This experiment proves how key factor three (the temperature) really does affect the activity of Chymosin and how the enzyme works at particular temperatures.
According to the kinetic theory, when the temperature of the surroundings increases, the molecules will move faster and the rate at which the product is made will also increase because the enzyme and substrate molecules meet more often.
Above 40 degrees Celsius the rate of reaction stops rising, and even though the enzymes might be causing a reaction to occur, they are doing it at a slower rate.
At 60oC the temperature becomes too high and the ionic bonds that hold the enzyme in shape, break. When this happens it is said that the enzyme is denatured. Once an enzyme has been denatured, it cannot be reversed.
Hypothesis
I predict that the mixture being held at room temperature will not react at all, or if it does the change will too slight to notice. My reason for that prediction is that there will not be sufficient heat to supply the molecules with kinetic energy. In my opinion no change will occur.
I think that the test tubes being kept at thirty degrees Celsius will change slightly, but I still don’t think there will be enough heat for the milk to coagulate. However, I’m certain the contents of the test tubes held at 40o C and 50oC will react considerably. I think that the mixture in the 50o C water bath will react faster than that in the 40oC bath, simply because there is more kinetic energy.
I think the tests at 60oC will not show any major change in the thickness and texture of the milk. The heat will be too strong, and cause the enzymes to denature, therefore giving no reaction.
Assuming that they are similar to the actual results of the experiment, these predictions tell me that the line graph of the results would resemble a curve with a high peak and very steep sides.
Planning – Pilot test
As part of our preparation, a pilot test was carried out to decide upon the final arrangements of the main experiment.
Five different temperatures were decided to test the milk and rennet: room temperature, 30, 40, 50, and 60 degrees Celsius. There is nothing higher than 60oC because any higher and the enzymes would almost certainly be denatured. There is also nothing below room temperature such as 10oC or 0oC because there would be no kinetic energy to activate the Chymosin (Rennin). It would be acceptable to test at those temperatures if the purpose of the test were to find methods of preserving the milk, but we just want to know which conditions the Rennet works best in, and at 0oC the milk would freeze, and stop any reaction occurring at all.
Water baths are being used to ensure the test tubes are kept at the right temperature for the duration of the experiment.
For the pilot test, the amount of milk used in each tube was 5cm3, and the amount of rennet used was 2cm3. For the main experiment the ratio of milk to rennet was decided upon as 8:2, therefore in each test tube there would be 8cm3 of milk, and 2cm3 of Rennet. This is a much simpler ratio which is easy to remember and saves any confusion whilst measuring out the quantities. It also uses more milk because I feared that the concentration of rennet with just 5cm3 of milk was too high.
Different people could perceive that the milk was clotted at one point, when another might disagree that it had not completely coagulated yet. For this reason, my partner and I agreed that we should stop the stopwatch when the milk was so clotted that it wouldn’t run out of the test tube when turned upside down. If it did, then it would not be quite ready, and we would wait for longer until it stayed at the bottom of the test tube, just as a jellied substance would.
It was also decided that for each temperature there would be one test and at least 2 repeats, calculating a total of 15 tests. It would have been preferable if a higher number of repeats could be carried out to ensure that our results were as correct as they could be. However we decided that perhaps this would not be the best idea as we found ourselves rushing to complete the experiment with just the two repeats. The period within which we wanted to complete the experiment was only one hour and ten minutes long. By rushing the experiments to fit in more repeats, it could cause us to make errors and misjudgements, therefore making our results unreliable.
It is important that the rennet and the milk do not mix prior to the point they should, which is when both are at the correct temperature. To make sure they don’t mix, when measuring the milk out, it cannot be put into a tube that has just contained rennet and vice versa. The test tubes must either be thoroughly washed before using them again, or fresh tubes must be used each time.
Graduated pipettes are used to measure out the milk and the rennet, because it is very important that the measures are exact, and I think that the pipettes are the most accurate way to do it. I am going to be as careful as I can and check that as much of the milk that possibly can be squeezed out of the pipette is removed, because it is also important that one measure doesn’t have slightly more than another.
