21 minutes reading time (4204 words)

    Why Does Your Apple Go Brown?

     Part 1: The Problem of Enzymic Browning in Foods

    When most, but not all, fruit and plant tissue is bruised, cut, or damaged it rapidly turns brown or even black; this is seen when you eat an apple or peel a potato. This discolouration is caused by an enzyme called diphenol oxidase (DPO) and this enzyme catalysed reaction is usually highly undesirable from the point of view of the consumer and food processor, or the biochemist trying to investigate of plant metabolism. By contrast in some other situations such as the manufacture of tea, coffee, cocoa or cider this enzymic browning is an essential part of the process; it's also an important part of the plant's chemical defense system! However not all browning of foods is caused by enzymes; the golden brown of crusty bread is due to complex chemical reactions between sugars and proteins (the "Maillard reaction") which also contributes to flavour.
    In this article I should like to tell you about some of our research into the problem of enzyme-catalysed browning and its control.

    Enzymes involved in fruit browning

    It is now generally accepted that enzymic browning in is brought about by the action of an enzyme system variously known as diphenol oxidase (hereafter DPO), catecholase, polyphenoloxidase or tyrosinase. DPOs are very common in most fruits, leaves and many fungi but they are found also in animals where they transform the amino acid 3:4-dihydroxyphenylalanine (DOPA) into the dark melanin pigments of skin and hair. Albinos are unpigmented because they lack this enzyme.
    For the biochemist isolating active DPOs from plant tissues is very difficult because both the enzyme and its substrate are present, but do not react, in the intact cell. However, as soon as the cell's internal organisation is damaged then enzyme and substrate interact to yield reactive quinone compounds. These quinones subsequently react in a soft of "kamikaze" reaction with DPO itself and also with other proteins and enzymes to give complex brown 'melanin' pigments thereby causing their inactivation.

    Thus it also becomes necessary for the investigator to adopt special precautions to prevent this enzyme inactivation and it is customary to add reducing agents such as ascorbic acid (Vitamin C), cysteine or sodium metabisulphite when attempting to isolate plant enzymes. Similarly these protective compounds have been used to prevent enzymic browning in foods.

    Modes of action of diphenol oxidases

    All DPOs contain copper atoms which help the enzyme's catalytic activity and the major DPO activity in plants is the oxidation of a range of o-dihydroxy-phenols to the corresponding σ-quinones as shown in Figure 1.1 below. Notice that the first stage of the reactions is reversible and it is for this reason that ascorbic acid and other reducing agents can prevent enzymic browning by reducing the colourless o-quinones back to their parent phenols. Unfortunately when all the ascorbic acid is consumed the o-quinone is no longer reduced and so it then undergoes oxidative polymerisation to yield brown-black melanin pigments; this is why your apple goes brown!

     Nevertheless the addition of ascorbic acid (Vitamin C) is still one of the commonest and safest methods for the commercial control of enzymic browning in fruit juices and similar products. These o-quinones are highly reactive compounds and may also couple with amino acids and proteins which enhances the final brown coloration produced; this happens in mushrooms to form the characteristic chocolate-brown colour.


    Assay of diphenol oxidases

    The simplest, but least accurate, method of assaying DPO activity is to record the final colour yield when the enzyme is incubated with a suitable substrate such as catechol, 4-methyl-catechol or DOPA. Unfortunately this simple procedure is wide open to error since it is measuring the end-product of a sequence of reactions rather than the initial reaction. Nevertheless this simple assay technique has proved adequate for useful comparative studies of the browning levels of different fruit varieties(1) and similar problems. Investigations of some aspect of enzymic browning in fruits and vegetables using this simple procedure could form the basis of some good Science & Technology Fair projects in biochemistry or food technology.

    In research labs the commonest method of DPO assay is by measurement of the rate of O2-uptake by means of an O2-electrode and this method is routinely used in the author's laboratory.

