As Pectinase is an enzyme it is affected by the normal factors such as Temperature, PH, Inhibitors and Concentration – which is the factor I have chosen to investigate.
Hypothesis
The apple exposed to the strongest concentration of Enzyme Pectinase will produce the highest yield of apple juice of its optimum amount.
Prediction
I predict that the strongest concentrations of the enzyme pectinase added to the apple will produce a highest yield of juice and will release the apples optimum amount of juice quicker than that of a weaker concentration. I believe this because a higher concentration of enzyme means that there will be more enzymatic activity upon the apple and reactions will take place faster breaking the pectin molecules thus releasing the juice quicker and more efficiently.
Outline Method
To measure out the peeled, sliced apple into equal proportions into 5 separate beakers and add the pectinase in 5 varying concentrations. Then warm them in a 40C water bath for 20 minutes, then filter each sample and record at 2-minute intervals the amount of juice that is produced for a period of 12 minutes.
Input Variable
I am going to change the concentration of the enzyme Pectinase.
Output Variable
I will measure the volume of fruit juice produced after five-minute intervals for each type of fruit that I use. To do this I will use the scale on the 100cm measuring cylinder that the mixture is filtered into, after each interval.
Control variables
To make this a fair test, the following factors must be kept constant each time the experiment is performed:
Prelimary Experiment
To work out the concentrations of Pectinase that would be most effective to use and to test the efficiency of my planned experiment I ran a prelimary experiment. This helped to ensure the smoothness and timing of my main practical thus insuring accurate and reliable results.
Prelimary Method
Prelimary Procedure
- Peel and chop apples, weigh into 4 equal quantities. Put into 4 separate beakers.
- Measure out 4 varying 4ml concentrations by measuring in a 5ml syringe differing amounts of enzyme and distilled water and then adding them together, adding a different concentration to each beaker with apple.
- Stir each beakers contents with clean glass rods. Keeping them separate.
- Incubate the beakers in a water bath at 40C for 20 minutes.
- Filter the juice from the apple pieces, using coffee filter papers in funnels placed in measuring cylinders.
- Record the volume of juice obtained from each lot of apple at 5minute intervals.
Prelimary experiment. Observations
From conducting the Pilot experiment it enabled me to establish the concentrations of enzyme, which I believe I should use. These are 100%, 75%, 50%, 25% and 0% concentration of enzyme. In my pilot experiment however I only allowed for 4 concentrations of pectinase to be investigated excluding a 0% concentration of enzyme. I will now include a 5th sample, as a 0% concentration of enzyme is vital to the experiment to allow comparisons of with and without the enzyme pectinase making my results more reliable.
Another aspect of my experiment, which I have now decided to change following the pilot run, is that I shall record the volume of juice obtained at intervals of 2 minutes rather than 5 as I feel this will show a more accurate rate of juice production.
I was also able to distinguish the mass of apple that should be used for each experiment, 30kg. When I filtered the apple juice, I observed that the filter papers were absorbing some of the apple juice to prevent this from happening in my main experiment and from giving unreliable results I shall dampen the filter paper first with water. Distilled water must be used throughout the experiment as tap water contains impurities, which could affect the results from my experiment.
I shall now go on to conduct my main experiment.
Method
Procedure
- Peel and chop apples, weigh into 5 equal quantities 30kg. Put into 5 separate beakers.
- Measure out 5 4ml concentrations. 100% 4ml Pectinase 0ml D.Water
75% 3ml Pectinase 1ml D.Water
50% 2ml Pectinase 2ml D.Water
25% 1ml Pectinase 3ml D.Water
0% 0ml Pectinase 4ml D.Water
Add to apple 30kg quantities.
- Stir each beakers contents with clean glass rods. Keeping them separate.
- Incubate the beakers in a water bath at 40C for 20 minutes.
-
Filter the juice from the apple pieces, using coffee filter papers in funnels placed in measuring cylinders.
- Record the volume of juice obtained from each lot of apple at 2minute intervals.
This experiment should then be repeated at least 3 times so averages can be obtained and more reliable results gained.
NB: When filtering the apple juice, it is important to dampen the filter papers before as this can result in inaccurate results as some of the juice may be absorbed by the filter paper. Dampening them first should prevent this.
Risk Assessment
Pectinase – potential allergies, may irritate eyes and skin.
Knife – sharp object can cause injuries.
Juice prepared in this way should NOT be consumed. The proportion of enzyme used is far greater than that employed in commercial production, where typically 130cm3 of enzyme is added for every ton of apples.
Control measures
Eye and hand protection to be worn.
Ensure bags and tools are not an obstruction.
Take care when handling the knife, putting it in a safe place when not in use.
Remedial Measures
If incase of skin contact flush with water
If incase of eye contact flush with water
Enzyme spills to be wiped up promptly and the area to be washed with water
Return residues to Technician.
Results Tables and Graphs
Averages
Analysis
My results show that Pectinase enzyme can increase the yield of juice obtained. As I predicted the stronger concentrations of pectinase produced the most juice from the apple.
