WO2003097858A1 - Identification method for protecting products - Google Patents

Identification method for protecting products

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Publication number
WO2003097858A1
WO2003097858A1 PCT/EP2003/004799 EP0304799W WO03097858A1 WO 2003097858 A1 WO2003097858 A1 WO 2003097858A1 EP 0304799 W EP0304799 W EP 0304799W WO 03097858 A1 WO03097858 A1 WO 03097858A1
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WO
Grant status
Application
Patent type
Prior art keywords
component
product
enzyme
identification
reaction
Prior art date
Application number
PCT/EP2003/004799
Other languages
German (de)
French (fr)
Inventor
Christel Adomat
Tilo Weiss
Heiner Brinkmann
Ralf Reifferscheidt
Matthias Gabler
Original Assignee
Henkel Kommanditgesellschaft Auf Aktien
Gabler, Julika
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date

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Classifications

    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/34Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
    • C12Q1/37Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase involving peptidase or proteinase
    • CCHEMISTRY; METALLURGY
    • C12BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
    • C12QMEASURING OR TESTING PROCESSES INVOLVING ENZYMES, NUCLEIC ACIDS OR MICROORGANISMS; COMPOSITIONS OR TEST PAPERS THEREFOR; PROCESSES OF PREPARING SUCH COMPOSITIONS; CONDITION-RESPONSIVE CONTROL IN MICROBIOLOGICAL OR ENZYMOLOGICAL PROCESSES
    • C12Q1/00Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
    • C12Q1/001Enzyme electrodes
    • C12Q1/004Enzyme electrodes mediator-assisted

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 METHOD FOR PRODUCT PROTECTION

The invention relates to an identification method for a product, comprising the steps of: a) selecting at least one system comprising at least the components A and B, of which one component in the product is included, or is added thereto, b) identification of the product component contained or added with the aid of the other component, wherein the component a is at least one enzyme and the component B at least one substrate.

The increasing imitation of branded products and the subsequent sale of these counterfeit products aimed at significant economic damage and affected often due to lower quality of the image of the brand product in question. For this reason, a label of the product that can be used to identify clearly, but hardly or only to imitate with considerable effort, desired.

Such labels of products for product protection have so far been limited mainly to the label of the package. It will only be protected as a whole, with the actual product in the packaging itself can not be identified.

For direct labeling of the product itself biological marker systems have already been described.

In WO 90/14441 a method is described for product protection, in which the product is added to a nucleic acid sequence that is collected for identification and detected. Preferably, the method 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, wherein a nucleic acid sequence is added to the product, which is then detected by hybridization with a complementary nucleic acid sequence.

In both the above cases, the identification of the added substances is associated with a considerable expenditure on equipment, which is the one hand, error-prone and on the other hand time-consuming, and thus does not allow a rapid identification of the product.

The international patent application WO 89/07272 describes a method for labeling of a substance can be added at the lowest concentrations of a marker substance 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 there by immunoaffinity chromatography and detected.

The addition of fluorescent and / or luminescent compounds alone is simple to prove by appropriate illumination, but relatively non-specific, unless a device is technically complex wavelength-dependent identification is performed.

The object of the invention is therefore to provide an alternative method for identification of products, on the one hand hardly or only difficult to reproduce, but on the other hand easily and inexpensively detected.

This object is achieved by an identification process for a product, comprising the steps of: a) selecting at least one system comprising at least the components A and B, of which one component in the product is included, or is added thereto, b) identification of the component contained in the product or added with the aid of the other component, wherein the component a is B is at least one enzyme and the at least one component substrate.

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

One of the components A or B is included in the product or added to the product. Is already one of the components A or B contained in the product, and this component can be used for identification. To the other component must then be selected accordingly. This has the advantage that no further addition of component A or B must occur to the product. Thus created by another labeling method changes in the product properties or the manufacturing costs can be avoided.

