Enzymatic Functioning in Bromelain from Pineapple Juice

Enzymatic process in Bromelain from Pineapple JuiceWilliam NguyenIntroductionAn enzyme is a biological qualityicle accelerator that accelerates special chemical reactions. It converts substrates into specific products. Most enzymes are proteins and hence diagnose a tether-dimensional structure of folded polypeptide chains. Enzymes afford an optimal temperature and pH consort in which they function most yieldively. If varietys occur that remove the enzyme stunned of this shed it whitethorn non function effectively. This principle forms the basis of this essayal program where the increasing temperature results in the denaturing of the enzymes rig in bromelain and thus lowering its catalytic effectiveness.Bromelain is a protein extract present in the fruit, leaves and stems of genus Ananas plants (Ananas comosus). Although it is present in each(prenominal) fractures of the ananas plant, the stem is the most bromelain rich dismantle that similarly makes it the mo st common commercial source of bromelain (Rowan, Buttle Barrett, 1990, p.869-875)1.The crude sedimentary extract from the genus Ananas plant fruit is known as bromelain. It is in concomitant a commixture of different thiolendopeptidases and otherwise components like phosphatases, glucosidases, peroxidases, cellulases, glycoproteins, carbohydrates and protease inhibitors (Maurer, 2001, p.1234-1245)2. It is also known that the enzymes in the bromelain extract begin to change when instigate everywhere 80C (Jutamongkon Charoerein, 2010, p943-948)3. Since bromelain is essentially a mixture composed of umteen different enzymes, its substrate spectrum is very broad. It extends from synthetic low molecular amides and dipeptides up to high molecular substrates such as fibrin, gelatin, matterin and bradykinin (Cooreman, 1978, p.107-121)4. Pineapples are an slight source of the trace element manganese which acts as an essential cofactor for umteen of the enzymes open in brome lain. In addition, thiamine also acts as a cofactor in enzymatic reactions central to energy production in bromelain (Joy, 2010, p.670-686)5.Historically, bromelain has been extracted and utilize for medicinal purposes by the natives of South and Central America. In modern society, bromelain has been proved to be effective at tenderising meat and as such, is sold commercially as powdered meat tende upraisers. It has also earned universal acceptableness as a phytotherapeutical drug because of its history of safe use and no side effects. In 1972, conclusive evidence that bromelain prevents aggregation of blood thrombocytes was account by Heinicke et al (1972, p.844-845)6. Bromelain also displays an anti-inflammatory and analgesic effect on soft tissue injuries especially in rheumatoid arthritis and osteoarthritis. It has also been found to dramatically reduce post-operative swelling and pain due to these properties (Leipner, Iten Saller, 2001, p.779-789)7. Additionally, bromelain applied locally as a cream in a lipid swinish has shown beneficial characteristics in the debridement of eschar from severe burns and in the acceleration of healing. It has been commercially approved in Europe and sold chthonic the trade name NexoBrid (Hauck, Chang Klein, 1983, p.124-134)8.Aim and HypothesisThis audition aims to qualitatively identify the enzyme mathematical process found in commercially sold can pineapple succus exploitation background information provided by previous experiments. The rate at which gelatin is degraded in this experiment by the bromelain provides an indication of the rate of enzyme activity. The recent pineapple adjudicate is known to be rich in bromelain and as such, will hypothetically result in a significant kernel of gelatin degradation. Boiling the pineapple succus sample over 80C will result in the denaturing of enzymes and should display little to no degradation of the gelatin. The canned sample in question whitethorn not display any degradation as it has undergone processing that may assume disturbed the natural functioning of the enzymes in bromelain.Materials15 essay supplys12 petri dishes3 experiment tube racks3 metal tongs3 plastic bottles1 thermometer1 timer1 macropipette1 pale yellowHot irrigate baths (35oC, 45oC, 50oC, 60oC and 90-100oC)1 stirring rod1 shave1 ruler1 fine black marker1 water bath1 electronic balance1 pineapple1 pineapple juice bottle100g gelatin powderMethods fragmentise A Determining the period of enzymatic functioning in canned pineapple juicePreparation phase/Pre-labPlace gelatin and live water into 250mL jug and mix with stirring rod until gelatin has change statePour equal amounts into 12 label petri dishes and place in the placid room to raise for a dayMake 3 pits in each dish by extracting gelatin using a straw