Proteins C1 - Common Protein Sources

What are the 6 common protein sources in food science?

Milk, meat, eggs, fish, cereals, and seed proteins (e.g. soybean).

Why does each protein system's properties vary?

Each protein system is unique and its properties depend on the process(es) used to extract or isolate it.

What percentage of fluid milk is protein?

Approximately 3.5% (fluid milk is ~12% total solids).

How do milk proteins rank nutritionally compared to other proteins?

High nutritive value (good amino acid balance), surpassed only by egg albumin.

What essential amino acid are milk proteins a particularly good source of?

Lysine.

What are the two main groups of milk proteins?

Caseins and whey proteins.

How is casein defined in terms of isoelectric point?

Casein is a general term for milk proteins with an isoelectric point at pH 4.5.

How is casein isolated from milk?

By adjusting the pH of milk to 4.5 using acid causing casein to precipitate.

What are whey proteins?

The proteins that remain in solution (the filtrate) after casein is precipitated at pH 4.5.

What acid is most commonly used in acid precipitation of milk proteins?

TCA (Trichloroacetic Acid).

After acid precipitation what salt treatment separates lactoglobulin from lactalbumin?

0.5 saturation with (NH4)2SO4 or saturation with MgSO4 precipitates lactoglobulin; lactalbumin remains in filtrate.

Besides acid precipitation how else can casein be separated from milk?

By rennet action or by saturation with sodium chloride.

In acid precipitation of total milk protein (3.2%), how much casein and whey protein are obtained?

Casein ~2.5%, whey protein ~0.57%.

What percentage of total milk protein do caseins represent?

83%

What percentage of total milk protein do whey proteins represent?

17%

Name the 5 casein fractions and their % of total milk protein.

αs1 (36%), αs2 (9%), β (21%), κ (12%), γ (4%).

What is the most abundant whey protein fraction and its % of total milk protein?

β-Lactoglobulin at 10%.

What % of total milk protein is α-Lactalbumin?

2–3%.

What % of whey proteins does β-Lactoglobulin represent?

58%

What % of whey proteins does α-Lactalbumin represent?

13%

Name all whey protein fractions and their % of whey.

β-Lactoglobulin 58%, α-Lactalbumin 13%, Immunoglobulins 12%, Serum albumin 6%, Minor proteins 12%.

Who first distinguished casein and lactalbumin and lactoglobulin in 1877?

O. Hammarsten.

What key property of casein did Hammarsten's separation procedure establish?

Casein is insoluble in weakly acidic media.

What did Hammarsten's procedure involve?

Skim milk diluted then acidified with acetic acid — casein flocculates while whey proteins stay in solution.

What type of protein is casein and what amino acid bears the modification?

Casein is a phosphoprotein; phosphate is attached to the amino acid serine.

What is the unique 3-amino acid phosphorylation sequence in casein?

Ser-X-A, where X is any amino acid and A is either glutamic acid or serine-phosphate.

Who proved in 1939 that casein has three fractions using electrophoresis?

Mellander.

What are the three casein fractions identified by Mellander?

α, β, and γ-casein.

What is γ-casein?

Proteolytic fragments of β-casein.

What is the molecular weight of αs1-casein?

23.6 kDa.

What is the molecular weight of αs2-casein?

25.2 kDa.

What is the molecular weight of β-casein?

24 kDa.

What is the molecular weight of κ-casein?

19 kDa.

What are the genetic variants of αs1-casein?

A, B, C, D, E.

What is the isoelectric point range of αs1-casein?

pH 4.92–5.35.

What is the isoelectric point range of β-casein?

pH 5.20–5.85.

What is the isoelectric point range of κ-casein?

pH 5.77–6.07.

Which casein has the highest phosphorus content?

αs2-casein at 1.4%.

What is the phosphorus content of αs1-casein?

1.1%.

What does the notation αs1-CN B-8P mean?

αs1-casein, genetic variant B, with 8 phosphorylated serine residues.

What is the net charge of αs1-casein at pH 6.5?

Net negative charge of 22.

Why does αs1-casein have no apparent secondary structure?

Its polypeptide chain contains 8.5% proline distributed uniformly which disrupts secondary structure formation.

What is notable about αs2-casein's phosphorylation?

It has the highest number of phosphorylations and a low proline content.

How many amino acids does β-casein have and how many genetic variants?

209 amino acids; 5 genetic variants.

Why does β-casein have surfactant properties?

Its N-terminal segment is highly hydrophilic (high negative charge) and C-terminal is highly hydrophobic — like a surfactant molecule.

What is the physical form of casein in milk?

Relatively large, nearly spherical particles called micelles (20–300 nm diameter).

What ion is critical for casein micelle stability?

Ca2+ (calcium ions).

What happens to casein micelles if Ca2+ is removed by dialysis?

Micelles dissociate; they will re-form if Ca2+ is added back.

What causes the opaque whiteness of milk?

Casein micelles scatter light — an optical effect that makes milk appear white.

What is the diameter range of casein micelles?

10–300 nm, with a mean of 150 nm.

What is the approximate volume of a casein micelle with 150 nm mean diameter?

1.6 × 10^6 nm³.

How do casein micelle sizes compare to fat globules?

Micelles are smaller (10–300 nm) than fat globules (100–10,000 nm).

How many monomers are in an average casein micelle?

About 25

How highly solvated are casein micelles?

1.9 g water per g protein — making them porous.

What % of total casein is present as monomers (serum caseins) in milk?