In step b) the presence of the component contained in the product or added is detected by means of the other component of the system and thus identifies the product.

Another object of the present invention is an identification method for a product, comprising the steps of: a) selecting at least one system comprising at least the components A, B and C, of ​​which at least one component in the product is included, or this is added b) identification of the component contained in the product or added with the aid of the other component (s), wherein the component a is at least an enzyme component B at least one substrate and the component C at least one the enzyme reaction-accelerating substance, in particular a cofactor, mediator and / or activator.

Under the enzyme reaction accelerating substance such substances are to be understood in the context of this invention, which promote the progress of the enzyme reaction by their presence or even permit only.

Advantageously, the conversion of the substrate by the enzyme occurs particularly significantly slowed or substantially not in the absence of the enzyme reaction-accelerating substance. Therefore, the respective components A and B in the absence of component C can be entirely used together in the product. With the help of the component C then the identification takes place in step b). It is also possible that the component C is included in the product or is added and the components A and B in step b) can be used to identify the component C. The specificity of the detection is enhanced by the three-component system.

According to a particularly preferred embodiment, the enzyme reaction can be used accelerating material in particular a co-factor, a mediator and / or activator.

Cofactors are substances that are required in addition to the enzyme (if necessary, the apoenzyme, the pure protein content of the enzyme) and the substrate or the substrates for the process of enzymatic catalysis. They are, inter alia, also referred to as coenzymes and differ from the substrates in that they can be regenerated in a short cycle, usually in a single reaction step. Therefore, many co-enzymes occur in two or more different forms and are effective as electron carriers or carriers of molecular groups. As so-called co-substrates bring some cofactors, such as NAD, certain enzymes for conversion of the substrate back to the original state, so the next catalytic cycle can take place. Cofactors are often reversible and non-covalently bound to the enzyme. However, there are also cofactors such. As heme, which can occur covalently bound to the enzyme and are then often referred to as a prosthetic group. As cofactors may be used, for example: cations of iron, copper, zinc, calcium, manganese, cobalt, potassium or sodium, biotin, heme, FAD, FMN or thiamine pyrophosphate.

As mediators, in particular such substances are understood to mediate the catalytic conversion of the substrate by the enzyme, in which the mediators themselves serve as the primary enzyme substrate. The enzyme converts the mediator into an activated form. This activated form of the mediator then causes the reaction of the substrate, wherein the mediator ia is completely regenerated. In the net reaction doing so, the substrate is converted by the enzyme, while the mediator remains unchanged.

Such a reaction chain is commonly observed in redox enzymes. The mediator is displaced by the reaction with the enzyme in an activated state with high oxidation and reduction potential. In the subsequent reaction of the mediator with the substrate, the substrate is oxidized or reduced.

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

For example, the placement of the enzyme reaction on the various mediators are known for polyphenol oxidases. These include 1-hydroxybenzotriazole (HBT), Violuonsäure, 2,2'Azino-bis- (3-ethylbenzthiazoline-6- sulfonic acid (ABTS), 2-nitroso-1-naphthol-4-sulfonic acid (HNNS) or methylsyringate. Polyphenol advantageously exhibit in the reaction with a suitable substrate and mediator color changes, often therefore can be observed easily and inexpensively. in addition to the aforementioned co-factors such substances are to be understood as activators in the context of this invention, which by their presence the catalytic with the naked eye, and accelerate implementation. such activators, for example, metal ions, such as calcium, magnesium, copper, nickel or iron, or chelating agents to be.

Glutathione (L-γ-glutamyl-L-cysteinylglycine, glutathione-SH) is a tripeptide, which passes through reversible oxidation to the disulfide, and thus represents a buffer system for the redox state. For example, peroxides and radicals are reduced under catalysis of selenium-containing glutathione peroxidase. Likewise, glutathione is a cofactor of the glutathione-S-transferase.

