Summary:

Unlike inorganic catalysts, enzymes are very specific organic molecules. Each type of enzyme acts on only one particular compound, known as its substrate. The substrate briefly binds with the enzyme, and in the process is changed. Each enzyme has a unique three dimensional shape, including a surface groove called an active site, which fits its target substrate much like a key fits in a lock. Other substances that dont fit can't enter the active site and no reaction occurs. If the shape of an enzymes active site is altered, or the cofactor removed, an enzyme can no longer work. Enzymes are protein catalysts, which control specific chemical reactions in living systems (plants and animals). Enzymes are active at low concentrations and are substrate specific. The enzyme rennin catalyzes the coagulation of casein in milk, but is not effective in any other chemical reaction.

Enzymes are protein polymers that possess the ability to specifically recognize biological molecules, bind to them, and catalyze a chemical reaction. In contrast to non-protein catalysts, enzymes are specific catalysts. They usually react with only one substrate. Since all biochemical reactions are enzyme catalyzed, many different enzymes must exist. An Escherichia coli bacterium, one of the simplest biological organisms, has more than 1,000 different enzymes working at various times to catalyze the reactions necessary to sustain life of the bacterium. The complex molecules that are contained in food provide the energy needed by living organisms to carry out all life functions. These molecules are not useful to the organism unless they are first broken down into smaller, simpler forms through digestion. Digestion involves the hydrolysis (breakdown) of proteins to amino acids, starches to monosaccharides, and fats to fatty acids and glycerol. Unfortunately, hydrolysis at body temperature occurs at a rate that is too slow to be useful to the organism. To speed up (catalyze) the hydrolysis reaction, living organisms produce and use enzymes.

INTRODUCTION Appearance, flavour, texture and nutritional value are four attributes considered by consumers when making food choices. Appearance which is significantly impacted by colour is one of the first attributes used by consumers in evaluating food quality. Colour may be influenced by naturally occurring pigments such as chlorophylls, carotenoids and anthocyanins in food, or by pigments resulting from both enzymatic and non-enzymatic reactions. Enzymatic browning is one of the most important colour reactions that affects fruits, vegetables and seafoods. It is catalysed by the enzyme polyphenol oxidase (1,2 benzenediol; oxygen oxidoreductase,

EC1.10.3.1) which is also referred to as phenoloxidase, phenolase, monophenol oxidase, diphenol oxidase and tyrosinase. Enzymatic browning is one of the most studied reactions in fruits, vegetables and seafoods. Researchers in the fields of food science, horticulture, plant and postharvest physiology, microbiology, and even insect and crustacean physiology have studied this reaction because of the diversity of its impact in these systems. Polyphenol oxidases are believed to play key physiological roles both in preventing insects and microorganisms from attacking plants and as part of the wound response of plants and plant products to insects, microorganisms and bruising. As fruits and vegetables ripen, their susceptibility to disease and infestation is increased due to a decline in their phenolic content. Phenoloxidase enzymes endogenous to fruits and vegetables, catalyse the production of quinones from their phenolic constituents. Once formed, these quinones undergo polymerization reactions, leading to the production of melanins, which exhibit both antibacterial and antifungal activity and assist in keeping the fruit and/or vegetable physiologically wholesome. Fresh cut fruits and vegetables usually suffer from enzymatic browning, and washing with water is not effective to prevent discoloration. Enzymatic browning is a key quality problem for fruits

