[go: up one dir, main page]

WO1999011815A1 - Test method for peroxidase - Google Patents

Test method for peroxidase Download PDF

Info

Publication number
WO1999011815A1
WO1999011815A1 PCT/EP1998/005498 EP9805498W WO9911815A1 WO 1999011815 A1 WO1999011815 A1 WO 1999011815A1 EP 9805498 W EP9805498 W EP 9805498W WO 9911815 A1 WO9911815 A1 WO 9911815A1
Authority
WO
WIPO (PCT)
Prior art keywords
porous
test
peroxidase
sample
test material
Prior art date
Application number
PCT/EP1998/005498
Other languages
French (fr)
Inventor
Jennifer Cloke
Carl Erik Hansen
Sylviane Reymond
Original Assignee
Societe Des Produits Nestle S.A.
Priority date (The priority date is an assumption and is not a legal conclusion. Google has not performed a legal analysis and makes no representation as to the accuracy of the date listed.)
Filing date
Publication date
Priority claimed from GB9718272A external-priority patent/GB2328742A/en
Priority claimed from GBGB9807772.0A external-priority patent/GB9807772D0/en
Application filed by Societe Des Produits Nestle S.A. filed Critical Societe Des Produits Nestle S.A.
Priority to AU94384/98A priority Critical patent/AU9438498A/en
Publication of WO1999011815A1 publication Critical patent/WO1999011815A1/en

Links

Classifications

    • 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
    • C12Q1/26—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase
    • C12Q1/28—Measuring or testing processes involving enzymes, nucleic acids or microorganisms; Compositions therefor; Processes of preparing such compositions involving oxidoreductase involving peroxidase
    • 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
    • C12Q2326/00—Chromogens for determinations of oxidoreductase enzymes
    • C12Q2326/30—2,2'-Azinobis (3-ethylbenzothiazoline-6-sulfonic acid), i.e. ABTS