when setting has complete constitute, measure and prove the diameter of the pit do using a black marker on to the bottom of a petri dishRepeat step 4 f or all petri dishesBuy a can of pineapple juice and extract 15mL of the juice into a plastic bottle labelled canned pineapple juicePurchase a rattling pineapple and cut into slices usingJuice all pineapple slices and boil half the juice collectedPlace 15mL of boiled juice in a bottle labelled boiled pineapple juice and 15mL into a bottle labelled spanking pineapple juiceproteolytic gelation phaseDivide the gelatin-petri dish samples up into 4 gatherings realise, boiled, canned and fresh. start the controlled petri dish absent of pineapple juicePipette 3 drops of the labelled juices into corresponding petri dishes. Allocate one team member per unsettled quantity group to minimise qualitative discrepanciesCover the petri dishes and allow reactions to occur for 120 minutes at room temperaturePost-proteolytic gelation phaseRemove cover from petri dishes and describe/ track record physical differences of each group.Tip any remaining juices out of the petri dishesDraw new circles a round the post-liquid pits, measure and record the new diameters into results table function B Measuring the effect of temperature on enzymatic functioning in bromelainPreparation phase/Pre-labPlace gelatin and hot water into 250mL beaker and mix with stirring rod until gelatin has dissolved take on the gelatin to set in the cool room for a dayCut out five 1.0 x 1.0cm third powers of gelatin using a razor markAllocate each cube a number from 1-10Weigh and record the saddle of each cube into the results tableProteolytic gelation phase trail five test tubes from 1-5 with a black markerPlace 10mL of fresh pineapple juice into each test tubeHeat test tube 1 using a water bath heated at 35oC for 10 minutesRemove the test tube from the water bath, using tongs, and place into test tube rackAllow the test tube to cool charge to room temperature by using a thermometerRepeat steps 3-5 using test tubes 2-5 at temperatures 45oC, 50oC, 60oC and 90oC. Placing test tubes in a bucket of crushed ice may quicken the cool processPlace allocated cubes of gelatin into respective tubes and allow to sit for 20 minutesSeparate juice from the gelatin cubes and rinse with distilled waterAllow gelatin cubes to completely dryWeigh the gelatin cubes and record variances in results tableResults gameboard 1. Degree of enzymatic functioning in various(a) types of pineapple juice shelve 1 depicts data taken from part A of the experiment. Three trials were run for each type of juice to secure reliable results. It is apparent that there is a oversized change in the diameter of the fresh pineapple juice samples compared to the other types tested. Whilst canned pineapple juice displayed a lower floor of change, the boiled sample thus far seemingly gained gelatin. The control sample did not display any changes in the diameter of the pit. melt down of a function 1. Step 1 of the proteolytic gelation phase (Part A)Figure 2. Step 3 of the proteolytic gelation phase (Part A) preserve Observa tionsMany circulate bubbles were formed during the pipetting of juices into the gelatin pitsThe diameter of the gelatin pits appeared to remain the same after(prenominal)ward a period of 120 minutes for canned, control and boiled pineapple juiceVisible changes in hole size observed in fresh pineapple juice samplesTable 2. Effect of temperature on enzymatic functioning in bromelainTable 2 depicts data taken from part B of the experiment. Like part A, three trials were made for each variable to ensure reliable data. As temperature applied to the juice increases, it is evident that the degree of weight deviation of the gelatin decrease. However, test tube 2 displayed a result that deviated from this trim down as the weight loss (%) dropped then bloom once again. The control sample displayed the highest amount of weight loss of the gelatin cube. The course of study (not inclusive of the control) is represented in the figure below.Figure 3. Effect of temperature on enzymatic fun ctioning in bromelainFigure 4. Step 3 of proteolytic gelation phase (Part B)Figure 5. Step 4 of proteolytic gelation phase (Part B)Recorded ObservationsFresh pineapple juice retained a cloudy, yellow food coloring with froth formation on the surface of the juiceDuring the cooling period, the pineapple pulp separates from the liquid and forms two distinct layersGelatin cubes that were dropped into the test tubes rise to the top of the solution with pulp sediments resting on the bottom of the test tubeDiscussionPart AIt was supported by the results in Table 1 that fresh pineapple juice displayed a larger degree of enzymatic functioning compared to the other three sample types (boiled, canned