WO2011070491A1 - Composition and method for temperature monitoring chilled and frozen products - Google Patents
Composition and method for temperature monitoring chilled and frozen products Download PDFInfo
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- WO2011070491A1 WO2011070491A1 PCT/IB2010/055567 IB2010055567W WO2011070491A1 WO 2011070491 A1 WO2011070491 A1 WO 2011070491A1 IB 2010055567 W IB2010055567 W IB 2010055567W WO 2011070491 A1 WO2011070491 A1 WO 2011070491A1
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- LIVNPJMFVYWSIS-UHFFFAOYSA-N silicon monoxide Chemical class [Si-]#[O+] LIVNPJMFVYWSIS-UHFFFAOYSA-N 0.000 description 1
- 229910052814 silicon oxide Inorganic materials 0.000 description 1
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- MAYCICSNZYXLHB-UHFFFAOYSA-N tricaproin Chemical compound CCCCCC(=O)OCC(OC(=O)CCCCC)COC(=O)CCCCC MAYCICSNZYXLHB-UHFFFAOYSA-N 0.000 description 1
- 230000005641 tunneling Effects 0.000 description 1
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Classifications
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
- G01K3/00—Thermometers giving results other than momentary value of temperature
- G01K3/005—Circuits arrangements for indicating a predetermined temperature
-
- C—CHEMISTRY; METALLURGY
- C12—BIOCHEMISTRY; BEER; SPIRITS; WINE; VINEGAR; MICROBIOLOGY; ENZYMOLOGY; MUTATION OR GENETIC ENGINEERING
- C12Q—MEASURING 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
- C12Q1/00—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions
- C12Q1/34—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving hydrolase
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01K—MEASURING TEMPERATURE; MEASURING QUANTITY OF HEAT; THERMALLY-SENSITIVE ELEMENTS NOT OTHERWISE PROVIDED FOR
- G01K2207/00—Application of thermometers in household appliances
- G01K2207/02—Application of thermometers in household appliances for measuring food temperature
- G01K2207/04—Application of thermometers in household appliances for measuring food temperature for conservation purposes
-
- G—PHYSICS
- G01—MEASURING; TESTING
- G01N—INVESTIGATING OR ANALYSING MATERIALS BY DETERMINING THEIR CHEMICAL OR PHYSICAL PROPERTIES
- G01N2333/00—Assays involving biological materials from specific organisms or of a specific nature
- G01N2333/90—Enzymes; Proenzymes
- G01N2333/914—Hydrolases (3)
- G01N2333/924—Hydrolases (3) acting on glycosyl compounds (3.2)
Definitions
- the present invention relates to the field of freezing temperature-sensitive articles, in particular foods.
- a storage temperature of at least -16°C must be maintained. If, however, a temperature of -10°C to -15°C is exceeded, the storage life of many products is no longer ensured; the frozen ware is considered to be initially thawed, although it still appears to be frozen externally.
- freezers are equipped with built-in thermometers which display the temperature at a defined site of the cold chamber, or a conventional thermometer is placed on or near the chilled ware and removed from the refrigerator in order to read it. In these cases there is no guarantee that the temperature of the chilled ware is measured exactly.
- the convection of air in the cold chamber can be prevented, and so even in the case of faultless functioning of the apparatus, at some points of the cold chamber the minimum temperatures required for the storage life of the products can be exceeded.
- the exceedance of the storage life temperature cannot be recognized, and so spoilage of the products cannot be counteracted by prompt removal and immediate consumption.
- the risk that frozen products are slightly thawed unnoticed is particularly great, for example, if a relatively large amount of ware to be chilled is introduced warm into the freezer. Therefore, it is desirable to have available a temperature indicator which, for each food package or a group of packs, displays whether they were adequately cooled during their entire storage time.
- Temperature indicators known in the prior art can detect interruptions in a chilling/freezing chain.
- the measurement principle is usually based on physical, chemical, microbiological or enzymatic processes.
- differing reactions are obtained to an interruption in the chilling/freezing chain such as, e.g. color change, color development or a migration of a dye along a scale.
- CTI Crohn's disease
- Temperature- or temperature-time indicators have been known for a relatively long time in the prior art.
- the system "3M MonitorMark”, for example, is based on the diffusion of a blue ester along a scale. Exceedance of a defined threshold temperature leads to an irreversible melting of the ester, wherein the entire time-temperature history can be determined (T.P. Labuza (2000) The search for shelf life - An update on continued efforts in understanding practical strategies for determining and testing the shelf life of food products. Food Testing Analysis).
- the commercially available "VITSAB” indicator is based on an enzymatic hydrolysis of tricaproin by a pancreas lipase. The enzyme and the substrate solution comprising a pH indicator are separated from one another in a two-chamber system.
- the label is activated by a pressure on the label, wherein the thin barrier between enzyme and substrate solution is broken.
- the warmer the outside temperature the more rapid is the mixing of the two solutions and the more rapid is the enzymatic hydrolysis.
- the formation of capric acid leads to a pH fall and thus to a color change from green to yellow.
- a disadvantage of this system is the inactivation of the enzyme at a temperature of above 40°C, which can lead to faulty functioning of the indicator (T. Labuza, Bin Fu, Use of Time/Temperature Integrators, Predictive Microbiology, and Related Technologies for Assessing in the extent and impact of temperature abuse on meat and poultry products, Journal of Food safety 15 (1995); 201 -227; S. Tsoka, P.S.
- Taoukis Time Temperature Integrators for chilled food shelf life monitoring using enzyme-substrate systems, Food Biotechnology 12 (1 &2) (1998), 139-155).
- the "fresh check" system is based on the polymerization of a colorless acetylene or diacetylene monomer to give a colored polymer. The length and therefore the color intensity are temperature-dependent. The reaction proceeds in the center of the label, wherein the darker circle around the light surface shows the reference ring.
- the system has the disadvantage that the labels are active from the time point of their manufacture and must be stored at -25°C (P.S. Taoukis, T . Labuza (1989) - Applicability of time-temperature indicators as shelf life monitors of food products. J.
- the "OnVu label” is based on the activation of a dinitrobenzylpyridine (DNBP) label with UV light.
- DNBP dinitrobenzylpyridine
- the blue coloration is based on a light-induced proton transfer reaction in which the blue photo product returns slowly to the base state by "thermally active tunneling" and becomes colorless again in the process. After expiry of the storage life calculated for the chilled ware and in the case of interruptions in the chilling chain, a (virtually) complete decolorization of the label can be seen.
- the active imprint In order to prevent repeated activation of the label, the active imprint must be covered by an adhesive UV-impermeable film (J.
- the objective technical problem of the invention was to provide a method which can detect an interruption in the chilling chain as simply, reliably and inexpensively as possible.
- the inventors have developed an enzyme-based CTI which, in the form of a liquid (also termed "smart ink”) can be printed onto paper-based packages - shortly before freezing. Gross failures of a freezing chain should be indicated by this means.
- an indicator for temperature monitoring a chilled or frozen product which indicator comprises: a. at least one enzyme which catalyzes the conversion of a polysaccharide to a monosaccharide,
- the enzyme activity is detectable only after application to a polysaccharide carrier matrix in the presence of water and in a temperature range in which the product is in the thawed state.
- the indicator is attached to a polysaccharide, preferably cellulose, and is present in a dried state.
- the color indicator comprises a chromogenic substrate for a peroxidase, or for a hydrolase, in particular a perhydrolase.
- the invention further relates to a method for producing an indicator according to the invention.
- the invention further relates to a method for temperature monitoring a chilled or frozen product, which method comprises applying an indicator according to the invention to the product, and monitoring the enzyme activity by means of the color indicator, wherein positive detection of the enzyme activity is indicative of an interruption of the chilling or freezing of the product.
- the indicator is applied in a temperature range in which the enzyme from a) and/or the enzyme from b) and/or the mixture from c) are inactive.
- the invention further relates to a package for storing a chilled or frozen product, wherein the package is provided with at least one fixed or removable indicator according to the invention.
- the indicator is arranged at least between one outer layer which is translucent to visible and/or UV light, and preferably water-impermeable, and/or one inner layer facing the product and impermeable to the indicator.
- the invention relates to the use of a package according to the invention for storing chilled or frozen products.
- Microtiter plate (MTP) with the test set-up of the "smart ink” formulation row 1 : only Onozuka cellulase; row 2: only beta-glucosidase; row 3: Onozuka cellulase and beta- glucosidase; row 4: glucose standard; last column: no enzyme, water control Figure 3
- Row 1 only Onozuka cellulase
- row 2 only beta-glucosidase
- row 3 Onozuka cellulase and beta-glucosidase
- last column no enzyme, water control
- Row 1 44 g of phenol red
- row 2 88 g of phenol red
- indicator means a device, an article, and/or a material which can change in a detectable manner depending on the temperature to which it is exposed.
- the change can be of a physical or chemical nature or both, and may be detected by means of human sight, sense of smell/feel and/or with the aid of electrical, chemical or radiation-based analytical equipment.
- temperature monitoring describes monitoring the chilling or freezing chain for readily spoilable products, for example foods, vaccines and blood and also for other applications. In connection with the present invention, this is taken to mean, in particular, monitoring whether a chilled, in particular frozen, product is present or has been present in the thawed state, that is has been warmed to a temperature of at least minus 15°C, at least minus 5°C, preferably at least 0°C, particularly preferably at least 10°C, very particularly preferably to a temperature of at least 15°C.
- the expression "chilled product” describes a product which must be stored at a temperature of about minus 5°C to plus 10°C, preferably from about minus 3°C to about plus 8°C, particularly preferably 0°C to plus 5°C.
- the expression "frozen product” relates to a product which must be stored at a temperature of about minus 80°C to minus 5°C, preferably minus 50°C to minus 8°C, particularly preferably minus 30°C to minus 10°C, very particularly preferably from about minus 25°C to minus 15°C.
- the German expression "felgekuhlt" [frozen] is equivalent to the German expression “gefroren” or "eingefroren”.
- polysaccharide in the context of the present invention, comprises natural polysaccharides, synthetic polysaccharides, polysaccharide derivatives, modified polysaccharides and also corresponding mixtures thereof.