Definitions

  • This invention relates to a test method for detecting the presence of or .for determining the amount of peroxidase or pseudoperoxidase in a sample. According to one embodiment, the invention relates to a test method for determining the degree of blanching of an edible plant product.
  • the degree of blanching of edible plant products is important to food companies, for example in the production of frozen vegetables.
  • vegetables are under-blanched, residual enzymes can produce off-flavours on storage even after the vegetable has been frozen.
  • over-blanching of a vegetable can have adverse effects on taste and texture.
  • peroxidase catalyses the oxidation of many organic compounds by hydrogen peroxide and in the case of a chromogenic substrate, i.e. where oxidation of the organic compound leads to the development of a colour, this can be used to detect the presence of peroxidase which in turn is related to the degree of blanching.
  • a number of tests of this type have been developed to evaluate blanching with the colour change being evaluated by eye, for example in a spot test, or being measured spectrophotometrically .
  • a test based on o- tolidine hydrochloride as the chromogenic substrate is available commercially in which a sample of the vegetable product is contacted with a test paper impregnated with o- toluidine hydrochloride and a blue colour develops in the presence of residual peroxidase which colour is, in turn, related to the degree of blanching.
  • test paper has considerable advantages in terms of convenience, but the known method based on o-toluidine hydrochloride is unsatisfactory in a number of respects especially as o-toluidine hydrochloride is carcinogenic.
  • One object of the present invention is to provide an improved method for determining the degree of blanching of an edible plant product, for example a vegetable product.
  • pseudoperoxidases are haemoproteins and many true peroxidases are also haemoproteins, the distinction being whether or not the protein can be classified as an enzyme.
  • Some breakdown products of haemoproteins as well as some organic complexes of heavy metal ions also show pseudoperoxidative activity.
  • the presence of pseudoperoxidative activity is a characteristic of blood and the presence of this activity has been used as the basis of a forensic test for the presence of blood.
  • the pseudoperoxidative activity of blood has also been used as the basis of a method for detecting adulteration of meat products with blood and of a method for classifying meat.
  • the present invention provides a method for detecting the presence of, or for determining the amount of, peroxidase or pseudoperoxidase activity in a sample by contacting the sample with a porous test material impregnated with a chromogenic substrate for peroxidase or pseudoperoxidase and detecting the presence of, or determining -the amount of, peroxidase or pseudoperoxidase activity in the sample as a function of the colour change of .
  • the porous test material characterised in that the chromogenic substrate is 2 , 2 ' -azino-di-[3-ethylbenzthiazoline sulfonate (6)] diammonium salt and the test is carried out in the presence of hydrogen peroxide.
  • the present invention provides a test method for determining the degree of blanching of an edible plant product, preferably a vegetable product, by contacting a sample of the edible plant product with a porous test material impregnated with a chromogenic substrate for peroxidase and determining the degree of blanching as a function of the colour change of the porous test material characterised in that the chromogenic substrate is 2,2'- azino-di- [3-ethylbenzthiazoline sulfonate (6)] diammonium salt and the test is carried out in the presence of hydrogen peroxide.
  • the method is a forensic test for blood.
  • the compound 2 , 2 ' -azino-di- [ 3-ethylbenzthiazoline sulfonate (6) ] diammonium salt (CAS number 030931670) is available commercially from a number of suppliers, for example Sigma and Boehringer Mannheim where the compound is referred to under the trade name ABTS®.
  • the compound is available in the form of crystals which are soluble in water and organic solvents such as methanol.
  • the present invention can be applied to any edible plant product which has been blanched, for example prior to freezing.
  • the term "edible plant product” includes whole vegetables or fruits and parts of vegetables and fruits in a form in which they are processed for use as a food, for example cut or diced vegetables, vegetable leaves, tips and spears, cut and diced fruits.
  • the term also includes juice extracted from an edible plant product where the peroxidase content of the juice is related to the degree of blanching of the plant product- from which it has been extracted.
  • vegetables include pea, carrot, bean, soy bean, spinach, onion, pepper, potato, leek, swede, brassicas such as Brussels sprout, cauliflower and broccoli, squash, sweet corn, turnip, mange tout, asparagus and cereals.
  • fruit include tomato and apple.
  • the edible plant material is a vegetable material.
  • the method according to the invention can be carried out using a porous test material, such as a test strip, for example of paper such as filter paper, which has been impregnated with 2 , 2 ' -azino-di- [ 3-ethylbenzthiazoline sulfonate (6)] diammonium salt.
  • a porous test material such as a test strip
  • paper such as filter paper
  • Test strips can be produced on a small scale by immersing the porous material, for example a filter paper in a solution of 2 , 2 ' -azino-di- [ 3-ethylbenzthiazoline sulfonate (6)] diammonium salt in a suitable solvent such as methanol drying the porous material and then cutting it into strips (for example 15mm x 15mm) .
  • a suitable solvent such as methanol
  • the present invention provides a porous test material suitable for testing a sample of an edible plant product, for example a vegetable product, which comprises a porous material, for example paper, preferably in the form of a strip, impregnated with 2 , 2 ' -azino-di- [ 3- ethylbenzthiazoline sulfonate (6) ] diammonium salt.
  • a porous test material suitable for testing a sample of an edible plant product, for example a vegetable product, which comprises a porous material, for example paper, preferably in the form of a strip, impregnated with 2 , 2 ' -azino-di- [ 3- ethylbenzthiazoline sulfonate (6) ] diammonium salt.
  • the method according to the present invention is generally applied to a sample of an edible plant product, such as a vegetable product, prior to freezing.
  • the method can be applied to an already frozen edible product, in which case the method is carried out on a thawed sample of the frozen edible plant product.
  • a sample of the edible plant product is contacted with a porous test material impregnated with 2 , 2 ' -azino-di- [ 3- ethylbenzthiazoline sulfonate (6) ] diammonium salt, preferably in the form of a paper test strip as described above, and the reaction started by adding hydrogen peroxide.
  • the hydrogen peroxide is generally used as a solution in . a suitable buffer, for example 0.5M acetate buffer pH 4.5.
  • the reaction is generally carried out at room temperature and the sample must be left for an appropriate period of time, generally a few seconds to a few minutes, preferably 20 to 60 seconds, to allow the colour to develop.
  • blanching should be carried out to a degree where the product contains little or no residual peroxidase. In other cases, particularly where there is concern that over-blanching may adversely affect taste and texture, some level of residual peroxidase may be desirable.
  • the test will need to be optimised for the edible plant product being tested.
  • the degree of blanching required will need to be correlated to the colour development in the substrate and this can be done by suitable preliminary experiments.
  • the quantity of hydrogen peroxide present is neither limiting nor is it present in an excess which is inhibitory, the time taken for perceptible colour to develop and the extent to which that colour develops will both be related to the amount of peroxidase present in the edible plant product and can be used as the basis for the test.
  • the test is intended to check that blanching has been carried out at least to a required level, i.e. the concern is under-blanching
  • the intensity of the green colour which develops on the test strip can be determined by eye, optionally in comparison with suitable standards, or spectrophotometrically with the result being compared to a standard curve, and the intensity used to determine the degree of blanching.
  • the present invention has the advantage that it provides a semi-quantitative method for estimating the peroxidase content of an edible plant product, such as a vegetable product, by means of which a result can be obtained within a short time of a few seconds to a few minutes, for example 20 to 60 seconds, after contact with the sample. Accordingly, the method is well suited to the control of the processing of edible plant products, for example the freezing of vegetables. Thus once the test has been optimised, it can be used on a routine basis to check that vegetables being processed, for example frozen, are within an acceptable specification in terms of blanching.
  • test strips for example test papers, can be prepared and used in a similar manner.
  • the test can be used to detect the presence of blood in a forensic situation based on the characteristic pseudoperoxidative activity of blood, the ability of the sample to bring about a colour change in the chromogenic reagent being taken as indicative of the presence of blood.
  • Hydrogen peroxide solution was prepared containing 40mM hydrogen peroxide in 0.5M acetate buffer at pH 4.5. Peas were tested after blanching and prior to freezing. A sample pea was removed and squashed. The squashed pea was then pressed against a test paper strip prepared as above, a drop of hydrogen peroxide solution was added and the colour change in the filter paper was observed. Initially the impregnated filter paper was white to pale green but in the presence of peroxidase and hydrogen peroxide a marked change in colour took place and the colour became green to dark green. The level of blanching of the pea was considered acceptable if no perceptible colour development occured in less than about 40 seconds.
  • Juice is extracted from a test Brussels sprout and 1 drop of juice, 1 drop of 500 mM citrate/phosphate buffer, pH 5.0 and 1 drop of 40 mM hydrogen peroxide (see Example 1) were mixed on a test paper as described above.