    Substrate specificity of o-diphenol oxidases

    All o-DPO's require the basic o-dihydroxyphenol structure for activity so that catechol is the simplest , but not necessarily the best, substrate; 4-methyl-catechol is the fastest! The formulae of some natural and synthetic substrates are shown in Figure 2. However the nature of, and position of, any substituent groups has profound effects on the rate of substrate oxidation and studies of these problems have helped shed light on the nature of the interaction between the substrate and active-site for σ-DPOs(2).

     The commonest natural substrate for this reaction is chlorogenic acid which is widely distributed in many plants. Probably the next commonest substrates are catechin and epi-catechin which are also of widespread occurrence. Many workers have investigated the substrate specificity of the o-DPO's from different plant sources. For example, a relative of DOPA, 3:4-dihydroxyphenylethylamine (dopamine), is the chief substrate in bananas whilst DOPA is the natural substrate in the leaves of broad beans (Vicia faba L.). Results from the author's studies of apple σ-DPO support the view that the nature of the side-chain is critical and therefore may play a part in the enzyme/substrate interaction.

    It was found also that substrates such as DOPA were oxidised relatively slowly by apple DPO (40-60% of rate for chlorogenic acid) yet their final colour yield was greater because of the darker nature of their polymeric end-product. Thus any investigations of substrate kinetics/specificity based on colour yields would be highly suspect!

    Inhibitors of diphenol oxidase activity

    Many reagents inhibit DPO activity and studies with them have provided valuable insight into the mode of action of this enzyme. However only a limited number of DPO inhibitors are considered acceptable on grounds of safety and/or expense for use to control enzymic browning during food processing and this would seem to be a fertile field for continuing research.

    Inhibitors of DPO may conveniently be grouped according to their mode of action; this maybe by chelation of the prosthetic group, competition for the substrate, or by interaction with the products of reaction. DPOs utilise Cu2+ as a tightly bound prosthetic group so that the first category includes many Cu-chelating agents; thus carbon- monoxide, cyanide, Na diethyldithiocarbamate (DIECA), and Na azide, will inhibit these enzymes.

     Inhibition by thiols and reducing agents

    One of the commonest methods of controlling enzymic browning, both in industry and the laboratory, is by the addition of reducing agents such as SO2, metabisulphite and/ or ascorbic acid. These compounds prevent browning by reducing the enzymically formed quinones back to their parent o-diphenols; they are therefore consumed in the process (Fig 1). The former two agents may give rise to off-flavours or corrosion problems if used to excess but these, and ascorbate, are still probably the method of choice for food processing operations because of their lack of toxicity and relatively low cost.
    By contrast other -SH compounds may combine chemically with the o-quinones to form a stable, colourless product thus permanently preventing further oxidation to brown pigments. However, these thiols can only combine with o-quinones when the molar ratio exceeds a critical value, usually 1.5:1, but provided this critical level is exceeded then they should, in theory provide permanent protection against enzymic browning. Experiments by Walker and Reddish (3) confirmed this idea and showed that cysteine could be used to prevent the browning of apple products for over 24 hours without introducing undesirable off-flavours.

    Polymeric and other diphenol oxidase inhibitors

    During the past decade various polymeric materials such as nylon powder or types of polyvinylpyrrolidone (PVP and PVPP). They have also proved to be useful in the extraction of plant enzymes because of their ability to inhibit enzymic browning and concomitant enzyme inactivation. Poly-Clar AT (PVPP) is an insoluble, high MW, grade of PVP, which is also much used for the prevention of hazes in beer, but in this case browning is probably prevented because phenolic compounds are very strongly bound to Poly-Clar AT and are thus removed from the reacting system.

