Chymosin, known also as rennin, is a proteolytic enzyme (made up of protein), which is most stable between pH 5.5 and 6.0 hence it is stable in milk, which is at pH 5.56
Hypothesis
My hypothesis is that the chymosin enzyme in calf rennet will be most efficient at clotting milk at temperatures between 39C to 41C, which I believe to be its optimum temperature. I predict that at this temperature the calf rennet will clot the most milk. At temperatures much higher than 40C and much lower than 40C, the least amounts of milk will clot, as chymosin reactivity will be very limited.
As the temperature increases the kinetic energy of the molecule will also increase, resulting in increased
movement of both enzyme and substrate molecules. The increased movement of the molecules results in
more collisions between the substrate and enzymes, hence increasing the chance of successful conversions of substrate into product. So as the temperature rises the rate of enzyme-catalysed reaction also will increase, which I have hypothesised. But beyond an optmium temperature the high temperature begins to change the shape of the molecule altering the shape of the active site. The active site is a region on an enzyme that binds to the substrate, but when this site is altered due to an increase in temperature the substrate can not bind with the enzyme slowly decreasing the rate at which the enzyme catalyses the reaction (rate at which the milk is coagulated). Once the higher temperatures are used this causes denaturation which is irreversible as it is the destruction of the tertiary structure of the enzyme. The destruction of the tertiary structure leads to the change of shape of the enzyme, resulting in the inactivity of the active sites. I hypothesis the optimum temperature will be between 39C and 41C which I have estimated researching though average enzyme optimum efficiency.
Method
Key Variables:
- For all individual experiments, the temperature will be kept constant at the temperature been tested
Controlled Variables:
- Calf rennet concentration will be the same for all experiments
- Milk type (whole milk, semi-skimmed) will be kept the same for all experiments
- PH of milk will be same for all experiments (buffer solution will be used if pH is compromised)
- Repeat each experiment at least three times
Equipment:
Water bath
Ice
2 Pipette
Test tube rack
Calf rennet
Whole milk
2 Thermometer
Scale (electronic)
8 test tubes (x3 for three attempts on each experiment)
Muslin cloth
Safety goggles
kettle
Procedure:
I will take 8 test tubes each containing 10cm3 of whole milk (ph 5.56), then place each one separately into a water bath until the milk has reached the temperature that I will be investigating on. I will measure the temperature of the milk using a thermometer. I will be measuring the rate of reaction of the rennet by heating and cooling to the following temperatures: 10C, normal room temperature (~27C), 30C, 40C, 50C, 60C, 70C and 80C.These wide range of results will enable me to compare my results to the rate of clotting of the rennet (chymosin) against the rate of reaction of the rennet at room temperature to what extent temperature affect the reaction. Once the milk will have reached the required temperature I will add 1cm3 of calf rennet using a pipette for accuracy. As soon as I add the rennet I will start the stopwatch, because rennet works extremely quickly under the right conditions the milk will clot instantly if in the right conditions. After 4 minutes I will stop the stop watch and remove the milk (liquid-solid) and place on a muslin cloth, where I will drain away requiring liquid in the substance. Once there is only clotted milk left I will weigh the remains and record it.
I will attempt the experiments at least three times to achieve a fair set of results within the time given.
Equipment layout:
Risk Assessment:
- Calf rennet (chymosin enzymes)
Are in standardized solution form. Enzymes are biologically active proteins so should be handled with care. May also irate the skin, eyes or mucous membrane
- Avoid direct contact
- Avoid inhalation
-
Clean up spillages immediately
-
DO NOT ALLOW TO DRY AND GENERATE DUST- after spillage
Pilot test:
In order to determine a suitable range of temperatures I could use I carried out a pilot test of the experiment.
Results of pilot tests:
Using 1cm
The two rows highlighted in red is the range that holds the enzymes optimum temperature.