Landscapes

  • Chemical & Material Sciences (AREA)
  • Organic Chemistry (AREA)
  • Life Sciences & Earth Sciences (AREA)
  • Zoology (AREA)
  • Wood Science & Technology (AREA)
  • Proteomics, Peptides & Aminoacids (AREA)
  • Health & Medical Sciences (AREA)
  • Engineering & Computer Science (AREA)
  • Microbiology (AREA)
  • Biochemistry (AREA)
  • Physics & Mathematics (AREA)
  • Molecular Biology (AREA)
  • Biotechnology (AREA)
  • Biophysics (AREA)
  • Analytical Chemistry (AREA)
  • Immunology (AREA)
  • Bioinformatics & Cheminformatics (AREA)
  • General Engineering & Computer Science (AREA)
  • General Health & Medical Sciences (AREA)
  • Genetics & Genomics (AREA)
  • Measuring Or Testing Involving Enzymes Or Micro-Organisms (AREA)
  • Investigating Or Analysing Biological Materials (AREA)

Abstract

A method for detecting the presence of, or for determining the amount of, peroxidase or pseudoperoxidase activity in a sample by contacting the sample with a porous test material impregnated with a chromogenic substrate for peroxidase or pseudoperoxidase and detecting the presence of, or determining the amount of, peroxidase or pseudoperoxidase activity in the sample as a function of the colour change of the porous test material by contacting the sample with a porous test material, for example in the form of a test paper, impregnated with 2,2'-azino-di-[3-ethylbenzothiazoline sulfonate (6)] diammonium salt as a chromogenic substrate for peroxidase in the presence of hydrogen peroxide and detecting the presence of or determining the amount of peroxidase or pseudoperoxidase activity as a function of the colour change of the chromogenic substrate. Preferably the method is used for determining the degree of blanching of vegetables such as peas, and fruits, for example prior to freezing.