- the expression includes chemical compounds which are made up of a multiplicity (several hundred to thousands) of monosaccharide units per molecule and can comprise straight or branched chains. The molecular weights thereof typically are in the range over 5000 and can reach up to millions of Daltons.
- polysaccharides are naturally occurring polymers such as glycogen, starch (amylose and amylopectin) and starch derivatives, cellulose, hemicellulose such as, e.g.
- the starch-based material which is suitable for the invention can be any desired starch.
- Such starch types comprise those of any desired plant type including corn, potato, sweet potato, flour, rice, tapioca, sago, millet.
- the expression likewise comprises conversion products the basis of which is the abovementioned starches, for example dextrins, which are produced by acid hydrolysis or the action of heat, and also derivatized or crosslinked starches.
- the expression "monosaccharide” defines organic chemical compounds having at least three carbon atoms.
- the expression comprises glyceraldehyde; erythrose; threose; ribose; arabinose; xylose; lyxose; allose; altrose; glucose; mannose; gulose; idose, galactose; talose.
- the monosaccharide is glucose.
- oxidation designates a chemical reaction in which a substance to be oxidized (electron donor) releases electrons. Another substance (oxidizing agent) accepts the electrons (electron acceptor) and is reduced thereby. Both reactions together are understood as partial reactions of a redox reaction .
- Enzymes which catalyze an oxidation/reduction reaction are known to those skilled in the art under the name “oxidases”, wherein molecular oxygen (0 2 ) acts as an electron acceptor which is reduced to water (H2O) or hydrogen peroxide (H2O2).
- H2O water
- H2O2O2O2 hydrogen peroxide
- color indicator designates a substance using which the course of a physical, chemical, microbiological, or preferably, enzymatic process may be followed or the state of a physical, chemical, microbiological, or preferably, enzymatic, system may be characterized, wherein a change of state is signaled by a one-colored or two-colored change of color.
- pH-indicators acid-base indicators
- redox indicators metal indicators
- metal indicators adsorption indicators
- fluorescence indicators chemiluminescence indicators.
- the color change of an indicator is due to the occurrence of structurally differing forms (e.g.
- chromogenic substrates or indicator enzyme substrates are known to those skilled in the art. These are taken to mean chromogenic substances which can participate in particular enzymatic reactions either as electron donor or as electron acceptor and which in the course of this reaction change their color.
- the enzyme peroxidase catalyzes the conversion of hyd rogen peroxide to water. If the donor molecu le is a chromogenic substrate, oxidation thereof causes a detectable color change.
- substrates are, for ⁇ -galactosidase, the galactosides of resorufin, chlorophenolate or nitrophenol.
- peroxidase substrates are resorufin and triarylimidazoles.
- TMB 3,3',5,5'-tetramethylbenzidine
- pNPP colorless p-nitrophenylphosphate
- benzenoid form colorless p-nitrophenoxide form
- Chromogenic substrates suitable for the present invention comprise, without being restricted thereto, 4-chloro-1 -naphthol (4-CN), ortho- phenylenediamine (OPD), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), d iaminobenzid i ne (DAB) , 3, 3'-dimethyloxybenzidine (ortho-dianisidine or ODN), 5-aminosalicylic acid (5AS), and 3,3',5,5'-tetraalkylbenzidines such as, for example, 3,3',5,5'-tetramethylbenzidine (TMB).
- Other chromogenic substrates comprise different fluorescent and chemiluminescent compounds. A list of chromogenic substrates with the possible color changes is shown in Table 2.
- the indicator system according to the invention is suitable for gross failures within a freezing chain and does not indicate time and temperature history.
- the system is based on a cascade of enzymes, matched to one another, which does not display a reaction until at room temperature. Short interruptions of the freezing chain, which can result during transfer of the goods, for example, are thus not detected.
- the invention first relates to an indicator for temperature monitoring a chilled or frozen product, which indicator comprises the components:
- the indicator according to the invention is first in an inactive state before it is applied to the product which is to be chilled or frozen. The indicator is only activated when the following conditions are complied with:
- the indicator is brought into contact with a polysaccharide specific for the enzyme (a) and/or
- the indicator is transferred to an aqueous medium and to a temperature range at which the enzymes in (a), (b) and optionally (c) possess a catalytic activity.
- a precondition of the invention is therefore firstly that the components in (a), (b) and (c) are free from monosaccharides, in particular glucose, since otherwise an undesired premature enzyme reaction takes place.
- the enzymes in (a), (b) and optionally (c) typically only possess their catalytic activity when a temperature optimum favorable for their activity is reached.
- the temperature optimum for enzymes is typically in a temperature range from at least 0 degrees Celsius, that is to say in a temperature range in which a product to be monitored is known to be present in the thawed state.
- this temperature range is between 0 degrees Celsius and 30 degrees Celsius, particularly preferably in the range between 5 degrees Celsius and 25 degrees Celsius, and still more preferably between 10 degrees Celsius and 20 degrees Celsius.
- the indicator according to the invention is suitable in particular for products, the storage life of which is known to be dependent on a defined temperature in a range from below 0 degrees Celsius. Any type of temperature variations during storage can cause a (faster or slower) color change.
- the enzymes in (a), (b) and optionally (c) have their catalytic activity only in an aqueous medium, that is to say that they have no activity in the dry state or in the frozen state.
- the components of the indicator are stored after their mixing in an anhydrous dry state.
- the indicator In order to keep the indicator in the inactive state before it is applied to the frozen products, the indicator must be frozen or dried within only a few minutes, in such a manner that the indicator is present in an anhydrous state and/or in a temperature range unfavorable for activity. Preferably, freezing or drying proceeds within 1 to 1 0 minutes, particularly preferably within 2 to 5 minutes.
- An ice condenser can ensure that the water vapor precipitates in a targeted manner at a site remote from the ware.
- Common drying methods which come into consideration for the present invention are: infrared or microwave drying, convection drying, spray drying, freeze drying (lyophilization), vacuum drying, drying with supercritical gases, spray-freeze drying.
- the drying operation should proceed extremely thoroughly in order to remove as comprehensively as possible even traces of remaining water.
- the chosen drying temperature can vary greatly. For example, in spray drying, for a short time - usually a few seconds - temperatures of up to about 150-170°C can be reached. It is known to those skilled in the art what temperatures are expedient for the respective method without denaturing the enzyme or protein solution to be dried.
- the polysaccharide is present as a carrier matrix on which the indicator according to the invention comprising the components a), b) and c) is applied for temperature monitoring.
- carrier matrix comprises any desired material onto which the components of the indicator can be applied and which either consists completely of polysaccharides or which can comprise polysaccharides.
- Such materials can be synthetic or natural chemical substances or substances of biological origin.
- the matrix materials comprise, without being restricted thereto, glass and other silicon oxides, polystyrene, polypropylene, polyethylene, poly(vinylidene fluoride), polyurethane, polyalginate, polysulfone, polyvinyl alcohol), acrylonitrile polymers, polyacrylamide, polycarbonate, polypentene, polypentane, nylon, amyloses, gelatin, modified (e.g.
- polysaccharide is cellulose or derivatives thereof.
- Cellulose is a naturally occurring polysaccharide of beta-linked glucose units.
- the carrier matrix on which the indicator is applied must be frozen or dried in the course of a very few minutes.
- the freezing or drying proceeds within 1 to 10 minutes, particularly preferably within 2 to 5 minutes.
- common methods such as warming, infrared irradiation, microwave treatment can be used.
- the polysaccharide or the polysaccharide carrier matrix is composed of cellulose or cellulose-comprising or cellulose-based materials.
- water-soluble cellulose ethers such as, e.g. carboxymethylcellulose, and also alkyl- and hydroxyalkyl celluloses, for example methylcellulose, hydroxypropylymethylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxybutylmethycellulose, and ethyl hydroxyethyl cellulose.
- cellulose comprises all simple and commercially available forms of cellulose such as, e.g. wood pulp, cotton wool, hemp, ramie fibers, or regenerated forms such as, e.g. rayon.
- the polysaccharide carrier matrix is paper.
- paper in the context of the present invention, comprises flat or differently shaped materials of fiber or fibrous cellulosic material typically of natural origin. Paper can also be made of synthetic cellulose fibers, regenerated cellulose and of recyclable waste paper. In addition, combinations of cellulosic and synthetic materials are suitable for the present invention. Card likewise is encompassed by the term paper.
- the indicator according to the invention for example, malate oxidase (EC 1 .1 .3.3), glucose oxidase (EC 1 .1 .3.4), hexose oxidase (EC 1 .1 .3.5), L-gulonolactone oxidase (EC 1.1.3.8), galactose oxidase (EC 1.1.3.9), pyranose oxidase (EC 1.1.3.10), L-sorbose oxidase (EC 1.1.3.11 ), N-acylhexosamine oxidase (EC 1.1.3.29), D-arabinono-1 ,4-lactone oxidase (EC 1.1.3.37), D-mannitol oxidase (EC 1 .1 .3.3), glucose oxidase (EC 1 .1 .3.4), hexose oxidase (EC 1 .1 .3.5), L-gulonolactone oxidase (EC 1.1
- enzyme (b) which catalyzes the oxidation of a monosaccharide is a glucose oxidase.
- glucose oxidase relates to an oxidase enzyme of the class EC 1.1.3.4, a dimeric protein which catalyzes the oxidation of beta-D-glucose to D- glucono-1 ,5-lactone, wherein the latter is hydrolyzed to gluconic acid and simultaneously molecular oxygen is reduced to hydrogen peroxide (H2O2).
- Hydrogen peroxide in turn, with the action of the second enzyme as explained hereinafter, oxidizes a substance which in the process changes color (redox indicator).
- the indicator apart from the enzymes a) and b), further comprises a mixture c) which is suitable for detecting the enzyme activity of a) and/or b).
- This mixture comprises according to the invention a color indicator, as defined, and typically a corresponding indicator enzyme which is able to effect a change of color of the color indicator.
- an “indicator enzyme” is taken to mean any enzyme, the enzyme activity of which can be determined by reaction with one or more substrates for this enzyme.
- all known indicator reactions which utilize an enzyme-substrate reaction can be used.
- these include, for example, those in which a detectable product is formed directly, but also those in which the product of the reaction first leads to a detectable signal in further reaction steps.