Up to 10%.

What % of casein proteins aggregate into micelles and complexes?

90%

What two factors mainly determine the extent of casein aggregation?

Ca2+ concentration and pH.

What happens to casein complexes dialyzed against a chelating agent?

Equilibrium shifts completely to monomers.

Name the 3 forces holding casein micelles together.

1) Hydrophobic interactions (minimal below 5°C), 2) Electrostatic interactions (Ca2+ and calcium phosphate bridges), 3) Hydrogen bonds.

How do calcium ions act as electrostatic bridges in caseins?

Ca2+ bridges between phosphoserine clusters and acidic residues, promoting aggregation and precipitation.

At what temperature are hydrophobic interactions in casein micelles minimal?

Below 5°C.

What type of phosphate (besides ester phosphate) does casein contain?

Colloidal phosphate (calcium phosphate), which helps maintain structural integrity of the casein micelle.

What is unique about κ-casein's solubility versus other caseins in the presence of Ca2+?

κ-casein is the only casein that remains soluble in the presence of Ca2+ at concentrations found in milk.

How does κ-casein protect other caseins from precipitating?

Association of αs1, αs2, and β-casein with κ-casein prevents their coagulation in the presence of Ca2+ ions.

Which casein is most sensitive to Ca2+ precipitation?

αs2-casein (most sensitive), then αs1, then β-casein (least sensitive among the three).

What are the two main proposed models for casein micelle structure?

The coat-core model (most popular) and the subunit/submicelle model.

In the coat-core model what is in the core?

The calcium-sensitive αs-caseins form the core.

In the coat-core model what covers the core?

A layer of κ-casein.

What is the function of the κ-casein coat in the coat-core model?

To protect the micelle from insolubilization by calcium ions.

What is a limitation of the coat-core model?

It does not explain the position of the colloidal calcium phosphate.

In the submicelle model where are κ-casein molecules located?

On the surface of the submicelles.

In the submicelle model how does κ-casein prevent further aggregation?

Its hydrophilic C-terminal domains protrude like hairs from the surface, sterically blocking aggregation.

How thick is the κ-casein surface layer on submicelles?

Approximately 5 nm thick.

How many casein molecules make up a submicelle?

10–100 casein molecules.

What does CMP stand for in the sub-micelle model?

Casein macro peptide (the hairy layer from κ-casein).

What is rennin (chymosin) and where does it come from?

A proteolytic enzyme obtained from the fourth stomach of the calf, with specificity for κ-casein.

What specific site does rennin attack on κ-casein?

Rennin cleaves the peptide bond between Phe-105 and Met-106.

What sequence does chymosin recognize in κ-casein?

The sequence from His-98 to Lys-111.

What two fragments result from chymosin cleavage of κ-casein?

Para-κ-casein (residues 1–105) and a glycomacropeptide (residues 106–169).

What happens to para-κ-casein in the presence of Ca2+?

It precipitates.

What happens to the released glycomacropeptide?

It is soluble and goes into the whey.

Why does rennin cause coagulation of the whole micelle?

By cleaving κ-casein, the micelle-stabilizing coat is removed, so Ca2+ now causes the remaining caseins to coagulate (curd formation).

What is the underlying principle of rennet in cheese making?

Rennin cleaves κ-casein → micelles destabilize → caseins coagulate into curd separating from whey.

What is paracasein?

Casein obtained by rennet (chymosin) action — named to differentiate it from acid/isoelectric casein.

How are acid/isoelectric casein and paracasein physically similar?

Both are not temperature sensitive — they can be boiled without denaturing.

What is the first step in cheese making?

Formation of paracasein (rennin cleavage of κ-casein leading to curd formation).

Name food applications of casein/caseinates.

Protein enrichment and stabilization in processed meats, baked products, candies, cereals, ice creams, whipping creams, coffee whiteners.

Name non-food industrial uses of casein/caseinate.

Paper sizing (for ink adhesion), waterproof glue, water-repellent textiles, and production of certain plastics.

What are the two methods to achieve casein coagulation for recovery?

Acidification (acid coagulation) or enzymatic treatment (protease enzymes).

How is acid coagulation of casein achieved industrially?

Lactic acid fermentation or direct addition of acids (HCl, H2SO4, lactic acid, H3PO4) at 35–50°C to pH 4.2–4.6.

What is the isoelectric point of casein?

pH 4.6–4.7.

In enzymatic coagulation for casein recovery what enzymes are used and at what temperature?

Chymosin and pepsin at 45°C; then heated to 65°C to inactivate the enzyme.

What are the two dry products of acid and rennet casein processing?

Acid casein and rennet casein (dried in a whirlwind drier after centrifugation and washing).

How is sodium caseinate or calcium caseinate produced?

Casein is dispersed in water (20–25% w/w), treated with alkali (NaOH or Ca(OH)2) at 89–90°C, pH 6.2–6.7, then spray-dried.

Define whey proteins.

Proteins that remain in solution after coagulation of caseins.

What is the most prevalent protein in whey?

β-Lactoglobulin.

What is the molecular weight of β-Lactoglobulin?

18.3 kDa.

What is the isoelectric point of β-Lactoglobulin?

pH 5.34.

What are the two major genetic variants of β-Lactoglobulin found in North America?

Variants A and B.

How do β-Lactoglobulin variants A and B differ?

B has Gly at position 64 and Ala at 118; A has Asp64 and Val118 (2 point mutations).