GB2328742A

GB2328742A - Test-paper impregnated with the diammonium salt of ABTS for testing blanching of vegetables

Info

Publication number: GB2328742A
Application number: GB9718272A
Authority: GB
Inventors: Sylviane Marie-Louise Reymond; Jennifer Denise Cloke; Carl Erik Granholm Hanson
Original assignee: Societe des Produits Nestle SA; Nestle SA
Current assignee: Societe des Produits Nestle SA; Nestle SA
Priority date: 1997-08-28
Filing date: 1997-08-28
Publication date: 1999-03-03
Also published as: GB9718272D0

Abstract

A test method for determining the degree of blanching of vegetables such as peas, and fruits prior to freezing, by contacting a sample of the vegetable or fruit with a porous test paper, impregnated with 2,2'-azino-di-[3-ethylbenzthiazoline sulfonate (6)] diammonium salt (ABTS) as a chromogenic substrate for peroxidase in the presence of hydrogen peroxide and determining the degree of blanching as a function of the colour change of the chromogenic substrate.

Description

TEST METHOD This 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 otolidine hydrochloride as the chromogenic substrate is available commercially in which a sample of the vegetable product is contacted with a test paper impregnated with otoluidine 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 conveniencs, but the known method based on o-tolidine hydrochloride is unsatisfactory in a number of respects including the high cost of test papers. An 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.

According to one aspect, 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-t3-ethylbenzthiazoline sulfonate (6)) diammonium salt and the test is carried out in the presence of hydrogen peroxide.

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&commat;. The compound is available in the form of crystals which are soluble in water and organic solvents such as methanol. 2,2'-Azino-di-t3- 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-[3ethylbenzthiazoline 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 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 400C 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 occurred 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. 5p1 of this solution each was added to 1.5 x 1.5 cm test papers (for example Schleisher & 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 O Units peroxidase activity/g fr w.

Claims

1. 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.

2. A method according to claim 1 wherein the edible plant product is a vegetable product.

3. A method according to any of claim 2 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.

4. A method according to any of claims 1 to 3 wherein the porous test material is a paper test strip.

5. A method according to any of claims 1 to 4 applied to an edible plant product prior to freezing.

6. A method as claimed in any of claims 1 to 5 wherein hydrogen peroxide is used in the form of a solution in a buffer.

7. A method as claimed in any of claims 1 to 6 wherein an acceptable level of blanching in the edible plant product is determined based on the time taken for perceptible colour to develop.

8. 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.

9. A test material according to claim 8 wherein the porous material is paper.

10. A test material according to claim 8 or 9 wherein the porous material is in the form of a strip.