The Effects of Temperature on the Rate of Clotting Milk and Rennet

The Effects of Temperature on the Rate of Clotting Milk and Rennet

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The Effects of Temperature on the Rate of Clotting Milk and Rennet


Introduction
------------

The following experiment investigates the effects of different
temperatures on a mixture of rennet and whole milk. On having the
choice between testing the mixtures reactions at various temperatures,
or testing the mixture with various amounts of concentration of
rennet, my partner and I decided upon the first option. We made this
decision as we felt it would be valuable to our scientific knowledge
if we had a better understanding of how different temperatures can
effect the behaviour of an enzyme, such as Rennin, which is also known
as Chymosin. Our scientific knowledge tells us that enzymes work most
efficiently at specific temperatures, and this experiment helps us to
discover exactly which temperatures they are.

It is important to remember that the Rennet was mixed with milk, which
is perhaps one of the most important sources of nutrition in the
world, and drunk by billions of people everyday. It is particularly
important to babies and growing children. It provides:

· Calcium, to build strong bones and teeth

· Protein, to build and repair muscle tissue

· Potassium, to help regulate our body's fluid balance

· Vitamin A

And many other useful vitamins and nutrients which help to maintain a
healthy body.

As wonderful a necessity that milk is, it is also an extremely
perishable food. Milk is usually stored in the fridge, because it
preserves better at a low temperature, but even so, once it has passed
its sell by date, it is no longer suitable for consumption. Although
the milk itself does not have a very long life, other foods and some
dairy products can be made using it. Cheese would be the main example
of this, which can be produced simply by the curdling of milk. Rennin,
found in the substance rennet, is a milk-coagulating enzyme capable of
assisting in the production of cheese. Therefore the temperatures at
which the milk and rennet coagulate best at in this experiment, are

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going to be the most suitable temperatures for manufacturing cheese.

Rennet can be obtained from the stomachs of young milk-fed mammals,
namely calves where the lining of the fourth stomach (the abomasum) is
a good source of the substance. Rennet contains the enzyme named
Chymosin (Rennin). Chymosin plays a vital part in the digestion of
milk inside the calf's stomach. It's job is to coagulate or curdle the
milk, and if this wasn't done then the milk would flow rapidly through
the calf's digestive system, not allowing the absorption of important
proteins.

Rennin having the ability to coagulate milk means that it was widely
used in cheese making, as I mentioned before. A few of the cheese made
by using animal rennet are:

*English Stilton
*Blarney Irish
*Blue Danish
*Cheddar Canadian Sharp
*Cheddar Tillamook
*Cheddar Wisconsin Aged
*English Cotswold
*Blue Castello
*English Huntsman
*Cheddar Wisconsin Black Wax
As cheese is made in such vast amounts in today's world, there are not
sufficient calf's stomachs to supply the amount of Rennet required, so
alternative sources have been found. For example there are liquid
forms and tablet forms of Rennet which don't actually contain
Chymosin, but any form of enzymatic preparation that clots milk is
called "Rennet".

The Rennet used in this experiment however is animal rennet, which
does contain the enzyme Chymosin. By having the scientific knowledge
prior to this investigation that enzymes are biological catalysts, it
is clear that the Chymosin will be capable of speeding up a reaction
of some sort.

Enzymes can be used in both anabolism (the build up of simple
chemicals into complex ones) and catabolism (the breakdown of complex
chemicals into simpler ones). Enzymes are most commonly used in
Catabolism, this particularly applies to animal digestive systems.

Most enzymes are proteins, and they can alter the rate of reaction
without themselves being changed because they have catalytic
properties. There are four key factors that affect enzyme activity and
they are:

1. The concentration of the enzyme

2. The concentration of the substrate

3. The temperature the enzyme is held at

4. The pH

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

Time (s)

Units/second

Temperature (oC)

First Test

Repeat 1

Repeat 2

Mean average

Rate (2d.p)

23 (room temp)

no reaction

no reaction

no reaction

0

0

30

no reaction

no reaction

no reaction

0

0

40

186

190

215

197

5.08

50

123

160

144

142.3

7.03

60

no reaction

no reaction

no reaction

0

0



Results- Graph Number 1
=======================



[IMAGE]
=======


[IMAGE]
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.
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