Ubiquinones (Coenzyme Q) are also co-factors, which may serve as electron and proton transfer agent. the ubiquinones are differentiated, depending upon the number of linked isoprene units in the side chain or on the number of C-atoms. Particularly well known is the coenzyme Q10, which carries 10 isoprene.

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

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

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

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

Coenzyme A (CoA) plays in the metabolism an important role as a carrier of acyl groups, particularly acetyl groups. An important methyl Gruppendonor is the S-adenosylmethionine (S-adenosine-5'-yl-methioninium, Ademethionin).

Tetrahydrofolic acid (N - [(6S) -5,6,7,8-Tetrahydropteroyl] -L-glutamic acid) is also known as coenzyme Q, and transfers one-carbon body (Ci).

Lipoic acid ([(R) -5- (1,2-dithiolan-3-yl) valeric acid also known as thioctic acid) as a coenzyme is z. As the pyruvate Dehydrogenasekomplexes known. Continue to form lipoic acid with dihydrolipoic and in accordance with liponamide dihydrolipoamide (R-6,8-dimercaptooctanoic acid) important redox pairs that can also act as biological antioxidants.

Pyridoxal-5'-phosphate plays transamination and

Decarboxylation and other important metabolic pathways play an important role. As more cofactors may also Nukleotidphosphate such. B. nucleotide triphosphates are used. As nucleotides, in particular adenosine, cytosine, uridine, and guanidine may be mentioned here. Particularly preferred are adenosine 5'-triphosphate (ATP) and the corresponding di- and monophosphate. The Nukleotidphosphate often act as Phosphogruppenüberträger as well as energy suppliers for a variety enzymatically catalyzed reactions.

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

According to another particularly preferred embodiment, the cofactor is selected from the group of non-covalently bound cofactors. This has the advantage, that the cofactor need not be added (together with the enzyme) in the product. A covalently bound cofactor could be solved by appropriate conditions of the enzyme out. Especially for colored cofactors such. B heme, is the presence of the cofactor in the product 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.

Under a genetically modified enzyme having an altered enzymatic activity, in this context, is meant in particular an enzyme that is modified by genetic engineering or by means of genetic engineering methods so that its enzymatic activity in contrast to the wild type to be designated enzyme is changed.

As enzymatic activity is the catalytic action of the enzyme, that is measurable under defined conditions increase the rate of reaction due to the presence of the enzyme understood as the difference of the turnover of the catalyzed and uncatalyzed reaction in a given period. As a measured variable while a Katal used (cat), the substrate conversion in mol per second. A change in the activity of the enzyme, for example, 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 from this measured value.

Than wild-type in this sense both the naturally occurring native enzyme, that is produced for example by bacteria as part of their metabolism, as well as an enzyme is understood, having the same enzymatic activity and has a substantially same amino acid sequence as occurring in the original organism enzyme , Including z can. For example, be understood to mean those enzymes which have been modified for ease of purification, for example, at the C- or N-terminus, without the activity in comparison with the isolatable from the source organism enzyme is changed.

In particular, such enzymes are not to be construed as wild type in which a mutation, in particular a point mutation (an amino acid variation) results in an increase or decrease in the enzymatic activity.

Under a genetic modification or alteration of an enzyme is induced (random or targeted) mutation in the sense of the invention, ie the change of at least one amino acid within the amino acid sequence of the enzyme to understand. The induction of random mutation may be effected by selective pressure or a certain mutagen.

The genetic modification or alteration of an enzyme often found by replacement or deletion of at least one nucleotide in the gene sequence coding for the corresponding enzyme, instead. Thereafter, the enzyme is expressed in a suitable expression system. Other methods for the generation of genetically modified enzymes in addition to the introduction of targeted or spontaneous mutations the error-prone PCR or gene shuffling.

