TEST FOR CROP DAMAGE

Abstract: The invention relates to a test method for crop damage which comprises the steps of providing a sample of the crop to be tested, removing the skin of the crop, and assaying the presence or absence of a crop cell damage indicator which is preferably the activity of an enzyme.

Description: TEST FOR CROP DAMAGE The present invention relates to a test for detecting crop damage and in particular to a test for damage to root vegetables, especially potatoes. The invention will be described using potatoes as an exemplary crop. Bruising is a natural response to cell damage by impact. Such impact damage usually takes place when the potatoes are lifted from the ground during harvesting, or when the potatoes are transported and/or processed. Processing of potatoes to make a variety of products such as crisps and shaped foods for oven cooking has increased rapidly in recent years. There is therefore a need for high quality ie. undamaged crops for use in such processing. It has been estimated that 30 to 50 million pounds sterling worth of potatoes are discarded as a result of damage by bruising each year in the United Kingdom alone. A considerable problem with damage to potatoes by bruising is that the damage is not normally revealed until after the potato skin (periderm) has been removed, for example by peeling. Until now, only laborious tests for potato damage by bruising have been developed. A common test involves placing potatoes at around 25 °C for 18 to 24 hours to stimulate bruise development. A delay of 24 hours for test results is unacceptable in large scale processing facilities. Hence, the conventional apparatus is not only expensive but also slow. The present invention aims to overcome the above and other problems of conventional crop damage test methods. According to the invention there is provided a test method for detecting crop damage which comprises the steps of providing a sample of the crop to be tested, removing the skin of the crop, and assaying the presence or absence of a crop cell damage indicator. By "assaying" we mean some form of analytical intervention so that the term does not cover known methods consisting only of visual inspection of natural bruise development at a given temperature. The term "damage indicator" is intended to include substances released from damaged cells and other physiological changes associated with cell damage, for example enzyme activities, oxygen uptake, utilisation of phenolic substrates in the cell vacuole etc. Preferably, the damage indicator is an enzyme, especially an oxidising enzyme, the activity of which can be assayed to indicate the presence or absence of damage to the crop. The most preferred enzyme for use in the invention as a damage indicator is the enzyme responsible for the initial browning reaction in damaged plant materials, henceforth referred to as polyphenoloxidase (PPOase). However, the same enzyme is also referred to as phenolase, catecholase, o-diphenol oxidase or tyrosinase depending on its phenolic substrate. (See Walker J.R.L. , & Ferrar P.H., Chemistry & Industry, 836-839, 16 October 1995; the disclosure of which is incorporated herein by reference.) Undamaged fruit and vegetables do not brown because, although both the enzyme and its substrates are present, they are separated by membranes within the cell, oxygen tensions are low and so the reaction does not take place in the intact cell. However, as soon as the cell's internal organisation is disrupted the enzyme and substrates interact to give reactive quinone compounds. These quinones combine with proteins and amino acids resulting in complex black/brown "melanin" pigments which are seen as a bruised area of the crop. 0 2 , PPOase Reducing agents σ-Dipheno! o-Quinone Melanin Although an oxygen electrode provides a very sensitive and rapid means of assaying PPOase activity, colourimetric methods are preferred for their ease of use in the hands of unskilled persons. In a preferred embodiment of the invention die crop is tested for the presence of PPOase by applying a substrate of the enzyme which is oxidised to produce a coloured product. Hence, in this embodiment crop damage is revealed as a coloured area of the crop. Preferably, the organic substrate is a phenolic compound. Preferred phenolic compounds for use in the method of the invention include diphenols. Most preferred phenolic compounds include one or a combination of two or more of 4-methyl catechol, catechol, chlorogenic acid, dopamine, L-DOPA (3,4 dihydroxy