Fruits and browning - testing some antioxidants

Transcription

1 Association of Danish Biologists (FaDB) Claudia Girnth-Diamba, Karen Lunden, Hanne Thomsen, Liselotte Unger, Lykke Thostrup, Michael Bom Frost, Lone Brinkmann Sørensen, Marie Kielsgaard CG, Solroed Gymnasium, Solroed Center 2, DK 2680 Solroed Strand, Denmark Fruits and browning - testing some antioxidants Do you want brown fruit juice? We are testing some methods and chemicals to prevent enzymatic browning in certain fruits and vegetables. Aim The purpose of the experiment is to investigate what can prevent enzymatic browning of fruits and some vegetables. In the experiment we want to test 3 substances with antioxidant capability and test how effective they are. The most effective substance is the one that prevents browning at a very low concentration. Furthermore since browning is due to enzyme activity denaturising the enzyme by heat treatment is investigated too. When plants get wounds, for example by insect bites, the content of the damaged cells get in contact with the oxygen in the air. The plant cells contain phenols that get polymerized by enzyme activity into brown substances similar to melanin the polymer that can be found in brown skin of humans and the dark exoskeleton of insects. This reaction needs oxygen as cofactor. Substances that catch the oxygen before it can be used by the enzymes are known as ANTIOXIDANTS. Introduction When plants get wounds the cell content gets in contact with the oxygen from the air. The oxygen reacts with some compounds like phenols, situated in the cell vacuole, and the enzymes transform them to a dark polymer. The reaction is catalysed by enzymes which are called phenolases (or phenol oxidases). When you make juice from plants this reaction is not desirable because we don t want to drink brown juice. You can stop this enzymatic browning by destroying the enzyme by boiling the fruits or vegetables. You can also stop the reaction by antioxidants like ascorbic acid, citric acid, sodium sulfite (or sodium disulfate). Their function is to collect and react with the oxygen before the browning process.is starting. There is a difference however between adding antioxidants to the food to keep it fresh and eating antioxidants as nutritional supplements. Oxygen is the cause of many damages in living organisms. Oxygen atoms are created during biochemical reactions. These atoms are called radicals and they can destroy organic compounds by creating dangerous peroxides. The best known peroxide is hydrogen peroxide, H 2 O 2. The enzyme catalase, which is to be found in all cells, destroys hydrogen peroxide by transforming it into water and oxygen.the formation of peroxides in living organisms cause damage to organic substances and may even be the cause of cancer. The browning of food items from plants (and fungae like champions) is the result of the enzyme phenol oxidase. In insects the enzyme causes the browning and hardness of their cuticula. It also protects from UV-light as in humans. In higher animals and in humans the enzyme works in the brown cells (melanocytes) of the skin, which too protects us from damage caused by UV-light. This is different in plants. The brown surface occurs when the plant is damaged. The enzyme phenol oxidase is situated in the cell wall and when wounded it comes in contact with the cell liquid and the browning process starts but only when oxygen is present. The function of the antioxidants is to catch the oxygen before it can be used by the enzyme. In this way the oxidation of phenols and the browning is prevented. The theory is that the melanin formation in plants in contrast to its function in animals protects wounds against infections from bacteria and fungus. Some melanins however we do like in plants. We know that the browning process of tobacco leaves during fermentation is caused by the same enzymes and compounds. This is where tobacco gets its special taste (which some of us enjoy despite health risks). The brown compounds in tea are also the result of the reaction of phenol oxidases with phenols, like for example tannins.