- the substrate in such reactions which are followed by further reaction steps can be, for example, in turn a support material, to which a further indicator enzyme is bound.
- Reactions which are particularly expedient are those in the course of which a color change takes place or a colored compound is formed or disappears.
- Such enzymes and associated substrates are known to those skilled in the art.
- the mixture expediently comprises the reagents which are required for determining the concentration of the indicator enzyme.
- these reagents comprise, for example, an indicator-enzyme substrate or chromogenic substrate as defined above.
- Indicator-enzyme substrates or chromogenic substrates are compounds which, via catalysis by the indicator enzyme, effect a detectable change. They are preferentially cleavable or components of a redox system. The detection of the substrate or of the product of reaction thereof with the indicator enzyme can be performed, for example, colorimetrically, fluorimetrically or else electrochemically. Furthermore, the reagents can comprise, if required, pH buffer substances, stabilizers, activators etc. The type of indicator-enzyme substrate and also the further components depend on the indicator enzyme to be determined and are known to those skilled in the art.
- the indicator enzyme is a peroxidase and consequently the chromogenic substrate is a redox indicator for a peroxidase (see definitions and Table 2).
- the dye used is ABTS.
- concentration of the ABTS is selected such that it corresponds to the upper limit of the measurement range.
- the concentration of ABTS is in a range from 0.05 mg/ml to 10 mg/ml, particularly preferably in a range between 0.5 and 7 mg/ml, very particularly preferably in a range between 0.7 and 3 mg/ml.
- an oxidase is used in a coupled system with the peroxidase.
- the glucose oxidase converts glucose with oxygen to form gluconic acid, wherein H2O2 is formed as byproduct, the concentration of which is determined using a chromogenic substrate such as ABTS and peroxidase.
- the peroxidase can be used in this case in concentrations high enough that the H2O2 is reacted quantitatively in the course of a very few minutes - and up to several hours - as soon as the indicator is activated.
- the indicator enzyme of the mixture (c) is a perhydrolase and consequently the chromogenic substrate is a redox indicator for a perhydrolase (see definitions and table 1 ).
- Enzymes designated “hydrolases”, in particular “perhydrolases”, which are also known under “haloperoxidases”, are enzymes which, in the presence of hydrogen peroxide (H2O2), catalyze the formation of sufficient amounts of peracids in order to make possible accompanying halogenation reactions (enzymes of classification EC 3.).
- H2O2 hydrogen peroxide
- the reagents in (c) in addition comprise halides, preferably potassium bromide, and a color indicator, preferably phenol red.
- peracids can be detected using ABTS and potassium iodide (Pinkernell, U.
- a nucleic acid encoding the SEQ ID NO.1 can be expressed in bacteria, preferably in Escherichia coli, and purified. Methods for expressing and purifying heterologously expressed proteins in bacteria are sufficiently known to those skilled in the art (see, in particular, "Molecular Cloning: A Laboratory Manual”; Sambrook and Russell, CSHL Press 2001 , ISBN 978-087969577-4).
- the present invention further relates to a method for producing an indicator for temperature monitoring a chilled or frozen product, which method comprises mixing a. at least one enzyme which catalyzes the conversion of a polysaccharide to a monosaccharide with
- c a mixture which is suitable for detecting the enzyme activity of a) and/or b) and which comprises a color indicator.
- the method additionally comprises applying the components a), b) and c) to a polysaccharide carrier matrix.
- the indicator is applied directly to the package of the product to be frozen.
- Methods for applying labels of various materials such as, for example, paper, to packages are familiar to those skilled in the art.
- labels are applied by means of labeling machines which apply the labels to the package either mechanically or manually or by means of air pressure. See:
- the package is made of paper or card, as is the case in common frozen products such as pizza, frozen vegetables, frozen fruit, the package itself acts as the polysaccharide carrier matrix.
- the polysaccharide carrier matrix After application of the components a), b), c), is immediately dried or immediately frozen. Common drying methods have been described above.
- the present invention further relates to a method for temperature monitoring a chilled or frozen product, which method comprises
- positive detection of the enzyme activity is indicative of an interruption of the chilling or freezing of the product.
- the polysaccharide carrier matrix comprising the indicator becomes moist or aqueous owing to the liquid formed during thawing and is transferred into a temperature range at which the enzymes (a), (b) and (c) of the indicator according to the invention exhibit their catalytic activity, and so the above described enzyme cascade which may be detected on the basis of the change of color of the color indicator is started.
- the application is carried out under conditions under which no enzyme activity is detectable. Therefore, the product can be packaged in the non-chilled temperature range, the indicator can be applied to the package and both together can be frozen. Alternatively, an already frozen product can be charged into a package which comprises the indicator according to the invention. Subsequently the product is again stored under frozen conditions.
- the invention further relates to a package for storing a chilled or frozen product wherein the package is provided with at least one indicator according to the invention, which indicator can be fixed or removable.
- package designates a container which is suitable for storing consumer goods or products, wherein the container is not restricted to a certain shape or size and can be made of metal, glass, plastic, polystyrene, paper (card) or mixed forms thereof.
- Package which comes into consideration includes cans, cartons, dishes, trays, pockets, envelopes, wrapping films, freshness-retaining films and the like.
- the indicator is arranged on the package so as to be externally visible.
- the package material does not already itself consist of polysaccharides or comprise same, such as paper materials, the package is provided with an indicator which has been previously applied to a polysaccharide carrier matrix.
- the indicator according to the invention can be applied to the package by means of a number of methods which are known to those skilled in the art, for example in the manner of a label.
- the indicator in this case can be arranged at least between one outer layer which is translucent to visible and/or UV light, and preferably impermeable to water, and/or one inner layer facing the product and impermeable to the indicator.
- the indicator If the indicator is to be removable, it can be provided in a small pouch having an outer layer and an inner layer made of customary package materials.
- a polysaccharide carrier matrix comprising the indicator is placed into a suitable material in the form of a small bag or envelope. The bag is then sealed by welding or other suitable methods and can then be applied to the package.
- Suitable materials for application are adhesive glue materials in the form of glue strips which are typically used for such purposes.
- the outer layer facing inspection should comprise a flexible, transparent plastic film material.
- materials are known to those skilled in the art and comprise materials such as polyester, polycarbonate, polyethylene, polypropylene, polyamide, polyurethane, polyvinyl chloride) or the like.
- the indicator can be applied to a fresh product package or enclosed therein before the package comprising the product is frozen. The product is then frozen and the indicator displays whether the package or the product was present in the thawed state, even if it has been frozen again after the thawing.
- the invention further relates to the use of a package according to the invention for storing chilled or frozen products. Frozen or chilled products can comprise preserved blood, foods, vaccines, medicaments, blood products, implants, diagnostics, eye drops, contact lenses and chemicals.
- the type of the package must be selected according to the product.
- Particular advantages of the present invention are that the ideal composition can be achieved by using appropriate enzyme and substrate concentrations.
- the system can be used very flexibly and its components - including the enzymes - may be obtained inexpensively.
- an inert indicator as a liquid formulation which only needs to be printed onto the package during the package process.
- the enzyme formulation can be applied to a glucose-free label and this can be dried, and so a storable label is provided.
- the user or person skilled in the art e.g. packaging manufacturer, food manufacturer
- the invention will be described in more detail with reference to the examples hereinafter, without being restricted thereto.
- Example 1 Compositions according to the invention Smart Ink for the peroxidase label:
- the peroxidase label functions by releasing glucose from wood pulp by cellulases. Glucose oxidase oxidizes the liberated glucose to D-gluconolactones and H2O2. A peroxidase, with the aid of H2O2, oxidizes the model substrate ABTS to its chromophoric form. This causes a color change from colorless/light green to dark green.
- the first and third lines of the microtiter plate of figure 2 show the complete Smart Ink formulation. Even at the highest enzyme concentrations, no green coloration of the solution is visible even after two days. The slightly yellowish color of the solution is due to the inherent color of the cellulose stock solution and not to the presence of glucose (see second line with only ⁇ -glucosidase). In the bottom line a glucose dilution series is shown in order to quantify the sensitivity of the system. In the right hand column there is situated the negative control (water instead of cellulose). No reaction takes place here, neither in the Smart Ink MTP nor in the wood pulp MTP (see figure 2).
- the wood pulp MTP (figure 3) shows that the cellulase Onozuka RS was able to hydrolyze the wood pulp very effectively and was able to liberate sufficient glucose for the further enzymatic reaction steps of the cascade.
- a first staining began even after 15 minutes (RT, not shown here). After 30 minutes, the degree of staining of the wood pulp pieces was already far advanced (up to 1.6 U/ml Onozuka RS).
- the ⁇ -glucosidase alone could not release glucose from the wood pulp used and thus could not initiate staining by the ABTS by means of the enzyme cascade.
- the mixture of the Onozuka and ⁇ -glucosidase showed no improvement of the system and is therefore not absolutely necessary.
- Example 3 Testing various enzymes which can catalyze the degradation/conversion of polysaccharides to monosaccharides.
- the remaining 35 glucose-comprising formulations were purified with a desalting column (Hi-Trap, from GE - isocratic run, 100 mM Na acetate, pH 5.5) and thereafter tested on a wood pulp MTP. In this manner, 18 enzyme formulations were able to be additionally identified as positive.
- the perhydrolase label likewise glucose is liberated from wood pulp by cellulases.
- the glucose oxidase again oxidizes the liberated glucose to D-gluconolactones and H2O2.
- the n ext step acetate - used as a concentrated buffer system - is perhydrolyzed by a perhydrolase using H2O2.
- the resultant peracetic acid oxidizes potassium bromide in situ to potassium hypobromide, which in turn brominates phenol red to bromophenol blue. This causes a color change from yellow to blue.
- a stock solution of 150 g/ml was produced from the perhydrolase.
- two cellulases were tested. Firstly, the cellulase Onozuka RS, which had proved to be very effective in the experiments for the peroxidase label and secondly, the glucanase/xylanase mixture Optimash BG (from Genencor), which, after desalting, was likewise active in the wood pulp MTP test. Test experiments showed, however, that only the glucanase/xylanase mixture Optimash BG was functional in this system.