Description

TEST METHOD FOR PEROXIDASE
This invention relates to a test method for detecting the presence of or .for determining the amount of peroxidase or pseudoperoxidase in a sample. According to one embodiment, the invention relates to a test method for determining the degree of blanching of an edible plant product.
The degree of blanching of edible plant products, such as vegetable products, is important to food companies, for example in the production of frozen vegetables. Thus, if vegetables are under-blanched, residual enzymes can produce off-flavours on storage even after the vegetable has been frozen. In addition, in some cases over-blanching of a vegetable can have adverse effects on taste and texture.
The endogenous enzyme peroxidase is present in relatively large quantities in most vegetables and has been used by vegetable processors as a means of determining the degree of blanching of vegetable products. Peroxidase catalyses the oxidation of many organic compounds by hydrogen peroxide and in the case of a chromogenic substrate, i.e. where oxidation of the organic compound leads to the development of a colour, this can be used to detect the presence of peroxidase which in turn is related to the degree of blanching.
A number of tests of this type have been developed to evaluate blanching with the colour change being evaluated by eye, for example in a spot test, or being measured spectrophotometrically . In addition a test based on o- tolidine hydrochloride as the chromogenic substrate is available commercially in which a sample of the vegetable product is contacted with a test paper impregnated with o- toluidine hydrochloride and a blue colour develops in the presence of residual peroxidase which colour is, in turn, related to the degree of blanching. The use of a test paper has considerable advantages in terms of convenience, but the known method based on o-toluidine hydrochloride is unsatisfactory in a number of respects especially as o-toluidine hydrochloride is carcinogenic. One object of the present invention is to provide an improved method for determining the degree of blanching of an edible plant product, for example a vegetable product.
Other proteins which are not enzymes can also catalyse the oxidation of organic compounds by hydrogen peroxide and these proteins can be referred to as pseudoperσxidases . Many pseudoperoxidases are haemoproteins and many true peroxidases are also haemoproteins, the distinction being whether or not the protein can be classified as an enzyme. Some breakdown products of haemoproteins as well as some organic complexes of heavy metal ions also show pseudoperoxidative activity. The presence of pseudoperoxidative activity is a characteristic of blood and the presence of this activity has been used as the basis of a forensic test for the presence of blood. The pseudoperoxidative activity of blood has also been used as the basis of a method for detecting adulteration of meat products with blood and of a method for classifying meat.
According to one aspect, the present invention provides a method for detecting the presence of, or for determining the amount of, peroxidase or pseudoperoxidase activity in a sample by contacting the sample with a porous test material impregnated with a chromogenic substrate for peroxidase or pseudoperoxidase and detecting the presence of, or determining -the amount of, peroxidase or pseudoperoxidase activity in the sample as a function of the colour change of. the porous test material characterised in that the chromogenic substrate is 2 , 2 ' -azino-di-[3-ethylbenzthiazoline sulfonate (6)] diammonium salt and the test is carried out in the presence of hydrogen peroxide. According to one embodiment, the present invention provides a test method for determining the degree of blanching of an edible plant product, preferably a vegetable product, by contacting a sample of the edible plant product with a porous test material impregnated with a chromogenic substrate for peroxidase and determining the degree of blanching as a function of the colour change of the porous test material characterised in that the chromogenic substrate is 2,2'- azino-di- [3-ethylbenzthiazoline sulfonate (6)] diammonium salt and the test is carried out in the presence of hydrogen peroxide.
According to another embodiment, the method is a forensic test for blood.
The compound 2 , 2 ' -azino-di- [ 3-ethylbenzthiazoline sulfonate (6) ] diammonium salt (CAS number 030931670) is available commercially from a number of suppliers, for example Sigma and Boehringer Mannheim where the compound is referred to under the trade name ABTS®. The compound is available in the form of crystals which are soluble in water and organic solvents such as methanol. 2 , 2 ' -Azino-di- [ 3- ethylbenzthiazoline sulfonate (6) ] diammonium salt is green to pale grey green in colour (depending on its source) but develops a deeper green colour on reaction with hydrogen peroxide in the presence of peroxidase.
The present invention can be applied to any edible plant product which has been blanched, for example prior to freezing. As used herein, the term "edible plant product" includes whole vegetables or fruits and parts of vegetables and fruits in a form in which they are processed for use as a food, for example cut or diced vegetables, vegetable leaves, tips and spears, cut and diced fruits. The term also includes juice extracted from an edible plant product where the peroxidase content of the juice is related to the degree of blanching of the plant product- from which it has been extracted. Examples of vegetables include pea, carrot, bean, soy bean, spinach, onion, pepper, potato, leek, swede, brassicas such as Brussels sprout, cauliflower and broccoli, squash, sweet corn, turnip, mange tout, asparagus and cereals. Examples of fruit include tomato and apple. Preferably the edible plant material is a vegetable material.
The method according to the invention can be carried out using a porous test material, such as a test strip, for example of paper such as filter paper, which has been impregnated with 2 , 2 ' -azino-di- [ 3-ethylbenzthiazoline sulfonate (6)] diammonium salt.
Test strips can be produced on a small scale by immersing the porous material, for example a filter paper in a solution of 2 , 2 ' -azino-di- [ 3-ethylbenzthiazoline sulfonate (6)] diammonium salt in a suitable solvent such as methanol drying the porous material and then cutting it into strips (for example 15mm x 15mm) . For larger scale production, conventional methods for the production of impregnated strips of porous material, for example paper, can be used.