    Inhibition of o-DPOS by substrate analogues

    Several years ago the author noticed that apples that had been attacked by the blue- mould fungus (Penicillium expansum) did not display the brown, dead tissue characteristic of most diseased fruit. A detailed investigation of this problem (4) eventually revealed that the juice from the soft, rotted tissue contained a number of simpler phenolic acids such as p-coumaric, caffeic and lerulic acids together with patulin a toxic mould metabolite. What was interesting was that this juice inhibited enzymic browning and this prompted more detailed studies of the inhibitory effects of a wide variety of phenolic acids upon apple o-DPO. It was hoped that these experiments could shed light on the mechanism of enzyme action and enzyme inhibition. Cinnamic acids (Figure 1.3) were found to be powerful inhibitors of apple o-DPO and inhibitory action decreased in the order cinnamic acid > p-coumaric acid > ferulic acid > m-coumaric acid > o-coumaric acid >> benzoic acids. It was also significant that sinapic (3:5-dimethoxy-4-hydroxycinnamic) and hydro-cinnamic (phenylpropionic) acid together with the lower homologues of p-coumaric acid were all without inhibitory action. These findings told us something about the nature of the active site of the enzyme and it seemed that the unsaturated side-chain of the cinnamic acid derivatives was essential for inhibition of o-DPO action.

    Enzymic browning in apricots

    Most of the work described above was investigated using apple DPO although it is of general applicability. I would now like to describe some of our research with apricots (5).
    Using tests with specific substrates and inhibitors we found that apricots were unusual in that they exhibited two types of DPO activity; both catecholase and laccase. Both enzymes acted on the naturally present substrates chlorogenic acid and catechin.

    From the commercial point of view we found the best way to prevent browning during the drying of apricots was by means of a pre-drying dip in 1% Na metabisulphite which left virtually undetectable residual SO2 levels. By contrast use of cinnamic acid of ascorbic acid had little effect; the latter actually enhanced browning which leads us to suspect that non-enzymic, Maillard type browning is the main cause of darkening in dried apricots(5).

    Phenolases and our daily bread

    Although this review is concerned primarily with fruits our work on wheat phenolics may also be of interest. A former research student, Dr John McCallum, investigated the biochemistry of phenolics in wheat and flour and found that both o-DPO activity and phenolic content varied widely between different red and white wheat cultivars and between different milling flour streams. In all cases these correlated significantly with flour colour and bread crumb colour. Thus there is a clear involvement of phenolics in the final colour, and therefore customer acceptance, of bread and other wheaten products. These phenolics also seem to be involved in the key problem of dormancy in wheat.

    The practical control of enzymic browning in food processing

    During the preparation of many fruits and vegetables for canning or other processing operations the prevention of enzyme-catalysed browning is a major problem and it is unfortunate that the majority of the DPO inhibitors discussed in the previous sections are not suitable for use in foodstuffs. In practice food processors faced with this problem usually rely on an early heat-treatment stage to inactivate DPO's and other enzymes but some o-DPOs are relatively heat stable, for example, apple o-DPO possessed a half life of 12 minutes at 70°C. Nevertheless it is essential to take positive action to prevent oxidative browning until the DPO's are denatured.
    The inhibitors of enzymic browning most frequently used in industry include acid or brine dips, ascorbic acid and SO2, either as the gas or sodium metabisulphite. Ascorbic acid is widely used to prevent the oxidative browning of fruit juices prior to pasteurisation whilst it may be also be added to acid dips used for the pretreatment of peeled or sliced fruit. Likewise SO2, or metabisulphite, may be used to control the browning of sliced fruit and vegetables before drying, as for example in the manufacture of apple rings. However excess SO2 may have undesirable effects such as the generation of off-flavours.

    The author (7) published a novel method for the prevention of enzymic browning in apple juice based on earlier fundamental studies of the inhibition of apple o-DPO by cinnamic acids. Different amounts of cinnamic, p-coumaric or ferulic acids were added to freshly prepared opalescent apple juice and the mixtures aerated to promote browning. Addition of quite low concentrations of these acids, especially cinnamic acid, provided effective long-term control of browning. The exact quantity of inhibitor required depended upon the level of phenolics of the particular fruit or variety. Typically less than 0.01% (= 10ppm) of cinnamic acid or its more soluble sodium salt was enough to prevent the browning of the juice from Granny Smith apples.

    The author has not investigated the question of possible toxicity of these cinnamic acids but it is well known that they are widely distributed either free or as esters in essential oils (such as oil of cinnamon, etc.). It would therefore seem unlikely that there would be any health hazards associated with the use of cinnamic acids to control enzymic browning.

