Investigating the Effect of Lipase Concentration on the Breakdown of
Fat in Milk
INTRODUCTION:
Enzymes are proteins which can catalyse chemical reactions without changing themselves. The
enzyme lipase breaks down the fat in dairy products such as full-cream milk for people who are
lactose intolerant. Lipase acts on its specific substrate, lipids produces fatty acids. If enzyme
concentration increases, random collisions between the substrates and active sites of enzyme
increase due to the increasing amount of active sites which allow more collisions to happen, so
the rate of breakdown of lipids to simpler substances will increase. During the experiment,
sodium carbonate solution and pH indicator phenolphthalein will be added ahead of lipase
enzyme; when the pH is below 8.2, phenolphthalein will turn from pink to colourless. Sodium
carbonate solution is added in order to turn the solution alkaline and the indicator pink then
back to colourless after lipase is added to catalyse the chemical reaction and speed up the
breakdown of lipids into fatty acid in the milk. Fatty acid produced then is going to neutralise
the solution with sodium carbonate solution added previously; phenolphthalein will finally turn
from
pink
back
to
colourless.
AIM:
In this experiment I will investigate the effect of increasing enzyme concentration on the
activity of the enzyme lipase that will be allowed to act on the same amount of milk and the
time taken for the phenolphthalein to turn from pink to white compared.
HYPOPTHESIS:
With higher lipase concentrations, there will be more active sites in the solution to be fit in by
the substrates, which means more collisions between the active sites and the substrates and
quicker breakdown of lipids in the milk within shorter period of time. At enzyme concentration
of 1% or 2%, reactions can happen at a relatively slower pace, with lipids broken down and
phenolphthalein turning from pink to white over a relatively longer time period. At 3%, enzyme
activity will speed up with lipids broken down and phenolphthalein turning from pink to white
over a relatively shorter time period. At 4% or 5%, enzyme activity will happen significantly
faster.
APPARATUS:
1.
2.
3.
4.
5.
6.
7.
8.
9.
10.
11.
12.
Lipase
solution
at
1
test
dropping
5
4
concentration
measuring
1
full-fat
cylinders
of
stopwatch
1
sodium
4
carbonate
beakers
solution
Marker
distilled
at
of
5%
tube
pipettes
milk
10ml
(±0.5ml)
(±1s)
stirrer
phenolphthalein
0.5
mol
dm3
pen
water
200ml
(±5ml)
VARIABLES:
Independent
Variables:
1. Enzyme concentration- effectiveness of enzyme concentration will be tested at 1%, 2%, 3%,
4%, and 5%. Enzyme lipase at the concentrations of 1%, 2%, 3%, 4% will be made by mixing
lipase at 5% and distilled water; all these solutions will be contained in different beakers to
keep them pure. In order to make 1% lipase, for each 1ml lipase, 4ml distilled water will be
added and poured into a beaker; to make 2% lipase, for each 1ml lipase, 3ml distilled water will
be added; to make 3% lipase, for each 1ml lipase, 2ml distilled water will be added; in order to
make 4% lipase, for each 1ml lipase, 1ml distilled water will be added.
Controlled
Variables:
1. Volume of the milk used for every test will be kept constantly at 5ml; substrate concentration
will be kept the same since full-cream milk will be used. Different volumes of milk will allow
different amount of substrates to be contained in the milk. Therefore, more substrates
contained in the milk will take it more time for the enzyme to break all the fats while the
amounts
of
lipase
and
sodium
carbonate
solution
stay
the
same.
2. pH of the substrate – the identical amount of 7ml sodium carbonate solution, which is
alkaline, will be added in the test tube; if not, more or less fatty acids will be produced and
needed to neutralise the solution and for the phenolphthalein to turn from pink to colourless.
3. Volume of lipase solution – 3ml lipase will be added to for each test. By keeping this the
same, the amount of active sites of enzyme will be equal in all the cases, therefore allowing
results to be fair. If more were placed in one of the test tubes, then the rate of activity of the
enzyme at different concentrations will be wrongly judged, leading to incorrect conclusion.
4. Temperature will be kept consistent at room temperature - All tested will be done in the
Biology laboratory with the windows shut to make sure the temperature will not change during
the tests. The higher the temperature, the shorter the time will be needed for the chemical
reactions to happen and phenolphthalein to turn from pink to colourless.
Dependent
Variable:
1. The time for the phenolphthalein to turn from pink to colourless will be taken using the
stopwatch.