phenylalanine), pyrogallol, catechin and caffeic acid. It will be appreciated that factors affecting the choice of phenolic substrate will include its availability and price and the speed of its reaction with the enzyme to produce a detectable product. Clearly, the quicker the reaction the quicker the test results. The optimum concentration of a particular substrate for the enzyme at a given temperature can be readily determined using standard optimisation experiments ie. assay enzyme activity at a range of substrate concentrations and plot a graph to show the concentration which gives the highest activity. For example, in a preferred embodiment of the invention the presence of the damage indicator PPOase in peeled potatoes can be detected by a colour change using the phenolic substrate 4-methyl catechol in less than twenty minutes. This is a vast improvement over conventional test methods which take from 18 to 24 hours to complete. Preferably, the test method further comprises applying an enzyme inhibitor to the crop in an amount effective to inhibit the development of background colouration during the course of the test due to minor damage during crop skin removal (referred to as "cut-damage" hereinafter). Such cut-damage produces less cell damage compared to that produced by bruising (impact-damage). Applying an enzyme inhibitor to slow PPOase activity as described above, prevents cut-damage from obscuring areas of impact-damage. Hence, the application of an enzyme inhibitor makes the test results clearer. The amount of enzyme inhibitor effective to inhibit the development of colouration due to cut-damage can be readily redetermined by carrying out a series of tests in which the inhibitor is applied at different concentrations to a peeled crop sample. The effective amount referred to above is the amount which inhibits colouration due to cut-damage, whilst allowing colouration due to impact-damage to develop. As reviewed by Walker & Ferrar (1995) ibid. , many reagents inhibit PPOase activity and can be grouped according to their mode of action. This may be by chelating the Cu-prosthetic group, competing with the substrate or interacting with the reaction products. For example, as little as lOμM salicylhydroxamic acid (SHAM) completely and specifically inhibits apple and potato PPOases. (Allan, A.C. , & Walker, J.R.L., Phytochem. , 1988, 27, 3075-76.) Brine dips can also be used, as the chloride ion is a competitive inhibitor of PPOase and brine solutions are cheap. Acidulents are particularly preferred, as they lower the product's pH and, hence decrease PPOase activity. Citric acid is a preferred acidulent. The enzyme catalysed reaction which leads to browning is reversible and as it is an oxidation reaction, can be reversed with a reducing agent. A preferred reducing agent is ascorbic acid (vitamin C). Sulphite compounds can also be used to inhibit PPOase. These inhibit browning by a variety of mechanisms, including complexation with quinones to form colourless sulpho-quinones such as sodium metabisulphite. The sulphur amino acids, such as cysteine can be used as PPOase inhibitors. According to Walker & Ferrar, ibid, 1995, recently, a derivative (N-acetyl-L-cysteine) has been reported to be as effective as sulphites at inhibiting browning in juices, apples and potatoes. (Friedman, M., & Bautista, F.F. , J. Agric. Food Chem., 1995, 43, 69-76.) The naturally produced tripeptide glutathione (γ-L-glutamyl-L-cysteinyl- glycine), which acts as a natural browning inhibitor in grapes, (Macheix, J.-J., Sapis, J.-C , & Fleuriet, A., Crit. Rev. Food Sci. Nutr., 1991 , 30, 441-86) is also effective. Cinnamic acids are also PPOase inhibitors and their inhibitory action stems from their close structural similarity to the enzyme's natural phenolic substrates. Cinnamic acids and their derivatives and homologues occur naturally in most plant foods. In addition to the cinnamic acids, many aromatic and aliphatic carboxylic acids inhibit PPOase. Potentially useful commercial inhibitors include sorbic, crotonic and kojic acids. (Chen, J.S. , Wei, C.