and control). This is pictured through the change in the diameter of the gelatin pit. Enzymes in bromelain get to to break down the gelatin and the larger the pit, the greater the functioning of those enzymes. The fresh pineapple juice sample showed an 18.18% change in the diameter of the gelati n pit.The canned pineapple juice had a lower degree of change (5.49%). This may have stemmed from the preparation and processing of the juice that may have altered the integrity of the bromelain in the juice. It may have been cognitive content to heating system to sterilise the juice that may have caused the denaturing of the enzymes obligated for the dislocation of gelatin.The boiled pineapple juice presented data that goes against the principle of bromelain breaking down gelatin. With a change in diameter of -1.18%, it has actually gained gelatin after the proteolytic gelation phase. Since it is known that bromelain is trustworthy tho for the degradation of gelatin, it is impossible for it to let on gelatin. Thus, this miscalculation may only have resulted from human error in marking and measuring the diameter. The marker used in this specific experiment produced rather thick lines relative to the small diameters that were measured. Use of a better tipped marker may have m inimised the chances of such inaccuracies to occur.The control sample involved a set of gelatin plates with no pineapple juice added. This was made to ensure that the pineapple juice was responsible for the degradation of the gelatin. If the control displayed a change in the diameter of the gelatin pits, then the experiment would be deemed invalid as more than one factor contributes to the degradation of gelatin besides bromelain. In accordance to the results, the control group displayed no changes to the gelatin pits.Part BPart B of the experiment explores the effect of temperature on the enzymatic functioning of bromelain. From the data recorded in Table 2, an increase in temperature applied to the juice corresponds to the decrease in the functioning of the enzymes as depicted through the amount of weight the gelatin cube lost(p). This trend is illustrated in Figure 1.Whilst there is an overall decrease in weight loss associated with increased temperature, there is an outlier at 45oC. Although this variable was retell thrice, it still remained inconsistent relative to the data set. Such an error may have occurred with the apparatus provided on the day of the experiment where there were only three water baths provided (37oC, 50oC and 90oC). The experiment design took into account the equipment available that was provided in the laboratory manual that offered water baths at 35oC, 45oC, 50oC, 60oC and 90oC. Hence, maintaining the pineapple juice at 45oC and 60oC using the apparatus provided was the most probable cause of the error. In the case of the 45oC variable, it may have been heated too strongly that resulted in the denaturing of legion(predicate) more enzymes compared to at 50oC and 60oC. To improve upon this error, water baths with temperatures corresponding to those give in the laboratory manual should be provided to ensure accurate quantitative data.As mentioned previously in the introduction, it is known from past experiments that the enzymes in bromelain begin to denature very quickly at 80oC (Jutamongkon Charoerein, 2010, p943-948)3. This experiment confirms the statement as the 90oC variable experienced a steep decline in weight lost that indicates a large degree of enzyme denaturing.Naturally, most enzymes have a temperature range that it functions most effectively at. However, this graph only portrays temperatures 35oC, not covering a large range of temperatures that is able to point out the optimum temperature. Again, this was not able to be accounted for in the experimental design due to the hold in apparatus provided. From the trend line in Figure 1, a unidimensional decrease is shown. However, a bell curve is usually representative of the optimum temperature where very low temperatures result in decreased enzyme functioning and decreased weight loss. As the temperature increases the enzyme reaches its optimum level of functioning and results in a large amount of gelatin degradation. With temperature reaching hi gher levels, the enzymes begin to denature and cease to function which brings the levels of weight loss down again. Relative to this experiment, the optimum temperature is ideally oC. In addition, the control displayed the greatest amount of weight loss compared to all the variables that were tested (36.14%). The control was not applied any heat but rather subject to room temperature which is 20oC. This reinforces the notion that the optimum temperature of the enzymes responsible for the breakdown of gelatin is oC. To validate this