WO1999011815A1 - Procede de test de la peroxydase - Google Patents

Procede de test de la peroxydase Download PDF

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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
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WO
WIPO (PCT)
Prior art keywords
porous
test
peroxidase
sample
test material
Prior art date
Application number
PCT/EP1998/005498
Other languages
English (en)
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/fr

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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.

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  • 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

L'invention porte sur un procédé de détection de la quantité de peroxydases ou pseudoperoxydases, ou de détermination de leur activité dans un échantillon. Ce procédé consiste à mettre en contact l'échantillon avec un matériau de test poreux imprégné d'un substrat chromogène destiné aux peroxydases ou pseudoperoxydases; à détecter la quantité de peroxydases ou pseudoperoxydases ou déterminer leur activité dans l'échantillon en fonction du changement de couleur du matériau de test poreux en mettant en contact l'échantillon avec un matériau de test poreux, se présentant, par exemple, sous la forme d'un papier de test imprégné d'un sel de 2,2'-azino-di-3[3-éthylbenzothiazoline sulfonate (6)] diammonium comme substrat chromogène des peroxydases en présence de peroxyde d'hydrogène; à détecter la présence de peroxydases ou pseudoperoxydases ou déterminer leur activité en fonction du changement de couleur du substrat chromogène. Ce procédé est de préférence utilisé pour déterminer le degré de blanchiment des légumes tels que les petits pois, et des fruits, par exemple avant leur congélation.
PCT/EP1998/005498 1997-08-28 1998-08-25 Procede de test de la peroxydase WO1999011815A1 (fr)

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 (fr) 1999-03-11

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Family Applications (1)

Application Number Title Priority Date Filing Date
PCT/EP1998/005498 WO1999011815A1 (fr) 1997-08-28 1998-08-25 Procede de test de la peroxydase

Country Status (2)

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

Cited By (1)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CN103073522A (zh) * 2013-01-14 2013-05-01 江西师范大学 2,2'-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐的合成方法

Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1026986A (fr) * 1974-10-24 1978-02-28 General Foods Corporation Methode perfectionnee pour envelopper les epis de mais
US4521511A (en) * 1982-09-22 1985-06-04 Enzyme Technology Company Catalyzed colorimetric and fluorometric substrates for peroxidase enzyme determinations
WO1987007718A1 (fr) * 1986-06-02 1987-12-17 Lawrence Paul J Reactifs de test et procedes permettant de deceler la presence de sang dans les feces
JPH01101898A (ja) * 1987-10-13 1989-04-19 Taunzu:Kk ペルオキシダーゼ活性測定用試薬
EP0475692A1 (fr) * 1990-09-06 1992-03-18 Lifescan, Inc. Bandelette d'essai pour la détermination visuelle de la concentration de glucose dans le sang
GB2250819A (en) * 1990-12-14 1992-06-17 Nat Res Dev Myocardial infarction test
EP0735369A1 (fr) * 1995-03-27 1996-10-02 Lifescan, Inc. Système chimique de mesure du temps pour la lecture directe de bandes de test

Patent Citations (7)

* Cited by examiner, † Cited by third party
Publication number Priority date Publication date Assignee Title
CA1026986A (fr) * 1974-10-24 1978-02-28 General Foods Corporation Methode perfectionnee pour envelopper les epis de mais
US4521511A (en) * 1982-09-22 1985-06-04 Enzyme Technology Company Catalyzed colorimetric and fluorometric substrates for peroxidase enzyme determinations
WO1987007718A1 (fr) * 1986-06-02 1987-12-17 Lawrence Paul J Reactifs de test et procedes permettant de deceler la presence de sang dans les feces
JPH01101898A (ja) * 1987-10-13 1989-04-19 Taunzu:Kk ペルオキシダーゼ活性測定用試薬
EP0475692A1 (fr) * 1990-09-06 1992-03-18 Lifescan, Inc. Bandelette d'essai pour la détermination visuelle de la concentration de glucose dans le sang
GB2250819A (en) * 1990-12-14 1992-06-17 Nat Res Dev Myocardial infarction test
EP0735369A1 (fr) * 1995-03-27 1996-10-02 Lifescan, Inc. Système chimique de mesure du temps pour la lecture directe de bandes de test

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 (zh) * 2013-01-14 2013-05-01 江西师范大学 2,2'-联氮-二(3-乙基-苯并噻唑-6-磺酸)二铵盐的合成方法
CN103073522B (zh) * 2013-01-14 2017-06-06 江西师范大学 2，2’‑联氮‑二(3‑乙基‑苯并噻唑‑6‑磺酸)二铵盐的合成方法

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Publication number Publication date
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WO1999011815A1 (fr) Procede de test de la peroxydase

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