-L , & Marshall, M.R. , J. Agric. Food Chem. , 1991, 39, 1897-1900.) Walker & Ferrar, ibid, 1995, also mention mat the well-established role of ascorbic acid as a short-term anti-browning treatment has led to efforts to increase its effectiveness by derivatisation. (Sapers, G.M. , et al, J. Food Sci., 1989, 54, 997-1002.) For example, fatty acid esters of ascorbic acid are effective in inhibiting browning in apple juice. Similarly di- and tri-phosphate derivatives have been investigated and found to be ineffective in juice, but effective on cut surfaces of apples where it is assumed that the phosphate group is hydrolysed by acid phosphatases. In a comparative study of ascorbic acid against dehydroascorbic acid, iso- ascorbic acid (erythorbic acid), ascorbic acid-2-phosphate and ascorbic acid-2-sulphate, none were as effective as ascrobic acid in inhibiting a phenolase bioassay, (Hsu, A.F. et al, ibid, 1988, 53, 765-71) although erythorbic acid proved effective in certain model systems. (Sapers, G.M. , et al, J. Food Sci., 1989, 54, 997-1002; and Sapers, G.M. , & Ziolkowski, M.A. , ibid, 1987, 52, 1732-3.) Exemplary PPOase inhibitors from Walker & Ferrar, ibid, 1995: Group I, Reducing agents Ascorbic acid, S0 2 , sodium metabisulphate, thiogly collate, mercaptoethanol , 2-mercaptobenzothiazole . Group II, Copper-chelating agents Sodium diethyldithiocarbamate (DIECA), carbon monoxide, cyanide, tropolone. Group III, Quinone couplers Cysteine, glutathione, penicillamine, benzenesulphinic acid, S0 2 , sodium metabisulphite. Group IV, Substrate analogues Cinnamic acid, p-coumaric acid, ferulic acid. Group VI, Other inhibitors Salicylhydroxamic acid (SHAM), 4-hexyl-resorcinol, kojic acid, sorbic acid, PVP and PNPP, cyclodextrins, halides. Other enzyme activities which can be used as damage indicators in the method of me invention include those of superoxide dismutase (SOD) and catalase. When oxygen gains entry into biological systems it is suffϊciendy reactive to form active oxygen species, such as singlet oxygen and the superoxide anion, which are potentially toxic to cells due to their ability to react with unsaturated lipids in membranes. These radicals are quenched in vivo by anti-oxidants and enzymes, including SOD and catalase. Activities of these enzymes increase as a result of impact damage. They can both be assayed in a de-salted cell-free potato extract spectrophotometrically. SOD generates hydrogen peroxide from superoxide radicals and the assay is based on colour formation at 595nm according to the amount of superoxide present. Hence, colour formation is lessened in the presence of the enzyme in this competitive assay. Catalase breaks down the hydrogen peroxide formed by SOD and the rate of enzyme activity can be followed by recording the change in absorbance at 240nm. Both enzymes thus measure a response to oxidative stress. Since oxidative stress can be caused by factors other than impact-damage, catalase and SOD activity is a less specific and therefore less preferred indicator of cell damage than PPOase activity. Oxygen uptake can also be used as an indicator of cell damage since bruising reactions consume oxygen. Bioluminescence can be used to measure oxygen uptake very accurately. This very sensitive process is based on die luciferin/luciferase oxidation, in which the substrate luciferin is oxidised by the enzyme luciferase in the presence of ATP and the amount of light produced is proportional to the concentration of oxygen present. However, mis is an extremely sensitive biological indicator of oxygen and can detect respiration, and so is unlikely to be specific to bruising reactions. In other words, oxygen uptake is not a very specific indicator of cell damage for bruising. Whilst the above indicators of cell damage can be used according to the invention, we have found that the most specific and therefore the best indicator of bruising is PPOase activity. In a second aspect die invention relates to the use of a phenolic compound as a substrate for an enzymic damage indicator in a test method according to die invention. In a third aspect, the invention relates to the use of an enzyme inhibitor in a test method according to the invention. In a fourth aspect the invention relates to the use of a phenolic compound and/or an enzyme inhibitor in the manufacture of a kit for carrying out a test method according to the invention. In a fifth aspect, there is provided a kit which is preferably portable, comprising a container housing means for carrying out the method of the invention as described previously. Preferably, reagents for carrying out the method of the invention are provided in the form of a kit comprising one or more receptacles containing me reagents, preferably a phenolic compound as a substrate for an enzymatic damage indicator and optionally one or more receptacles containing an enzyme inhibitor, preferably an acidulent. Preferred embodiments of the invention will now be described to provide non-limiting examples, with reference