statement, the experiment should be repeated again but with a wider range of temperatures to account for the bell curve trend that should occur to identify the optimum temperature.Reflective CommentsThis experiment on bromelain was performed by three psyches including myself. The first part of this assessment task was performed individually as per instructions. After we completed this task, we got in concert and planned the experimental design. Each some body was assigned a role in this activity where I was tasked with end the risk assessment and drawing scientific diagrams. The other two collaborators were responsible for completing the methodology and the list of materials and equipment. When we had completed our tasks, we discussed each part together to come to a mutual agreement. Many modifications were made to all move that were essential to keeping our experimental design valid. Designing this experimental protocol with my classmates was a pleasant experience as each one of us were committed and contributed to the project. During this part of the project, no significant difficulties were faced as we all worked through our tasks smoothly. To me, teamwork was already a notion that I valued strongly, heretofore this activity of getting together and completing such a large amount of work in so little time has definitely set that notion in stone.The barbel to allocating tasks to different members was also through on the day o f the experiment. An action plan was formulated with all the roles and clock of the tasks that have been allocated and given to each member of the group. A great resolve for doing this is so that we can minimise human error in repetitious tasks. For example, I had a role in heating the juices in the given hot water baths. If two of us were to heat it, one may have a different way of regulating the temperatures 45oC and 60oC (the temperatures not given) than the other member. Hence, we assigned such roles to be consistent with the method. It also maximises productivity so that all of us are working rather than one person work with the other looking on to see if they can help.The specific tasks that I have been allocated on this day was majority of Part B of the experiment. I was tasked with cutting the gelatin cubes, weighing and recording their weights, pipetting of juices into test tubes and heating the juices. After gelatin cubes have been dropped in and timed by other member, I removed, washed and dried the gelatin for the weighing process. In among my tasks where I had time I lent my assistance to my team members. At the end of the experiment we packed up the equipment and cleaned the lab bench so that we can sit down and discuss our results. The only minor line experienced in this teamwork was the action plan. Some of the times allocated to specific tasks were under or overestimated. Although this is not necessarily a problem as we were provided three hours to conduct our experiment, it resulted in the clumping up of tasks where one member waited for another to give the sack their task to continue his/her own.Overall, I am very happy and cheerful with the outcomes of our team project (possibly the best team project I have ever had). With minimal difficulties and great results, I would definitely proceed to approach teamwork like this in future projects. However, this method of teamwork is only suitable if all team members are committed to their ro les. If one does not pull their weight and not complete their allocated roles, the group could fall apart. Hence, it would be best for me to pay back myself a group of dedicated individuals in future projects.ReferencesRowan AD, Buttle DJ Barrett AJ, 1990. The cysteine proteinases of the pineapple plant, Biochem J, Volume 226, pp.869-875.2 Maurer HR, 2001. Bromelain Biochemistry, pharmacology and medicinal use, CMLS Cell gram molecule Life Sci, Volume 58, pp.1234-1245.3 Jutamongkon R Charoerein S, 2010. Effect of Temperature on the constancy of Fruit Bromelain from Smooth Cayenne Pineapple, Kasetsart J, (Nat. Sci.), Volume 44, pp.943-948.4 Cooreman W, 1978. Bromelain, In pharmaceutical enzyme Properties and Assay Methods, R Ruyssen and A Lauwers (Eds), E Story Scienta Scientific make Co. Gent/Belgium, pp.107-121.5 Joy PP, 2010. Benefits and uses of pineapple, Pineapple Research Station (Kerala untaught University), Vazhakulam, pp.670-686.6 Heinicke RM, Van der Wal M and Yok oyama MM, 1972. Effect of bromelain on human platelet aggregation, Experientia, Volume 28, pp.844-845.7 Leipner J, Iten F and Saller R, Therapy with proteolytic enzymes in rheumatic disorders, Biol Drugs, 2001, 15 (12), pp.779-789.8 Hauck JC, Chang CM and Klein G, 1983. Isolation of an effective debriding agent from the stems of pineapple plants, Int J Tissue React, Volume 5, pp.125-134.