According to another aspect, the present invention provides a porous test material suitable for testing a sample of an edible plant product, for example a vegetable product, which comprises a porous material, for example paper, preferably in the form of a strip, impregnated with 2 , 2 ' -azino-di- [ 3- ethylbenzthiazoline sulfonate (6) ] diammonium salt.
The method according to the present invention is generally applied to a sample of an edible plant product, such as a vegetable product, prior to freezing. Alternatively, the method can be applied to an already frozen edible product, in which case the method is carried out on a thawed sample of the frozen edible plant product.
In order to carry out the method according to the invention, a sample of the edible plant product is contacted with a porous test material impregnated with 2 , 2 ' -azino-di- [ 3- ethylbenzthiazoline sulfonate (6) ] diammonium salt, preferably in the form of a paper test strip as described above, and the reaction started by adding hydrogen peroxide. The hydrogen peroxide is generally used as a solution in . a suitable buffer, for example 0.5M acetate buffer pH 4.5. The reaction is generally carried out at room temperature and the sample must be left for an appropriate period of time, generally a few seconds to a few minutes, preferably 20 to 60 seconds, to allow the colour to develop.
The degree of blanching required varies with the edible plant product and the precise nature of the processing to which it is to be subjected. Thus in some cases blanching should be carried out to a degree where the product contains little or no residual peroxidase. In other cases, particularly where there is concern that over-blanching may adversely affect taste and texture, some level of residual peroxidase may be desirable.
In any particular case, the test will need to be optimised for the edible plant product being tested. Thus the degree of blanching required will need to be correlated to the colour development in the substrate and this can be done by suitable preliminary experiments. Provided that the quantity of hydrogen peroxide present is neither limiting nor is it present in an excess which is inhibitory, the time taken for perceptible colour to develop and the extent to which that colour develops will both be related to the amount of peroxidase present in the edible plant product and can be used as the basis for the test.
Where the test is intended to check that blanching has been carried out at least to a required level, i.e. the concern is under-blanching, then it may be most convenient for the test to be based simply on the time-required for perceptible colour to develop in the test strip. Alternatively, the intensity of the green colour which develops on the test strip can be determined by eye, optionally in comparison with suitable standards, or spectrophotometrically with the result being compared to a standard curve, and the intensity used to determine the degree of blanching.
The present invention has the advantage that it provides a semi-quantitative method for estimating the peroxidase content of an edible plant product, such as a vegetable product, by means of which a result can be obtained within a short time of a few seconds to a few minutes, for example 20 to 60 seconds, after contact with the sample. Accordingly, the method is well suited to the control of the processing of edible plant products, for example the freezing of vegetables. Thus once the test has been optimised, it can be used on a routine basis to check that vegetables being processed, for example frozen, are within an acceptable specification in terms of blanching.
The present invention can be applied as a test for blood in a similar manner to that described above for determining the degree of blanching of a vegetable product. In particular, test strips, for example test papers, can be prepared and used in a similar manner.
The test can be used to detect the presence of blood in a forensic situation based on the characteristic pseudoperoxidative activity of blood, the ability of the sample to bring about a colour change in the chromogenic reagent being taken as indicative of the presence of blood.
The invention is illustrated further by the following examples : EXAMPLE 1
(a) Production of Test Papers
200 mg of crystalline 2 , 2 ' -azino-di- [ 3-ethylbenzthiazoline sulfonate (6)] diammonium salt (Sigma) was heated in 10 ml methanol in a closed bottle at 50°C until dissolved. A filter paper was then plunged into the solution at room temperature in a Petri dish, removed, and placed in a dark cupboard at room temperature until dry. The filter paper was then soaked in the solution again and dried in the same manner. When the filter paper was dry, it was cut into 15mm x 15mm strips and the strips were stored in a dark bottle.
(b) Testing of Peas
Hydrogen peroxide solution was prepared containing 40mM hydrogen peroxide in 0.5M acetate buffer at pH 4.5. Peas were tested after blanching and prior to freezing. A sample pea was removed and squashed. The squashed pea was then pressed against a test paper strip prepared as above, a drop of hydrogen peroxide solution was added and the colour change in the filter paper was observed. Initially the impregnated filter paper was white to pale green but in the presence of peroxidase and hydrogen peroxide a marked change in colour took place and the colour became green to dark green. The level of blanching of the pea was considered acceptable if no perceptible colour development occured in less than about 40 seconds.
EXAMPLE 2
(a) Preparation of Test Papers
20 mM 2 , 2 ' -azino-di- [ 3-ethylbenzthiazoline sulfonate (6)] diammonium salt was prepared in 50% aqueous methanol. 5μl of this solution each was added to -1.5 x 1.5 cm test papers (for example Schleicher & Schuell Fast No 300010) and the papers allowed to air dry. The papers can be stored for several months at 4°C in the dark.
(b) Testing of Brussels Sprouts
Procedure (i)
Juice is extracted from a test Brussels sprout and 1 drop of juice, 1 drop of 500 mM citrate/phosphate buffer, pH 5.0 and 1 drop of 40 mM hydrogen peroxide (see Example 1) were mixed on a test paper as described above.
Procedure (ii)
1 drop of 100 mM citrate/phosphate buffer, pH 5.0 and 1 drop of 40 mM hydrogen peroxide (see Example 1) were mixed on a test paper as described above and a slice of a test Brussels sprout placed on top.
In both cases the intensity of the green colour developed on the test paper was recorded and compared to a standard curve produced with a known available peroxidase. The method was found to be capable of distinguishing Brussels sprouts with different blanching times and containing 80, 40, 13, 6 and 0 Units peroxidase activity/g fr w.