According to a particular embodiment of the invention the enzyme is genetically modified so that it has a changed specific activity towards a substrate. The specific activity is directly related to the binding or reaction of a substrate with an enzyme and is used as substrate turnover in moles per second, and mass of the enzyme is calculated (kat / kg). As a reference for determining the change in specific activity of the wild type already described is used.

Particularly preferably, the genetically modified enzyme to a higher specific activity towards the substrate than the wild type. This has the advantage that a smaller amount of the enzyme sufficient to achieve a secure identification. The detection can be as cost-effectively and efficiently carried out.

In another particular embodiment of the method according to the invention the enzyme is genetically modified so that it has an altered enzymatic activity in the presence of an inhibitor and / or an activator as compared to a non-genetically modified enzyme (wild type). One of them independent modification of the specific activity of the enzyme is also possible here.

The characteristic that makes the genetically modified enzyme of an inhibitor or an activator regulate in a changed compared to the wild-type manner has the advantage that the authenticity of the enzyme can be detected more easily. For example, one contained in the product or this enzyme added can be less inhibited by an inhibitor. In such a case, despite the addition of an inhibitor is performed in identifying a bond, and if necessary conversion of the substrate. could then do not bind or convert the substrate comparable or counterfeit products without the enzyme with these specific changes. This difference will contribute in particular to more efficient and clearer identification. Accurate determination of the specific activity is not necessary then, as the observation that the catalyzed reaction to take place, despite inhibitor for an inexpensive rapid test is sufficient.

According to another embodiment of the inventive process the component contained in the product or added it is the component A. benefits of adding one or more enzymes is the product that they are both more difficult to completely identify in their amino acid sequence and heavy mimic as a substrate , It is particularly difficult to imitate genetically modified enzymes complete because the appropriate methods for the production of a defined by its amino acid sequence the enzyme are usually very time and cost intensive.

In detergents and cosmetic compositions, for example, enzymes are often added, which can be used within the inventive identification method as A component. Mostly these are enzymes that have been modified for their particular tasks by genetic engineering techniques and optimized. Examples of these are named for detergents and cleaners proteases, amylases, oxidases and lipases.

According to a further embodiment, the component contained in the product or added it is the component B. In particular, it may be desirable in cosmetics to use the substrate in the product, on the one hand, the substrates often have a lower sensitizing potential than the corresponding enzyme (e.g. saccharides) and certain enzymes can not be applied without other undesirable side effects on the other. So it is not convenient often to use proteases in cosmetics, such as in skin creams. For a corresponding identification of the product, it may therefore be more appropriate to add the substrate to the product, for example, a peptide is carried out its identification with the aid of a protease. According to another particularly preferred embodiment at least two enzymes are used as component A. This has the advantage that the identification of the product can be made more secure by two different enzymes are used for identification. This has the advantage that multiple parameters can be checked simultaneously in identifying, among other things, so that a particularly accurate and efficient identification of the branded product can take place.

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

According to a particularly preferred embodiment, component A is selected from the group of proteases, cellulases, lipases, oxidases, reductases, transferases, and amylases. These enzymes are used, inter alia, for example in washing and cleaning agents or cosmetics in order to improve the product properties. This results in a double benefit. On one hand, the added enzymes improve the product properties, on the other hand they are used for proper identification of the product. Particularly preferred in Waschmitteln- and cleaners doing the amylases and the proteases. Proteases often have different enzymatic activity with respect to different oligopeptides constructed. A different reaction, for example, can then easily be used for checking the authenticity of the product.

The reaction, binding, inhibition or activation of the reaction of suitable substrates (for example, 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 in particular results in highly visible color reactions, for example, can be easily detected with the aid of a corresponding spot tests and a color scale or by a spectrophotometer.

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

According to another particularly preferred embodiment, the component B is at least one peptide, in particular an oligopeptide. As oligopeptides is referred to the combination of several amino acids, usually from two to about ten, for example, of four amino acids. In particular, in detergents that already contain proteases, the identification can be as simple take place by the selection of, or the corresponding oligopeptides, as the rate of degradation and / or probabilities between the oligopeptides often depend strongly on the protease used.

