Lee Gwan Cheung, Phillip

11/1/17 and 11/8/17

A03 Wed PM 2:10PM – 5:00PM

N.D.

Lab 5&6 : Proteins in Food

I. Objective
In the first part, the purpose is to test whey and casein proteins’ absorbance for their
protein functionality under different temperatures and pH. Different concentrations of BSA are
used to find out the standard curve of protein absorbance. The second part is to investigate the
enzymatic inactivation after blanching at different time points (120s, 90s, 60s, 30s, 15s, 0s).
Guaiacol solution was used to react with the peroxidase in turnips for measurement. Both
experiments used Spectrophotometer to measure activity.

II. Introduction
Food usually contains a mix of proteins that have different functional properties.
Changes of external factors such as temperature and pH will alter the structure and also their
functionality. Therefore, it is important to evaluate the functionality of protein’s characteristics in
food. Most of the proteins will denature in high temperatures and proteins will inactivate in
extreme pH. Protein solubility is also an important criterion for determining the usefulness is the
interactions with water. Proteins depends on the composition and ratio of polar to nonpolar amino
acids. Solubility increases as polarity of the protein surface increases and decreases as molecular
weight increases. The net charge of the protein will be zero at isoelectric point where water
binding and solubility is at a minimum. In this lab, whey and casein are the two proteins that were
investigate the protein functionality in different temperatures and pH values. They are both exists
in milk where caseins has about 80% and whey has about 20% of the milk. Casein is sensitive to
pH because removal of glycomacropeptide (GMP) will coagulate hydrophobic micelles. As GMP
is neutralized by the positive ions, there will be precipitation at the isoelectric point. Whey is more
sensitive to heat than casein. The globular protein will unfold when heat is added. Due to structural
modification, it will eventually become denatured and insoluble. Bradford Assay is used to prepare
a standard curve by using known concentration of Bovine Serum Albumin (BSA). The standard
curve helps to find out the protein concentration of an unknown. This lab also used Coomasie
Brilliant Blue G-250 (in the Bradford reagent to indicate the presence of protein by changing its
color. It will change the color of the dye from reddish to a bluish color.
In the second part, it is important to measure enzyme activities in food because
enzymes play an important role for preservations of food in industry. There are several enzymes in
food that catalyze chemical reactions. In order to have a longer storage time, enzymes can be
thermally denatured since they are proteins. Blanching is a common technique in food industry
because it is simple and cheap. The product has to expose to hot water bath around 80°C to 100°C
for a specific time and freeze afterwards. Cooling helps to reduce bacteria growth and minimize
quality losses. Under-blanching may not inactivate all the enzymes and over-blanching may cook
the food or introduce losses due to tissue damage. The products need to be small uniform sized
pieces to ensure they are equally boiled. In the lab, turnip is used and it is rich in peroxidase.
Guaiacol is used to determine the enzymatic activity of blanched turnips. Tetraguaiacol is a
colored complex that will be formed after peroxidase reacting with four guiaicol.
In both labs, spectrophotometer is used to monitor color formation. In the first part,
595nm wavelength was set to determine the presence of bluish color that formed from Bradford
reagent. In the second part, 420 nm wavelength was set to determine the presence of violet color
that formed from tetraguaiacol. Spectrophotometer is a common tool in science industry to
measure the absorbance or transmittance of an unknown solution. It helps to identify the
unknown’s concentration and also helps to set up standard curve with known concentration
solution.
III. Procedure
A. Measurement of Standard Curve and Protein Samples
5 mL of Bradford reagent was added into 23 glasses in Bradford rack by using pumper.

