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Andre Smith

November 4, 2015
FST 101A-A06, Wednesday 9 AM
RH
LAB 6 Protein Functionality: Solubility and Foam Formation
I. Purpose/Objective
The purpose of this lab is to examine the solubility of casein and whey milk
proteins at different pHs and temperatures.
II. Introduction
The general properties of proteins can lead to functional properties in the food
itself and food processing, storing, and preparation. Some examples include curd
formation with casein, dough formation of wheat gluten, foaming in egg whites, and
texture from meat proteins. Food and protein structure is related to the functionality
of the proteins. Egg white proteins unfold from their original conformation and allow
for foam formation. While the previously mentioned proteins contribute to the
properties of their corresponding foods, many proteins have more than one functional
attribute. Food scientists are interested in proteins with multiple functional attributes
so that they may be manipulated to form better or more consistent products.
Basic understanding of the structural functions of a particular protein are vital for
studying the relationship between structure and function. Typically, the primary
secondary and tertiary structures, molecular size and shape, charge distribution, and
intra- and intermolecular bonding of the protein must be understood to determine its
properties alone, but not necessarily its function in a food because foods are typically
a mixture of proteins. Many functional properties of proteins are dependent on
solubility properties and interactions with water. Protein solubility increases with
polarity of the protein surface and decreases as molecular weight increases.
Milk proteins consist of casein and whey proteins. Caseins are phosphoproteins
and precipitate at a pH of 4.6. After removal of the precipitated casein proteins, the
remainder are considered whey proteins and are soluble at pH 4.6. They both exist in
milk in different phases due to their structural and functional properties. Casein exists
as micelles and whey exists as groups of heat-sensitive globular proteins in solution.
The solubility of casein and whey can be determined by creating a standard curve.
In this experiment, Bovine Serum Albumin (BSA) was used in a Bradford Assay to
generate a curve. Determining the solubility of the proteins can help food scientists
understand what functions they can have in food products.
III. Procedure
The procedure for Lab 6 is found in Principles of Food Composition, Laboratory
Manual, FS&T 101A (2015) page 53.
Modifications include:
Instead of 0.8 mg/ml bovine serum albumin, 1.0 mg/ml was used.
Actual [protein]: Casein: 0.50 mg/ml and Whey: 0.75 mg/ml
IV. Data
Table 1: Group Nine and Group Tens Standard Curve Data
Group Nine Group Ten
T1 H2O 1.0 mg/ml BSA [BSA] (mg/ Abs. at 595 Std. Abs. at 595 Std.
(ml) (ml) ml) nm Dev. nm Dev
0.100 0.000 0.00 0.000 0.000 0.000 0.000
0.090 0.010 0.10 0.173 0.008 0.133 0.008
0.080 0.020 0.20 0.364 0.002 0.310 0.018
0.040 0.060 0.40 0.611 0.018 0.578 0.000
0.020 0.080 0.80 1.035 0.049 0.963 0.011

Group Nine's Standard Curve


1.200
y = 1.2694x + 0.0556
1.000 R = 0.9852
Absorbance @ 595 nm

0.800

0.600

0.400

0.200

0.000
0.00 0.20 0.40 0.60 0.80 1.00
[BSA] (mg/ml)

Fig. 1: Group Nines Standard Curve with BSA Bradford Assay.


Group Ten's Standard Curve
1.200

y = 1.2039x + 0.0354
1.000
R = 0.9865
Absorbance @ 595 nm
0.800

0.600

0.400

0.200

0.000
0.00 0.10 0.20 0.30 0.40 0.50 0.60 0.70 0.80 0.90
[BSA] (mg/ml)

Fig. 2: Group Tens Standard Curve with BSA Bradford Assay.

Table 2: Group Nines Whey Protein and Group Tens Casein Protein Absorbance
Data.
Group Nine's Whey Protein
pH Abs. (Heat) Abs. (No Heat) Mean Std Dev
2.68 0.426 0.446 0.436 0.014
3.47 0.422 0.430 0.426 0.006
4.57 0.468 0.464 0.466 0.003
5.54 0.418 0.454 0.436 0.025
6.32 0.576 0.498 0.537 0.055
7.52 0.648 0.458 0.553 0.134
8.51 0.666 0.656 0.661 0.007
Group Ten's Casein Protein
2.76 0.205 0.229 0.217 0.017
3.64 0.390 0.422 0.406 0.023
4.63 0.016 0.021 0.019 0.004
5.47 0.420 0.442 0.431 0.016
6.51 0.112 0.502 0.307 0.276
7.32 0.167 0.364 0.266 0.139
8.82 0.043 0.508 0.276 0.329
Table 3: Solubility of whey and casein proteins at different temperatures and pHs.
Group Nine's Whey Protein
pH %Solubility (Heat) %Solubility (No Heat)
2.68 50 53
3.47 50 51
4.57 56 55
5.54 49 54
6.32 70 60
7.52 80 54
8.51 83 81
Group Ten's Casein Protein
2.76 28 32
3.64 59 64
4.63 -3.2 -2.4
5.47 64 68
6.51 13 78
7.32 22 55
8.82 1.3 79

Whey Protein %Soluility vs. pH


90
80
70
60
% Solubility

50
40 Heated
30 Non Heated
20
10
0
2 3 4 5 6 7 8 9
pH

Fig. 3: %Solubility of Whey Protein at different temperatures and pHs.