and vegetables such as peeled and sliced apple and potato. This browning reaction resulted from the oxidizing enzymes, such as polyphenol oxidase, which is released from broken cells. Enzymatic browning does not occur in intact plant cells since phenolic compounds in cell vacuoles are separated from the polyphenol oxidase which is present in the cytoplasm. Once tissue is damaged by slicing, cutting or pulping, however, the formation of brown pigments occurs. Both the organoleptic and biochemical characteristics of fruits and vegetables are altered by pigment formation. The rate of enzymatic browning in fruit and vegetables is governed by the active polyphenol oxidase content of the tissues, the phenolic content of the tissue, pH, temperature and oxygen availability within the tissue. Polyphenol oxidase catalyses the oxidation of phenols to o-quinones, which are highly reactive compounds. O-quinones thus formed undergo spontaneous polymerization to produce highmolecular-weight compounds or brown pigments (melanins). These melanins may in turn react with amino acids and proteins leading to enhancement of the brown colour produced. Many studies have focused on either inhibiting or preventing polyphenol oxidase activity in foods. Various techniques and mechanisms have been developed over the years for the control of these undesirable enzyme activities. These techniques attempt to eliminate one or more of the essential components (oxygen, enzyme, copper, or substrate) from the reaction. i) The elimination of oxygen from the cut surface of fruits or vegetables greatly retards the browning reaction. Browning however occurs rapidly upon exposure to oxygen. Exclusion of oxygen is possible by immersion in water, syrup, brine, or by vacuum treatment. ii) This copper prosthetic group of polyphenol oxidases must be present for the enzymatic browning reaction to occur. Chelating agents are effective in removing copper. iii) Inactivation of the polyphenol oxidases by heat treatments such as steam blanching is effectively applied for the control of browning in fruits and vegetables to be canned or frozen. Heat treatments are not however practically applicable in the storage of fresh produce. iv) Polyphenol oxidase catalyses the oxidation of phenolic substrates such as caffeic acid, protocatechuic acid, chlorogenic acid, and tyrosine. Chemical modification of these substrates can however prevent oxidation. v) Certain chemical compounds react with the products of polyphenol oxidase activity and inhibit the formation of the coloured compounds produced in the secondary, non-enzymatic reaction steps, which lead to the formation of melanin. Many techniques are applied in the prevention of enzymatic browning. Relatively new techniques, such as the use of killer enzymes, naturally occurring enzyme inhibitors and ionizing radiation, have been explored and exploited as alternatives to heat treatment and the health risks associated with certain chemical treatments. Processing technologies applied in the control of enzymatic browning in fruits and vegetables are now reviewed.

MATERIALS AND PROCEDURES MATERIALS Potato 60 ml of 1% thiourea 0.01g of sodium sulfite 0.12g of dipotassium phosphate 60 ml of 1% ascorbic acid 60 ml of 1% citric acid 60 ml of 1% sodium metabisulphate 60 ml of 1% sodium chloride APPARATUS Spectrophotometer Buchner funnel and filter paper, Whatman No. 1 filter paper Blender Aspirator vacuum Beaker Filtration flask Test tube PROCEDURES i. Peel apples and cut into uniform thin slices. Divide the sliced apple into four lots of 30.0g each. Place one lot into a beaker containing 60 ml of distilled water, which will serve as control. ii. Place the second lot into a beaker containing 60ml of distilled water with 1% citric acid. iii. Place the third lot into a beaker containing 60ml of distilled water with 1% of ascorbic acid. iv. Place the fourth lot into a beaker containing distilled water with 1% of sodium metabisulphate. v. Place the fourth lot into a beaker containing distilled water with 1% of sodium chloride.

vi. After 30 minutes, homogenize the content of each beaker in a blender, and filter through a Buchner funnel into a filtration flash under aspirator vacuum using Whatman No. 1 filter paper. vii. Attach the funnel to the filtration flask, turn on the aspirator, wet a piece of filter paper using a filter paper, and carefully place the filter paper in the Buchner funnel. viii. Pour apple tissue from the blender into the Buchner funnel and continue filtration until several milliliters of filtrate have been collected. ix. Place 1 ml of each filtrate in five different test tubes each containing 5 ml of water and mix. x. Transfer contents of each test tube to a cuvette and read optical density at 475nm using a spectrophotometer. RESULTS AND DISCUSSION Data reporting: Discussion: QUESTIONS 1. Outline the reactions that occur as potato turn brown. Which intermediate is being detected by the spectrophotometer? 2. Describe the effect of each treatment on discoloration. 3. What is the ingredient in fresh cut fruits which decreases browning?was it effective in decreasing browning?