40mL of whey protein was put into seven 50mL beakers by pumpers. The seven beakers were
adjusted pH by HCl and NaOH to pH 2.5, pH 3.5, pH 4.5, pH 5.5, pH 6.5, pH 7.5, pH 8.5.
5mL of the whey protein from different pH were put into the room temperature test tubes and
heated test tubes (10-23). The test tubes that needed to be heated were placed into 70°C water
bath for 10 minutes. After 10 minutes, the heated test tubes with cloudy solution were placed
into labeled conical tubes and centrifuge for 15 minutes.
Type I water and 1mg/mL BSA solution were put into tube 1-9 as follows.
Tube Type I water 1mg/mL BSA Bradford (mL) Final [BSA]
(mL) (mL) (mg/mL)
1 0.100 0 5 0
2 0.090 0.010 5 0.1
3 0.090 0.010 5 0.1
4 0.080 0.020 5 0.2
5 0.080 0.020 5 0.2
6 0.060 0.040 5 0.4
7 0.060 0.040 5 0.4
8 0.020 0.080 5 0.8
9 0.020 0.080 5 0.8

Test tubes 1-9 were covered with parafilm and were inverted gently. Then, they were placed
in the test tube rack for 5 minutes. At the same time, 0.1mL of the appropriate protein
solution was transferred into correspond tubes in the Bradford rack by appropriate serological
pipette. Them, they were inverted gently with the parafilm. All the tubes were measured
absorbance in order by Spec 20 at 595nm. Data were recorded and analyzed.
Same procedure was done by using casein protein instead of whey protein.

B. Preparation of turnips
For safety precaution, gloves were used for cutting. A turnip was cut into a cube
shape in order to cut off the stem and skin. Then, the turnip was cut into smaller pieces cubes
for about ¼ to ½ inch cubes. The cubes were weighed to 25±1g by electronic balance, then
they were put into a cheese cloth. The weighing process was repeated 5 times for a total of
six. The turnip pouches were made by using rubber bands. Each pouch was labelled with
group number and blanch time (120s, 90s, 60s, 30s, 15s, 0s). The pouches were blanched one
by one from the highest time to the lowest. After each blanching, the pouch was removed
from boiling water and put into ice bath immediately.

C. Extraction of Enzymes
150 mL of Type I water and one pouch of turnips were put into the blender and
was blended for 30 seconds. At the same time, the cheese cloth was squeezed to dry and was
put on the beaker. The blended material was put on the dried cheesecloth. This steps were
repeated for each timepoint by using corresponding cheesecloth and labeled beaker. The
cheesebloth was disposed after filtering.

D. Measurement of enzyme activity

3mL of peroxide assay was added to 7 glass tubes (blank, 120s, 90s, 60s, 30s, 15s
and 0s) by pumper. 125µL of water was added to the blank test tube. The blank test tube was
placed into Spec 20 at 420nm for zeroing the Spec 20. The measuring of enzyme was done
one by one in the order of highest time point to lowest time point. 125µL of 120s time point
enzyme was added to the 120s test tube and was mixed very quickly with gloved finger. After
mixing, it was put into Spec 20 for recordings. Different intervals’ absorbance were
measuring according to the lab manual page 51. The steps were repeated for the other 5
enzymes.
The procedure followed for the experiment is found in “FS&T 101A-Food Chemistry
Laboratory Fall 2017 Lab Manual” (2017), Lab 5: protein Solubility, Lab 6; Thermal
Inactivation, p.39-40, p.46-50.
IV. Data
Table 1: Concentration, absorbance values, volumes of water, BSA and Bradford of Group 3 and
Group 10. Each group make their known concentration of BSA solution and test for the
absorbance of each concentration. This helps to make the standard curve for BSA.

Tube Type 1 water 1mg/ml Bradford [BSA] Group 3 Group 10

(mL) BSA (mL) (mL) (mg/mL) Absorbance Absorbance
1 0.10 0 5 0 0.000 0.000
2 0.09 0.01 5 0.1 0.200 0.144
3 0.09 0.01 5 0.1 0.207 0.185
4 0.08 0.02 5 0.2 0.332 0.283
5 0.08 0.02 5 0.2 0.235 0.355
6 0.06 0.04 5 0.4 0.280 0.556
7 0.06 0.04 5 0.4 0.444 0.596
8 0.02 0.08 5 0.8 0.830 1.020
9 0.02 0.08 5 0.8 0.790 1.000

Table 2: Absorbance data of Whey (Group 3) and Casein (Group 10). All the absorbance
were measured by Spec 20 at 595 wavelength. It indicated the amount of proteins after
treatment with different pH and temperatures.

