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Test of Proteolytic Enzyme Concentration in Pineapple Juice

Introduction

The aim of this experiment was to test how the concentration of proteolytic enzyme in pineapple juice changes with different days of ripening.
The proteolytic enzymes belong to a large group of enzymes known as proteases or proteinases. Proteases belong to the class of enzymes known as hydrolases, which catalyse thereaction of hydrolysis of various bonds with the participation of a water molecule. Proteases are enzymes that digest long protein chains into short fragments, broken the peptide bonds.
The proteolytic enzymes are found both in animals and in vegetables. In animals, these enzymes have important biological processes, including: protein digestion, the blood clotting, and differentiation oftissues. His role in the digestive process is essential to the absorption because hydrolysing proteins (breaking) into amino acids, it can be absorbed and reused by the body.
On vegetables, proteolytic enzymes are involved in processes of maturation, germination, differentiation and morphogenesis, oxidative stress processes, among others. Some enzymes involved in fruit ripening, such as papain (papaya)and bromelain (pineapple), can be extracted in large quantities and therefore represent a significant economic importance.
On this experiment are possible to measure the concentration of the enzyme (bromelain) present on the juice. At the early stages of fruit development, the presence of the enzyme was very low, but its level increases rapidly and remained high until the ripening (BALDINI etal., 1993).
The proteolytic enzymes also have a wide application commercial / industrial, as part of many laundry detergents and among the three largest groups of industrial enzymes. In food industry, proteolytic enzymes are widely used for softening meat, clarification of beer and softening leather. In the pharmaceutical industry, these proteolytic enzymes are used in medicines for digestibilitydisorders, and also anti-inflammatory
The technique of this experiment was to monitor the concentration of the enzyme using exposed photographic film, where the exposed film consists of black silver granules embedded in gelatin on a transparent backing of cellulose. When the protein is digested by the enzyme, the black silver granules will fall off leaving the clear backing. Taken the time forthis reaction and using different days of ripening was possible to determinate the concentration of the enzyme. The bromelain is most activity at acid pH environment, around 4.0, and the better temperatures are between 37°C and 40°C (Taussig, S.J. and S. Batkin., 1988). For this environment the experiment was made inside a water bath with temperature constant at 37°C and the juice was mix with buffersolution at pH 4.0 and one tube with just buffer solution was used as control.

Method

* Labelled 4 test tubes with the days of ripening: 0, 5, 10, 15 and one tube as a Buffer.
* Added 5cm3 of appropriate juice into labelled tubes.
* Added 5cm3 of buffer solution into one test tube.
* Placed all the tubes into a water bath at 37°C and left 5 minutes to equilibrate thetemperature. Meanwhile cut 5 strips of 1 cm of photographic film.
* After 5 minutes was passed, added one strip of film into each tube and started mark the time (make sure the strips are fully submerged).
* With every 2 minutes stir each tube and observe the state of the film. Lift the film out with the aid of a glass rod and gently wipe it to check the state of the film, remember to rinse anddrying the rod between tubes to avoid contamination.
* Recorded in a table the time taken for all the black granules to fall from the film and intermediate stages.
* Keep observations until all the protein has been digested in each tube containing pineapple juice or as long time permits.
Note: The juice was provide in advance and was extracted from pineapple at 4 different stages of...
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