Casein Protein %Solubility vs. pH
90
80
70
60
%Solubility

50
40 Heated
30 Non Heated
20
10
0
-10 2 3 4 5 6 7 8 9
pH

Fig. 4: %Solubility of Casein Protein at different temperatures and pHs.

V. Calculations
Calculating final whey and casein concentrations:
Whey: The final whey protein concentration can be determined using the line equation on the
Group Nines Standard Curve graph. Y= absorbance. X= Concentration in mg/ml. Using
whey protein absorbance data for the heated tube at pH 2.68:
y=1.2694x+0.0556 x=(y-0.0556)/0.9852
x=(0.426-0.0556)/0.9852 = 0.376 mg/ml
0.376 mg/ml x 1g/1000mg = 0.000376 g/ml
0.000376 g/ml x 100/100 = 3.76 x 10-2 g/100ml

Casein: The concentration of the casein protein can be found using the same method that was
used for whey protein, except using the line equation on Group Tens Standard Curve graph.
Using casein protein absorbance data for the heated tube at pH 2.76:
y=1.2039x+0.0354 x =(y-0.0354)/1.2039
x=(0.205-0.0354)/1.2039= 0.141 mg/ml
0.141 mg/ml x 1g/1000mg = 0.000141 g/ml
0.000141 g/ml x 100/100= 1.41 x 10-2 g/100ml
Calculating %Solubility
% Solubility can be determined by dividing the final protein concentration by the original
protein concentration and multiplying by 100. The original concentration of whey protein is
0.75 mg/ml (0.075g /100 ml). The original concentration of casein protein is 0.50 mg/ml
(0.050g/100ml)
Using the concentration of the heated tube of whey protein at pH 2.68:
3.76 x 10-2 g/100ml x 100 = 50%
0.075 g/100ml

Using the concentration of the heated tube with casein protein at pH 2.76:
1.41 x 10-2 g/100ml x 100 = 28%
0.050 g/100 ml

VI. Results/Discussion
The Bradford Assay standard curves were required in determining protein
concentration in this experiment, as they generated line equations that were manipulated
to calculate the concentrations. The R2 value for both group nine and group tens standard
curves are greater than 0.985, which shows a high level of accuracy. Errors in absorbance
data for the standard curves could potentially come from smudged cuvettes or not
measuring the BSA properly.
The isoelectric point of a protein can affect solubility. As the pH moves further from
a proteins isoelectric point, it becomes more soluble. Whey protein is expected to be
soluble at pH 4.6 and casein is expected to precipitate around pH 4.6 and the graph data
agrees with both of these expectations. Whey protein is not heat stable, and the graph for
group nines whey protein %solubility shows that the protein becomes less soluble with
heat around pH 5 and higher, however it was not expected to be more soluble at a higher
temperature. At pH 5.54, the solubility decreases in the heated sample, but then begins to
increase and is actually more soluble than the nonheated samples. Casein is expected to
be heat stable, but group tens %solubility graph for casein actually shows the opposite,
with the heated tubes of casein becoming much less soluble than the non-heated tubes
around pH 4.5 and higher. Perhaps the tubes were heated too long or there were
consistent difficulties with cleaning the cuvettes before placing them in the
spectrophotometer. Group ten also had a low absorbance reading for their casein tubes at
pH 4.63. pH 4.6 is the pH at which casein precipitates, however, the absorbance was so
low that the calculated %solubility values came out to be negative. While the idea that
casein precipitates at this pH and is more soluble as the pH deviates from the isoelectric
point is shown by the graph, the negative value could be a result of the
spectrophotometers output.

VII. Conclusion
The purpose of this experiment was to use the Bradford Assay to generate a standard
curve to calculate the %solubility of casein and whey protein. Whey is globular and
generally soluble in milk while casein precipitates at a pH of 4.6 and is more soluble at
pHs further than this value.

VIII. Questions
1. Review your data and identify the pH at which whey and casein proteins are least soluble.
Describe the condition of the protein at this pH, which causes this point of minimum solubility.
Casein protein was least soluble at a pH of 4.63. At this pH, the phosphoproteins precipitate. It
cannot be determined if the heated or nonheated samples in this experiment were more soluble,
because they both have negative %solubility values. Whey protein was least soluble at a pH of
5.54. The heated sample had a lower %solubility at 49%. Whey is not heat stable. As it is heated,
the protein begins to unravel and becomes less soluble.
2. At their respective points of minimum solubility, contrast the degree of solubility between
whey and casein proteins for both heated and nonheated protein solutions. What was the impact
of heat treatment? Discuss how amino acid composition and secondary, tertiary, and quaternary
structure influences the degree of solubility for each type of protein.
Lowest %Solubility (Heated) Lowest %Solubility (Non Heated)
Whey Protein 49 % (pH 5.54) 51% (pH 3.47)
Casein Protein -3.2 % (pH 4.63) -2.4% (pH 4.63)

Casein is found in milk as micelles that have a charged glycomacropeptide (GMP) tail. The GMP
tail is neutralized at pH 4.6 which causes the casein to precipitate. For casein, the lowest %
solubility is found at this pH for both the heated and non heated samples. Casein is heat stable, so
temperature is not expected to affect the solubility. Whey protein is globular in milk and is
expected to be much more soluble than the casein. When heat is applied to whey protein, it
becomes denatured and insoluble. It is not heat stable and the lowest solubility is found in one of
the heated samples.