2 Materials Used by each group or person Chemicals and solutions: 100 ml of the following solutions: 1% ascorbic acid solution (Vitamin C) 1% citric acid solution 1% sodium sulphite NaSO 3 (you can also use sodium disulfide, Na 2 S 2 O 5 ) prepare them the same day (the concentration is weight per volume = 1g in 100 ml of water) you can prepare a stem solution of 10% and dilute 10 ml + 90 ml (1%) and 1 ml + 99 ml (0,1%) Other materials Apples, potatoes, bananas and/or other colourless fruits or vegetables 2 beakers (100 ml) made of heatproof glass, for experiment 1 6 plastic cups with at least a 100 ml volume for experiment 2 A kitchen grater White plastic plates or white kitchen paper Cooking plate Measuring cylinder Plastic spoons for stirring and other manipulations Kitchen strainer (not absolutely necessary - you can try using a spoon) Procedure Some important remarks This procedure and the material are meant for 1 type of fruit per group or person. The amount of fruit that you need is the size of 3 table tennis balls. Remember to prepare more solutions when a group wants to investigate several types of fruit or vegetable at the same time. Use 10% stem solutions just 100 ml of a 10% stem solution is enough for 8-9 preparations. Procedure part 1 Heat deactivation of browning enzymes Prepare 1 beaker containing 100 ml cold water. Warm 100 ml of water in another beaker on the cooking plate. When the water is boiling you can start with the next step. Do not start before. Grate quickly your fruit (fruits) without peel. Part it into 3 equal portions, each having the size of a table tennis ball. Add one portion quickly to the boiling water, turn down the heat. Put another portion in cold water. Leave both there for 10 minutes. Take a third portion and leave it on a plate with no further treatment. After 10 min take the shredded fruits out of the cold and the hot water with a spoon (or use a strainer) and place them each on white kitchen paper or plates. Check after 30, 60 and 90 minutes whether there is any colour change. Colour change is evaluated by a scale, where white is 0 and completely brown is 5. Procedure part 2 Testing 3 types of antioxidants Prepare 100 ml of the 6 solutions: a 1,0% solution and a 0,1% solution of each of the 3 substances. Grate 1 portion of fruit for each solutioni. e. 6 portions totally per type of fruit or vegetable and add it quickly to the solutions. Stir and wait for 5 minutes. Take the grated fruits out with a spoon (or use a strainer) and place them each on white kitchen paper or a plate. Check after 30, 60 and 90 minutes whether there is any colour change. Colour change is evaluated by a scale, where white is 0 and completely brown is 5.

3 Results Evaluate the colour change on a scale of 0 to 5-0 being no colour change and 5 being the highest grade of brown (grated fruit left for 90 minutes in air only). If you are not sure about the grading you can prepare a scale made of tea, since tea s colour is also based on polyphenols. You can also take photographs for later evaluation but remember that artificial light in the laboratory might give you a wrong impression of the colour. Present the colour grade after 90 minutes as stick diagrams comparing the 1% and the 0,1% solutions. Add the controls i.e. the hot and cold water treated and not treated as comparison. Results part 1 TREATMENT 30 min 60 min 90 min none 10 min in cold water 10 min in boiling water Results part 2 TREATMENT 30 min 60 min 90 min 1,0% citric acid 0,1% citric acid 1,0% ascorbic acid 0,1% ascorbic acid 1,0% sodium suphite 0,1% sodium sulphite

4 Guide to the protocol: Fruits and browning - testing some antioxidants Do you want brown fruit juice? We are testing some methods and chemicals to prevent enzymatic browning in certain fruits and vegetables. Pedagogical tips The experiment is quite simple and does not need any special laboratory experience or chemical insight. It would however be an advantage if the students had an introduction to antioxidants. It is our intention that the experiment leads to a discussion about antioxidants as food preservatives and about which antioxidants are relevant to add. A further perspective could be to discuss whether it is appropriate to use antioxidants to enrich food (so-called functional foods). Discussion Ask the pupils to find information in school books and/or on the internet about the three substances and discuss the properties and the possible harm of using them as antioxidants in food items after all they ARE food additives, whether they are natural substances or chemicals. Pictures of results The figure below shows the browning of potatoes after different treatments with antioxidants. Conclusion For conclusions you can ask the following questions: What is the best substance used for preventing browning? Explain why. But what is the healthiest substance to use is there a difference? Explain your choices. What are the reasons for using different concentrations? Can you explain this by referring to your results? It can happen, that one antioxidant is even more effective than cooking try to explain that finding. If you have used different types of fruit: are there differences if yes, explain why. Open ended investigations You can test different types of fresh fruit or vegetables except for strongly coloured ones. 0,1% ascobic acid 0,1% citric acid 0,1% sodium sulphite light brown dark brown light, no colour change