According to another embodiment of the identification method of the invention at least one inhibitor may be used together with one of the components in addition as a further component. The use of an inhibitor together with one of the components A, B or C, if necessary, advantageously leads to the fact that the authenticity verification can be carried out even more clearly.

For example, when the component A is used in the product, it is advantageous to use the inhibitor together with component B in the detection reaction, otherwise the activity of the enzyme, for example in washing and cleaning agents, is reduced. The genetically modified enzymes which are used as component A may be modified, for example, that they have an increased resistance to an otherwise specific inhibitor. For example, the protoporphyrinogen oxidase indicates by a specific genetic modification increased resistance to the inhibitor Oxifluorofen (2-chloro-4-trifluoromethylphenyl-3-ethoxy-4-nitrophenyl ether). The advantage of such a change is that as the genetically modified enzyme can be very well distinguished from other similarly acting enzymes.

According to a further embodiment of the present invention, the identification of the component B takes place in step b) by reaction thereof binding, inhibition of the reaction and / or activation of the reaction with the component A, optionally in the presence of at least one component C instead. For example, the identification of component B may also take place through the inhibition of the reaction catalyzed by component A reaction. Furthermore, the substrates can be moved by the reaction of substrates with the enzyme absorption maxima so that the reaction can be monitored with the naked eye or a spectrophotometer easy. Also, the binding of the component A to an immobilized component B by the refractive index change can be detected.

According to a particular embodiment of the identification method of the invention, the product in step b) by identifying at least two different components B by reaction thereof, 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 is C instead. For example, have different activities a protease to various oligopeptides. The ratio of the per unit time unreacted substrates allows one another a much more accurate and still less expensive authenticity check when the analysis of only one component B. Advantageously, makes the determination of the ratio of the activities of the determination of the specific activity of the enzyme (kat / kg) superfluous, which would otherwise ia with the isolation of the enzyme is coupled (to determine its mass) of the product. This method still has to be improved by greater safety of the test result by the determination of the ratio of at least two conversion reactions to identify the brand product advantage.

According to another particular embodiment, the reaction, binding, inhibition of the reaction and / or activation of the reaction of the component B by the component A calls optionally in the presence of at least one component C in step b) a color reaction, a fluorescent signal or its change, a different spectroscopically measurable effect, an electrochemical potential change, a detectable change in the size of one of the components (eg. for example, the cleavage of a peptide into smaller components) or an odor effect produced. Advantage of a color reaction is that a positive result can be detected for example by a distinct visible color change with the naked eye. A fluorescence signal is easily detected using a UV lamp. As a further spectroscopically measurable effects complicated apparatus methods such as NMR, IR, MS or GC-MS include to take spectroscopy. Electrochemical potential changes can be detected by simple sensors easily without any outlay on equipment. Electronic noses, can track the changes of odors, are also suitable to detect an odor effect, for example the release of a fragrance. According to a further preferred embodiment of the invention, the component B carries a marker group. These markers group serves to make the reaction of the component B by the component A and whose binding better or easier detectable. As markers for example, dyes, chromophoric groups, chromogens, fluorescent dyes and isotopes can be used (in particular, radioisotopes). As chromogens of the invention are to be understood those groups which do contain chromophore groups, but do not absorb in the visible region in the frame. By the enzyme reaction dyes may arise from these groups by introducing auxochromes. Suitable as auxochromes substituents having free electron pairs, such as, for example, - NR 2, -OR, -COOH or -SO 3 H suitable. The radioactivity is then suitable above all as a marker when the component A is used in the product and in specific cases, other means of identification are not sufficient. As a marker of the invention are to be understood especially those groups whose presence can be detected easier by their property or properties or the variation of these characteristics in mind. For example, suitable p-nitroanilide or 5-bromo-4-chloro-3-indolyl phosphate nitroblue Tetrazoleum is (BCIP / NBT) 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) by reacting, binding, inhibition of the reaction and / or activation of the reaction of component A with component B is changed, if necessary, in the presence of component C. Including, for example, a shift of the absorption peak particularly in the visible or ultraviolet range, an increase (or decrease) or shift in fluorescence, an increase (or decrease) or displacement in the extinction coefficient or other detectable property to understand.

