Milk proteins Flashcards
Protein functionality refers to:
physical and chemical properties that influence the performance of proteins in food systems during processing, storage, preparation, and consumption.
Ignoring nutrition and other bioactivity!
Performance = solubility, thermal stability, gelation, emulsifying, foaming, fat binding, water binding, …
List the functional roles of food proteins in food systems.
[list some of]
- Solubility
- Viscosity
- Water binding
- Gelation
- Cohesion-adhesion
- Elasticity
- Emulsification
- Fat and flavour binding
What is the mechanism of solubility?
Hydrophilicity
What is the mechanism of viscosity?
Water binding; hydrodynamic size and shape
What is the mechanism of gelation?
Water entrapment and immobilization, network formation
What is the mechanism of cohesion-adhesion?
Hydrophobic, ionic, and hydrogen bonding
What is the mechanism of elasticity?
Hydrophobic bonding; disulfide crosslinks
What is the mechanism of emulsification?
Adsorption and film formation at interfaces
What is foaming?
Interfacial adsorption and film formation
What is the mechanism for fat and flavour binding?
Hydrophobic bonding; entrapment
Where do we get milk proteins from?
What does milk look like under a microscope?
Casein micelles ~50-100 nm in diameter
Describe the composition of milk.
Describe the concentration of proteins in bovine milk.
Much more casein than whey protein.
Describe the structure of casein.
- Family of phosphoproteins (phosphorylated via post-translational modification)
- Functionally defined as proteins that precipitate ≤ pH 4.6
- Intrinsically disordered proteins
Describe the properties of casein proteins. [4]
- Number of proline residues: disrupt secondary structures, leading to flexible, open conformation
- Number of intra-molecular -s-s- bonds: Lack disulfide cross-linkages, meaning casein will not form rigid 3D structures, but instead will remain highly hydrated and flexible.
- Phosphorus content: Phosphate groups attached to serine residues; contributes to casein negative charge and ability to bind calcium
- Sensitivity to calcium: Highly sensitive, which impacts ability to form micelles; calcium bridges help hold micelles together; kappa-casein which is less sensitive, stabilizes micelles by preventing uncontrolled precipitation (too much free calcium can lead to aggregation and precipitation - curd formation)
- Isoelectric point: Caseins have a low pI, meaning they will retain a negative charge in milk which has a pH ~6.7.
These properties make caseins unique among milk proteins, allowing them to form micelles, stabilize emulsions, and function as excellent nutritional and structural components in dairy-based products.
Compare alpha-s1-casein, beta-casein, and kappa-casein.
- αs1-Casein: Major casein protein (~40% of total casein), plays a key role in calcium binding and micelle formation, highly hydrophobic, and involved in cheese curd formation.
- β-Casein: More surface-active, contributes to micelle hydration, forms aggregates in cold milk (cold gelation).
- κ-Casein: Acts as a stabilizer, prevents micelles from aggregating; crucial in cheese-making as rennet cleaves it, triggering coagulation.
Compare alpha-s1-casein, beta-casein, and kappa-casein functions in milk and role in cheese-making.
Describe alpha-s1-casein.
- Highly negative (24 at pH 6.7)
- Crucial to binding calcium nanoclusters.
Describe beta-casein.
- Greater charge in N-terminus.
- β-Casein has a highly hydrophilic N-terminus (positively charged) and a hydrophobic C-terminus.
The greater charge in the N-terminus of β-casein is critical for its solubility, micelle stabilization, temperature-dependent dissociation, emulsification properties, and digestion behavior, making it functionally distinct from other caseins.
Describe kappa-casein.
- Ser149 is phosphorylated (SerP149), contributing to κ-casein’s negative charge.
- The C-terminal region of κ-casein contains negatively charged glutamic acid (Glu) residues. Contributes to the hydrophilic nature of the C-terminal tail, allowing κ-casein to interact with water and stabilize micelles. This region extends into the surrounding liquid, forming a protective shell around micelles. The hydrophilic C-terminus is cleaved by chymosin during cheese-making, triggering curd formation.
- The glycosylation of Thr133 makes κ-casein more hydrophilic, reinforcing its stabilizing role at the micelle surface.
- When chymosin (rennet) cleaves κ-casein, it cuts the molecule at Phe105-Met106. The N-terminal fragment (para-κ-casein, residues 1-105) remains attached to the casein micelles and stays in the curd.
This allows the casein micelles to aggregate into a gel, forming the structure of cheese. Para-κ-casein is hydrophobic, which helps it contribute to curd formation. - The C-terminal fragment (residues 106-169), which contains the highly charged, glycosylated region, is released into the whey after chymosin cleavage. This soluble glycomacropeptide (GMP) does not participate in curd formation and remains in the whey fraction.
- By removing the hydrophilic, stabilizing C-terminal region, chymosin destabilizes the casein micelles, allowing them to aggregate into curds.
Significance of:
SerP at 149
Kappa-casein
Limited phosphorylation keeps κ-casein surface-active and less sensitive to calcium.
Significance of:
Glu towards C-terminus
Kappa-casein
Creates a strong negative charge, stabilizing micelles by electrostatic repulsion.
Significance of:
O-glycosylation at Thr133
Kappa-casein
Enhances water solubility, contributes to steric hindrance, and stabilizes casein micelles.
Significance of:
Hydrophilic C-terminus (q = -11)
Kappa-casein
Forms a protective shell around micelles, preventing aggregation.
