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NAMES: Patrice Burrows, Eddlytany Donne, Karla Hinshelwood, Shenelle Biggs CLASS: BIO 301, 1700 DUE DATES

& TIME: January 30, 2014 @ 3:00 p.m

TITLE: The Examination of Bromelian Activity in Gelatin Under different Conditions OBJECTIVES: The purpose of this experiment was to determine if fresh or canned pineapple juice affects the formation of gelatin and if pH or temperature had an effect on enzyme activity. HYPOTHESIS: If fresh pineapple juice is added to the gelatin then it will take longer for the gelatin to form compared to the canned pineapple juice because it contain bromelian that break down gelatin proteins preventing the gelatin from forming. If fresh pineapple juice is heated then gelatin will form because temperature is going to denature the bromelian in the pineapple juice. If bromelian is exposed to different pHs then gelatin formation will be slower because pH wont prevent the enzymes from breaking down the gelatin formation. INTRODUCTION: Enzymes are biological catalyst that increases the rate of chemical reactions taking place within living cells without themselves being changed. The reactant of an enzymes-catalyzed reaction is the as a substrate (Palmer, 2001).The substrates are converted into different molecules known as products at the end of the chemical reaction. Enzymes work by lowering the activation energy of the reaction thus the rate of the reaction increases. As a result, products are formed faster. Enzymes are substrate specific, which results from the enzymes unique three dimensional shape (Understanding How Enzymes Work, p. 111). Enzymes are normally facilitated by other factors. Cofactors are normally non-organic metal that aid in the action of enzymes. Enzyme cofactors are molecules that work by altering the shape of an enzyme so as to make it participate effectively during an enzymatic reaction (Fox, 2011). Most enzymes within the body have optimal conditions at which they perform their work. The major proteinase present in pineapple is Bromelian. Bromelian breaks down proteins to their amino acid by hydrolysis (Understanding How Enzymes Work, p. 112). Gelatin is a

protein known to be obtained from collagen (Understanding How Enzymes Work, p. 112). This laboratory examined the effect pineapple has on gelatin formation and the effect of pH and temperature on enzyme activity. MATERIALS AND METHODS: Bromelian and Gelatin When testing for the effect of bromelian on the formation of gelatin, a beaker was half filled with water and placed on a hot plate before the actual experiment. An ice bath was prepared and recorded to about 37 F. After the water began to boil the temperature was decreased and two packs of gelatin are added. The solution is left on hot plate to stay warm. Next three test tubes were placed on a tube rack. Three ml of the warm gelatin was added to the three test tubes. The tubes were labeled water, fresh, and canned accordingly. Using separate syringes two ml of each substance was added to the correct tube; water in the water tube, fresh pineapple juice in the fresh tube, and canned pineapple juice in the canned tube. That step was rapidly done mixed, and placed in iced water. The water test tube was observed carefully due to the fact that it should solidify between five and ten minutes. After all three solidified it was observed and recorded. Bromelian and Temperature When testing for the effect of temperature on the rate of bromelian activity, a beaker filled half way was prepared at 70 F. Three test tubes were placed on a tube rack labeled water, cold, and hot. Two ml of water was added to the water test tube, 2ml of fresh pineapple juice added to the test tube labeled cold and hot. Next, test tubes labeled water and cold are left in room temperature. The test tube labeled hot was placed into the 70F water bath for five minutes. After time the tube was removed and 3ml of warm gelatin was added. All three test tubes where placed in the ice bath. They were taken out as soon as the tube containing water and gelatin had solidified. The tubes were than observed and recorded. Bromelian and pH When testing for the effect of pH on the rate of bromelian activity, six test tubes were placed on a test rack. In tube 1 it contained 2 ml of water, tube 2 contained 2 ml of fresh pineapple juice, tube 3 contained 1 ml of HCL and 1 ml of fresh pineapple juice, tube 4 contained 1 ml HCL and