to die accompanying Figure 1 which is a schematic showing use of a preferred kit of the invention. Example 1 - Rapid test for bruising in potato tubers (5. tuberosum) Cultivar selection Maincrop cultivars employed in the test were Cara, Maris Piper, Romano and Pentland Dell. These are representative of general purpose potato tubers. Record is from the same seasonal group, although this cultivar is intended for crisp manufacturing. In order to represent the seasonal group, second earlies, the cultivar Estima was utilised. Pre-treatment Pre-treatment involved storage or chilling of the tubers at 10°C and 5°C, respectively. This 24 hour conditioning process standardised the tubers prior to impact. The extent of internal damage was greater when tuber temperature was low. Mechanical injury Morphological differences between the stem and bud ends of the tuber give rise to differences in susceptibility to bruising. The stem end is known to bruise more easily on impact because cells found in the stem end are usually larger and have thicker cell walls and are therefore developmentally older than die bud end. To induce tissue damage at me stem end a 235g ram bolt was released from a height of 300mm. On impact the tuber received 0.7 joules of energy. The rounded end of the bolt was coated with ink to mark the exact area of impact. 30 minute interim The mbers were left at room temperature for 30 minutes. This period was intended to simulate the approximate time between harvesting the potato tubers and employment of the rapid detection test for bruising. Enzyme inhibitor (acidulent) treatment: Immersion in 2% citric acid (w/v) A blackspot due to bruising is usually located below the periderm (skin) and extends 5- 10mm in diameter. Therefore, when the stem end was removed, the slice did not exceed 5mm iickness. This was to ensure that the cut surface had the exact area of impact-damaged cells exposed. Subsequently, the cut surface was immersed in 2% citric acid (w/v) for 15 minutes. It was found during the optimisation of 1 % ascorbic acid (w/v) and 2% citric acid (w/v) antioxidant diat ascorbic acid was not an essential component of the mixture. The optimum citric acid concentration was 2% and this alone slowed oxidation in die cut-damaged cells and allowed oxidation to occur in the impact-damaged cells. Treatment with PPOase (impact-damage indicator) substrate: Immersion in lOmM 4-Methylcatechol buffered to pH 5.0 The cut surface was immersed in lOmM 4-methyl catechol for 2 minutes. An initial polargraphic investigation into the enzymology of PPOase was conducted using Clark-Type oxygen electrodes to provide a mediod for identifying phenolic substrates which are useful in the test mediod of the invention. Di- and trihydroxy phenolic compounds were screened and compared to the tuber's naturally occurring substrates, tyrosine and chlorogenic acid. 4-medιyl catechol was oxidised at 152% of die rate for chlorogenic acid. Optimisation experiments concluded that 4-methyl catechol had the highest specific activity at 25-30 °C, at lOmM concentration and at pH 5.0. Colour development The tubers were left at room temperature and colour development was recorded after 10 and 20 minutes. The rate at which colouration appears is cultivar dependent, but die times given are long enough for any potato cultivar so far examined. Example la - Selection of phenolic substrates DOPA Stem-end samples of potato tubers are homogenised in a buffer eg. lOOmM MES at pH 6.0 and desalted by passage tlirough Sephadex PD10™. The extract is used as a crude enzyme preparation in a polargraphic enzyme assay performed using a Clark-type electrode. Enzyme activity is expressed as micromoles of oxygen taken up per minute per milligram of protein. The namral substrate for PPOase in potatoes is tyrosine, whilst the most abundant phenol is chlorogenic acid. The former is present in about ten times the level of the latter, but is a much slower reactant in the PPOase activity assay. It is preferred that the phenolic substrate produces an activity of at least 20% and preferably more than 50% , especially 70% or more, than that produced by chlorogenic acid. Table 1 results of polarographic tests with a range of phenolic compounds as potential PPOase substrates Skilled persons will appreciate that a combination of two or more phenolic substrates and/or two or more enzyme inhibitors might increase the speed and/or sensitivity of the test for a particular crop type. Example 2: Maincrop cultivars Estima, Marfona and Mar is Piper were chosen because