    More recently the compound 4-hexyl-resorcinol has been patented under the name "EverFresh"TM as an alternative to sulphites for the control of enzymic browning or "blackspot" in shrimps and other crustaceans.

    "Natural" DPO inhibitors

    I'd now like to consider a new approach to the problem of control of enzymic browning and the avoidance of the use SO2 which is coming under increasing suspicion. In 1969 I found that cultures of the "blue-mould" fungus Penicillium expansum secreted "expansin" an, as yet unidentified, inhibitor of apple o-DPO and I suggested that this could be a factor in overcoming the fruit's natural defence mechanisms thus facilitating infection by phytopathogenic fungi. We have maintained our interest in this phenomenon and have screened a wide range of microorganisms for their ability secrete DPO inhibitors. Such compounds could have commercial potential as "natural" DPO inhibitors to replace SO2.

    Beneficial forms of enzymic browning

    So far browning has been presented as a problem for the food processor but not all cases of enzyme-catalysed browning are undesirable. In certain instances such as the manufacture of tea, coffee, or cocoa, these reactions are essential to the manufacturing processes but only brief descriptions of these can be included in this review.
    Probably the best studied example of beneficial browning is that concerned with the biochemical changes that take place during the manufacture of black tea. These have been investigated by a number of workers who have isolated and studied tea-leaf o-DPO and shown that it plays an important role in the "fermentation" stage of tea manufacture. Studies with tea-leaf o-DPO showed that its major natural substrates were catechin, epi-catechin and epi-catechin gallate; their o-quinone oxidation products were precursors of the more complex theaflavins and thearubigins which give tea its colour.

    The beverage quality of coffee has been shown to be related to the level of o-DPO of the green coffee beans whilst o-DPO's also play a part in the development of the final colour of processed cacao beans which contain large amounts of phenolic constituents such as epicatechin. Powerful o-DPO's were found in fresh cacao beans and their pericarp.

    Enzymic browning is responsible in whole, or in part, for the characteristic brown colour of certain dried fruits such as dates, prunes and sultanas. During the manufacture of cider and perry (fermented pear juice) and white wine the action of o-DPOs upon the naturally occurring phenolics and tannins leads to their subsequent condensation and polymerisation; reactions which may have an important role in aiding subsequent clarification processes as well as removing unwanted astringency.

    In conclusion it is fitting to quote from a review by Mathew and Parpia (10) who concluded that "That greatest challenge for food technologists is to evolve more economical and easier methods or preventing undesirable browning during commercial handling and processing of food".

    General reading

    Macheix J-J. Fleuriet, A. & Billot, J. (1990) Fruit phenolics. CRC press, Boca Raton, USA
    Vamos-Vigyazo, L (1981) Polyphenoloxidase and peroxidase in fruits and vegetables. CRC Critical Reviews in Food science and Nutrition 15 49-127
    Walker, JRL (1975) The Biology of plant phenolics,studies in Biology, No54, Edward Arnold, pp 46-49
    Walker, JRL (1995). 'Enzymic Browning in Fruits; its biochemistry and its Control'. In 'Symposium on Enzymic Browning and its prevention in Foods'. Amer.Chem.Soc. Symposium series No. 600, pp8-22.
    Walker, JRL & Ferrar,PH. 1998 Review; Diphenol oxidases, enzyme-catalysed browning and plant disease resistance. Biotech. Genetic Eng. Rev.15;457-498