According to a particularly preferred embodiment, the change in the property of the marker group in a color reaction, a fluorescent signal, another spectroscope ish measurable effect of 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 quickly and easily to the corresponding identification. It can be mixed the component contained in the product with the / the other component (s), wherein a color reaction makes the identity of the product as visible color change. For non-obvious color changes in a corresponding displacement of the extinction coefficient can be also detected by means of an analyzer. Such devices, especially those for the UV / VIS range, can be used anywhere.

A further possibility for product marking provides enzymatically-controlled release of aroma by reacting marker group or substrate. Electronic scanners that can detect such odor effects produced and the observed reaction towards optimizing.

In addition, the combination of component A and B and optionally of component C results from the individuality of the possibility of further information about the product to be protected to encode. For example, different batches of a product with different components, for example, various enzymes or enzyme mixtures are equipped. When tested for authenticity not only the manufacturer but also the batch is to be determined directly on the product.

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

Detectable markers often fluorescent groups, for example, Cy 2, Cy3 be beyond (Amersham Life Sciences, Inc. Arlington Heights, USA) or Cy-5 (Amersham Life Sciences, Sun), FITC (fluorescein isothiocyanate), CT (5, (6) - carboxytetramethylrhodamine N-hydroxysuccinimide ester (Molecular Probes Inc., Eugene, USA)), TRITC (tetramethylrhodamine-5,6-isothiocyanate (Molecular Probes Inc. so) or FLUOS (5 (6) -carboxyfluorescein-N -hydroxysuccinimidester (Boehringer Mannheim, Mannheim, Germany)). Alternatively, chemiluminescent groups or radioactive labels such as 35 are S, 32 P, 33 P, 125 I, is used. When the component a is an enzymatically active molecule, such as an alkaline phosphatase, acid phosphatase, peroxidase, horseradish peroxidase, beta-D-galactosidase or glucose oxidase, a number of chromogens are known for each of these enzymes, which can be implemented instead of the natural substrate either colored or fluorescent products n. Examples of such chromogens are given in Table 1 below.

Table 1

enzymes Chromogenic

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

3-O-Methylfluorescein, flavone-3- Disphosphattriammoniumsalz (*), p-Nitrophenylphosphatdinatriumsalz

2. peroxidase tyramine (*)

3- (p-hydroxyphenyl) propionic acid (*), p-hydroxyphenethyl alcohol (*), 2,2'-azino-bis- (3-ethylbenzothiazolin- sulfonic acid) (ABTS) ortho-phenylenediamine, o-dianisidine, 5 aminosalicylic acid, p-Ucresol (*), 3,3'-Dimethyloxybenzidin, 3-methyl l-2-benzoth iazol in hyd Razon, tetramethylbenzidine

3. horseradish peroxidase H 2 O 2 + H 2 O 2 + Diammoniumbenzidin tetramethylbenzidine

4. ß-D-galactosidase o-nitrophenyl-beta-D-galactopyranoside, 4-methylumbelliferyl-ß-D-galactoside

5. ABTS glucose, glucose and thiazolyl

* fluorescence

According to another preferred embodiment, wherein the product to be protected by washing and / or cleaning agents, cosmetic products or adhesives. Not only luxury goods, but also consumer goods are more and more affected by product. Therefore, it is necessary to use simple and inexpensive identification method to prove the authenticity of the corresponding product. Advantageously, are in cosmetics or detergents and / or cleaning agents already enzymes, in particular those which are genetically modified and therefore can be used without extensive modifications or directly as components of the detection system. This results in a correspondingly cost-effective way for product protection without the product to add other ingredients.