When the test tubes are removed from the water bath to be inspected for any reaction that may have occurred, it will be done very quickly. It must be done quickly because as soon as the tube leaves the water, it will begin to cool which could slow down the reaction. Another reason to be as quick as possible is that the stopwatch will continue to time the reaction, even whilst the tubes are removed from the bath. If the tubes are out of the bath for any reasonable length of time then the times we have recorded for the tubes being inside the bath would be incorrect.
It is just as important that we are careful as well as quick. Rushing to check the mixtures could mean that some splashes out of the tube.
As it was believed that there would be no or little reaction at certain temperatures, there has to be a limit as to how long their progress should be monitored. If there was no reaction occurring after fifteen minutes, then the stopwatch will be stopped and the test confirmed as to have given “no reaction”.
The pilot test itself proved to be quite successful and a good practice for the real thing. . There was a reaction for all of the mixtures maintained at thirty, forty, and fifty degrees Celsius. However no reaction occurred at room temperature, or at sixty degrees Celsius. It came as a surprise to me that there was such a good reaction at 30oC seeing as it was quite close to the room temperature of 24oC which had no reaction whatsoever.
I did find it quite challenging to complete all of tests in the given time, and perhaps some of the times I recorded weren’t as accurate as they could have been because there were several tests taking place at the same time in different water baths. For the main experiment, the water baths will be situated closer together so that it is easier to move from one to the other to check on the reactions. I know now that either my partner or myself must check all of the tubes at least every minute. If not then the milk in one tube may have clotted whilst we are attending to another, and the times that we record will be wrong.
I am able to arrange my time more carefully in the main practical because I now have a good understanding of which temperatures are going to be causing the reactions and after what approximate time.
After the experiment is completed and all of the results are collected, I am going to draw up a results table clearly showing the results of the original tests and their repeats. I will then draw a line graph to show the relationship between the temperature and the time taken to react. It will also show which temperature the enzyme worked best in and gave the quickest reaction.
Finally I will draw a curve of the rate of reaction against the temperature. By drawing a rate of reaction curve it will show exactly which temperatures the Chymosin had the highest rate of reaction, which is different to the time taken to react. A curve for temperature against time can be quite misleading because one might look at it and automatically assume that the highest point was the quickest reaction, when in fact it’s the opposite. To react at the quickest speed would mean that the reaction would have taken the shortest time and therefore be the lowest point of the curve not the highest. By drawing a rate of reaction curve, it should give a positive correlation, and then it is more obvious at a glance which temperature gave the quickest reaction.
Apparatus
The apparatus used in both the pilot test and the main experiment was as follows:
- Stopwatches
- Thirty test tubes (Or fifteen that were thoroughly washed and used again)
- Four water baths (the fifth test was done in room temperature)
- Graduated pipettes
- Full fat milk
- Rennet
- Test tube stands
- A Black wax pencil
- Thermometers
- Glass beakers
Method
The first things to be done were to tie back the hair and put on the lab coats. The apparatus was collected and arranged on the laboratory benches, and a notebook was also available to write down the exact times the reactions took, and any problems that occurred.
The water baths were switched on and set to the different temperatures. A large measure of the whole milk was poured into a glass beaker straight its container. From there, 8cm3 was removed using a graduated pipette and then located in a test tube. This process occurred three times so that there were three test tubes standing in the rack ready for the room temperature experiment. The room temperature was recorded by the thermometer as being 23oC.
2cm3 of rennet was measured out and then poured into one of the test tubes containing milk. As soon as the rennet reached the milk it was given a quick stir by a thermometer and the stopwatch was activated.
Using the black wax pencil, the test tube rack was labelled with “room temp test” so that those tubes would not be confused with any of the others. Rather than waiting for the first test to finish, the two repeats began soon after using two additional stopwatches to measure the time. After fifteen minutes, no reaction occurred, and the negative result was recorded.
Whilst the test at room temperature was taking place, several more measurements of both milk and rennet were put into test tubes and stood in the rack. Three test tubes containing milk and three containing rennet were then carried over to the water bath, which was heated to a temperature of 30oC. Each had a thermometer stood in it, and once they had all risen to 30oC they were ready to be mixed together. The rennet inside one tube was then carefully poured into another tube carrying milk, and the stopwatch was activated. The repeats at this temperature began shortly after the first test, and the results were recorded.