    References

    1. Walker, JRL (1962) Studies on the Enzymic Browning of Apple Fruit, NZ. J.Sci.5; 316-24
    2. Walker, JRL & Wilson, EL.(1975) studies on the Enzymic Browning of Apples. III.Inhibition by Phenolic acids. J. Sci.Fd. Agric.26; 1825-32
    3. Walker, JRL & Reddish, CES.(1964) Note on the use of cysteine to prevent Browning in Apple Products. J. Sci. Food Agric. 12; 902-4.
    4. Walker, JRL (1969) Inhibition of the Apple Phenolase System through infection by Penicillium expansum. Phytochem. 8; 561-6
    5. Dijkstra, Lucette & Walker, JRL (1990) Control of Browning in Dried Apricots. Food Technology in NZ.25(8), 34-85.
    6. McCallum, JA & Walker, JRL (1990). O-Diphenol oxidase Activity, phenolic Content, and Colour of NZ Wheats, Flours and Milling Streams. J.Cer.Sci. 12;83-96.
    7. Walker, JRL (1976) The Control of Enzymatic Browning in Fruit Juices by Cinnamic Acids. J. Food Technol. 11; 341-6.
    8. Walker, JRL (1970) Phenolase Inhibitor from Cultures of Penicillium expansum which may play a part in Fruit Rotting. Nature 221; 298-9.
    9. Ferra, PH & Walker, JR. (1999) Phytopathogens as sources of novel diphenol oxidase inhibitors. J.Food Biochem, 23, 1-15.
    10. Matthew, AG & Parpia, HAB (1971) Food browning as a polyphenol reaction. Adv Food Res 19;75-145.

    Part 2: Some Practical Experiments

    In the first part of this article I discussed some of the biochemistry involved with enzyme- catalysed browning in fruits and its control. This can form the basis of a study of the properties of diphenol oxidases (DPOs) or an investigation of the problem of enzymic browning in food processing. I should now like to outline some simple experiments you could do for yourself; it is to be hoped that this information will also help you if you choose this theme for a Science & Technology Fair project.

    Materials

    Enzyme source: any fruit or vegetable which goes brown when cut or bruised could be used for these experiments but apples, bananas, pears, potatoes, avocados and mushrooms are all good sources of DPO.
    Chemicals: most of the chemicals mentioned should be available through local laboratory supply houses. More specialised phenolic compounds can be obtained from Sigma Chemical Co.(PO Box 14 508, St Louis, MO 63178-9916, USA) or Fluka AG, Switzerland.

    Experimental

    1. Properties of DPO

    For a simple demonstration of DPO activity in fruits and vegetables you can apply a few drops of 0.1% catechol or 3:4-dihydroxyphenylalanine (DOPA) solution to the surface of the freshly cut fruit; the formation of a brown zone is indicative of DPO action.
    However, if you want to make a more quantitative study you should prepare a solublised enzyme preparation by grinding about 20g plant tissue (eg potato) in a pestle and mortar with 100mL 0.05M Na fluoride (caution POISON). You may add a little coarse sand to assist homogenisation. Filter the resultant slurry through fine muslin or white nappy liner tissue (n.b. nappy liner tissue is ideal for coarse filtration) and use the liquid as a source of crude potato DPO for the following experiments.

    1.1 Demonstration of enzyme action

    Set up a series of test tubes containing the following reagents:

     Mix the tubes thoroughly at 5 min intervals to ensure adequate O2 transfer; use a vortex mixer if you have one.


    Record any changes in colour for each tube at 5 min intervals using an arbitrary 0-5 scale. If you are lucky enough to have access to a colorimeter you should clarify the reaction mixtures by filtering or centrifugation and then record the absorbance using a blue filter.

    How does altering the amounts of enzyme and substrate (catechol) affect the reaction?

    1.2 Effect of temperature

    Enzymes, being proteins, are denatured or destroyed by high temperatures and this is the basis of the blanching and pasteurisation processes used in food preservation, sterilisation etc. Thus, during the manufacture of fruit juices, DPO activity can be halted by a heat treatment step.
    To investigate the heat stability of your DPO preparation set up a series of pairs of tubes #1 and #2 as for the previous experiment (1.1) but leave out the substrate and incubate them at different temperatures from 0-100°C for 5 or 10 minutes. Cool rapidly at room temperature, add 1.0mL substrate to alternate tubes to start the DPO reaction and compare final colour/enzyme activity.

    At what temperature was DPO activity lost? Why is this sort of experiment important to food technologists?

    1.3 Effect of pH

    Enzymes have an optimum pH for activity. Reducing the pH by dipping apple slices in 0.5% citric acid solution or lemon juice is often used to provide a temporarily halt to enzymic browning during processing.
    Prepare a range of 0.1M-phosphate-citrate buffers between pH 4.0 - 7.6. Use them to repeat the first experiment to determine the optimum pH for your DPO preparation.