Claims

1. A method for detecting the presence of, or for determining the amount of, peroxidase or pseudoperoxidase activity in a sample by contacting the sample with a porous test material impregnated with a chromogenic substrate for peroxidase or pseudoperoxidase and detecting the presence of, or determining the amount of, peroxidase or pseudoperoxidase activity in the sample as a function of the colour change of the porous test material characterised in that the chromogenic substrate is 2 , 2 ' -azino-di- [3-ethylbenzthiazoline sulfonate (6) ] diammonium salt and the test is carried out in the presence of hydrogen peroxide.
2. A test method for determining the degree of blanching of an edible plant product by contacting a sample of the edible plant product with a porous test material impregnated with a chromogenic substrate for peroxidase and determining the degree of blanching as a function of the colour change of the porous test material characterised in that the chromogenic substrate is 2 , 2 '-azino-di- [3-ethylbenzthiazoline sulfonate (6)] diammonium salt and the test is carried out in the presence of hydrogen peroxide.
3. A method according to claim 2 wherein the edible plant product is a vegetable product.
4. A method according to any of claim 3 wherein the vegetable product is pea, carrot, bean, soy bean, spinach, onion, pepper, potato, leek, swede, Brussels sprout, cauliflower broccoli, squash, sweet corn, turnip, mange tout, asparagus or a cereal.
5. A method according to any of claims 2 to 4 applied to an edible plant product prior to freezing.
6. A method as claimed in any of -claims 2 to 5 wherein an acceptable level of blanching in the edible plant product is determined based on the time taken for perceptible colour to develop.
7. A method according to claim 1 for the detection of blood.
8. A method according to any of claims 1 to 7 wherein the porous test material is a paper test strip.
9. A method as claimed in any of claims 1 to 8 wherein hydrogen peroxide is used in the form of a solution in a buffer.
10. A porous test material suitable for testing a sample of an edible plant product, which comprises a porous material impregnated with 2 , 2 ' -azino-di- [ 3-ethylbenzthiazoline sulfonate (6) ] diammonium salt.
11. A porous test material suitable for testing a sample for the presence of blood, which comprises a porous material impregnated with 2 , 2 ' -azino-di- [ 3-ethylbenzthiazoline sulfonate (6)] diammonium salt.
12. A test material according to claim 10 or 11 wherein the porous material is paper.
13. A test material according to any of claims 10 to 12 wherein the porous material is in the form of a strip.
PCT/EP1998/005498 1997-08-28 1998-08-25 Test method for peroxidase WO1999011815A1 (en)

