Calcium caseinate has an isoelectric point of pH 4.6. This means it is insoluble in solutions with a pH less than 4.6. The pH of milk is 6.6, therefore, casein has a negative charge at this pH and is solubilized as a salt. If an acid is added to milk, the negative charges on the outer surface of the casein micelles are neutralized, by protonation of the phosphate groups. The casein micelles disintergrate and the casein (the neutral protein) precipitates because it is no longer polar, with the calcium ions remaining in solution

1. Casein extraction: A precipitation
reaction
• . By Dr. Robert D. Craig, Ph.D.

2. A precipitation reaction
• http://nutrafoodingredients.com/products/pr
otein/acid-casein

3. For CHM 111 and CHM 141
• Metal salts can be used at low concentrations
to precipitate enzymes and nucleic acids from
solutions. Polyvalent metal ions frequently
used are Ca2+, Mg2+, Mn2+ or Fe2+.

4. Casein extraction
• Casein extraction

5. The 21 amino acids

6. A zwitterion-

7. What is a zwitterion?
• The chemistry of amino acids is complicated
by the fact that the -NH2 group is a base and
the -CO2H group is an acid.
• In aqueous solution, an H+ ion is therefore
transferred from one end of the molecule to
the other to form a zwitterion (from the
German meaning hybrid ion).

8. The isoelectric point
• The isoelectric point (pI) is the pH of a solution
at which the net primary charge of a protein
becomes zero.
• At a solution pH that is above the pI the
surface of the protein is predominantly
negatively charged and therefore like-charged
molecules will exhibit repulsive forces.

9. Is used to focus DNA in analysis
fragments have a charge and respond
to pH changes

10. The isoelectric point
• Likewise, at a solution pH that is below the pI,
the surface of the protein is predominantly
positively charged and repulsion between
proteins occurs. However, at the pI the
negative and positive charges cancel

11. The isoelectric point
• Isoelectric point the negative and positive
charges cancel, repulsive electrostatic forces are
reduced and the attraction forces predominate.
The attraction forces will cause aggregation and
precipitation. The Isoelectric point of most
proteins is in the pH range of 4-6.
•
• . The precipitation of casein during
cheesemaking, or during production of sodium
caseinate, is an isoelectric precipitation

12. Precipitation
• Precipitation by means of acidification can be
considered in terms of simple chemistry as
follows, R being the casein protein:
• H2N-R-COO- + H+ → +H3N-R-COO
• casein
• micelle acid casein
• (pH = 6.6) (pH = 4.6)
• Colloidal dispersion Insoluble particles

13. How to extract from milk
• Can adjust pH
• Make then precipitate as they exist in a
specific buffer range

14. .

15. .

16. .Will precipitate proteins as ions go to
hydration layer

17. Protein precipitates
• Protein precipitates left in the salt solution can
remain stable for years-protected from
proteolysis and bacterial contamination by the
high salt concentrations. Ammonium sulfate
salt cannot be used in solutions that have pH >
8 because the ammonium ion has a buffering
effect on the solution. Sodium citrate[is a good
alternative for solutions above pH 8.