Significance of:
Para-kappa-casein
Forms the structural component of cheese curd after chymosin cleavage.
Significance of:
Glycomacropeptide
Kappa-casein
Enters the whey fraction, has prebiotic properties, and is used in nutrition.
What is the role of chymosin in cheese making?
Initiates cheese-making by cleaving κ-casein, causing micelle destabilization and curd formation.
Para-kappa-casein stays with the curd; glycomacropeptide is released in the whey.
What do all the different models of casein micelles have in common?
- Interior occupied by Ca2+-sensitive
proteins (α-s1, α-s2, β-caseins) - κ-casein oriented to surface, with (–ve) charged C-terminus facing outwards = ‘hairy’ negative surface
- κ-casein C-terminus is glycosylated = steric hindrance prevents clumping
What is a casein micelle?
Particles of caseins, Ca2+, CCP & H2O; colloidal calcium phosphate, CCP
The interior of a casein micelle is:
Occcupied by calcium-sensitive proteins (alpha-S1; alpha-S2, and beta-caseins)
Which casein is oriented to the surface of a casein micelle?
- Kappa-casein, with (–ve)
charged C-terminus facing outwards - “hairy” negative surface
Describe the role of kappa-casein C-terminus glycosylation in casein micelles.
Steric hindrance prevents clumping
Describe the nanocluster casein model.
- Closely matches small-angle neutron scattering data
Describe how neutron scattering has been used to determine the structure of a casein micelle.
**'CCP Invisible' (Colloidal Calcium Phosphate Invisible)**
* At a specific D₂O/H₂O ratio, the scattering power of CCP matches that of the solvent, making it effectively "invisible."
* This means only the casein protein structure is visible, so we can study the protein matrix without interference from the CCP phase.
**'Casein Invisible'**
* At a different D₂O/H₂O ratio, the scattering power of casein proteins matches that of the solvent, so caseins become invisible.
* In this case, only the CCP nanoclusters are visible, allowing researchers to analyze their distribution inside the micelle.
Describe SANS analysis of casein micelles.
- Compare calculated and experimental SANS profiles at increasing %D2O
- Each graph represents SANS intensity vs. scattering angle (or q-value) at a specific D₂O percentage.
- As D₂O increases, the scattering contrast changes, allowing selective visualization of casein vs. CCP (colloidal calcium phosphate).
- At Low %D₂O (~0%) → Casein scatters strongly, CCP is more visible.
- At Intermediate %D₂O (~40-60%) → CCP and casein have similar scattering power, so their contrast is reduced (one component becomes “invisible”).
- At High %D₂O (~100%) → Casein becomes more transparent, highlighting the CCP nanoclusters.
Caseins are coagulated by heat.
True or False?
False.
Gives cheese its unique melting profile.
Caseins are not coagulated by heat.
True or False?
True.
Gives cheese its unique melting profile.
What happens to casein micelles in cheese-making?
Compare curd and whey.
- Bacteria ferment the lactose: lactic acid (lowers pH)
- Casein micelles clump together @ pH < 4.6 (30 degrees C):curd
- Chymosin: hydrolysis of κ-casein removes hydrophilic tail (FM106), produces a firmer cheese; fat trapped in casein matrix
- Serum drains away = whey proteins (soluble)
Describe whey proteins.
Notice that whey proteins have more ability to form disulfide cross-linkages, and have a higher pI than caseins.
This means they are more likely to form rigid, globular structures that remain soluble in whey during cheese production.
How do the number of cysteine residues, S-S bonds, and isoelectric points (pI) differ between whey and casein proteins?
Whey Proteins:
- More cysteine residues → Can form intramolecular & intermolecular disulfide (S-S) bonds.
- Globular & heat-sensitive → Denature and aggregate when heated.
- Higher pI → Precipitate near pI during heat treatment.
Casein Proteins:
- Few/no cysteine residues → No disulfide bonds.
- Flexible, random coil structure → Stable in milk, forms curds with acid or rennet.
- Lower pI → Precipitate in cheese-making at pH < 4.6.
Describe the proteins found in whey.
Largest component is beta-lactoglobulin protein
Describe beta-lactoglobulin.
- Binds vitamin A and non-polar compounds
- Absent from human milk
- Major cow’s milk allergen
How does β-lactoglobulin’s structural state change with pH, and why is this important?
- pH < 3.5 → Monomer (prevents aggregation, affects solubility).
- pH > 3.5 → Dimer (most stable in milk, impacts heat sensitivity).
- pH > 3.7 → Octamer (promotes gelation, relevant in food gels).
- pH > 5.4 → Dimer (dominant in milk, affects whey protein functionality).
- pH > 7.5 → Monomer (reduces thermal stability, alters processing properties).
🔹 Relevance:
Affects heat stability, solubility, and gelation in dairy products.
Describe alpha-lactalbumin.
- 4 disulfides; very stable (Tm> 80 degrees C)
- homologous to lysozyme
- binds Ca2+
- plays role in lactose synthesis
Describe precision fermentation of milk proteins using yeast or fungi.
- Select and modify host organism
- Produce the recombinant milk protein
- Purify the recombinant milk proteins
- Make reassembled casein micelles
- Produce dairy products like cheese
Can milk proteins be produced with plants?
Apparently yes?
Can milk proteins be produced with microbes?
i.e., not those in the cow’s gut
Apparently yes!