Estima bruises readily, whilst Marfona bruises less and Maris Piper bruises least. The test procedures of the invention were optimised for each cultivator as follows: Immerse five washed and lightly peeled tubers in 5 % aq, citric acid pH 5.0 for 17 minutes (Marfona) or 20 minutes (Estima). For Maris Piper immersion was optimised at 17 minutes in 4% citric acid. Transfer tubers to a 10 mM aq. solution of 4 methyl catechol buffered to pH 5.0 for 1.5 minutes. Gently dry the mbers eg with paper towels and set aside in a well ventilated area at room temperature. Note colour development after 30 and 60 minutes as purple/pink dopachrome formation reveals bruised areas. The chemicals were used in 1 litre volumes and each batch remained functional after approximately 100 tubers had been tested at room temperature. Colour development was most apparent when the tuber temperature was 15°C or below. The test was less effective with tubers pretreated to room temperature (23.5 °C). It will be appreciated that optimisation of the methods of the invention for particular crops is within the skill of a worker in this field of technology. Example 3: kit for carrying out the method The kit preferably comprises a colander (1), ie. a pan with a perforated bottom for sfraining or rinsing samples, which is dimensioned to fit into a container for a phenolic substrate (S) of an enzymic damage indicator. Advantageously, the kit further comprises a container for an enzyme inhibitor (I). Use of the kit is extremely simple. A sample (2) of the crop to be tested is peeled and placed in the colander (1) which is immersed in an aqueous solution of the phenolic substrate (S) for two minutes or so. Bruising is detected as a black/brown area of the crop. In the preferred embodiment shown in Figure 1 , the colander (1) containing the peeled crop sample (2) is first immersed in a solution of an enzyme inhibitor (I) for a suitable period of time eg. 15 minutes.

Claims: CLAIMS 1. A test method for detecting crop damage which comprises the steps of providing a sample of the crop to be tested, removing the ' skin of the crop, and assaying the presence or absence of a crop cell damage indicator. 2. A method as claimed in Claim 1 , wherein the damage indicator is an oxidising enzyme activity. 3. A method as claimed in Claim 2, wherein the enzyme is a polyphenoloxidase . 4. A method as claimed in Claim 2 or 3, wherein activity of the enzyme on a substrate leads to formation of a coloured product. 5. A method as claimed in Claim 3 or 4, wherein the substrate is a phenolic compound selected from one or more of 4-methyl catechol, catechol, chlorogenic acid, dopamine, L-DOPA, pyrogallol, catechin and caffeic acid. 6. A method as claimed in Claim 5, wherein the phenolic compound is 4-methyl catechol and/or catechol. 7. A method as claimed in any one of Claims 2 to 6, wherein the method further comprises the step of applying an amount of an enzyme inhibitor effective to show enzyme activity in cut-damaged, but not impact-damaged cells. 8. A method as claimed in Claim 7, wherein the inhibitor is selected from one or more of brine, acidulents and reducing agents. 9. A method as claimed in Claim 8, wherein the acidulent is citric acid. 10. A method as claimed in Claim 8, wherein the reducing agent is ascorbic acid. 11. A method as claimed in any preceding claim, wherein the crop is a root vegetable. 12. A method as claimed in Claim 11 , wherein the root vegetable is a potato. 13. Use of a phenolic compound as a substrate for an enzymic damage indicator in a test method as claimed in any preceding claim. 14. Use of an enzyme inhibitor in a test method as claimed in any preceding claim. 15. Use of a phenolic compound and/or an enzyme inhibitor in die manufacture of a kit for carrying out a test method as claimed in any one of Claims 1 to 12. 16. A kit for carrying out a test method as claimed in any one of the preceeding claims comprising a container, which is preferably portable, housing means for assaying the presence of a damage indicator in a sample of a peeled crop. 17. A test method for detecting crop damage substantially as described herein with reference to one or more of the accompanying examples. 18. A test kit substantially as described herein with reference to one or more of the accompanying examples.

Inventors: COBB ANDREW HOWARD, COBB, ANDREW, HOWARD
Applicants: COBB ANDREW HOWARD, UNIV NOTTINGHAM TRENT, COBB, ANDREW, HOWARD, NOTTINGHAM TRENT UNIVERSITY
Application year: 1998
Main classification: C12Q1
IPC codes: C12Q1