- the perhydrolase stock solution (150 pg/ml) was diluted in a microtiter plate 1 :2 from left to right.
- 20 ⁇ of perhydrolase dilution and 20 ⁇ of Optimash BG total protein concentration 13.3 mg/ml were added to 180 ⁇ of reaction solution.
- 20 ⁇ of perhydrolase dilution and 20 ⁇ of Optimash BG total protein concentration 13.3 mg/ml
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Abstract
An indicator for temperature monitoring a chilled or frozen product, which indicator comprises an enzyme which catalyzes the conversion of a polysaccharide to a monosaccharide, and enzyme which catalyzes the oxidation of the monosaccharide formed, and a mixture which is suitable for detecting the enzyme activities and which comprises a color indicator. In addition, a method for producing the indicator, a method for temperature monitoring and a package provided with the indicator thereof.
Description
Composition and method for temperature monitoring chilled and frozen products
FIELD OF THE INVENTION
The present invention relates to the field of freezing temperature-sensitive articles, in particular foods.
BACKGROUND OF THE INVENTION
In the frozen storage of products, e.g. foods, if a relatively long storage life is to be ensured, a storage temperature of at least -16°C must be maintained. If, however, a temperature of -10°C to -15°C is exceeded, the storage life of many products is no longer ensured; the frozen ware is considered to be initially thawed, although it still appears to be frozen externally. Usually, freezers are equipped with built-in thermometers which display the temperature at a defined site of the cold chamber, or a conventional thermometer is placed on or near the chilled ware and removed from the refrigerator in order to read it. In these cases there is no guarantee that the temperature of the chilled ware is measured exactly. In addition, by stacking and packing the chilled ware, the convection of air in the cold chamber can be prevented, and so even in the case of faultless functioning of the apparatus, at some points of the cold chamber the minimum temperatures required for the storage life of the products can be exceeded. For the user of the frozen ware, the exceedance of the storage life temperature cannot be recognized, and so spoilage of the products cannot be counteracted by prompt removal and immediate consumption. The risk that frozen products are slightly thawed unnoticed is particularly great, for example, if a relatively large amount of ware to be chilled is introduced warm into the freezer. Therefore, it is desirable to have available a temperature indicator which, for each food package or a group of packs, displays whether they were adequately cooled during their entire storage time. Temperature indicators known in the prior art can detect interruptions in a chilling/freezing chain. The measurement principle is usually based on physical, chemical, microbiological or enzymatic processes. Depending on the system, differing reactions are obtained to an interruption in the chilling/freezing chain such as, e.g. color change, color development or a migration of a dye along a scale.
Indicators known to those skilled in the art as "partial history" integrators do not display a reaction until a defined threshold temperature has been exceeded. "Full-history" integrators react continuously and display from the time point of activation the entire temperature-time history. This system functions independently of a threshold temperature (J.H. Wells, R.P. Singh (1987) - A graphical Interpretation of time-temperature related quality changes in frozen food. J. Food Sci. 52, 436-439). According to Taoukis and Labuza (Proc. Of the International Symposium Quimper Froid, 1997, 291 -297), indicators are classified into three groups: (i) "Critical Temperature Indicators" (CTI) - when a defined temperature is
exceeded, an irreversible change in the label takes place; (ii) "Critical-Time-Temperature Indicators" (CTTI) - display a cumulative temperature-time history above a defined threshold temperature; (iii) "Time-Temperature Indicators" (TTI) - are activated and then display the entire temperature-time history.
Various demands are made of such freshness indicators, also known as "labels" to those skilled in the art. What is primarily of importance is the stability of the indicator. This is taken to mean a stable formulation before activation and subsequently linear behavior. The indicator should not be able to be influenced by external factors such as, e.g., UV light and moisture. For the food producer, the label must particularly be able to be used conveniently and simply. Simple application of the label to the package is a condition in order to integrate a novel system as easily as possible into the packaging process and in order to avoid additional costs such as the purchase of a new expensive labeling machine. For the consumer, the label must be easily understandable and the degree of freshness detected must be able to be seen at a glance.
Temperature- or temperature-time indicators have been known for a relatively long time in the prior art. The system "3M MonitorMark", for example, is based on the diffusion of a blue ester along a scale. Exceedance of a defined threshold temperature leads to an irreversible melting of the ester, wherein the entire time-temperature history can be determined (T.P. Labuza (2000) The search for shelf life - An update on continued efforts in understanding practical strategies for determining and testing the shelf life of food products. Food Testing Analysis). The commercially available "VITSAB" indicator is based on an enzymatic hydrolysis of tricaproin by a pancreas lipase. The enzyme and the substrate solution comprising a pH indicator are separated from one another in a two-chamber system. The label is activated by a pressure on the label, wherein the thin barrier between enzyme and substrate solution is broken. The warmer the outside temperature, the more rapid is the mixing of the two solutions and the more rapid is the enzymatic hydrolysis. The formation of capric acid leads to a pH fall and thus to a color change from green to yellow. However, a disadvantage of this system is the inactivation of the enzyme at a temperature of above 40°C, which can lead to faulty functioning of the indicator (T. Labuza, Bin Fu, Use of Time/Temperature Integrators, Predictive Microbiology, and Related Technologies for Assessing in the extent and impact of temperature abuse on meat and poultry products, Journal of Food safety 15 (1995); 201 -227; S. Tsoka, P.S. Taoukis, Time Temperature Integrators for chilled food shelf life monitoring using enzyme-substrate systems, Food Biotechnology 12 (1 &2) (1998), 139-155). The "fresh check" system is based on the polymerization of a colorless acetylene or diacetylene monomer to give a colored polymer. The length and therefore the color intensity are temperature-dependent. The reaction proceeds in the center of the label, wherein the darker circle around the light surface shows the reference ring. The system has the
disadvantage that the labels are active from the time point of their manufacture and must be stored at -25°C (P.S. Taoukis, T . Labuza (1989) - Applicability of time-temperature indicators as shelf life monitors of food products. J. Food Sci., 54 (4), 783-788). What is called the "OnVu label" is based on the activation of a dinitrobenzylpyridine (DNBP) label with UV light. The blue coloration is based on a light-induced proton transfer reaction in which the blue photo product returns slowly to the base state by "thermally active tunneling" and becomes colorless again in the process. After expiry of the storage life calculated for the chilled ware and in the case of interruptions in the chilling chain, a (virtually) complete decolorization of the label can be seen. In order to prevent repeated activation of the label, the active imprint must be covered by an adhesive UV-impermeable film (J. Kreyenschmidt (2003) - Modellierung des Frischeverlustes von Fleisch sowie des Entfarbeprozesses von Temperatur- Zeit- I ntegratoren zu r Festleg u n g von Anforderungsprofilen fur die produktbegleitende Temperaturuberwachung, Diss. Universitat Bonn [Modeling of the loss of meat freshness and also the decolorization process of temperature-ti me i ntegrators for esta bl ish i n g req u i rement profi les for prod uct- accompanying temperature monitoring, dissertation, University of Bonn]).
Because of frequent press reports on abuses in food storage, the maintenance of the freezing chain has again become a topical theme. The usual commercially available temperature-indicator systems have the disadvantage that they require special storage conditions in order not to become prematurely activated. In addition, the visualization of a temperature change on the package must be able to be carried out as far as possible without further steps on the part of the consumers.
SUMMARY OF THE INVENTION
Therefore, the objective technical problem of the invention was to provide a method which can detect an interruption in the chilling chain as simply, reliably and inexpensively as possible.
The inventors have developed an enzyme-based CTI which, in the form of a liquid (also termed "smart ink") can be printed onto paper-based packages - shortly before freezing. Gross failures of a freezing chain should be indicated by this means.
The object has therefore been achieved by the subject matter of the invention relating to an indicator for temperature monitoring a chilled or frozen product, which indicator comprises: a. at least one enzyme which catalyzes the conversion of a polysaccharide to a monosaccharide,
b. at least one enzyme which catalyzes the oxidation of the monosaccharide formed by means of a),
and
c. a mixture which is suitable for detecting the enzyme activity of a) and/or b) and which
comprises a color indicator,
wherein the enzyme activity is detectable only after application to a polysaccharide carrier matrix in the presence of water and in a temperature range in which the product is in the thawed state.
In a preferred embodiment, the indicator is attached to a polysaccharide, preferably cellulose, and is present in a dried state.
In further preferred embodiments, the color indicator comprises a chromogenic substrate for a peroxidase, or for a hydrolase, in particular a perhydrolase.
The invention further relates to a method for producing an indicator according to the invention. The invention further relates to a method for temperature monitoring a chilled or frozen product, which method comprises applying an indicator according to the invention to the product, and monitoring the enzyme activity by means of the color indicator, wherein positive detection of the enzyme activity is indicative of an interruption of the chilling or freezing of the product.
Preferably, the indicator is applied in a temperature range in which the enzyme from a) and/or the enzyme from b) and/or the mixture from c) are inactive.
The invention further relates to a package for storing a chilled or frozen product, wherein the package is provided with at least one fixed or removable indicator according to the invention.
Preferably, the indicator is arranged at least between one outer layer which is translucent to visible and/or UV light, and preferably water-impermeable, and/or one inner layer facing the product and impermeable to the indicator.
Finally, the invention relates to the use of a package according to the invention for storing chilled or frozen products. DESCRIPTION OF THE DRAWING
Figure 1
Flow diagram of the enzyme cascade of the indicator according to the invention Figure 2
Microtiter plate (MTP) with the test set-up of the "smart ink" formulation; row 1 : only Onozuka cellulase; row 2: only beta-glucosidase; row 3: Onozuka cellulase and beta- glucosidase; row 4: glucose standard; last column: no enzyme, water control
Figure 3
Test set-up peroxidase label in microtiter plate comprising cellulose tissues after incubation for 30 min (a) and 6 hours (b) at room temperature. Row 1 : only Onozuka cellulase; row 2: only beta-glucosidase; row 3: Onozuka cellulase and beta-glucosidase; last column: no enzyme, water control
Figure 4
Analysing the various enzyme formulations for the presence of glucose.
Figure 5
Test set-up perhydrolase label in microtiter plate containing cellulose tissues after incubation for 3 hours (a) and 6 hours (b) at room temperature. Row 1 : 44 g of phenol red; row 2: 88 g of phenol red; last column of a) and b): no enzyme, water control.