This method was repeated at the three remaining water baths in exactly the same way. The test tubes were washed thoroughly when some more were needed. Once the entire experiment was completed, and all of the results were recorded, any other dirty equipment was washed too, and the benches were cleared of any apparatus.
Results – Table
Results- Graph Number 1
Graph Number 2
-The rate of reaction is the reciprocal of the time taken. The mean time was taken and divided by a thousand each time to find it. The rate is how fast a reaction takes place.
Analysis
At room temperature and 30oC, the temperature was obviously not high enough to stimulate the kinetic energy of the molecules, because there was no reaction. These results proved to be completely different to those from the pilot test, which showed a reaction from the 30oC test as well as a reaction from the other two. The only difference in the milk after the fifteen minutes when it was declared that no reaction had occurred was that it seemed to be a little thicker, but there was no change in appearance at all.
It was apparent that no reaction would occur at room temperature soon after the test began. The liquid did not change in appearance at all, and didn’t seem to be thicker either. It was checked regularly, but nothing happened, so the timer was stopped after fifteen minutes for all three tests. The only difference I could say there was, was that the mixture seemed a little more watered down, but I can only assume that was because the rennet is not as thick a liquid as the milk.
Conclusion
The final results showed that there was no reaction in three out of the five experiments. The mixture of the milk and Rennet only coagulated at 40oC and 50oC. At room temperature and 30oC, no reaction occurred. However, the tests in the 30oC water bath did show a slightly thicker mixture even though the milk did not clot. The fastest time for the milk to clot was at 50 degrees Celsius.
Evaluation
Although a lot of planning and thought went into the experiment to make sure it was as fair a test as possible, there are still ways of improving the method, and things that I would do differently if I was doing the same experiment again.
The fact that the results differed quite noticeably from those of the pilot test is a problem that needs to be answered. I think the reason that the mixture in the 30oC water bath clotted in the pilot test but not in the main experiment is because the concentration of rennet was so much higher. It was at a ratio of 5:2 rather than 8:2, which means that for every five parts of milk there were 2 parts of rennet which is a high concentration. This means that the rennet must be capable of causing a reaction at 30oC, but only with sufficient concentration of the enzyme. The only other thing I can think of to explain it is that the rennet solution had been interfered with prior to the test and diluted somewhat, but that is very unlikely as it was taken straight from the bottles in the science laboratory.
If I were to repeat this experiment then I would take tests of more temperatures but all around the same area. The area that I’m not sure about it the 30oC area, so perhaps I would do the same test at 28oC, 30oC, 32oC, 34oC and 36oC and take notice of the changes occurring between those temperatures. This would help me to understand what temperature it is that the Chymosin starts to curdle the milk, because at the moment I only have a very vague idea. All I know is that it must be somewhere above 23oC and below a temperature around 35oC because by the time the temperature has reached 40oC the reaction is reasonably fast and could even be slowing down.
I was unsure before the experiment whether the two repeats would be enough to show exactly which results were correct and which ones may have been incorrect. There was no real need to worry about this as the curve for temperature against time shows that there are no particular results that stand out, they are all in a similar position. This is a good result because it shows that all the repeats took approximately the same amount of time to react and were therefore quite accurate.
The curve for the rate of reaction also turned out quite well and showed a good positive correlation which is what I had hoped for.
Using the graduated pipettes did make the measuring of the milk and rennet easier than just pouring into a measuring beaker. However, the difficult was squeezing all of its contents into the tube. The milk in particular would stick to the sides, and when the pipette was pinched at the top to try and force out the last drops, bubbles of air appeared, and the milk remained on the sides. There was no way of getting every last bit of the milk out of the pipette, but my partner and I tried our best to persevere for as long as we could just waiting for it to drip out on its own accord. Next time I need to use the graduated pipette, I’ll practice beforehand and try to find a way of removing as much of the liquid as possible.
My hypothesis turned out to be reasonably accurate, and in the main practical the milk clotted at the same temperatures as I predicted it would. I did think that there would be no reaction at 30oC because I assumed it would be too low a temperature. This was a correct prediction in as much as it matched the final results, but it did not match the results of the pilot test, which had a higher concentration of rennet.