Enzymatic activity of protease in fruit juice on gelatin and the reaction of different PH and temperatures on the bromelain within pineapple juice PDF

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Enzymatic activity of protease in fruit juice on gelatin and the reaction of different PH and temperatures on the bromelain within pineapple juice.

Introduction Enzymes are very significant for biochemical reactions, they enable reactions to speed up and provide an alternative reaction pathway. In most cases enzymes are selective, catalyzing only specific reactions (Alexander, Mathie and Peters, 2011). This selectivity is because of the shape of enzymes in molecules. For this experiment two main substances were of significance, Gelatin and Pineapple. Gelatin’s origins are from tendons, bones and ligaments in fauna. As it is from living origins it contains collagen which is a primary protein from cartilage, bones and nails. It also contains a range of amino acids which are the building blocks for the proteins these can include Lysine, Tryptophan and valine (Gelita.com, 2017). On the other hand, Pineapple from the bromeliad family is from flora origins, Pineapple is a tropical sweet fruit that is rich in manganese as well as high levels of vitamin C. In addition to this it also has high levels of the protease enzyme bromelain in the stem as well as the fruit (Salma Omar, et al. 1977). Bromelain is a protein digestive enzyme and in its clinical application, bromelain is used for its anti-inflammatory properties, to treat ulcerative colitis, removing dead and damaged skin from tissue after a serious burn and more recently been found to possibly help autoimmune conditions such as rheumatoid arthritis (Taussig and Batkin, 1988). However, when it comes to gelatin if the protease enzyme is placed with gelatin it will be degraded and will loose the ability to solidify. As bromelain has many uses globally it is important to find the optimum pH and temperature for it to work effectively (Cheung and LiChan, 2017). Other fruits that were tested in this experiment was Kiwi, Apple and orange, further research into protease shows that MEROPS database give a clear number of known protease in each fruit. Pineapple has 9, Kiwi has 6, Apple has 983 and orange has 731. This enabled us to test if the number of proteases in each fruit sample had an effect on the level of proteolytic activity (Ebi.ac.uk, 2017). It was then tested bromelains effect under different conditions which extended from pH 0 to 14 and 0 °C to 100 °C whilst it was in a gelatine mix. It also became clear that the pineapple pulp juice had a buffering capacity of around 4pH and could buffer the mixture of solution that it was added in, this was more clearly the test on the impact effect on the buffered environments.

Aim The aim of the experiment was to investigate if large numbers of protease in fruit can influence catalytic efficiency in regards to the fruits proteolytic enzymes as well as observing the effect that buffering and temperature effects the enzyme function and in aid of that determining the ideal range of pH and temperature for the enzymes to work effectively. Materials Taken for Biol1004 practical manual semester 2. National University, pg. 28 • • • • • • • •

Gelatine Test tube pipet pump with serological pipette Micropipette with disposable tips Water baths ( 40 °C, 55 °C, 70 °C, 100 °C and 0 °C) Pureed fresh fruit from apple ,kiwi, orange and pineapple) Distilled water 2M NaOH 2M HC1

Method Part A: Enzymes that digest proteins To begin all test tubes for this part of the practical were labelled from A1 to A5. Next 3.5gms of gelatin was added to a conical flask, 50 ml of boiling water was then added however 5% of the gelatin did not dissolve it was then mixed for 2:30 minutes by hand, 20 ml of cold water was then added and swirled for 10 seconds. Whilst this occurred the following juices and water, apple juice, orange juice, Kiwi juice and pineapple juice, it was noted that there was some pulp on the side of the test tubes. 10 ml of gelatin mix was then added to all test tubes, it is important to note that as it was done one by one A1 had the longest to set and A5 had the shortest duration. They were removed after 10:35 minutes Part B: The effect of temperature on enzyme function Test tubes were labeled B1 to B5 then 3ml of pineapple juice was added to each of the test tubes. Once completed the test tubes were placed in the following locations - B1 to 0 °C ice box - B2 left at room temperature at 22°C - B3 to 40 °C water bath - B4 to 70 °C water bath - B5 to 100 °C water bath Each test tube was left in place for 11 minutes. Whilst the pineapple juice sat, the gelatin mixture was prepared as it was in part A. Following this and once the test tubes were removed from there individual conditions, 10 ml of the gelatin mixture was added to each test tube then mixed well however 3% did not mix. Following this the test tubes were placed in icebox for 25 minutes.

