Cases and exercises
• Digital case study emulsions
1. Many food products are emulsions. An emulsion is a mixture of two mutually insoluble liquids
prepared in such a manner that one liquid is dispersed as droplets in the other liquid. Both
liquids have a large interface between the two different phases. Emulsions are
thermodynamically unstable so given enough time, phase separation will occur.
2. Two types of emulsions are: water in oil (W/O) and oil in water (O/W). Indicate for the following
food products whether they are W/O or O/W emulsions.
- Milk is a(n) O/W emulsion.
- Butter is a(n) W/O emulsion.
- Mayonnaise is a(n) O/W emulsion
3. You have seen that emulsions are thermodynamically
unstable. The main processes which cause the instability are
shown below. Often these processes occur simultaneously.
Fill in the name of each process below the picture.
1. Flocculation (network)
2. Flocculation (clusters)
3. Coalescence
4. Creaming
5. Ostwald ripening
4. Part about destabilization
169
,An emulsion can be made by homogenization. During homogenization, the interface between water
and oil increases. If the interface is not stabilized, phase separation will occur over time. Stabilization
of an interface can be done by a surface-active agent. Proteins are surface-active agents because they
are amphiphilic, meaning they have both hydrophobic and hydrophillic parts. The following questions
will focus on proteins as a surface-active agent.
5. To stabilize your emulsion, you would like to use a protein. Casein and whey protein are
available. Both are milk proteins, but they differ with respect to various properties.
- Casein is a random coil protein. This means that they nest easier at the interface because
they do not need to unfold. Whey proteins are globular proteins, which have several
hydrophobic patches exposed when folded, however most of the hydrophobic core only
becomes exposed when the protein unfolds. This happens at the interface, but is a relatively
slower process than that for a random coil.
170
,6. Statements – true or false
- The solubility of a protein is affected by the pH. true
- Proteins are best soluble at their pI value. false
- When pH = pI, there are no charges present on a protein. false
- When pH > pI, the protein has a net negative charge. true
- Amino acids have side groups which may contribute to the overall charge of the protein.
true
- At pH = 2.3, casein (pI = 4.6) is net positively charged. true
7. The pI of a protein can be determined by monitoring the solubility at different pH values.
After adjustment of the pH, the suspension is centrifuged resulting in a supernatant (soluble
proteins) and a pellet (insoluble proteins). The amount of soluble protein in the supernatant is
quantified by doing a Bradford analysis, in which the amount of protein is measured
spectrophotometrically at 595 nm (blue colour). The measured absorbance is plotted on a
calibration curve with the protein standard bovine serum albumin (BSA), after which the
amount of protein can be extrapolated.
In the table below the results are shown for the protein standard (BSA) at one specific pH
value. Plot the calibration curve in Excel.
- When your protein solutions contains 15 mg/mL, how many times should you at least dilute
your sample to fit into the linear part of the calibration curve? 19 times
o Correct, the answer is at least 19 times. When plotting the standard curve for BSA it
is noticeable that the upper part of the curve is not linear. Up to a concentration of
0.8 mg/ml the response of the absorbance shows linearity. To have the lowest error
the concentration of the sample should fall within the linear part of the curve.
- If the protein sample is not diluted sufficiently, the protein concentration will be
o The protein concentration is underestimated because the absorption signal is
saturated above the concentration of approximately 0.8
mg/mL
8. In the previous question you have seen that the charge and solubility
of a protein depend on the pH. In the figure below you can see the
solubility of a protein plotted against the pH of the solution.
- The pI of the protein in this solubility curve equals 5.4. When the
pH of the solution is equal to the pI of the protein the net charge on
the protein will be zero, which will result in proteins that aggregate.
o Emulsion stability is related to repulsion between oil
droplets. This repulsion is caused by electrostatic repulsion
due to charge on the droplets. The charges are introduced
by proteins that cover the interface of the oil droplet.
171
, 9. The time has come for you to choose which of the two available proteins you want to use. The
sauce which is to be produced should have a pH of 4.5. The iso-electric points of each protein
are shown in the table below.
Which protein would you choose to make your new sauce?
Hint: Think about the solubility profile of each protein
- Whey protein is suitable, because it is soluble at pH 4.5, whereas
casein is not. should know the properties of these proteins
10. You have chosen to use whey protein as a surface-active agent (and thus stabilizer). You have
obtained four different whey protein concentrates (from the same source). Before testing the
effect of whey protein on the emulsification properties, you decided to measure the solubility of
the four concentrates as a function of pH. The results are shown below.
Apparently you have obtained four different concentrates. Indicate for each concentrate what
happened to it, and how this relates to the pI.
11. You have seen that chemical reactions have a major effect on the the solubility profile of whey
proteins. In Q9 you have selected whey protein because it is soluble at pH 4.5. But solubility is
not the only important thing. There should also be electrostatic repulsion between the proteins,
and hence the oil droplets which they cover, so that the emulsion is more stable.
- The iso-electric point of sample 3 is at pH = 5.0.
- After hydrolysis, the solubility at pH (choose any value) = 6 is lower compared to the heated
concentrate.
- Indicate for the whey protein concentrates if they would be suitable as a surface-active
agent in your emulsion (pH = 4.5).
