Four Milk Portions and One Isoelectric Point

Portion 1 (40 °C)

• At pH 6.7, casein micelles carry a net negative charge from ionised carboxyl and phosphate groups. Electrostatic repulsion keeps the micelles apart.
• Adding acid supplies H+ ions, which protonate the carboxylate and phosphate groups. As the pH falls towards 4.6, the net charge approaches zero.
• At the isoelectric point, electrostatic repulsion disappears, so hydrophobic interactions and calcium phosphate bridging cause the micelles to aggregate and form curd.

Portion 2 (5 °C)

• The same pH change is eventually reached, but the low temperature slows molecular motion and weakens hydrophobic interactions, which are entropy-driven and stronger at higher temperature.
• Acid diffusion through cold milk is also slower, so the local pH may not fall evenly.
• Result: incomplete aggregation, seen only as cloudiness.

Key point: the isoelectric point explains why precipitation occurs, but temperature explains the rate and extent. Both factors must be used, because pH alone does not account for the difference between the two portions.

Ammonium sulfate (portion 4)

• The salt raises the ionic strength of the solution and competes with the protein for water molecules. The hydration shell is stripped and protein molecules associate, so they "salt out".
• This is a physical, reversible effect: the protein's native charge distribution and structure are largely retained.
• Removing the salt by dilution or dialysis restores the hydration shell, so the precipitate redissolves.

Acid curd (portion 1)

• Acid brings the protein to its isoelectric point, where the net charge is zero. Aggregation is driven by hydrophobic interaction and by loss of the calcium phosphate that helps stabilise the micelle.
• The resulting curd is a collapsed, aggregated network, not simply dehydrated native protein. Plain water cannot restore the charge because it does not change the pH.
• Note the qualification: acid casein can be redissolved by adding alkali to raise the pH well above 4.6. The point is that water alone is ineffective, whereas salt removal is sufficient for the salting-out precipitate.

Contrast: salting out preserves charge; isoelectric precipitation abolishes it.

Portion 3 (boiled milk)

• Heating to near 100 °C denatures the whey proteins, especially beta-lactoglobulin. Denatured whey proteins expose sulfhydryl and hydrophobic groups and complex with casein micelles, particularly with kappa-casein on the micelle surface.
• This alters the surface of the micelles and the calcium phosphate equilibrium. The result is a weaker, finer curd and a lower yield of recoverable curd under the same acid treatment.
• The effect is a heat-induced change in protein structure, not a change in the isoelectric point itself.

Recovery from portion 1

1. Filter the curd through muslin and wash with water to remove lactose and salts.
2. Dissolve the curd in dilute sodium hydroxide or sodium bicarbonate to raise the pH to about 7; the casein becomes charged and dissolves.
3. Reprecipitate by adding acid slowly to pH 4.6.
4. Filter, wash, and dry at low temperature.

Confirmation tests

• Biuret test: violet colour with copper sulfate in alkaline solution, indicating peptide bonds.
• Xanthoproteic test: yellow colour with concentrated nitric acid, indicating aromatic residues.
• A nitrogen determination would confirm that the product is protein rather than residual carbohydrate or fat.