DEFINITIONS
The expression "indicator" means a device, an article, and/or a material which can change in a detectable manner depending on the temperature to which it is exposed. The change can be of a physical or chemical nature or both, and may be detected by means of human sight, sense of smell/feel and/or with the aid of electrical, chemical or radiation-based analytical equipment.
The expression "temperature monitoring" describes monitoring the chilling or freezing chain for readily spoilable products, for example foods, vaccines and blood and also for other applications. In connection with the present invention, this is taken to mean, in particular, monitoring whether a chilled, in particular frozen, product is present or has been present in the thawed state, that is has been warmed to a temperature of at least minus 15°C, at least minus 5°C, preferably at least 0°C, particularly preferably at least 10°C, very particularly preferably to a temperature of at least 15°C.
The expression "chilled product" describes a product which must be stored at a temperature of about minus 5°C to plus 10°C, preferably from about minus 3°C to about plus 8°C, particularly preferably 0°C to plus 5°C. The expression "frozen product" relates to a product which must be stored at a temperature of about minus 80°C to minus 5°C, preferably minus 50°C to minus 8°C, particularly preferably minus 30°C to minus 10°C, very particularly preferably from about minus 25°C to minus 15°C. In the context of the present invention the German expression "tiefgekuhlt" [frozen] is equivalent to the German expression "gefroren" or "eingefroren".
The expression "polysaccharide", in the context of the present invention, comprises natural polysaccharides, synthetic polysaccharides, polysaccharide derivatives, modified polysaccharides and also corresponding mixtures thereof. The expression includes
chemical compounds which are made up of a multiplicity (several hundred to thousands) of monosaccharide units per molecule and can comprise straight or branched chains. The molecular weights thereof typically are in the range over 5000 and can reach up to millions of Daltons. Typically, polysaccharides are naturally occurring polymers such as glycogen, starch (amylose and amylopectin) and starch derivatives, cellulose, hemicellulose such as, e.g. xylan , mannan, (arabino)galactan, dextran, pectin, guar gum, scleroglucan, gum Arabic, agar, chitin, callose, xanthan, carob bean gum (carob gum), chitosan, algin, carrageenan, gellan, welan, rhamsan, curdlan, lignin, tamarind gum and pullulan. If the polysaccharide is starch, the starch-based material which is suitable for the invention can be any desired starch. Such starch types comprise those of any desired plant type including corn, potato, sweet potato, flour, rice, tapioca, sago, millet. The expression likewise comprises conversion products the basis of which is the abovementioned starches, for example dextrins, which are produced by acid hydrolysis or the action of heat, and also derivatized or crosslinked starches.
The expression "monosaccharide" defines organic chemical compounds having at least three carbon atoms. The expression comprises glyceraldehyde; erythrose; threose; ribose; arabinose; xylose; lyxose; allose; altrose; glucose; mannose; gulose; idose, galactose; talose. For the present invention it is particularly preferred if the monosaccharide is glucose.
The expression "oxidation" designates a chemical reaction in which a substance to be oxidized (electron donor) releases electrons. Another substance (oxidizing agent) accepts the electrons (electron acceptor) and is reduced thereby. Both reactions together are understood as partial reactions of a redox reaction . Enzymes which catalyze an oxidation/reduction reaction are known to those skilled in the art under the name "oxidases", wherein molecular oxygen (02) acts as an electron acceptor which is reduced to water (H2O) or hydrogen peroxide (H2O2). The resultant hydrogen peroxide acts as a component of the mixture c. of the indicator according to the invention in the context of the present invention as a further oxidizing agent which in combination with the "color indicator" is suitable for detecting the enzyme activity.
The expression "color indicator" designates a substance using which the course of a physical, chemical, microbiological, or preferably, enzymatic process may be followed or the state of a physical, chemical, microbiological, or preferably, enzymatic, system may be characterized, wherein a change of state is signaled by a one-colored or two-colored change of color. Depending on the application and indication principle, the prior art differentiates as follows: pH-indicators (acid-base indicators), redox indicators, metal indicators, adsorption indicators, fluorescence indicators, chemiluminescence indicators. The color change of an indicator is due to the occurrence of structurally differing forms (e.g. by protolysis or deprotolysis as a result of a pH change) or the reaction of the indicator with one of the components of the reaction system, forming a differently colored compound. Depending on the pH or the redox state, the light absorption of the color indicator changes,
which is detectable by a change of color in the visible spectral range. Such substances are also familiar to the person skilled in the art as "chromogenic" substances, i.e. substances which are capable of a change of color. The color change range designated is the pH range in which the change of color of the indicator can be followed visually and extends approximately over an interval of 1 -2 pH units. A selection of common color indicators which are known to those skilled in the art are summarized in Table 1 hereinafter.
Furthermore, "chromogenic substrates" or indicator enzyme substrates are known to those skilled in the art. These are taken to mean chromogenic substances which can participate in particular enzymatic reactions either as electron donor or as electron acceptor and which in the course of this reaction change their color. For example, the enzyme peroxidase catalyzes the conversion of hyd rogen peroxide to water. If the donor molecu le is a chromogenic substrate, oxidation thereof causes a detectable color change. Examples of such substrates are, for β-galactosidase, the galactosides of resorufin, chlorophenolate or nitrophenol. Examples of peroxidase substrates are resorufin and triarylimidazoles. For example, 3,3',5,5'-tetramethylbenzidine (TMB) is colorless in the reduced state, but blue in
the oxidized state and yellow in the diamine state. In the case of reactions under the influence of alkaline phosphatase, colorless p-nitrophenylphosphate (pNPP) is converted into its colorless p-nitrophenoxide form (benzenoid form), at alkaline pH from the benzenoid form to the quininoid form which is yellow. Chromogenic substrates suitable for the present invention comprise, without being restricted thereto, 4-chloro-1 -naphthol (4-CN), ortho- phenylenediamine (OPD), 2,2'-azinobis-(3-ethylbenzothiazoline-6-sulfonic acid) (ABTS), d iaminobenzid i ne (DAB) , 3, 3'-dimethyloxybenzidine (ortho-dianisidine or ODN), 5-aminosalicylic acid (5AS), and 3,3',5,5'-tetraalkylbenzidines such as, for example, 3,3',5,5'-tetramethylbenzidine (TMB). Other chromogenic substrates comprise different fluorescent and chemiluminescent compounds. A list of chromogenic substrates with the possible color changes is shown in Table 2.
Table 2:
The indicator system according to the invention is suitable for gross failures within a freezing chain and does not indicate time and temperature history. The system is based on a cascade of enzymes, matched to one another, which does not display a reaction until at room temperature. Short interruptions of the freezing chain, which can result during transfer of the goods, for example, are thus not detected.
Therefore, the invention first relates to an indicator for temperature monitoring a chilled or frozen product, which indicator comprises the components:
a. at least one enzyme which catalyzes the conversion of a polysaccharide to a monosaccharide,
b. at least one enzyme which catalyzes the oxidation of the monosaccharide formed by means of a),
and
c. a mixture which is suitable for detecting the enzyme activity of a) and/or b) and which comprises a color indicator,
wherein the enzyme activity is detectable only after application to a polysaccharide carrier matrix in the presence of water and in a temperature range in which the product is in the thawed state. Therefore, the indicator according to the invention is first in an inactive state before it is applied to the product which is to be chilled or frozen. The indicator is only activated when the following conditions are complied with:
(1 ) the indicator is brought into contact with a polysaccharide specific for the enzyme (a) and/or
(2) the indicator is brought into contact with a monosaccharide specific for the enzyme (b); and
(3) the indicator is transferred to an aqueous medium and to a temperature range at which the enzymes in (a), (b) and optionally (c) possess a catalytic activity. A precondition of the invention is therefore firstly that the components in (a), (b) and (c) are free from monosaccharides, in particular glucose, since otherwise an undesired premature enzyme reaction takes place. Secondly, the enzymes in (a), (b) and optionally (c) typically only possess their catalytic activity when a temperature optimum favorable for their activity is reached. The temperature optimum for enzymes is typically in a temperature range from at least 0 degrees Celsius, that is to say in a temperature range in which a product to be monitored is known to be present in the thawed state. Preferably, this temperature range is between 0 degrees Celsius and 30 degrees Celsius, particularly preferably in the range between 5 degrees Celsius and 25 degrees Celsius, and still more preferably between 10 degrees Celsius and 20 degrees Celsius.
Accordingly, the indicator according to the invention is suitable in particular for products, the storage life of which is known to be dependent on a defined temperature in a range from below 0 degrees Celsius. Any type of temperature variations during storage can cause a (faster or slower) color change.
Furthermore, it is known to those skilled in the art that the enzymes in (a), (b) and optionally (c) have their catalytic activity only in an aqueous medium, that is to say that they have no activity in the dry state or in the frozen state. Preferably, the components of the indicator are stored after their mixing in an anhydrous dry state.
In order to keep the indicator in the inactive state before it is applied to the frozen products, the indicator must be frozen or dried within only a few minutes, in such a manner that the indicator is present in an anhydrous state and/or in a temperature range unfavorable for activity. Preferably, freezing or drying proceeds within 1 to 1 0 minutes, particularly preferably within 2 to 5 minutes.
Drying methods for enzymes and proteins are known to those skilled in the art from the prior art. In order to remove water from a protein solution, water vapor must be generated
and transported away from the sample. For this purpose, those skilled in the art know three possibilities: (i) vaporization at temperatures above boiling point, (ii) evaporation below the boiling point, but above the triple point of water and (iii) sublimation from ice at low pressures. Principle (iii) is only used in freeze drying. The resultant water vapor can be removed by placing the gas situated above the ware in motion and removing it from the drying zone. This takes place by means of the use of fans, blowing in dry gases, with simultaneous removal of the water-vapor-saturated air by suction, or by evacuating the chamber. An ice condenser can ensure that the water vapor precipitates in a targeted manner at a site remote from the ware. Common drying methods which come into consideration for the present invention are: infrared or microwave drying, convection drying, spray drying, freeze drying (lyophilization), vacuum drying, drying with supercritical gases, spray-freeze drying.