1 ml of water, tube 5 contained 1 ml NaOH and 1 ml ml of fresh pineapple juice, and tube 6 contained 1 ml NaOH and 1 ml of water. As the substance was being poured in the test the tubes were labeled with what it contained a number to avoid any confusion. Every tube was then mixed carefully, swirling the substances, these sat for three minutes, after time 3 ml of gelatin was added to each tube. All 6 tubes were then placed in the ice bath and taken out when tube number one containing water and gelatin solidified. Each tube was observed then recorded. RESULTS: The experiment was done to determine how bromelian works. Bromelian was used to see how enzymes affected the formation of gelatin. In the first experiment, the results were water and canned pineapple juice were solidified and fresh pineapple juice was liquefied after being in the ice bath for five to ten minutes. Furthermore, in the second experiment the results were that hot and cold water were both liquefied and water became solid. In the third experiment, after testing and examining how pH would affect the production of bromelian reaction, the results was that water, HCl mixed with water, NaOH mixed with fresh pineapple juice were all solidified while fresh pineapple juice and NaOH mixed with water were both solidified with a little liquid being poured out at the top. Leaving HCl mixed with fresh pineapple juice as a liquid.

Table: 1 The Effect of Bromelian on the Formation of Jello TEST TUBE Water Fresh pineapple juice Canned pineapple juice RESULT Solidified Liquefied Liquefied

Table: 2 The Effect of Temperature on the Rate of Bromelian TEST TUBE Water Cold water Hot water RESULTS Solidified Liquefied Liquefied

Table: 3 The Effect of pH on the Rate of Bromelian TEST TUBE Water Fresh pineapple juice HCl and Fresh Pineapple Juice HCl and Water NaOH and Fresh Pineapple Juice NaOH and Water RESULT Solidified Solidified with liquid on top Liquefied Solidified Solidified Solidified with little liquid on top

DISCUSSION: In this experiment, many tests were examined to find out how the enzyme bromelian works. Boromelian is an enzyme that occurs in pineapples including the leaves and stem (Understanding How Enzymes Work, 112). The purpose of this lab was to examine how enzymes work, using bromelian and gelatin as the substrate. Temperature and pH were two factors examined to see how they affected enzyme activity. In the first experiment, water, fresh pineapple juice and canned pineapple juice were examined to see the effect it had on gelatin formation. The water and canned pineapple juice solidified quicker than the fresh pineapple juice. The water and canned pineapple juice solidified

faster because there were no enzymes present in water and in the canned pineapple juice the enzymes denatured. On the other hand, fresh pineapple juice had the bromelian enzyme which was still functional to break down the gelatin protein preventing the gelatin formation from happening (Understanding How Enzymes Work, 112). In the second experiment, water, cold water, and hot water were examined to see the effect of temperature on the rate of bromelian activity. Gelatin was added to three test tubes to see which substance would solidify the fastest. Cold water and hot water was still a liquid while water became a solid. The water solidified faster than the cold and hot water because no enzymes inside of it to prevent it from forming a gelatin formation. This shows that temperature has no effect on gelatin formation in a substance because the cold and hot water was still a liquid when gelatin was added to the test tubes. In the last experiment, five substances were examined to see the effect of pH on the rate of boromelian. Water, a mixture of HCl and water, a mixture of NaOH and fresh pineapple juice, and NaOH and water solidified faster than the fresh pineapple juice, and a mixture of HCl and pineapple juice.An acid or a base shouldnt have affected the formation of gelatin because there were no bonds being broken in the gelatin. As you can see when an acid is mixed with the gelatin, no bonds are being broken and it is easy for the gelatin setting to appear. The results were incorrect for the NaOH and fresh pineapple juice because it shouldnt have solidified. The test tube was probably left in the ice to long. REFERENCES: Fox, S. (2011). In S. Fox, Human Physiology (p. 92). New York: The Mgraw Hill Publishers. Palmer, T. (2001). An Introduction to enzymes. In T. Palmer, Enzymes: Biochemistry, Biotechnology and Clinical Chemistry (p. 3). England: Horwood Publishing Limited. (n.d.). Understanding How Enzymes Work. In Exploring Biology in The Laboratory (pp. 111112).