Another object of the invention is a test kit for the identification of the labeled substance by the novel identification methods, containing not contained in the product, the (s) component (s) and optionally further substances, component contained or in step b) to identify the product added are needed. For example, it can involve one or more substrates (component B) and optionally component C act, by which the additional component A is contained in the product is identified. The substances further optionally used in this kit may, for example inhibitors or as well as buffer systems for pH regulation, salts or solutions to adjust the ionic strength or marker to be associated with the component A, if necessary, indicating a reaction of the component B in the presence of component C ,

Another object of the invention is a test kit for identifying a product which contains the components A and B and, if appropriate component C. In addition, optional further substances may be present, which are necessary for the identification of the employed in the product component. This kit can be used to then, to enable a product having the components contained in the kit, and then detect with the / the other component (s) the presence of the added component in the product.

The following example illustrates the invention without limiting it to them.

Example 1 :

Determining the substrate-specific activity of proteases:

A stock solution of 1 mg / ml in 50% 2-propanediol / distilled water each (w / v) 1, recognized; of the tested proteases (EC 3.4.21.62 Savinase, Alcalase). Further dilution of the sample is carried out 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 recognized by dissolving 70 mg of tetrapeptide in 1 ml DMSO. Of 0.1 M Tris / HCl buffer pH 8.6 is preheated to 25 ° C. There are added to separate semi-micro cuvettes following solutions and then mixed:

Sample Approach A: sample preparation B: Blank:

10 ul of substrate solution A 10 .mu.l substrate solution B 10 .mu.l substrate solution, 10 .mu.l enzyme solution, 10 .mu.l enzyme solution 1000 ul O, 1M Tris pH 8.6 in 1000 .mu.l O, 1M Tris pH 8.6 in 1000 .mu.l of 0.1M Tris pH 8.6

After mixing, the absorbance of the reaction mixture at the spectrophotometer is monitored at 410 nm. The calculation of the protease activity in the solution (C = kat / l) using the following formula: C = AE / s * vkue * Fy [mol / (sx I)] ε 410 * V PiP * "with AE / s: extinction per second

VK OSR: volume of the solution in the cuvette

Fy: dilution factor of the enzyme solution ε 410: molar decadic extinction coefficient at 41 ONM d: the optical path through the cuvette = 1 cm

V P j P: einpipettiertes volume of the enzyme solution, the specific activity (kat / kg) is based on the mass of the enzyme used.

For the various substrates tetrapeptide A and B tetrapeptide following specific protease activities of the various proteases were determined by the method specified.

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

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

Example 2:

Detecting a color change:

On addition of 0.1 wt .-% of a mediator (methyl syringate, or 1-hydroxybenzotriazole) mCat to 2 wt .-% solution of 3,4-dihydroxybenzoic acid in water with polyphenol oxidase (0.3 / kg, concentrate, Denilite®, Novozyme) was observed a color change of the reaction solution from colorless to red-brown.

