* E-mail: buschj@lincoln.ac.nz. Biochemical Education 27 (1999) 171}173 Enzymic browning in potatoes: a simple assay for a polyphenol oxidase catalysed reaction J.M. Busch* Animal and Food Sciences Division, Lincoln University, P.O. Box 84, Canterbury, New Zealand Abstract A simple laboratory procedure is described for demonstrating the enzyme-catalysed reaction in the browning of potato. It requires a minimum of equipment and can be completed in a 3-h lab class.  1999 IUBMB. Published by Elsevier Science Ltd. All rights reserved. 1. Introduction This paper describes a student laboratory procedure designed to show the enzyme-catalysed reaction resulting in the browning of raw potato. Although several methods are available for recording enzymic browning in plants, they generally require the use of toxic chemicals (phenol) or expensive equipment (eg. spectrophotometers and oxygen electrodes) [1,2]. This method allows students to demonstrate enzymic browning in a semi-quantitative way using non-toxic chemicals. It can be completed in a 3-h laboratory class. For two years the basic experiment plus the problem solving exercises have been used as the basis on which food biochemistry students in our institution develop an experimental design to show the extent of enzymic browning in a particular potato cultivar. They have also developed feasible procedures that could be used by food industries to prevent enzymic browning. These students have reported favourably on this application of a simple practical experiment to problem solving in real-life situ- ations in the food industry [3]. 2. Background Many fruits and vegetables turn brown when cut or damaged surfaces are exposed to the air, with the reaction showing most clearly on light-coloured #esh. This browning occurs due to the oxidation and dehyd- rogenation of colourless polyphenols present in the plants. The initial reaction catalysed by polyphenol oxidase and produces reddish-brown o-quinones. These are highly reactive, and so they subsequently undergo a series of non-enzymic reactions [4,5] to yield insoluble black}brown melanin pigments (Fig. 1). Polyphenol oxidase is a very important enzyme for food chemists and processors because its action leads to major economic losses in fresh fruits and vegetables such as potatoes, lettuce and other leafy vegetables, apples, grapes, bananas and many tropical fruits [6]. Up to one-half of some tropical fresh fruits are lost because of browning [7]. Potential purchasers show consumer res- istance to the dark colour of the damaged products, the o!-tastes in juices and vegetables, and the resulting cha- nges in texture. This reaction is, however, exploited in the fermentation of tea leaves, co!ee beans, cocoa and tobacco, and in the colour of dried prunes, dates and raisins [7]. The enzyme is located in the plastids of plant cells and the phenolic substrates are stored in the vacuoles. This physical separation prevents any oxidation of the phen- olics in the undamaged living tissues [3]. The separation can be lost as a result of damage to the cell during harvesting (unintentional) and processing (intentional). Oxidation of the phenolics by polyphenol oxidase (PPO) then begins. The functions of PPOs in higher plants are not fully known but they are thought to play a key part in the plant's defence mechanism against disease causing micro-organisms and insect attack [7]. When microbial 0307-4412/99/$20.00#000  1999 IUBMB. Published by Elsevier Science Ltd. All rights reserved. PII: S 0 3 0 7 - 4 4 1 2 ( 9 9 ) 0 0 0 3 3 - 3 Fig. 1. The chemical reactions involved in browning. infection occurs the cell integrity is broken and the enzymic reaction takes place. An impervious scab of melanin forms and this acts as a physical anti-microbial barrier [3]. The quinones formed during the reaction are known to denature proteins in the invading microorgan- isms and the polymeric phenols complexes can act as inhibitors of microbial growth [3]. 3. Method Four 5 mm slices are cut with a knife, across the short axis of each washed but unpeeled potato (two blanks and two test slices). Slices are placed in individual Petri dishes and 100 l of control bu!er (0.1 M Tris-HCl/0.02%w/v SDS, pH 9.0) is spread over the whole surface of the blank slices using a hockey stick (a right-angled piece of glass rod). Substrate solution (100 l of 0.01 M disodium tyrosine/0.1 M Tris-HCl/0.02%w/v SDS, pH 9.0) is spread over the test slices. Lids are placed on the Petri dishes to minimise evaporation. All slices are incubated at 303C for 1 h before being examined for a black deposit of melanin. If the slices need to be kept for another laboratory session they must be placed in a freezer be- cause the enzyme remains active at low temperatures. The resulting area of black melanins on each slice can be ranked from 1 to 5, with &1' indicating no discolour- ation and &5' representing total discolouration of the slice. Alternatively, the darkening of a representative number of 1 cm  squares of a slice can be calculated using a 1 cm  template overlay. Each square that is completely black is counted and recorded as a percentage of the total num- ber of complete squares covered by the template. This method is adapted from the Speck Test for wheat cul- tivars [8]. A colour meter (e.g. Minolta chroma meter CR-210) can also be used to measure the colour of the slices. 