The drying operation should proceed extremely thoroughly in order to remove as comprehensively as possible even traces of remaining water. Depending on the drying method used, the chosen drying temperature can vary greatly. For example, in spray drying, for a short time - usually a few seconds - temperatures of up to about 150-170°C can be reached. It is known to those skilled in the art what temperatures are expedient for the respective method without denaturing the enzyme or protein solution to be dried.
It is particularly expedient for the present invention if the polysaccharide is present as a carrier matrix on which the indicator according to the invention comprising the components a), b) and c) is applied for temperature monitoring.
The expression "carrier matrix" comprises any desired material onto which the components of the indicator can be applied and which either consists completely of polysaccharides or which can comprise polysaccharides. Such materials can be synthetic or natural chemical substances or substances of biological origin. The matrix materials comprise, without being restricted thereto, glass and other silicon oxides, polystyrene, polypropylene, polyethylene, poly(vinylidene fluoride), polyurethane, polyalginate, polysulfone, polyvinyl alcohol), acrylonitrile polymers, polyacrylamide, polycarbonate, polypentene, polypentane, nylon, amyloses, gelatin, modified (e.g. crosslinked) gelatin, collagen, natural and modified polysaccharides as defined above, including dextrans and celluloses (e.g. nitrocellulose), agar and magnetite. For the present invention it is most favorable if the polysaccharide is cellulose or derivatives thereof. Cellulose is a naturally occurring polysaccharide of beta-linked glucose units.
In order to keep the indicator in the inactive state on the carrier matrix before it is applied to the frozen products, the carrier matrix on which the indicator is applied must be frozen or dried in the course of a very few minutes. Preferably, the freezing or drying proceeds within 1 to 10 minutes, particularly preferably within 2 to 5 minutes. In order to accelerate the drying operation, common methods such as warming, infrared irradiation, microwave treatment can be used.
In a further embodiment, the indicator according to the invention is applied to the carrier matrix by means of an inkjet printer which is suitable for packages. Printing systems which come into consideration for such purposes and are commercially available are described extensively, for example, on the Internet page http://www.sigtech- ag.ch/index.php?id=tintenstrahldrucker.
In a preferred embodiment of the indicator according to the invention, the polysaccharide or the polysaccharide carrier matrix is composed of cellulose or cellulose-comprising or cellulose-based materials. As a non-exhaustive list of examples thereof mention may be made of water-soluble cellulose ethers such as, e.g. carboxymethylcellulose, and also alkyl- and hydroxyalkyl celluloses, for example methylcellulose, hydroxypropylymethylcellulose, hydroxyethylcellulose, hydroxyethylmethylcellulose, hydroxybutylmethycellulose, and ethyl hydroxyethyl cellulose. Generally, the term cellulose comprises all simple and commercially available forms of cellulose such as, e.g. wood pulp, cotton wool, hemp, ramie fibers, or regenerated forms such as, e.g. rayon.
In a very particularly preferred embodiment, the polysaccharide carrier matrix is paper.
The name "paper", in the context of the present invention, comprises flat or differently shaped materials of fiber or fibrous cellulosic material typically of natural origin. Paper can also be made of synthetic cellulose fibers, regenerated cellulose and of recyclable waste paper. In addition, combinations of cellulosic and synthetic materials are suitable for the present invention. Card likewise is encompassed by the term paper.
It is understood that, depending on the selected polysaccharide, different enzymes or enzyme preparations (a) which can catalyze the conversion of a polysaccharide to a monosaccharide come into consideration. For the indicator according to the invention, for example cellulases, amylases, hemicellulases or xylanases come into consideration. Preferably, cellulases are used for the present invention. A list of commercially available enzymes which can degrade polysaccharides may be found in example 4, table 4.
It is understood, furthermore, that, depending on the monosaccharide resulting under (a), differing enzymes or enzyme preparations (b) which can catalyze the oxidation of a monosaccharide come into consideration. For the indicator according to the invention, for example, malate oxidase (EC 1 .1 .3.3), glucose oxidase (EC 1 .1 .3.4), hexose oxidase (EC 1 .1 .3.5), L-gulonolactone oxidase (EC 1.1.3.8), galactose oxidase (EC 1.1.3.9), pyranose oxidase (EC 1.1.3.10), L-sorbose oxidase (EC 1.1.3.11 ), N-acylhexosamine oxidase (EC 1.1.3.29), D-arabinono-1 ,4-lactone oxidase (EC 1.1.3.37), D-mannitol oxidase (EC 1 .1 .3.40), xylitol oxidase (EC 1 .1 .3.41 ), cellobiose dehydrogenase (EC 1.1.99.18), aldehyde oxidase (EC 1.2.3.1 ) can be considered.
In a preferred embodiment, enzyme (b) which catalyzes the oxidation of a monosaccharide, is a glucose oxidase. The name "glucose oxidase" relates to an oxidase enzyme of the class EC 1.1.3.4, a dimeric protein which catalyzes the oxidation of beta-D-glucose to D- glucono-1 ,5-lactone, wherein the latter is hydrolyzed to gluconic acid and simultaneously molecular oxygen is reduced to hydrogen peroxide (H2O2). Hydrogen peroxide in turn, with the action of the second enzyme as explained hereinafter, oxidizes a substance which in the process changes color (redox indicator).
For the function of the indicator it is necessary that the indicator, apart from the enzymes a) and b), further comprises a mixture c) which is suitable for detecting the enzyme activity of a) and/or b).
This mixture comprises according to the invention a color indicator, as defined, and typically a corresponding indicator enzyme which is able to effect a change of color of the color indicator.
An "indicator enzyme" is taken to mean any enzyme, the enzyme activity of which can be determined by reaction with one or more substrates for this enzyme. In particular, all known indicator reactions which utilize an enzyme-substrate reaction can be used. For this purpose these include, for example, those in which a detectable product is formed directly, but also those in which the product of the reaction first leads to a detectable signal in further reaction steps. The substrate in such reactions which are followed by further reaction steps can be, for example, in turn a support material, to which a further indicator enzyme is bound. Reactions which are particularly expedient are those in the course of which a color change takes place or a colored compound is formed or disappears. Such enzymes and associated substrates are known to those skilled in the art. Examples of indicator enzymes/substrates are -galactosidase/ -galactosides, peroxidase/peroxides, and also phosphatases/phosphates etc. In addition , the mixture expediently comprises the reagents which are required for determining the concentration of the indicator enzyme. These reagents comprise, for example, an indicator-enzyme substrate or chromogenic substrate as defined above.
Indicator-enzyme substrates or chromogenic substrates are compounds which, via catalysis by the indicator enzyme, effect a detectable change. They are preferentially cleavable or components of a redox system. The detection of the substrate or of the product of reaction thereof with the indicator enzyme can be performed, for example, colorimetrically, fluorimetrically or else electrochemically. Furthermore, the reagents can comprise, if required, pH buffer substances, stabilizers, activators etc. The type of indicator-enzyme substrate and also the further components depend on the indicator enzyme to be determined and are known to those skilled in the art.
In a very particularly preferred embodiment, the indicator enzyme is a peroxidase and consequently the chromogenic substrate is a redox indicator for a peroxidase (see definitions and Table 2). Preferably, the dye used is ABTS. The concentration of the ABTS is selected such that it corresponds to the upper limit of the measurement range. Thereby, with increasing enzyme concentration and correspondingly increasing H2O2 conversion rate, not only does the concentration of a colored product change, but also the ratio of two differently colored products and thereby the spectral composition of the color to be examined. Preferably, the concentration of ABTS is in a range from 0.05 mg/ml to 10 mg/ml, particularly preferably in a range between 0.5 and 7 mg/ml, very particularly preferably in a range between 0.7 and 3 mg/ml.
In the context of the present invention, an oxidase is used in a coupled system with the peroxidase. As described above, the glucose oxidase converts glucose with oxygen to form gluconic acid, wherein H2O2 is formed as byproduct, the concentration of which is determined using a chromogenic substrate such as ABTS and peroxidase. The peroxidase can be used in this case in concentrations high enough that the H2O2 is reacted quantitatively in the course of a very few minutes - and up to several hours - as soon as the indicator is activated.
In another embodiment, the indicator enzyme of the mixture (c) is a perhydrolase and consequently the chromogenic substrate is a redox indicator for a perhydrolase (see definitions and table 1 ).
Enzymes designated "hydrolases", in particular "perhydrolases", which are also known under "haloperoxidases", are enzymes which, in the presence of hydrogen peroxide (H2O2), catalyze the formation of sufficient amounts of peracids in order to make possible accompanying halogenation reactions (enzymes of classification EC 3.). If, for the indicator, a perhydrolase is used , the reagents in (c) in addition comprise halides, preferably potassium bromide, and a color indicator, preferably phenol red. Alternatively, peracids can be detected using ABTS and potassium iodide (Pinkernell, U. et al., Analyst, 1997, 122, 567-71 ), and also acetate as buffer system or a carboxylic acid ester derivative. In the case of carboxylic acid ester derivatives, lipases, proteases or esterases can be used which are known to be able to catalyze a perhydrolysis as a side reaction (WO 2009/067279; WO 20061 33079; Molecular basis of perhydrolase activity in serine hydrolases, Angewandte Chemie, International Edition (2005), 44(18), 2742-2746; Molecular cloning and expression of perhydrolase genes from Pseudomonas aeruginosa and Burkholderia cepacia in Escherichia coil, Biotechnology Letters (2006), 28(12), 849-856). In a preferred embodiment, a perhydrolase from Flavobacterium johnsoniae UW101 (EC 3.1.1.), see SEQ ID NO:1 , is used.
In order to obtain sufficient amounts of the enzyme, a nucleic acid encoding the SEQ ID
NO.1 can be expressed in bacteria, preferably in Escherichia coli, and purified. Methods for expressing and purifying heterologously expressed proteins in bacteria are sufficiently known to those skilled in the art (see, in particular, "Molecular Cloning: A Laboratory Manual"; Sambrook and Russell, CSHL Press 2001 , ISBN 978-087969577-4).