Claims

claims
1. Identification process for a product, comprising the steps of: a) selecting at least one system comprising at least the components A and B, of which one component in the product is included, or is added thereto, b) identification of the contained in the product or added component with the aid of the other component, wherein the component a is B is at least one enzyme and the at least one component substrate.
2. Identification process for a product, comprising the steps of: a) selecting at least one system comprising at least the components A, B and C, of ​​which at least one component in the product is included, or is added thereto, b) identification of the product component contained or added with the aid of the other component (s), wherein the component a is at least a substrate and the component C at least one the enzyme reaction-accelerating substance, in particular a mediator, co-factor and / or activator is at least one enzyme component B, ,
3. Identification method according to claim 2, characterized in that component C is at least one co-factor, in particular selected from the group consisting of glutathione, Nukleotidphosphate, ubiquinones, NAD, NADP, FAD and FMN.
4. The identification method of any 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 any one of claims 1 to 4, characterized in that the enzyme is genetically modified so that it has an altered enzymatic activity.
6. The identification method of claim 5, characterized in that the enzyme is genetically modified so as to be opposed to the substrate a modified, in particular having a higher specific activity.
7. The identification method of any of claims 5 or 6, characterized in that it comprises, in the presence of an inhibitor and / or an activator as compared to a non-genetically modified enzyme is a modified enzymatic activity.
8. Identification method according to one of the preceding claims, characterized in that contained in the product or it added component is the component A.
9. The identification method of any of claims 1 to 8, characterized in that contained in the product or added him component, component B.
10. The identification method according to any one of the preceding claims, characterized in that are used as component A at least two enzymes.
11. The identification method according to any one of the preceding claims, characterized in that are used as component B at least two substrates.
12. The identification method according to any one of the preceding claims, characterized in that the enzyme is selected from the group of proteases, cellulases, lipases, oxidases, reductases, transferases, and amylases, especially the proteases and peroxidases.
13. The identification method according to any one of the preceding claims, characterized in that the substrate is selected from the group of fatty acids, polysaccharides or peptides, especially the oligopeptides.
14. The identification method according to any one of the preceding claims, characterized in that additionally at least one inhibitor is used together with one of the components as a further component.
15. The identification method according to any one of the preceding claims, characterized in that the identification in step b) by reacting, binding reaction, inhibition of the reaction and / or activation of the reaction of the component B with the component A, optionally in the presence of component C takes place.
16. The identification method according to claim 15, characterized in that the identification in step b) by reacting, binding, inhibition of the reaction and / or activation of the reaction of at least two different components B comprising at least one component A, optionally in the presence of component C takes place.
17. The identification method according to any one of claims 15 or 16, characterized in that the reaction, binding, inhibition of the reaction and / or activation of reaction of component B with component A, optionally in the presence of component C, a color reaction, a fluorescent signal, a ( causing other) spectroscopically measurable effect, an electrochemical potential change or an odor effect.
18. The identification method according to any one of the preceding claims, characterized in that component B carries a marker group.
19. The identification method according to claim 18, characterized in that at least one property of the marker group in step b), in particular by reacting, binding reaction, inhibition of the reaction and / or activation of reaction of component B with component A, optionally in the presence of component C, is changed.
20. The identification method according to any one of claims 18 or 19, characterized in that the change in the property of the marker group to a color reaction, a fluorescent signal, a different spectroscopically measurable effect of an electrochemical potential change or odor effect results.
21. The identification method according to any one of the preceding claims, characterized in that it is the product to washing and / or cleaning agents, cosmetic products or adhesives.
22. A test kit for identifying a product according to any one of claims 1 to 21, characterized in that it contains the component (s) not contained in the product or added, and optionally other substances in step b) to identify the information contained in the product or added component (s) are needed.
23. A test kit for identifying a product according to any one of claims 1 to 21, characterized in that it contains the components A, B and, if appropriate component C and, optionally, further substances, component A being at least one enzyme component B at least one substrate and the component C at least one the enzyme reaction-accelerating substance, in particular a mediator, co-factor and / or activator.
PCT/EP2003/004799 2002-05-17 2003-05-08 Identification method for protecting products WO2003097858A1 (en)

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US8703068B1 (en) 2009-03-04 2014-04-22 The Procter & Gamble Company Counterfeit detection kit

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WO2009149702A2 (en) * 2008-06-14 2009-12-17 Helling Guenter Multi-layer degradable polymer system used as a security element
WO2009149702A3 (en) * 2008-06-14 2010-02-18 Helling Guenter Multi-layer degradable polymer system used as a security element
US8703068B1 (en) 2009-03-04 2014-04-22 The Procter & Gamble Company Counterfeit detection kit

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