METHOD:
*
The
apparatus
was
collected;
* The beakers labelled; Solutions of lipase respectively at concentrations of 1%, 2%, 3%, and 4%
were made using lipase at 5% and distilled water. * By using the dropper and measuring
cylinder, an amount of 5ml milk was placed in the test tube * By using the dropper and
measuring cylinder, 10ml phenolphthalein was added to the test tube * By using the dropper
and measuring cylinder, 7 ml sodium carbonate solution was added to the test tube * By using
the dropper and measuring cylinder, 3ml of 1% lipase was added into the test tube * As long as
the lipase was added, the button “start” on the stopwatch was pressed; and the timing started.
* After the phenolphthalein turns from pink to colourless, timing was over. * The time taken for
all the lipids to be broken down and the phenolphthalein to turn from pink to colourless was
recorded * Repeat the tests two more times at 1% enzyme concentration following the
methods and procedures stated above and finish the experiment at 2%, 3%, 4%, and 5% lipase
concentration in the same way * The times taken for the reactions to finish were compared.
Raw
Data
Collection
and
Processing
Concentration of Lipase Enzyme (%)| Time Taken for Phenolphthalein to Turn from Pink to
Colourless/s(±1s)| | 1| 2| 3| 1 | 390| 464| 369| 2 | 204| 226| 210| 3 | 140| 136| 131| 4 | 96|
95|
94|
5
|
74|
75|
73|
Sample
Calculation
for
Average
Value
When lipase enzyme concentration is at 1%, average time taken for Phenolphthalein to turn
from
pink
to
colourless:
Mean
=
Sum
of
test
volumenumber
=
=
=
of
tests
(390+464+369)÷3
1223÷3
407.7s
Processed
Data
Concentration of Lipase Enzyme (%) | Average Time Taken for Phenolphthalein to Turn from
Pink
to
Colourless/s(±0.1s)|
1
|
407.7|
2
|
213.3|
3
|
135.7|
4
|
95.0|
5
|
74.0|
Conclusion
All the equipment, raw materials, and solutions needed were properly prepared. All the
procedures and methods were appropriately routinely followed to do every single test. Most of
the equipment was examined carefully before being used to ensure the accuracy of the results
from the tests and the success of the experiment. The average times taken for the
phenolphthalein solution to turn from pink to colourless at each lipase enzyme concentration
were clearly indicated by dots on the separate graph paper. Each dot was linked by a smooth
down sloping curve, which showed the effect of the lipase enzyme concentrations on the
breakdown of the lipids contained in milk. In accordance with the curve and dots on the graph,
the results went as the hypothesis suggests: Enzyme concentration increases, random collisions
between the substrates and active sites of enzyme increase due to the increasing amount of
active sites which allow more collisions to happen, so the rate of breakdown of lipids to simpler
substances will increase. As lipase concentration goes up, the time taken for the lipids to be
broken down shortens while all the other factors being equal. As the graph shows, with 1%
lipase enzyme concentration, the average time is 407.7s; at 2%, the time was recorded as
213.3s; with 3% lipase, the time is 135.7s; it was 95.0s for 4% lipase; with 5% lipase, the time
taken
is
74.0s.
Evaluation
Sources of Errors; Limitations of Experimental Design; Suggestions for Improvements
1. Timing was not accurate; Limitations of human reaction produced uncertaint Use a datay;
logger
to
do
the
timing.
2. Volumes of each solution added were not exact as planned; Eyes were not parallel to the
lowest points of the solutions while using the measuring cylinders; Use measuring cylinder with
less
uncertainty
associated
with
them.
3. The solutions were not at the same concentrations constantly; Solutions were kept in the air
for too long so that they were oxidised; Keep all the solutions in the refrigerator and sealed.
4. Water alters the concentrations of solutions; Test tube was washed after each test was
finished, but was undried; Use dried test tubes for each of the following tests.
5. Amounts of lipids in the milk were not constantly equal; Full-fat milk used in the experiments
was not produced by the same manufacturer; Use the full-fat milk of the same brand.
6. Inadequate amounts of solution were used for each test; There was a hole in one pipette
dropper for one of the solutions involved in the experiment; Test the droppers with water
before
the
experiments
and
change
the
badly-made
one.
7. The solutions were not all mixed to the same extent; Test tube was shaken harder or more
slightly during each test; Use a stirrer to obtain the same effect of mixing the solutions.
8. The colour from pink to colourless was wrongly judged; The differences of colours were not
precisely judged by human eyes; Use a colorimeter to judge the colour of the solution.