Group 3 Whey Group 10 Casein

pH Abs. at Room Abs. of heated pH Abs. at Room Abs. of heated
Temperature protein (70 °C) Temperature protein (70 °C)
2.62 0.286 0.312 2.63 0.281 0.286
3.51 0.344 0.321 3.59 0.253 0.248
4.47 0.286 0.157 4.54 0.072 0.054
5.55 0.303 0.199 5.40 0.272 0.284
6.57 0.244 0.318 6.67 0.314 0.280
7.49 0.323 0.311 7.42 0.294 0.301
8.35 0.282 0.297 8.60 0.292 0.321
Table 3: Percent solubility of heated and non-heated proteins (whey and casein). The percent
solubility was calculated by the absorbance and the standard curve.

Whey Casein
pH Percent Percent pH Percent Percent
Solubility at Solubility Solubility at Solubility
Room after heated Room after heated
Temperature (%) Temperature (%)
(%) (%)
2.62 30.08 34.10 2.63 35.33 36.17
3.51 39.05 35.50 3.59 30.63 29.79
4.47 30.08 10.12 4.54 0.25 -2.77
5.55 32.71 16.62 5.40 33.82 35.83
6.57 23.58 35.03 6.67 40.87 35.16
7.49 35.81 33.95 7.42 37.51 38.69
8.35 29.46 31.78 8.60 37.18 42.04

BSA Standard Curve (Group 3)

0.9

0.8

0.7
Absorbance at 595nm

0.6
y = 0.8617x + 0.0916
0.5 R² = 0.9665
0.4

0.3

0.2

0.1

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
BSA (mg/mL)

Figure 1:BSA standard curve of Group 3. This is plotted by the data from Table 1. It used 2
data’s mean to find out each point. The standard curve help to find out the concentration of
the whey protein.
 BSA Standard Curve (Group 10)
1.2

y = 1.1916x + 0.0705
1 R² = 0.9957

Absorbance at 595nm 0.8

0.6

0.4

0.2

0
0 0.1 0.2 0.3 0.4 0.5 0.6 0.7 0.8 0.9
BSA (mg/mL)

Figure 2: BSA standard Curve of Group 10. This is plotted by the data from Table 1. It used 2
data’s mean to find out each point. The standard curve help to find out the concentration of
the casein protein.

Percent solubility versus pH for Casein Protein

45.00
40.00
35.00
30.00
Percent Solubility (%)

25.00
20.00 Room Temperature of
Casein Protein
15.00
Heated Casein Protein
10.00
5.00
0.00
-5.000.000 2.000 4.000 6.000 8.000 10.000

-10.00
pH

Figure3: Percent solubility of casein in different pH at different temperature. This figure

helps to compare the effects of temperature and pH to casein.
 Percent solubility versus pH for Whey Protein
45.00

40.00
Percent Solubility (%) 35.00

30.00

25.00
Room Temperature of Whey
20.00 Protein

15.00 Heated Whey Protein

10.00

5.00

0.00
0 2 4 6 8 10
pH

Figure 4: Percent solubility of Whey in different pH at different temperature. This figure

helps to compare the effects of temperature and pH to Whey.

Table 4: Absorbance of different blanch time at specific time intervals. It measures the
enzymes’ activity after blanching treatment. All the samples were measured at 495nm by
Spec 20.

Time (sec) 120 sec 90 sec 60 sec 30 sec 15 sec 0 sec

0 0 0 0 0 0 0
5 0.036 0.034 0.025 0.050 0.053 0.140
10 0.032 0.028 0.028 0.099 0.086 0.247
15 0.022 0.025 0.049 0.160 0.179 0.349
20 0.006 0.023 0.055 0.218 0.214 0.480
25 0.002 0.022 0.069 0.281 0.269 0.568
30 0.005 0.022 0.083 0.336 0.334 0.690
35 0.005 0.022 0.100 0.390 0.399 0.799
40 0.003 0.022 0.123 0.448 0.452 0.885
45 0.004 0.021 0.149 0.496 0.512 0.965
50 0.001 0.021 0.169 0.554 0.564 0.995
55 0.000 0.021 0.189 0.600 0.612 1.055
60 0.000 0.021 0.203 0.648 0.659 1.080
90 0.000 0.023 0.305 0.870 0.865 1.220
120 0.000 0.023 0.408 0.965 0.960 1.370
Table 5: Blanch time, slope, enzyme activity (EAU), ln (EAU and ln 9Vo0/Vot). Slope was
found by the trend line in figure 5. It helps to find out the enzyme activity, ln (EAU) and
ln(Vo0/Vot)