The present invention further relates to a method for producing an indicator for temperature monitoring a chilled or frozen product, which method comprises mixing a. at least one enzyme which catalyzes the conversion of a polysaccharide to a monosaccharide with
b. at least one enzyme which catalyzes the oxidation of the monosaccharide formed by means of a),
and with
c. a mixture which is suitable for detecting the enzyme activity of a) and/or b) and which comprises a color indicator.
Preferably, the method additionally comprises applying the components a), b) and c) to a polysaccharide carrier matrix. In a further embodiment, the indicator is applied directly to the package of the product to be frozen. Methods for applying labels of various materials such as, for example, paper, to packages are familiar to those skilled in the art. Typically, labels are applied by means of labeling machines which apply the labels to the package either mechanically or manually or by means of air pressure. See:
http://www.bluhmsysteme.com/
and also
www.neue-verpackung.de/ai/resources/0705dceb58a.pdf
If the package is made of paper or card, as is the case in common frozen products such as pizza, frozen vegetables, frozen fruit, the package itself acts as the polysaccharide carrier matrix.
For the storage and for avoiding premature activation of the indicator, the polysaccharide carrier matrix, after application of the components a), b), c), is immediately dried or immediately frozen. Common drying methods have been described above.
The present invention further relates to a method for temperature monitoring a chilled or frozen product, which method comprises
i. applying an indicator according to the invention to the product,
ii. and monitoring the enzyme activity by means of the color indicator,
wherein positive detection of the enzyme activity is indicative of an interruption of the chilling or freezing of the product.
In the case of an interruption of the chilling chain, i.e. if the product is thawed in an undesirable manner, the polysaccharide carrier matrix comprising the indicator becomes moist or aqueous owing to the liquid formed during thawing and is transferred into a temperature range at which the enzymes (a), (b) and (c) of the indicator according to the invention exhibit their catalytic activity, and so the above described enzyme cascade which may be detected on the basis of the change of color of the color indicator is started.
In a preferred embodiment, the application is carried out under conditions under which no enzyme activity is detectable. Therefore, the product can be packaged in the non-chilled temperature range, the indicator can be applied to the package and both together can be frozen. Alternatively, an already frozen product can be charged into a package which comprises the indicator according to the invention. Subsequently the product is again stored under frozen conditions. The invention further relates to a package for storing a chilled or frozen product wherein the package is provided with at least one indicator according to the invention, which indicator can be fixed or removable.
The expression "package" designates a container which is suitable for storing consumer goods or products, wherein the container is not restricted to a certain shape or size and can be made of metal, glass, plastic, polystyrene, paper (card) or mixed forms thereof. Package which comes into consideration includes cans, cartons, dishes, trays, pockets, envelopes, wrapping films, freshness-retaining films and the like. Preferably, the indicator is arranged on the package so as to be externally visible.
If the package material does not already itself consist of polysaccharides or comprise same, such as paper materials, the package is provided with an indicator which has been previously applied to a polysaccharide carrier matrix.
The indicator according to the invention can be applied to the package by means of a number of methods which are known to those skilled in the art, for example in the manner of a label. For protecting the indicator, the indicator in this case can be arranged at least between one outer layer which is translucent to visible and/or UV light, and preferably impermeable to water, and/or one inner layer facing the product and impermeable to the indicator. If the indicator is to be removable, it can be provided in a small pouch having an outer layer and an inner layer made of customary package materials. A polysaccharide carrier matrix comprising the indicator is placed into a suitable material in the form of a small bag or envelope. The bag is then sealed by welding or other suitable methods and can then be applied to the package. Suitable materials for application are adhesive glue materials in the form of glue strips which are typically used for such purposes.
The outer layer facing inspection should comprise a flexible, transparent plastic film
material. Such materials are known to those skilled in the art and comprise materials such as polyester, polycarbonate, polyethylene, polypropylene, polyamide, polyurethane, polyvinyl chloride) or the like. The indicator can be applied to a fresh product package or enclosed therein before the package comprising the product is frozen. The product is then frozen and the indicator displays whether the package or the product was present in the thawed state, even if it has been frozen again after the thawing. The invention further relates to the use of a package according to the invention for storing chilled or frozen products. Frozen or chilled products can comprise preserved blood, foods, vaccines, medicaments, blood products, implants, diagnostics, eye drops, contact lenses and chemicals. The type of the package must be selected according to the product. Particular advantages of the present invention are that the ideal composition can be achieved by using appropriate enzyme and substrate concentrations. The system can be used very flexibly and its components - including the enzymes - may be obtained inexpensively. It is possible to offer an inert indicator as a liquid formulation which only needs to be printed onto the package during the package process. Furthermore, the enzyme formulation can be applied to a glucose-free label and this can be dried, and so a storable label is provided. For the activation, the user or person skilled in the art (e.g. packaging manufacturer, food manufacturer) would apply water and subsequently cover the label with a protective film made of packaging materials familiar to those skilled in the art. The invention will be described in more detail with reference to the examples hereinafter, without being restricted thereto.
EXAMPLES Example 1 : Compositions according to the invention Smart Ink for the peroxidase label:
• Cellulase Onozuka RS from Trichoderma viride (Serva) - 2 U/mg - glucose free
(β-Glucosidase from almonds (Sigma) - 2.52 U/mg - glucose free)
· Glucose oxidase from Aspergillus niger Type II (Sigma) - 15.5 U/mg
• Peroxidase from soya bean (Sigma) - 90 U/mg (purpurogallin)
• ABTS (Roche)
Smart Ink for the perhydrolase label:
· Cellulase Optimash BG (Genencor) - 35 mg/ml
• Glucose oxidase from Aspergillus niger Type II (Sigma) - 15.5 U/mg
• Perhydrolase from Flavobacterium johnsoniae - 1.5 mg/ml
• Potassium bromide & phenol red
All components are formulated in Na acetate buffer (pH 5.5) as a stable stock solution (Smart Ink). By adding the described smart ink formulations to a polysaccharide carrier matrix, in particular wood pulp/cellulose (bleached birch sulfate paper - glucose free), the enzymatic cascade is activated (see figure 1 ).
Example 2: Peroxidase label
The peroxidase label functions by releasing glucose from wood pulp by cellulases. Glucose oxidase oxidizes the liberated glucose to D-gluconolactones and H2O2. A peroxidase, with the aid of H2O2, oxidizes the model substrate ABTS to its chromophoric form. This causes a color change from colorless/light green to dark green.
Stock solutions were produced from Onozuka cellulase and the β-glucosidase, each at 1000 U/ml. In the case of a combination of Onozuka and β-glucosidase, a ratio of 4:1 was selected. This mixture comprised 800 U/ml of Onozuka RS and 200 U/ml of β-glucosidase. The various enzyme solutions were in each case diluted in a microtiter plate from left to right 1 :2. 20 μΙ_ each of the individual dilutions were added to 180 μΙ of reaction solution (= Smart Ink MTP, figure 2). Per dilution, 30 μΙ of the Smart Ink MTP were added to an MTP with birch sulfate leaflets (= wood pulp MTP, see figure 3). Smart Ink for the peroxidase label:
100 mM Na acetate (pH 5.5)
10 g/ml Peroxidase
10 g/ml Glucose oxidase
1 mg/ml ABTS
Cellulase(s) - 1 :2 series of dilutions from left to right 20 μΙ
The first and third lines of the microtiter plate of figure 2 show the complete Smart Ink formulation. Even at the highest enzyme concentrations, no green coloration of the solution is visible even after two days. The slightly yellowish color of the solution is due to the inherent color of the cellulose stock solution and not to the presence of glucose (see second line with only β-glucosidase). In the bottom line a glucose dilution series is shown in order to quantify the sensitivity of the system. In the right hand column there is situated the negative control (water instead of cellulose). No reaction takes place here, neither in the Smart Ink MTP nor in the wood pulp MTP (see figure 2).
The wood pulp MTP (figure 3) shows that the cellulase Onozuka RS was able to hydrolyze the wood pulp very effectively and was able to liberate sufficient glucose for the further
enzymatic reaction steps of the cascade. A first staining began even after 15 minutes (RT, not shown here). After 30 minutes, the degree of staining of the wood pulp pieces was already far advanced (up to 1.6 U/ml Onozuka RS). The β-glucosidase alone could not release glucose from the wood pulp used and thus could not initiate staining by the ABTS by means of the enzyme cascade. The mixture of the Onozuka and β-glucosidase showed no improvement of the system and is therefore not absolutely necessary.
In a parallel experiment, a freshly treated wood pulp MTP in the course of 2-5 minutes at -20°C was frozen. After 5 days, the pulp plate was thawed. The green coloration was clearly visible after approximately 40 minutes at room temperature. This proved the application concept for a freeze indicator.
Example 3: Testing various enzymes which can catalyze the degradation/conversion of polysaccharides to monosaccharides.
Screening of commercially available cellulases was carried out. For this purpose, 40 candidates of interest to the system were studied (inter alia cellulases, cellobiases, amylases, hemicellulases and xylanases - see annex). All enzymes were tested for the presence of glucose in a preliminary experiment. The corresponding Smart Ink solutions were prepared and incubated overnight at RT. This means that 20 μΙ of the stock solution of each candidate were added in 180 μΙ of the reaction solution.
In this somewhat qualitative analysis, 5 enzyme formulations were identified which were absolutely glucose free (see figure 4, circled microtiter plate wells - top). In the case of the remaining 35 formulations, a slight to strong green coloration is displayed which indicates a somewhat unfavorable - for this application - presence of glucose.
Of the five glucose-free enzyme formulations, two gave a positive signal on wood pulp MTP: Novozyme cellulases Novo 342 & Novo 613.
The remaining 35 glucose-comprising formulations were purified with a desalting column (Hi-Trap, from GE - isocratic run, 100 mM Na acetate, pH 5.5) and thereafter tested on a wood pulp MTP. In this manner, 18 enzyme formulations were able to be additionally identified as positive.
Table 3.
