19. Identification method according to claim 18, characterized in that at least one property of the marker group in step b), in particular is changed by the reaction, binding, reaction, inhibition of the reaction and / or activation of the reaction of component B with component A, if appropriate in the presence of component C.

20. Identification method according to one of claims 18 or 19, characterized in that the change in the property of the marker group leads to a color reaction, a fluorescence signal, another spectroscopically measurable effect, an electrochemical potential change or an odor effect.

21. Identification method according to one of the preceding claims, characterized in that the product is a washing and / or cleaning agent, cosmetic agent or adhesive.

22. Test kit for identifying a product according to any one of claims 1 to 21, characterized in that it contains the component (s) not or not added to the product and optionally further substances which are used in step b) to identify those added or added to the product Component (s) are required.

23. Test kit for identifying a product according to one of claims 1 to 21, characterized in that it contains components A, B and optionally component C and optionally further substances, component A having at least one enzyme, component B having at least one substrate and component C is at least one substance which accelerates the enzyme reaction, in particular a mediator, cofactor and / or activator.