Blanching Time Slope Slope Enzyme ln(EAU) ln(VoO/Vot)

(sec) (au/sec) (au/min) Activity (EAU)
120 -0.0002 -0.0120 -12 0.0000 7.1670
90 2.00E-05 0.0012 1.2 0.1823 6.9847
60 0.0034 0.2040 204 5.3181 1.8489
30 0.0111 0.6660 666 6.5013 0.6657
15 0.0115 0.6900 690 6.5367 0.6303
0 0.0216 1.2960 1296 7.1670 0.0000
Figure 5: Turnip Peroxidase absorbance after blanch at different time intervals (120s, 90s, 60s, 30s, 15s and 0s). The absorbance was
measuered by Spec 20 at 495nm. Linear trendline and equation were plotted for each time interval with r2 > 0.90.
Figure 6: Initial Velocity versus blanch time. EAU was calculated with the slopes of each
trend line from figure 5.

Rate of Peroxidase Inactivation

8.0

7.0
Ln[VoT=0/Vo T=0,15,30,60,90,120)

6.0
y = 4.0474x - 0.6587
5.0 R² = 0.8916

4.0

3.0

2.0

1.0

0.0
0 0.5 1 1.5 2 2.5
-1.0

-2.0
Blanch Time (min)

Figure 7: Rate of Peroxidase inactivation of ln[Vot=0/Vot] versus time. The y-values was
calculated in Table 5. A trend line is plotted, but it is not a good linear line since its r2 is
0.8916. This graph also helps to calculate t1/2.
V. Calculations
Final [BSA] in mg/mL for tube 2:
0.1
= 0.1 𝑚𝑔/𝑚𝐿
(0.1 + 0.9)

Final Whey concentration at pH 2.62 at room temperature:

𝑦 = 0.8617𝑥 + 0.0916
0.286 = 0.8617𝑥 + 0.0916
𝑥 = 0.2256 𝑚𝑔/𝑚𝐿
Percent solubility of whey at pH 2.62 at room temperature:
𝐹𝑖𝑛𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
𝑥 100%
𝑂𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛

0.2256
𝑥 100% = 30.08%
0.75
Final casein concentration at pH 2.63 at room temperature:
𝑦 = 1.1916𝑥 + 0.0705
0.281 = 1.1916𝑥 + 0.0705
𝑥 = 0.1767
Percent solubility of casein at pH 2.63 at room temperature:
𝐹𝑖𝑛𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛
𝑥 100%
𝑂𝑟𝑖𝑔𝑖𝑛𝑎𝑙 𝑝𝑟𝑜𝑡𝑒𝑖𝑛 𝑐𝑜𝑛𝑐𝑒𝑛𝑡𝑟𝑎𝑡𝑖𝑜𝑛

0.1767
𝑥 100 = 35.33%:
0.5

Enzyme Activity (EAU) at 0 blanch time where slope of 0s trend line is 0.0216:
0.0216𝑚𝑖𝑛 60𝑠𝑒𝑐 1𝐸𝐴𝑈
𝑥 𝑥 = 1296 𝐸𝐴𝑈
𝑠𝑒𝑐 1𝑚𝑖𝑛 0.001𝑚𝑖𝑛/𝑠𝑒𝑐
The rate constant can be found from the slope of figure 7: k=4.0474

