Enzymic browning in potatoes

* 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.. Box 84, Canterbury, Ne

* 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#0 00  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 100ll 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 (100ll 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 discolourdiscolour-ation 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

a de"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: 100ll 4% citric acid, 100 ll 4% ascorbic acid,100ll 2% cysteine, 100 ll 1% sodium acid pyro-phosphate or 100ll 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