Part C : The effect of PH on enzyme function Test tubes were labelled from P1 to P5 then 4.5 ml of distilled water was added to each tube and then 0.5 of 2M HC1 was added to test tube p1. Then a 10 fold serial dilution followed in the rest of the test tubes. Then more test tubes were labelled as C1 to C6, enzyme essays was then prepared by adding 3ml of pineapple juice to the C1 to C6 test times. Following this 3ml of diluted HC1 was then added to each of the tubes C1- C6 respectively, each was then inversed to mix. 10ml of gelatin was then added to each tube C1 to C6 then each was put in icebox for 10 minutes.

Results Part A : Enzymes that digest proteins Kind of fruit juice Solid/ Liquid state Water Solid Apple juice Solid ( bubbles at top) Orange Solid Kiwi Liquid ( pulp had settled) Pineapple Liquid ( pulp had settled) Table 1: Effect on different fruit juices on gelatin To understand how proteolytic activity effects gelatin, different fruit juice pulps were added to a gelatin mixture to investigate each reaction. tube A1 which contained water evidently did not have any protease therefore it did not have an effect on the gelation within the gelatin, this enabled it to set solid. Tube A2( apple) and A3(orange) set completely which in an indication that there was a lower level of proteolytic activity of the protease therefore caused little disruption the the setting process within the gelatin. Finally A4 and A 5 containing Kiwi juice and Pineapple juice had high levels of proteolytic activity of the protease and through this the gelatin was unable to set and remained in a liquid state.

Part B: The effect of temperature on enzyme function Temperature 0 °C

Solid/ Liquid content Liquid

22 °C Liquid 40°C Liquid 70 °C Solid 100 °C solid Table 2. Effect different temperatures have on the activity of bromelain To investigate the effects of different temperatures on bromelain (enzymatic activity). Each sample of pineapple juice which contains bromelain was put into different temperatures as shown in table above. From these results it is clear that only when the pineapple juice was heated above 70 °C was the level of proteolytic activity low enough to form into a solid. The

other samples that were kept at lower temperatures all had a similar response and stayed liquid.

Part C: The effect of PH on enzyme function Tube

C1 C2 C3 C4 C5 C6 D1 D2 D3 D4 D5 D6

Expected pH after pH after adding pineapple juice after adding 2M NaOH and adding pineapple juice 2M HCI 0 0 1 3 2 3 3 3 4 3 7 3 14 10 13 3 12 3 11 3 10 3 7 3 Table 3: pH and the buffering effect of pineapple juice.

Solid/ liquid content

Solid ( partially set) Liquid Liquid Liquid Liquid Liquid Solid Liquid Liquid Liquid Liquid Liquid

After pineapple juice was added in each sample, the pH became 3 in all samples except C1 which had a pH of 0 and D1 which had a pH of 10. C1 and D1 had solid gelatin mixtures which is an indication that there is a lack of bromelain present within. C2-C6 and D2-D6 all had a stable pH of 3 and also remained in a liquid form, this is an indication that there is proteolysis as a result by bromelain which disabled the gelation from forming into a solid. This shows a significant buffering action of the pH. The results present in table 4 as well as table 3 indicated there is a relationship between the levels of enzymatic activity and pH.

Discussion This experiment determined if the number and intensity of protease in fruit (fruit juice pulp) can have an influence on catalytic efficiency of proteolytic enzymes. This effects were observed by changes in temperature and pH and from this an optimal range for the enzyme was investigated. Each individual part of the experiment (A,B and C) all had important roles to play in further developing knowledge on this. In part A it was observed that the efficiency of proteolytic enzymes within the fruit juices had a denaturing effect on the proteins within the gelatin. The highest levels of enzymatic activity of the protease was present in only two of the juices (Pineapple and Kiwi) as it remained in a liquid state. This did not only support the hypothesis for the experiment but it also supported a study conducted in 2011 by Ji-Hye Koak that suggested that fruits such as Kiwi’s have higher levels of enzymatic activity when compared to other fruits such as apples and orange (Koak et al., 2011). Results from part A can be found in table 1. In part B, the effect of different ranges of temperatures on the enzyme function was tested. The temperatures tested were 0°C, 22°C, 40°C,70°C and 100°C, this was tested with a mixture of gelatine with bromelain present. The results concluded that once it had reached 70° Celsius the fresh pineapple’s enzyme bromelain had become inacvtive with the two test tubes over 70° Celsius solidifying. This is supported by other wide spread experiments that have yielded the same results, in particular a journal article by Adu-Amanuka states” Incubation at 40°C showed no loss of fruit bromelain activity… whereas at 50°C almost 83% of activity remained. Incubation at 80°C for 8 min caused almost complete activity loss(AduAmankwa, 1997). Results from part B can be found in table 2.