5 Practical tips Safety guidelines There are no safety problems with the used chemicals. But just to be safe, do not eat the leftovers of the fruits no eating in the laboratory. Storage of materials All antioxidants get quickly oxidised by oxygen - naturally. They can not last long, especially Vitamin C. To be sure that they work properly, buy only small portions of ascorbic acid and citric acid and make fresh solution a few hour before the experiment. Disposal of waste Pack the remains in a plastic bag, close the bag with a firm nod and put it in a dust pin. The solutions can be poured out in the sink. Preparation and timing Preparation takes 45 min and the experiment takes 45 min, with additional 90 minutes to check the browning. Troubleshooting Be careful with fruits containing a lot of organic acids, they may not react test them in due time before the experiment. It can be difficult to judge the browning without a reference system. Because the brown colour in tea is almost the same as in fruits you can eventually make a reference system with different dilutions of tea. For example use as 100% one teabag in 200 ml boiling water in 5 minutes and dilutions 50%, 25%, 10% and 2% and use it for comparison. Other sources of information For more literature, both English and Danish se: Acknowledgement In 2005 the protocol has been developed for teacher education as part of the collaboration between FaDB (The Danish Association for Biologists), the Chemistry Teacher Association and Copenhagen University, Department of Life Science. The development of a teacher training course has been sponsored by the Danish Ministry of Education (GYM23 Reformprojekt 2004 projektnummer ) as part of a new reform for secondary school education. This material is based on the Danish material Hvorfor bliver frugten brun og kødet gråt? written by Claudia Girnth-Diamba, Karen Lunden, Hanne Thomsen, Liselotte Unger, Lykke Thostrup, Michael Bom Frost, Lone Brinkmann Sørensen, Marie Kielsgaard in collaboration with other scientist and public relations personal at Copenhagen University, Department of Life Science. The authors thank the University for the possibility to use this material for adaptation to the Volvox project. Thanks very much to our English colleagues for valuable help with the English translation. Thanks also to Bjørn Fahnøe and the pupils of Solroed Gymnasium for testing and revising the experiment since its publication in the Danish material. This protocol is part of the Volvox project (FOOD AND NUTRITION) and is founded under the Sixth Framework Program of the European Commission.

6 More about antioxidants Oxidative stress C-vitamin is an example of an antioxidant. There are also examples of antioxidants which are not vitamins. Antioxidants neutralize free radicals and other reactive oxygen- and nitrogen compounds. These compounds are created in the body under normal oxidative, metabolic processes like for example the burning of nutrients and in relation to sickness, smoking, poisonings, medicine, some kind of food, alcohol and radiation. These highly reactive compounds can be very destructive. They can change the structure and function of cell membranes, lipoprotein, protein, carbohydrate, RNA and DNA and more. Oxidative stress is therefore meant to contribute to the development of cardiovascular disease or cancer, amoung others. The interaction between antioxidants from vitamins and non-vitamins is important in order to get an optimal effect in destroying free radicals in the body. When antioxidants interact a synergy arises; i.e. an effect bigger than the sum of the function of each antioxidant. It is probably not enough to eat antioxidants as pills, therefore it is essential to eat varied (see below). Natural antioxidants in our food C-vitamin (ascorbic acid) from fruits and vegetables E-vitamin from vegetable oils, nut seeds, fat fish, yolk of egg, coarse cereals Carotenoids (for example beta carotene) - from red fruits and vegetables Flavonoids from yellow fruits and vegetables Phyto estrogenes from leguminous fruit (soy beans f.ex.) and rough cereals Manganese form green leaved vegetables, leguminous fruit, and rough cereals Zink from meat, milk products, rough cereals Cobber from pluck (highest contents) Selenol from fish, shellfish, egg and pluck A little chemistry on phenol oxidase The enzyme oxidises for example the essential amino acid tyrosine (chemical a phenol, having OH groups on a benzene ring), to DOPA (dihydroxyfenylalanin) under the uptake of oxygen. Therefore the old name for the enzyme was tyrosinase. DOPA is converted into dopachinon under the liberation of water. Besides tyrosine also other phenols can be transformed to this kind of chinons. Chinons are reacting further in a non enzymatic reaction to brown-black high-molecular compounds called melanins. Melanins are a network of many thousand units of different chinons.