Priority Applications (1)

Application Number Priority Date Filing Date Title
AU94384/98A AU9438498A (en) 1997-08-28 1998-08-25 Test method for peroxidase

Applications Claiming Priority (4)

Application Number Priority Date Filing Date Title
GB9718272A GB2328742A (en) 1997-08-28 1997-08-28 Test-paper impregnated with the diammonium salt of ABTS for testing blanching of vegetables
GB9718272.9 1997-08-28
GB9807772.0 1998-04-09
GBGB9807772.0A GB9807772D0 (en) 1998-04-09 1998-04-09 Test method

Publications (1)

Publication Number Publication Date
WO1999011815A1 true WO1999011815A1 (en) 1999-03-11

Family

ID=26312142

Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1998/005498 WO1999011815A1 (en) 1997-08-28 1998-08-25 Test method for peroxidase

Country Status (2)

Country Link
AU (1) AU9438498A (en)
WO (1) WO1999011815A1 (en)

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103073522A (en) * 2013-01-14 2013-05-01 江西师范大学 Synthesis method of 2,2' -biazoyl-di (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1026986A (en) * 1974-10-24 1978-02-28 General Foods Corporation Process for improved enwrapment of cob corn
US4521511A (en) * 1982-09-22 1985-06-04 Enzyme Technology Company Catalyzed colorimetric and fluorometric substrates for peroxidase enzyme determinations
WO1987007718A1 (en) * 1986-06-02 1987-12-17 Lawrence Paul J Fecal occult blood test reagents and methods
JPH01101898A (en) * 1987-10-13 1989-04-19 Taunzu:Kk Reagent for measuring peroxidase activity
EP0475692A1 (en) * 1990-09-06 1992-03-18 Lifescan, Inc. Visual blood glucose concentration test strip
GB2250819A (en) * 1990-12-14 1992-06-17 Nat Res Dev Myocardial infarction test
EP0735369A1 (en) * 1995-03-27 1996-10-02 Lifescan, Inc. Chemical timer for a direct-reading reagent test strip

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1026986A (en) * 1974-10-24 1978-02-28 General Foods Corporation Process for improved enwrapment of cob corn
US4521511A (en) * 1982-09-22 1985-06-04 Enzyme Technology Company Catalyzed colorimetric and fluorometric substrates for peroxidase enzyme determinations
WO1987007718A1 (en) * 1986-06-02 1987-12-17 Lawrence Paul J Fecal occult blood test reagents and methods
JPH01101898A (en) * 1987-10-13 1989-04-19 Taunzu:Kk Reagent for measuring peroxidase activity
EP0475692A1 (en) * 1990-09-06 1992-03-18 Lifescan, Inc. Visual blood glucose concentration test strip
GB2250819A (en) * 1990-12-14 1992-06-17 Nat Res Dev Myocardial infarction test
EP0735369A1 (en) * 1995-03-27 1996-10-02 Lifescan, Inc. Chemical timer for a direct-reading reagent test strip

Non-Patent Citations (4)

* Cited by examiner, † Cited by third party
Title
DATABASE WPI Section Ch Week 7811, Derwent World Patents Index; Class A35, AN 78-19741A, XP002085016 *
K. A. HOLM: "Automated determination of microbial peroxidase activity in fermentation samples using hydrogen peroxide as the substrate and 2,2'-azino-bis(3-ethylbenzothiazoline-6-sulfonate) as the electron donor in a flow injection system.", ANALYST, vol. 120, no. 8, August 1995 (1995-08-01), cambridge, uk, pages 2101 - 2105, XP002084293 *
O. L. HÖRER: "Algorithms for automated ultramicroanalysis by bichromatic paper disk spectrophotometry.", ROMANIAN JOURNAL OF MEDICINE - VIROLOGY, vol. 36, no. 3, July 1985 (1985-07-01), pages 165 - 173, XP002084294 *
PATENT ABSTRACTS OF JAPAN vol. 013, no. 315 (C - 619) 18 July 1989 (1989-07-18) *

