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Andre Smith

November 10, 2015


FST 101A-A06, Wednesday 9 AM
RH
LAB 7 Thermal Inactivation of Enzymes
I. Purpose/Objective
The purpose of this lab is to examine the denaturation of peroxidase in turnips
after blanching.
II. Introduction
Enzymes catalyze reactions that would otherwise take long periods of time to
occur, which is why they are important in biological systems. Enzymes in fruits and
vegetables allow the plant to mature which can lead to compromises in texture, color,
flavor, odor and nutritional value. Blanching, placing the food in boiling water then
into ice water, is one process of deactivating enzymes. When the enzymes are
inactivated, the maturation process in the fruits and vegetables can be slowed down.
Food scientists are concerned about the enzymatic activity in foods because they
can have an effect on shelf life and how the product looks and taste. Enzyme
inactivation can help prevent food from spoiling faster than it normally would. When
blanching, it important to place the product in ice water immediately after removing it
from the boiling water. There is potential for microbial growth in the product as it
cools from a high temperature to a cooler warm temperature. Nutrition
professionals can also be concerned about blanching as the product can cook and
have tissue damage if not cooled immediately after being in a hot water bath. There is
also the potential for the nutrient value of the food to decrease as the food cooks.
III. Procedure
The procedure for Lab 7 is found in Principles of Food Composition, Laboratory
Manual, FS&T 101A (2015) page 59.
IV. Data
Table 1: Group Nines Absorbance data at 470 nm. The top row represents how long
the particular sample was blanched and the first column represents the time that the
absorbance was recorded.
Time
(sec) 120 sec 90 sec 60 sec 30 sec 15 sec 0 sec
0 0 0 0 0 0 0
5 0.014 0.013 0.02 0.017 0.051 0.087
10 0.014 0.016 0.025 0.082 0.092 0.187
15 0.014 0.017 0.047 0.138 0.161 0.285
20 0.014 0.019 0.078 0.192 0.22 0.372
25 0.014 0.022 0.104 0.24 0.268 0.456
30 0.014 0.024 0.128 0.285 0.324 0.54
35 0.018 0.025 0.153 0.333 0.372 0.608
40 0.013 0.023 0.174 0.376 0.416 0.668
45 0.013 0.023 0.197 0.416 0.458 0.712
50 0.013 0.023 0.219 0.458 0.494 0.742
55 0.013 0.023 0.239 0.498 0.524 0.77
60 0.013 0.025 0.261 0.526 0.544 0.786
90 0.013 0.025 0.373 0.618 0.596 0.853
120 0.013 0.031 0.456 0.646 0.61 0.905

Absorbance vs. Time


1
0.9
0.8
Absorbance at 470 nm

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)

120 sec 90 sec 60 sec 30 sec 15 sec 0 sec

Fig. 1a: Absorbance of blanched samples.


Absorbance vs. Time
0.7

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)

120 sec 90 sec 60 sec 30 sec 15 sec 0 sec

Fig. 1b: Absorbance of blanched samples using only linear portions.

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)

Fig. 2: EAU vs blanch time.

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)

Fig. 3: Linear representation of enzyme inactivation using the natural logarithm of


EAU plotted against blanch time.

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 the rate constant


The rate constant is expressed as k. k can be found from the slope on the line equation on
figure 3. The slope represents the rate that the enzyme is becoming inactive. Due to first order
kinetics, the slope is k.
Slope = -0.0164
k= -(-0.0164) = 0.0164

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.