4. Discussion The black colour can occur all over the slice and/or in a ring about 5 mm inside the skin of the potato or in ade"ned semi-circular area out from the skin. In this case it usually re#ects mechanical damage (knocks and bruises) from rough handling since harvest. Some potato slices can show a pink colour instead of black. This pink colour is due to an o-quinone, an intermediate in the complete conversion to melanins. Allowing more time will let the reaction go to completion. The control slices show no colour change. The photograph shows a typical result (Fig. 2). It is important to note that there is con- siderable variation in the results obtained with this test and there are several reasons for this. Studies have shown that both the degree of darkening and the rate of darkening vary considerably from one potato variety to another, depending on the age and maturity of the potato as well as on the availability and levels of substrate [5,6,9]. The e$ciency of treatment to prevent melanin formation can vary according on the nature and concentration of inhibitor, sources of oxygen and substrate availability, pH and temperature [3]. These inherent variations mean that no one treatment is successful all the time so processors have to resort to chemical as well as physical means to prevent enzymic browning, even when using recommended &&non- browning'' commercial varieties of potatoes [10]. In po- tatoes, internal discolouration is of major concern to growers and processors due to the increased labour costs for sorting blemished and damaged tubers and the cost of preventing browning during processing [10]. Scientists in the food industry are currently investigating innovative ways to control or inhibit enzymic browning in foods (eg. gene manipulation for low polyphenol oxidase content of potatoes) [9]. 5. Experiments based on this simple test The types of treatments used can be divided into two broad types, chemical and mechanical. Chemical: Students can design experiments to show the e!ect of using chemical treatments to prevent en- zymic browning by trying any of the following pre-treat- ments: 100 l 4% citric acid, 100 l 4% ascorbic acid,100 l 2% cysteine, 100 l 1% sodium acid pyro- phosphate or 100 l 0.5% sodium bisulphite. The treat- ment chemical is applied to the potato slice and allowed to soak in before the substrate is added. Students need to be aware that any chemicals used need to be non-toxic and have no e!ect on the taste, #avour, texture and wholesomeness of the "nal product [3]. Mechanical: Students can use this procedure to inves- tigate the following questions: Do di!erent potato 172 J.M. Busch / Biochemical Education 27 (1999) 171}173 Fig. 2. Results for a control (left) and test (right) slice of potato. cultivars have browning di!erences? Do commercially harvested potatoes show higher levels of browning than hand harvested potatoes? Does storing potatoes for dif- ferent lengths of time (days or weeks) have an e!ect on the browning? Does storage temperature have an e!ect on browning? Does blanching the potato slices a!ect the browning? Does covering the potato slices with cling- wrap a!ect the level of browning? 6. Case studies (1) Students could investigate which industries are a!ec- ted either positively or negatively by this reaction (e.g. wine, fruit, grain or vegetable, including the pre- peeled potato industry). (2) Students could determine the feasibility of developing a test that could be used for quality control before purchase to ascertain whether plants have been damaged during harvest or transport to the factory. Such a test would need to be economically feasible. (3) Student could search the published literature to review what work has been done to breed plants, either by conventional plant breeding or gene manip- ulation, which have low levels of polyphenol oxidases [9]. 7. Conclusions The laboratory method described here can be used to as the basis for a number of other experiments to increase the students' understanding not only of how enzymes function, but also how this information can be applied practically in the food industry. References [1] C.W. Wrigley, Single-seed identi"cation of wheat varities: use of grain hardness testing, electrophoretic analysis and a rapid test paper for phenol reaction, J. Sci. Food Agric. 27 (1976) 429}432. [2] D.R. Marsh, T. Galliard, Measurement of polyphenol oxidase activity in wheat-milling fractions, J. Cereal Sci. 4 (1986) 241}248. [3] J. Zawistowski, C.G. Biliaderis, N.A.M. Eskin, In D.S. Robinson, N.A.M. Eskin (Editors), Oxidative Enzymes in Food, Elsevier Science, New York, 1991. [4] M A. Rouet-Mayer, J. Ralambosoa, J. Phipippon, Phytochemis- try 29 (1990) 2. [5] J.R. Whitaker, C.Y. Lee, Enzymatic Browning and its Preven- tion, ACS Symposium Series Vol. 600, Washington, (1995) pp. 3}7. [6] J.R. Whitaker, in: O. Fennama (Ed.), Food Chemistry, 3rd ed. Marcel Dekker, New York, 1996, pp. 431}530. [7] J.R.L. Walker, Enzymatic Browning and its Prevention, ACS Symposium Series,Vol. 600, Washington, 1995, pp. 8}22. [8] J.M. Busch, R.L. Hay, S.E. Holst, T.N. Lindley, M.P. Newberry, Proc. RACI Cereal Chemistry Division Conf. Adelaide, Australia, 1995, pp. 303}306. [9] C.W.B. Bachem, G J. Speckman, P.C.G. Van der Linde, F.T.M. Verheggen, M.D. Hunt, J.C. Ste!ens, M. Zabeau, J. Bio/technol. (1994) 1101}1105. [10] I.R. Gubb, J.C. Hughes, M.T. Jackson, J.A. Callow, Ann. Appl. Biol. 114 (1989) 579}586. J.M. Busch / Biochemical Education 27 (1999) 171}173 173 . the increased labour costs for sorting blemished and damaged tubers and the cost of preventing browning during processing [10]. Scientists in the food industry are currently investigating innovative ways. on the browning? Does storage temperature have an e!ect on browning? Does blanching the potato slices a!ect the browning? Does covering the potato slices with cling- wrap a!ect the level of browning? 6 to show the enzyme-catalysed reaction resulting in the browning of raw potato. Although several methods are available for recording enzymic browning in plants, they generally require the use of