After a time span of 12 minutes the average volume of 13.00ml had been released from the apple, which had been exposed to a 100% concentration of the enzyme pectinase. An average of only 2.50ml of juice was released from the apple not exposed to any pectinase, which is on average 10.50ml less than the volume released from the apple exposed to the 100% concentration.
The trends in my results are represented in my line graphs. It is clear to see that as the enzyme concentration decreases so does the amount of juice released decrease.
I think that the extraction of fruit juice is most effective when the concentration of enzyme is high, this is because there would be more enzyme to react with the pectin molecules in the apple breaking the bonds allowing the Cellulose to move more freely thus forming a liquid – apple juice.
The rate at which the juice was being released from the apple exposed to 100% concentration of pectinase according to my results was fastest in the first two minutes. It released 1.83ml of juice in that time.
Towards the final 12 minutes of my recordings the volume of juice being released was still on a steady increase, suggesting that the enzyme is still active and reacting with the pectin breaking the bonds within the apple cell wall. However the juice released without the addition of pectinase was also still on a steady increase, but with a far lower yield of juice being released, this also links with the biological knowledge as it states that Pectinase is produced during the natural ripening process of some fruits.
I feel that my hypothesis ‘The apple exposed to the strongest concentration of Enzyme Pectinase will produce the highest yield of apple juice of its optimum amount.’ Has been proven correct, however there are further investigations that can be done to backup my hypothesis.
Evaluation
I would consider my results to be fairly reliable as I repeated my experiments 3 times in order to obtain average results. I maintained the constants considerably well and took into account what would make the experiment unfair and give unreliable results.
I feel I was successful in proving my hypothesis, and there was not any anomalies in my results However the error bars do represent some variability between my results although the most significant variables in my data is that of a 3.50 difference on the lines representing 50% and 75% concentration juice extraction rate.
One trend that I did notice to be significant is that of the variability between the results from the apple added with 0% concentration. The variability between the results is very small, with there only being a 0.5 difference between the majorities of them. I feel that this may be due to no enzyme being present thus the results are steady and stable, as we know that enzymes can be affected by several factors including Temperature, Ph and Concentrations. Taking this into account I would conclude that to improve my experiment and to obtain more reliable results with less variability the experiments concerning the enzyme should be conducted as many times as possible in strict conditions recording and maintaining the conditions and constants thoroughly.
The most significant error in my method could be the fact that I did not rinse each lot of 30 kg of apple with distilled water before I added the pectinase enzyme. This means that the juice that had already come out of the fruit before the pectinase was added was not removed. This could have caused the volume of fruit juice measured at each interval to be higher than would normally be expected and would reduce the reliability of my results.
I also found out that sometimes it was difficult to measure the volume of fruit juice in the measuring cylinder due to the fact that the scale on it was not sensitive enough. This may have resulted in inaccurate measurements being taken and may explain the variability in my results.
The temperature of the water bath fluctuated sometimes and this will have affected the activity of the pectinase enzyme, more of the pectin molecules in the cell wall may have been broken down in one experiment than in another causing a higher average result than expected.
To improve this experiment I would take the readings for each separate 30 kg of apple one at a time. This would make the results more reliable as it would be easier to measure the total volume of fruit juice in the measuring cylinder after each 2-minute interval. This is an unpractical solution when the amount of time that the experiment was allowed to take place in is considered. I would also use a measuring cylinder with a smaller scale, for example one that measures from 0 to 50 cm3. This would reduce the inaccuracies that occurred when measurements were taken. Similarly, rinsing the chopped apple with distilled water before the pectinase enzyme is added would definitely make the results more reliable. This would ensure that the juice produced was due to the action of the pectinase enzyme.
To further improve my experiment I would also ensure that the surface area of the sliced apple was the same each time, as this can also affect my results because in one sample there may be more surface area for the enzyme to attack. This should be kept as a constant.
Another error in my method is that I did not take recordings of the results over a sufficient period of time. As it says in my background information:
‘Enzyme treatment commercially takes anything from 15 minutes to 2 hours depending on the exact nature of the enzyme, the dosage rate, the reaction temperature and the variety of apple used.’ Suggesting that the time enzyme treatment takes is dependant on several factors (the factor I was investigating was concentration (dosage) of enzyme), So I could have left the experiment going for a longer time period therefore acknowledging the time on average that enzyme treatment takes concerning concentration rate, gaining more reliable and detailed results.
There are many investigations into pectinase that I could have done. For example instead of changing the concentrations of the enzyme the temperature could be changed, the ph and the variety of apple used could also be explored. As different apple types may have varying amounts of pectin, and celluloses in their cell walls and this in turn could effect the production of fruit juice.
Another investigation could be one that investigates the differences in the effects of pectinase on the yield of fruit juice from different fruits such as pears, pineapples and oranges. You could also try replacing the pectinase enzyme with cellulose to see if this has the same effect as pectinase. The two enzymes could also be mixed together to see if using cellulose enzyme to break down the molecules of cellulose in the cell wall will effectively increase the yield of fruit juice obtained from fruits such as apples, which have a high proportion of cellulose in the cell wall.