From part C, only the proteolytic enzymes at a pH of 10 (D1) and a pH of 0 (tube C1) had the ability to solidify. Through the process of a 10-fold dilution each test tube should have had only 1pH unit between them (Biology.kenyon.edu, 2017). Furthermore, it was evident that the pineapple juice had a buffering effect and altered the pH of the solution to pH 3 rather than the range of pH which was expected, these included test tube C2-C6 as well as D2 to D6. The test tubes with a pH of 3 did not set and remained as a liquid, from this it can be inferred that pineapple had a buffer range of around 4.1 can act as a buffer to the environments it was added in to and works optimally within the buffer range limits (Corzo, Waliszewski and Welti-Chanes, 2012). Results from part C can be found in table 3. In conclusion, the aim of the experiment was successfully completed due to the fact that Part A and table 1 shows that only pineapple and kiwi only have enough protease to effectively digest proteins within the gelatin. Part B and table 2 indicated the ideal temperature range between 0°C and 70°C and then inactive after that. Finally, in part C and table 3 it is evident

that pineapple juice has the ability to be a buffer in pH environments between pH 2 to 13, in addition it is also evident that bromelain is unable to successfully work at a pH of 0 and 10. Furthermore pineapple has intensive proteolytic levels of the enzyme bromelain that cannot extend past the range of 0°C and 70°C and needs to be within a buffer range to work effectively.

Acknowledgements Lixinyu liu u6132930 Yashan Jiang u6381166 Cathy Gao

References Adu-Amankwa, P. (1997). EFFECT OF TIME OF HARVEST ON THE FRUIT QUALITY CHARACTERISTICS OF PINEAPPLE CV 'SMOOTH CAYENNE' IN TWO AREAS OF SOUTHERN GHANA. Acta Horticulturae, (425), pp.531-538. Alexander, S., Mathie, A. and Peters, J. (2011). ENZYMES. British Journal of Pharmacology, 164, pp.S279-S324. Biology.kenyon.edu. (2017). serial dilution. [online] Available at: http://biology.kenyon.edu/courses/biol09/tetrahymena/serialdilution2.htm [Accessed 15 Aug. 2017]. Cheung, I. and Li-Chan, E. (2017). Enzymatic production of protein hydrolysates from steelhead (Oncorhynchus mykiss) skin gelatin as inhibitors of dipeptidyl-peptidase IV and angiotensin-I converting enzyme. Journal of Functional Foods, 28, pp.254-264. Corzo, C., Waliszewski, K. and Welti-Chanes, J. (2012). Pineapple fruit bromelain affinity to different protein substrates. Food Chemistry, 133(3), pp.631-635. Ebi.ac.uk. (2017). MEROPS - the Peptidase Database. [online] Available at: https://www.ebi.ac.uk/merops/ [Accessed 15 Aug. 2017]. Gelita.com. (2017). What is gelatine? | GELITA. [online] Available at: https://www.gelita.com/en/knowledge/gelatine/what-is-gelatine [Accessed 15 Aug. 2017]. Koak, J., Kim, H., Choi, Y., Baik, M. and Kim, B. (2011). Characterization of a protease from over-matured fruits and development of a tenderizer using an optimization technique. Food Science and Biotechnology, 20(2), pp.485-490. Salma Omar., Ahmad Zaharudin Idrus. and Omar Abdul Razak. (1977). Extraction and activity of bromelain from pineapple. Serdang: Mardi. Sayre, R. (1972). The Enzyme Bromelin. The American Biology Teacher, 34(8), pp.449-451.

Taussig, S. and Batkin, S. (1988). Bromelain, the enzyme complex of pineapple (Ananas comosus) and its clinical application. An update. Journal of Ethnopharmacology, 22(2), pp.191-203.

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