Half- life:
ln(2)
= 0.1713 𝑚𝑖𝑛𝑠
4.0474
VI. Results and Discussion

In the protein test, a standard curve was done in every group. According Table 1, each
concentration has done for two times. The mean of each concentration was used to plot the dots in
Figure 1. This helps to minimize the error of the standard curve in order to achieve a better
standard curve for further calculations. Refer to figure 1, the r2 is 0.9665 while the r2 in Figure 2
is 0.9957. Therefore, the standard curve from figure 2 has a better linear line than figure 1. There
are several reasons. The solution did not mix completely before measuring the spectrophotometer.
The micro pipet or spectrophotometer did not work properly. The isoelectric point has a net
charge of zero and has the lowest solubility. The unfold protein is mainly nonpolar that will not
able form hydrogen bond with water. In the whey protein, the lowest solubility in room
temperature is 23.58% at 6.57 pH which is also known as the isoelectric point. In the casein, the
lowest percent solubility in room temperature is 0.25% which is at pH 4.54. It is also the pH for
the isoelectric point. The percent solubility calculated by the absorbance and standard curve is to
determine the presence of protein. It indicates the existence of protein under specific pH and heat
conditions. In figure 3, the casein of heated and room temperature have similar trend. They
dropped at the pH around 4 to 5. This indicates it is heat stable and sensitive to heat. In figure 4,
heated whey dropped a lot around pH 4.5. Therefore, whey is more heat sensitive than casein. As
results, heat treatment will unfold secondary, tertiary and quaternary structure. Heat can cleave
the peptide bonds and unfold the protein into primary structure.

In the enzyme lab, different blanch times were investigated and figure 5 has shown all of
them. According to figure 5, the longer the blanching time, the lower the slope and the less
enzymes react. It is same as the prediction that longer blanching time will inactivate enzymes
more effectively. Referring to figure 6, it has a concave curve that enzymatic activity units
decrease as blanching time increases. The more time for blanching, the more enzymes inactivate.
At 1.5 minutes blanching, the enzyme activity dropped to nearly zero. According to figure 7, it
showed the rate of inactivating peroxidase enzyme. The longer the blanching time, the higher the
rate of peroxidase. The slope, 4.0474, is the rate constant of the equation. The half-life,
0.1713mins, is the time that half of the original enzymes are inactivated. The half-life helps to
control the amount of inactivation of enzymes. In turnip, the optimum blanching time is 0.1713
minutes. The reason is that it almost lowered the enzyme activity to half of it and still remain
some enzymes in it. Over-blanching may cook the food and introduce excess loss of nutrients.
Overall, it is a smooth curve. However, the slopes of 15sec and 30sec are similar. The reason is
that the turnip cubes did not boiled evenly. Also, enzymes may tend to inactivate more efficiently
 after 30 seconds. In figure 5, the slope of 120seconds blanching time has a negative number. The
reason of it is that the absorbance was already close to zero and it had fluctuated slightly near
zero range. Therefore, the theoretical slope should be zero instead of negative values.

VII. Conclusions
From this lab, measuring the proteins’ or enzymes’ absorbance is a simple way to
find out the activity of the proteins. Different proteins have different sensitivity to temperature
and pH. Standard curve is essential for calculating unknown by using known protein
concentration. Spectrophotometer is an easy method to measure the absorbance and convert the
absorbance to find out the concentration of an unknown protein. The turnip experiments can
repeat each blanch time for 2 times and find out the average for a better result. The rate graph will
have a better r2 value. The protein test for first part can also repeat 2 times for a more accurate
result.
VIII. Post Lab Question
Lab 5:
1. By finding the lowest protein solubility from table 3, Whey: Room Temperature: pH6.57,
Heated Temperature: pH4.47. Casein: Room Temperature: pH4.54, heated temperature pH
4.54. The condition of the protein is at its isoelectric point which had unfold to primary
structure and had net charge of zero. It is less polar to form bond with water.
2. BSA is a stable protein that will not easily affected by external factors. It also prevents from
sticking to test tubes and other measuring tools. This can help for accurate measurement.

Lab 6
1. Half-life represents the decay decays by the same ration in the equal intervals of time.
2. It is not evenly boiled at 100 °C. For improvement, the water should be stirred to have an
equal temperature.
3. The most blanched sample has a lower rate of activity, so it is easier to measure compared to
the least blanched. This can practice the measuring technique before the quick rate of reaction.