Factors Which Will Affect the Experiment
1. Temperature of Milk - Like most chemical reactions, the rate of an enzyme-catalyzed reaction increases as the temperature is raised. A ten degree Centigrade rise in temperature will increase the activity of most enzymes by 50 to 100%. Variations in reaction temperature as small as 1 or 2 degrees may introduce changes of 10 to 20% in the results. In the case of enzymatic reactions, this is complicated by the fact that many enzymes are adversely affected by high temperatures. As shown in the graph, the reaction rate increases with temperature to a maximum level, and then abruptly declines with further increase of temperature. Because most animal enzymes rapidly become denatured at temperatures above 40·C, most enzyme determinations are carried out somewhat below that temperature.
2. pH Levels in the Milk – The reason for this can be explained by the following graph:
Extremely high or low pH values generally result in complete loss of activity for most enzymes. pH is also a factor in the stability of enzymes. As with activity, for each enzyme there is also a region of pH optimal stability. Therefore, we will use distilled water to maintain the pH.
- Amounts of Rennet used – With more rennet present in the concentration then less time will be taken for the reaction to occur due to the fact that there will be more active sites for the reaction to take place on.
- Amounts of Milk used – This will affect the experiment, as with more milk in the experiment, more reactions will need to take place with less active sites free due to the larger amount of milk. Thus making the time for the clotting to occur longer than it would normally be.
- When to decide the Milk has clotted – The milk when undergoing the enzyme activity goes through different stages. The clotting can be seen at different stages so it must be decided when the milk has clotted for the experiment. For this particular experiment it will be when the milk has all turned solid and the test tube will be shaken to test if the milk has all clotted.
Equipment
- 400ml Beaker - to use as a Water Bath
- Burette – To ensure accuracy in the experiment
- Syringe – For accuracy when measuring the milk and enzyme concentrations
- Test Tubes x5
- Pasteurised Milk
- Rennet
- Kettle - to heat water and maintain the 40ºC temperature in the water bath
- Stopwatch – to time the experiment
Method
We will take 2 cm³ of milk measured from a burette to ensure accuracy and put this in a test tube. Five test tubes will be filled up this way and labelled A, B, C, D and E. We will then use a syringe to measure 1 cm³ of a water and rennet mixture, and put this in test tube A. The test tube will be placed in a water bath, which has been pre-heated to 40°C with water from a kettle. We have decided on the temperature from our pilot study where we found out the best temperature to perform the experiment was at 40°C which maybe because the rennet is manufactured compared to natural ones which work best at 37°C. If the water is too hot when it comes out of the kettle, cold water can be added to cool it down quicker. When the test tubes are placed in the water bath they will be allowed to warm up before the stopwatch is started so the rennet is heated to optimum temperature before the experiment starts. Once the test tubes have heated up to, 40°C then the stopwatch will be started and the time will be recorded, it takes the milk to solidify in a curd. To make sure the time is taken from the start to when the milk is set completely, the test tube can be shaken around, to see if it has all set. In the first test tube the concentration of rennet added would be 2% In B, it will be 1.75, in C 1.5 in D 1.0 and in E, 0.5. The results will be taken down in a table. The experiment will be repeated 3 times and an average time will be taken to make the test more accurate.
To work out the concentrations of the rennet water solution we will use the following table:
Safety
Although no hazardous solutions or substances are being used, for safety precautions if any beakers or burettes fall or break, the glass will be cleared away quickly so as not to cause any harm. Safety glasses will be worn throughout the experiment as a precaution.
Fair Test
To ensure that the test remains fair the following precautions will be taken:
- The same amounts of milk, rennet and water mixtures will be used.
- The same amount of water will be used in the water bath and the same size beakers will be used.
- In each test tube, the milk will be left to solidify to the same point as each other.
Observations
Changes
When performing the experiment to keep the temperature at a constant 40°C required the addition of more water, which slowly made the water bath fill up. This meant that the water bath had to be tipped over the sink to lose some water.
Results Table
Reciprocal Results
Analysis
The graph of my results gives a straight line of best fit in a strong negative correlation. This shows us that as the concentration of the rennet increased, the time taken for the milk to clot decreased in direct proportion. We can therefore say that the more rennet present in the reaction speeds up the rate of reaction. We can see that when there was a 0.5% concentration of rennet on average over the three results the time taken in seconds was 2242 whereas when there was a 2% concentration it only took 256 seconds. The plotted reciprocal graph of the rate of reaction gave us a line of best fit with a positive correlation through (0,0). This showed us that as the concentration of the rennet increase the rate of the reaction increases in direct proportion, thus decreasing the time taken for the milk to fully clot. On the rate of reaction graph, we can also identify two anomalies. The reason for these maybe because the temperature may have varied by 1 or 2°C while monitoring another beaker of results, thus causing a slower or faster time
We can explain the increase in the rate of reaction because with more rennet present in the reaction it increases the number of active sites for the reaction to take place. The rennet breaks down bonds in the glycopeptides on the surface of the calcium caseinate (milk). This reaction is specified by the rennet with the “Lock and Key” action on the active sites. Therefore, with more active sites present more bonds can be broken at one time. This increases the rate of reaction, and eventually increasing the speed of the time taken for the milk to fully clot. It clots because the calcium caseinate is replaced by the insoluble calcium paracaseinate, thus forming the clotted milk. The increase in the rate of reaction and the time taken for the time clot are therefore directly proportional. This is because if there are twice as may active sites then the reaction will become twice the speed and the time taken for the milk to clot will occur in half the time.
“I predict that as the concentration of the enzyme, rennet is increased then the time taken for the milk to clot will decrease in direct proportion to the increase of the concentration. This is because rennet is a biological catalyst and will increase the rate of the lactose being chemically changed to lactic acid. When the concentration of the rennet is increased then more active sites on the enzymes will become available in the solution allowing more reactions to take place at once.”
My results support my prediction accurately. My prediction states that as the concentration of rennet increases then the time take decreases in direct proportion. This is the conclusion I have arrived at from studying the results and graphs. I can further conclude that the rate of reaction also affects the time taken for the milk to clot in direct proportion to the concentration of the rennet as well.
Evaluation