Concentrate 1: no
Concentrate 2: no
Concentrate 3: no
Concentrate 4: yes
172
• Digital case study emulsions
1. Many food products are emulsions. An emulsion is a mixture of two mutually insoluble liquids
prepared in such a manner that one liquid is dispersed as droplets in the other liquid. Both
liquids have a large interface between the two different phases. Emulsions are
thermodynamically unstable so given enough time, phase separation will occur.
2. Two types of emulsions are: water in oil (W/O) and oil in water (O/W). Indicate for the following
food products whether they are W/O or O/W emulsions.
- Milk is a(n) O/W emulsion.
- Butter is a(n) W/O emulsion.
- Mayonnaise is a(n) O/W emulsion
3. You have seen that emulsions are thermodynamically
unstable. The main processes which cause the instability are
shown below. Often these processes occur simultaneously.
Fill in the name of each process below the picture.
1. Flocculation (network)
2. Flocculation (clusters)
3. Coalescence
4. Creaming
5. Ostwald ripening
4. Part about destabilization
169
,An emulsion can be made by homogenization. During homogenization, the interface between water
and oil increases. If the interface is not stabilized, phase separation will occur over time. Stabilization
of an interface can be done by a surface-active agent. Proteins are surface-active agents because they
are amphiphilic, meaning they have both hydrophobic and hydrophillic parts. The following questions
will focus on proteins as a surface-active agent.
5. To stabilize your emulsion, you would like to use a protein. Casein and whey protein are
available. Both are milk proteins, but they differ with respect to various properties.
- Casein is a random coil protein. This means that they nest easier at the interface because
they do not need to unfold. Whey proteins are globular proteins, which have several
hydrophobic patches exposed when folded, however most of the hydrophobic core only
becomes exposed when the protein unfolds. This happens at the interface, but is a relatively
slower process than that for a random coil.
170
,6. Statements – true or false
- The solubility of a protein is affected by the pH. true
- Proteins are best soluble at their pI value. false
- When pH = pI, there are no charges present on a protein. false
- When pH > pI, the protein has a net negative charge. true
- Amino acids have side groups which may contribute to the overall charge of the protein.
true
- At pH = 2.3, casein (pI = 4.6) is net positively charged. true
7. The pI of a protein can be determined by monitoring the solubility at different pH values.
After adjustment of the pH, the suspension is centrifuged resulting in a supernatant (soluble
proteins) and a pellet (insoluble proteins). The amount of soluble protein in the supernatant is
quantified by doing a Bradford analysis, in which the amount of protein is measured
spectrophotometrically at 595 nm (blue colour). The measured absorbance is plotted on a
calibration curve with the protein standard bovine serum albumin (BSA), after which the
amount of protein can be extrapolated.
In the table below the results are shown for the protein standard (BSA) at one specific pH
value. Plot the calibration curve in Excel.
- When your protein solutions contains 15 mg/mL, how many times should you at least dilute
your sample to fit into the linear part of the calibration curve? 19 times
o Correct, the answer is at least 19 times. When plotting the standard curve for BSA it
is noticeable that the upper part of the curve is not linear. Up to a concentration of
0.8 mg/ml the response of the absorbance shows linearity. To have the lowest error
the concentration of the sample should fall within the linear part of the curve.
- If the protein sample is not diluted sufficiently, the protein concentration will be
o The protein concentration is underestimated because the absorption signal is
saturated above the concentration of approximately 0.8
mg/mL
8. In the previous question you have seen that the charge and solubility
of a protein depend on the pH. In the figure below you can see the
solubility of a protein plotted against the pH of the solution.
- The pI of the protein in this solubility curve equals 5.4. When the
pH of the solution is equal to the pI of the protein the net charge on
the protein will be zero, which will result in proteins that aggregate.
o Emulsion stability is related to repulsion between oil
droplets. This repulsion is caused by electrostatic repulsion
due to charge on the droplets. The charges are introduced
by proteins that cover the interface of the oil droplet.
171
, 9. The time has come for you to choose which of the two available proteins you want to use. The
sauce which is to be produced should have a pH of 4.5. The iso-electric points of each protein
are shown in the table below.
Which protein would you choose to make your new sauce?
Hint: Think about the solubility profile of each protein
- Whey protein is suitable, because it is soluble at pH 4.5, whereas
casein is not. should know the properties of these proteins
10. You have chosen to use whey protein as a surface-active agent (and thus stabilizer). You have
obtained four different whey protein concentrates (from the same source). Before testing the
effect of whey protein on the emulsification properties, you decided to measure the solubility of
the four concentrates as a function of pH. The results are shown below.
Apparently you have obtained four different concentrates. Indicate for each concentrate what
happened to it, and how this relates to the pI.
11. You have seen that chemical reactions have a major effect on the the solubility profile of whey
proteins. In Q9 you have selected whey protein because it is soluble at pH 4.5. But solubility is
not the only important thing. There should also be electrostatic repulsion between the proteins,
and hence the oil droplets which they cover, so that the emulsion is more stable.
- The iso-electric point of sample 3 is at pH = 5.0.
- After hydrolysis, the solubility at pH (choose any value) = 6 is lower compared to the heated
concentrate.
- Indicate for the whey protein concentrates if they would be suitable as a surface-active
agent in your emulsion (pH = 4.5).
Concentrate 1: no
Concentrate 2: no
Concentrate 3: no
Concentrate 4: yes
172