    1.4. Substrate specificity

    Enzymes often display exacting requirements towards the nature of their substrate and this can tell us something of the nature of the enzyme/substrate interaction. Thus DPO usually requires the basic catechol structure to be present for activity.

     Using the same protocol as for Expt 1.1 investigate the rate of reaction and nature of the colour produced when you use different types of substrate. The chemical formulae of some potential substrates are given in Figure 2.2. Do you find any correlation between structure and enzyme activity? Do you find different patterns for DPOs from different sources such as apples or mushrooms?

    1.5. DPO inhibitors; the control of browning

    Control of enzymic browning is of obvious importance in the processing of fruit juices etc. The chemicals most commonly used by industry to control browning are Na meta-bisulphite Na2S2O5 (or SO2) and ascorbic acid (Vitamin C). Note that some compounds, like ascorbic acid or metabisulphite prevent browning by preventing further reaction of the quines. By contrast compounds like DIECA act by blocking the action of the enzyme by reacting with the Cu prosthetic group which is needed for full catalytic activity.

    Using the basic catechol/DPO assay system you could investigate the ability of different concentrations (try 10-4 to 10-2M) of the following groups of chemicals to act as DPO inhibitors:

    a. Anti-oxidants; Ascorbic acid, (you could try Vitamin C tablets or lemon juice), Na2S2O5 or cysteine (an amino acid).
    b. Cu-reagents; DPO needs Cu atoms in its protein structure for activity and these reagents target this metal; thiourea, phenyl-thiourea, Na diethyldithiocarbamate (DIECA).
    NB. These reagents are toxic so do not be tempted to sample your products!
    c. Chloride ions; high levels of NaCl (about 0.1M) can limit enzymic browning.

    2. Experiments with juice and whole fruit

    If you wish to simulate home or factory conditions, rather than using a soluble enzyme preparation, you could try using thin slices of potatoes, or a fruit such as apples, and subject them to the various treatments described above. Similarly, you could make samples of fruit and vegetable juices using a domestic juicer and investigate different ways of preventing their browning.

    In this context it could be very interesting to compare different varieties of the same fruit; for example Granny Smith or Red Delicious apples brown much more than do Golden Delicious. You might like to try to find out if these differences are due to different levels of DPO enzyme, or phenolic substrate, or maybe both factors are involved?

    3. Experiments with Fungi

    Mushrooms and toadstools often contain DPO enzymes but their properties, such as substrate specificity, may differ from the DPOs of fruits. If they show strong activity with substrates like p-quinol and p-phenylene diamine they are probably "laccases" which are para-DPOs in contrast to the enzyme from apples etc which is an ortho-DPO or "catecholase" and likes its substrate's two hydroxy groups to be vicinal(side- by-side).

    3.1 Distribution of DPO in mushrooms

    Obtain a medium-sized mushroom and cut a thin (3-5mm) transverse slice right across the mushroom and through the stipe (stalk). Next, place this slice of mushroom tissue in a petri dish containing a few ml of 10mM 3:4-dihydroxyphenylalanine (DOPA). At five minute intervals examine the slice and note the order in which the different tissues show development of a red-brown colour. Try other substrates for activity; is a laccase present?3.2 Comparison of different fungiCompare DPO activity in different mushroom cultivars, wild mushrooms and toadstools. You could quantify these differences by using the procedures outlined in section 1.1. Similarly you could investigate ways and means of preventing enzymic browning in mushrooms.

    CAUTION: some toadstools are poisonous so handle them with care! Wash your hands thoroughly after your experiments.

    CONCLUSION

    I hope that students will find these ideas useful and that they will stimulate some "hands-on" research. Even today, control of enzymic browning is still a major problem in the food processing industry.

    REFERENCES

    Please refer to bibliography and references cited in Part I of this series.
    Western Australia Branch Bayliss Youth Lecture Or...
    The Structure of the Atom
     

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