0.7
0.6
0.5
0.4
0.3
0.2
0.1
0
0 20 40 60 80 100 120 140
Time (seconds)
y = 0.0177x + 0.0079
0.6 R = 0.9976
0.5
y = 0.0108x - 0.0038
Absorbance at 470 nm
0.4 R = 0.998
y = 0.01x - 0.0141
R = 0.9939
0.3
y = 0.0045x - 0.0089
0.2 R = 0.9809
y = 0.0026x
0.1 R = 1
y = 0.0028x
0 R = 1
0 5 10 15 20 25 30 35 40
-0.1
Time (seconds)
Table 2: Slopes from Fig. 1b for each sample and calculated EAUs and ln EAUs.
Blanch a.u. /
Time Slope min EAU ln EAU
0 0.0177 1.062 1062 6.968
15 0.0108 0.648 648 6.474
30 0.01 0.6 600 6.397
60 0.0045 0.27 270 5.598
90 0.0026 0.156 156 5.050
120 0.0028 0.168 168 5.124
Enzyme Inactivation
1200
1000
800
EAU
600
400
200
0
0 20 40 60 80 100 120 140
Blanch Time (seconds)
Enzyme Inactivation
8.000
7.000
y = -0.0164x + 6.7962
6.000 R = 0.92
5.000
ln EAU
4.000
3.000
2.000
1.000
0.000
0 20 40 60 80 100 120 140
Blanch Time (seconds)
V. Calculations
Calculating Enzyme Activity (EAU):
The slopes of each line in figure 1b are in absorbance units per second (a.u./sec). To find
EAU the slope must be converted to a.u./min then multiplied by a conversion factor.
Using the slope for 0 seconds of blanching in figure 1b:
Slope= 0.0177 a.u/sec
0.0177 a.u./sec (60 sec/1 min) = 1.062 a.u./min
1.062 a.u./min (1 EAU/(0.001 a.u./min)) = 1062 EAU
Calculating half-life:
Half-life in first order kinetics can be represented as ln(2)/k.
t1/2 = ln (2)/0.0164= 0.693/.0164 = 42.3 seconds
VI. Results/Discussion
The information given by figure 1 suggests that longer blanching times results in
lower enzyme activity. The enzyme activity is related to absorbance. In this experiment,
guaiacol was used as an indicator. Peroxidase catalyzes the reaction of colorless guaiacol
to brown tetraguaiacol. Therefore, the darker the color of the sample, the more peroxidase
activity there is. The sample that was blanched for 120 seconds was almost colorless and
the sample that was not blanched at all was the darkest brown and also had the highest
absorbance readings. Figure 1 shows that as samples were blanched longer they had
lower absorbance readings and lower enzyme activity. The blanched samples for 90 and
120 seconds have very similar absorbance readings, as you can barely see the points for
120 seconds underneath the points for 90 seconds. In the linear graph, figure 1b, the 120
second sample has almost the same slope as the 90 second sample where the R2 values
are greater than 0.90 at 0.0026 and 0.0028 respectively. The 120 second sample had 168
EAU and the 90 second sample had 156 EAU. Theoretically, the 90 second sample
should have a larger EAU as it was blanched less. Table 1 even shows that all but 2 of the
absorbance readings are greater than those from the 120 second sample, but when making
the linear graph, points were deleted to create lines with an R2 value greater than 0.90.
There could have also been error in the first few data points as the spectrophotometer was
adjusting to read the absorbance.
The slope in Figure 3 represents the rate of enzyme inactivation as each sample was
blanched. The reaction is first order, so the plot of the natural logarithm against the time
each sample was blanched will have a straight line. The half-life is calculated from the
slope. For group nine, the half-life was calculated to be 42.3 seconds. The half-life gives
insight into the efficiency of blanching by indicating how long it took for half of the
peroxidase enzyme to become inactive. This is an exponential trend, so at double the
half-life, 25% of the active enzyme will be left. Each food is different so they will have
different optimal blanching times depending on the present enzymes and food matrix.
The optimal blanching time can be difficult to determine from this data alone. There
are many factors to consider for the optimal blanching time. For the data gathered in this
experiment, the optimal blanching time could be about 80 seconds, which is about two
half-lives. There has to be a balance between enzyme inactivation and heating to the
point of tissue damage. In this experiment, tissue damage was not measured, so the
optimal blanching time is an estimate. The optimal blanching time is different for every
vegetable. Each vegetable has a different size, shape, texture, active enzymes and food
matrix. For example, it is difficult to compare turnips to celery or tomatoes as they all
have different shapes and textures.
VII. Conclusion
Blanching vegetables is one method to inactivate the enzymes that cause the vegetable to
mature. Inactivating enzymes proves to be useful for food preservation and prolonging
shelf life. Enzyme activity was measured by examining the activity of the enzyme
peroxidase on guaiacol. This method was useful, but one downfall was the inability to
measure the absorbance of the sample at t=0 when the sample was added to the cuvette.
VIII. Questions
1. What does the calculated half-life represent?
The half-life represents the amount of time it takes for half of the peroxidase to inactive by
blanching.
2. During the blanching, we have assumed that the enzyme was continuously exposed to 100
degrees Celsius. Was this the case? If not, how would you redesign the experiment to improve it?
While the water was visibly boiling, there was no thermometer to indicate what the temperature
of the water was at any given time. After blanching, the samples were in a room temperature
environment while the spectrophotometer was being set up. If there was more time, perhaps the
samples could each have thermometers and measurements could be taken once they reach certain
temperatures.
3. Why is the most blanched sample always the first to be measured, and the least blanched last?
The most blanched sample has the least amount of active enzyme. If the least blanched samples
were measured first, the active enzymes could contaminate the samples to follow.