Cited By (2)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103073522A (en) * 2013-01-14 2013-05-01 江西师范大学 Synthesis method of 2,2' -biazoyl-di (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt
CN103073522B (en) * 2013-01-14 2017-06-06 江西师范大学 Synthesis method of 2,2' -biazoyl-di (3-ethyl-benzothiazole-6-sulfonic acid) diammonium salt

Also Published As

Publication number Publication date
AU9438498A (en) 1999-03-22

Similar Documents

Publication Publication Date Title
Baldwin et al. Improving storage life of cut apple and potato with edible coating
Concellón et al. Characterization and changes in polyphenol oxidase from eggplant fruit (Solanum melongena L.) during storage at low temperature
Krokida et al. Effect of osmotic dehydration on color and sorption characteristics of apple and banana
Rocha et al. Characterization of polyphenoloxidase (PPO) extracted from ‘Jonagored’apple
Krokida et al. Effect of pretreatment on color of dehydrated products
Rocha et al. Polyphenoloxidase activity and total phenolic content as related to browning of minimally processed ‘Jonagored’apple
Rocha et al. Shelf life of minimally processed apple (cv. Jonagored) determined by colour changes
Benkeblia Phenylalanine ammonia-lyase, peroxidase, pyruvic acid and total phenolics variations in onion bulbs during long-term storage
Rao et al. Kinetics of thermal softening of foods–a review
Soares et al. Reduction of internal browning of pineapple fruit (Ananas comusus L.) by preharvest soil application of potassium
Walter Jr et al. Effect of substrate levels and polyphenol oxidase activity on darkening in sweet potato cultivars
Soliva‐Fortuny et al. Browning, polyphenol oxidase activity and headspace gas composition during storage of minimally processed pears using modified atmosphere packaging
Cabezas-Serrano et al. Effect of solution pH of cysteine-based pre-treatments to prevent browning of fresh-cut artichokes
US4105800A (en) Immobilized enzyme method to assess fish quality
González-Buesa et al. Suitability for minimal processing of non-melting clingstone peaches
Surya et al. Rapid methods for histamine detection in fishery products
Kamdee et al. Effect of heat treatment on ripening and early peel spotting in cv. Sucrier banana
Buta et al. Browning Inhibition of Fresh-cutAnjou',Bartlett', andBosc'Pears
Singh et al. Ways of measuring shelf-life and spoilage
Reeve Preliminary histological observation on internal blackspot in potatoes
Haque et al. High carbon-di-oxide modified atmospheric packaging on quality of ready-to-eat minimally processed fresh-cut iceberg lettuce
US8007852B2 (en) Method for production of frozen vegetables or fruits
Stewart et al. Blackheart development following chilling in fruit of susceptible and resistant pineapple cultivars
Velasco et al. Enzymes Responsible for Off‐Flavor and Off‐Aroma in Blanched and Frozen‐Stored Vegetables 1
Lemoine et al. Influence of a combined hot air and UV-C treatment on quality parameters of fresh-cut broccoli florets at 0 C

Legal Events

Date Code Title Description
AK Designated states

Kind code of ref document: A1

Designated state(s): AL AM AT AU AZ BA BB BG BR BY CA CH CN CU CZ DE DK EE ES FI GB GE GH HU IL IS JP KE KG KP KR KZ LC LK LR LS LT LU LV MD MG MK MN MW MX NO NZ PL PT RO RU SD SE SG SI SK SL TJ TM TR TT UA UG US UZ VN YU ZW

AL Designated countries for regional patents

Kind code of ref document: A1

Designated state(s): GH GM KE LS MW SD SZ UG ZW AM AZ BY KG KZ MD RU TJ TM AT BE CH CY DE DK ES FI FR GB GR IE IT LU MC NL PT SE BF BJ CF CG CI CM GA GN GW ML MR NE SN TD TG

121 Ep: the epo has been informed by wipo that ep was designated in this application
DFPE Request for preliminary examination filed prior to expiration of 19th month from priority date (pct application filed before 20040101)
NENP Non-entry into the national phase

Ref country code: KR

REG Reference to national code

Ref country code: DE

Ref legal event code: 8642

122 Ep: pct application non-entry in european phase
NENP Non-entry into the national phase

Ref country code: CA