No. Name Company Enzyme class
1 BAN 480L Novozym Glucanohydrolase
2 Celluclast 1.5 FG Novozym Cellulase
β-Glucanase,
Cellulase,
CelluFood AL 140 Biopract Xylanase
Cellulase Sigma Cellulase
Denimax 16000L
Dextrozym GA Novozym Gluco-Amylase
Delta
DP10 Ingridients Cellulase
Gluco-Amylase
Fermenzyme L400 Genencore Protease
Glucanase 1XL- Biocatalysts β-Glucanase, G01 1 L (Wales) Xylanase, Cellulase
Glucanase 5XL- Biocatalysts β-Glucanase, G015L (Wales) Xylanase, Cellulase
Glucanase, Protease,
Glucanex 200G Novozymes Chitinase, Cellulase
G-Zyme 480 Ethanol Genencore Gluco-Amylase
Liquozym SC DS Novozym a-Amylase
Multifect CX 2000L Genencor (NL) Cellulase
Novo 188 Novozym Cellobiase
Cellulase
Novo 342 Novozym Hemicellulase
Novo 476 Novozym Cellulase
Novo 50045 Novozym -
Novo 51008 Novozym Cellulase
Novo 51081 Novozym Cellulase
Novo 613 Novozym Endo-Glucanase β-Glucanase
Optimash BG Genencore Xylanase
Cellulase
Optimash XL Genencore Xylanase
Pulpzym HC Novozym Xylanase
Shearzyme 500L Novozym Xylanase
Spezyme Ethyl Genencore a- Amylase
Spezyme Fred Genencore a- Amylase
Spezyme HPA Genencore a- Amylase
29 Spirizym Fuel Novozym Gluco-Amylase
Glucanase
β-Glucanase little laminarinase
30 Submers Erbsloh Cellulase
31 Stargen 001 Genencore Amylase
32 Themamyl SC Novozym a-Amylase
Valley
33 Ultra Thin Research a-Amylase
Glucanase, Xylanase,
34 Viscoflow L Novozymes Cellulase
Cellulase
35 Viscozyme BG Novozym Xylanase
36 Viscozyme L Novozym
37 Viscozyme Wheat Novozym Enzyme mix
Xylanase,
VP 0965/4 ex Pectinase, Cellulase,
38 T.reesii Erbsloh Galoaktomannase
Delta
39 WXL 600 Ingri clients
40 Xylanase BASF Xylanase
Overall, in the abovedescribed experiments, 21 functional and commercially available enzymes were able to be identified. These preliminary experiments clearly show that the corresponding Smart Ink formulations for the peroxidase label are also functional using differing cellulases. For later enzyme combinations, this should offer a starting point for very flexible and inexpensive formulations.
Example 4: Perhydrolase label
In the case of the perhydrolase label , likewise glucose is liberated from wood pulp by cellulases. The glucose oxidase again oxidizes the liberated glucose to D-gluconolactones and H2O2. I n the n ext step acetate - used as a concentrated buffer system - is perhydrolyzed by a perhydrolase using H2O2. The resultant peracetic acid oxidizes potassium bromide in situ to potassium hypobromide, which in turn brominates phenol red to bromophenol blue. This causes a color change from yellow to blue.
A stock solution of 150 g/ml was produced from the perhydrolase. For the perhydrolase experiments, two cellulases were tested. Firstly, the cellulase Onozuka RS, which had
proved to be very effective in the experiments for the peroxidase label and secondly, the glucanase/xylanase mixture Optimash BG (from Genencor), which, after desalting, was likewise active in the wood pulp MTP test. Test experiments showed, however, that only the glucanase/xylanase mixture Optimash BG was functional in this system.
Smart Ink for the perhydrolase label:
1 M Na acetate (pH 5.5)
100 pg/ml Glucose oxidase
450 mM KBr
44 or 88 μΜ Phenol red
+ Optimash BG
+ Perhydrolase from F. johnsoniae
The perhydrolase stock solution (150 pg/ml) was diluted in a microtiter plate 1 :2 from left to right. Of the individual dilutions, in each case 20 μΙ of perhydrolase dilution and 20 μΙ of Optimash BG (total protein concentration 13.3 mg/ml) were added to 180 μΙ of reaction solution. Of the Smart Ink MTP - per dilution - 30 μΙ were added to an MTP with beach sulfate leaflets (= wood pulp MTP; see figure 5).
As soon as after 1 .5 h at room temperature, a clear staining of the wood pulp can be recognized of the two highest concentrations of the perhydrolase. The concentration of phenol red is not critically decisive for the sensitivity of the system (see figure 5). As control, as in the above described experiments, water was added to the reaction solution instead of perhydrolase and Optimash BG. This water control remained yellow (figure 5, far right). As a second control, in this experiment, only 20 μΙ of Optimash BG was added to the reaction solution (without perhydrolase) and of this formulation 30 μΙ was added to the wood pulp MTP. In this case after 48 h a very light blue stain was obtained which is suspected to be due to the presence of the resultant H2O2. Since the time in which the perhydrolase label displays a first stain is less than 2 hours, this side reaction is negligible.
Claims
1. An indicator for temperature monitoring a chilled or frozen product, which indicator comprises the components:
a) at least one enzyme which catalyzes the conversion of a polysaccharide to a monosaccharide,
b) at least one enzyme which catalyzes the oxidation of the monosaccharide formed by means of a),
and
c) a mixture which is suitable for detecting the enzyme activity of a) and/or b) and which comprises a color indicator,
wherein the enzyme activity is detectable only after application to a polysaccharide carrier matrix in the presence of water and in a temperature range in which the product is in the thawed state.
2. The indicator according to claim 1 , wherein the temperature range is between 0 degrees Celsius and 30 degrees Celsius, preferably in the range between 5 degrees Celsius and 25 degrees Celsius, and still more preferably between 10 degrees Celsius and 20 degrees Celsius.
3. The indicator according to claim 1 or 2, wherein the components a), b) and c) are attached to a polysaccharide carrier matrix.
4. The indicator according to any one of claims 1 to 3, wherein the monosaccharide is glucose.
5. The indicator according to any one of claims 1 to 4, wherein the polysaccharide is cellulose.
6. The indicator according to any one of claims 1 to 5, wherein the color indicator comprises a chromogenic substrate for a peroxidase.
7. The indicator according to any one of claims 1 to 5, wherein the color indicator is a chromogenic substrate for a hydrolase, in particular a perhydrolase.
8. A method for producing an indicator for temperature monitoring a chilled or frozen product, which method comprises mixing
a) at least one enzyme which catalyzes the conversion of a polysaccharide to a monosaccharide with
b) at least one enzyme which catalyzes the oxidation of the monosaccharide formed by means of a),
and with c) a mixture which is suitable for detecting the enzyme activity of a) and/or b) and which comprises a color indicator.
The method according to claim 8, wherein the method additionally comprises applying the components a), b) and c) to a polysaccharide carrier matrix.
A method for temperature monitoring a chilled or frozen product, which method comprises
(i) applying an indicator as defined in claims 1 to 7 to the product,
(ii) and monitoring the enzyme activity by means of the color indicator,
wherein positive detection of the enzyme activity is indicative of an interruption of the chilling or freezing of the product.
The method according to claim 10, wherein the application is carried out under conditions under which no enzyme activity is detectable.
12. A package for storing a chilled or frozen product, wherein the package is provided with at least one fixed or removable indicator as defined in claims 1 to 7.
13. The package according to claim 12, wherein the indicator is arranged so as to be externally visible.
14. The package according to claim 12 or 13, wherein the indicator is arranged at least between one outer layer which is translucent to visible and/or UV light, and preferably water-impermeable, and/or one inner layer facing the product and impermeable to the indicator.
15. The use of a package as defined in any one of claims 12 to 14 for storing chilled or frozen products selected from the group consisting of foods, preserved blood, vaccines, medicaments, blood products, implants, diagnostics, eye drops, contact lenses and chemicals.
Applications Claiming Priority (4)
Application Number | Priority Date | Filing Date | Title |
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US26747409P | 2009-12-08 | 2009-12-08 | |
EP09178356.3 | 2009-12-08 | ||
EP09178356A EP2333505A1 (en) | 2009-12-08 | 2009-12-08 | Compound and method for monitoring the temperature of cooled and deep frozen products |
US61/267,474 | 2009-12-08 |
Publications (1)
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WO2011070491A1 true WO2011070491A1 (en) | 2011-06-16 |
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PCT/IB2010/055567 WO2011070491A1 (en) | 2009-12-08 | 2010-12-03 | Composition and method for temperature monitoring chilled and frozen products |
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EP (1) | EP2333505A1 (en) |
WO (1) | WO2011070491A1 (en) |
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DE102017009507A1 (en) * | 2017-10-12 | 2019-04-18 | Giesecke+Devrient Currency Technology Gmbh | Method for monitoring the temperature or time of an object |
Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992005415A1 (en) * | 1990-09-18 | 1992-04-02 | Cambridge Consultants Limited | Time temperature indication |
EP1138777A2 (en) * | 2000-03-23 | 2001-10-04 | Konica Corporation | Temperature history indicator |
EP1607475A1 (en) * | 2003-03-20 | 2005-12-21 | Asahi Kasei Life & Living Corporation | Oxygen indicator and packaged material |
US20090226948A1 (en) * | 2004-08-11 | 2009-09-10 | Hans Reichert | Enzyme-based time temperature indicator |
Family Cites Families (2)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
CA1043676A (en) * | 1974-05-10 | 1978-12-05 | Baxter Travenol Laboratories | Method and composition for confirming the heat sterilization of parenteral solutions and the like by measurement of total heat energy exposure |
US20040265440A1 (en) * | 2002-09-16 | 2004-12-30 | Agcert International, Llc | Food borne pathogen sensor and method |
-
2009
- 2009-12-08 EP EP09178356A patent/EP2333505A1/en not_active Withdrawn
-
2010
- 2010-12-03 WO PCT/IB2010/055567 patent/WO2011070491A1/en active Application Filing
Patent Citations (4)
Publication number | Priority date | Publication date | Assignee | Title |
---|---|---|---|---|
WO1992005415A1 (en) * | 1990-09-18 | 1992-04-02 | Cambridge Consultants Limited | Time temperature indication |
EP1138777A2 (en) * | 2000-03-23 | 2001-10-04 | Konica Corporation | Temperature history indicator |
EP1607475A1 (en) * | 2003-03-20 | 2005-12-21 | Asahi Kasei Life & Living Corporation | Oxygen indicator and packaged material |
US20090226948A1 (en) * | 2004-08-11 | 2009-09-10 | Hans Reichert | Enzyme-based time temperature indicator |
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