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1"
Structure of the Casein
Supramolecule
Otherwise known as the Casein Micelle
Prof. Donald J. McMahon
Western Dairy Center
Utah State University
2"
What is the biological function
of milk?
• Caseins are milk proteins produced in the mammary
glands.
• Function of milk is to provide the neonate with the
nutrients required for growth and energy.
• Milk by necessity needs to be
• High in protein
• High in fat
• High in calcium and phosphate (higher than solubility limit)
• Low in viscosity
• Mammary glands must avoid calcification to function.
3"
How is this achieved by milk?
• Viscosity challenge met by packaging proteins as
supramolecules of colloidal particle size.
• Solubility and Calcification challenge met by rapid
binding of calcium phosphate by caseins into colloidal
particles
• By precipitating the calcium phosphate inside the
colloidal particles.
• Genetic imperative of caseins is to maintain calcium
binding and avoid pathological mammary gland
calcification
• even at the expense of optimum amino acid nutrition
• Caseins are flexible molecule without any fixed
structure
• and have rapid calcium binding properties.
4"
What are Caseins
• Major protein group in milk: ~ 80%
• Four major types in cows milk: αs1- αs2- β- and κ-caseins
• All have significant hydrophobic (water hating) regions
• They like to bind to each other to avoid contact of their
hydrophobic regions with water
• Three of them (αs1- αs2- β-casein) have unique clusters of
phosphorylated-serine amino acids
• They rapidly bind to calcium
• It makes them sensitive to calcium
• κ-casein is partially glycosylated and has no phosphoserine
clusters
• (-)ly charged hydrophilic domain
• (+)ly charged hydrophobic domain
• Insensitive to calcium
5"
What are the properties of the
Casein Supramolecule?
• Occur as spherical colloidal aggregates
• Size: 20-600 nm diameter, average about 150 nm
• About 2/3 of calcium and phosphate in milk is contained in
these spherical particles
• Plus significant amounts of Mg and citrate
• About 4 g water per g protein included in particles
• Average particle would contain about 10,000 protein molecules
• Particles held together by combination of
• Hydrophobic interactions
• Electrostatic interactions (including Ca-bridging)
• Colloidal stability of the supramolecule maintained via
• Charge repulsion
• Steric Stabilization
6"
What is a Supramolecule?
A supramolecule is defined as:
• 2 or more molecular entities
• held together and organized
• by means of non covalent binding interactions
7"
Models of Casein Aggregate Structure
• Colloidal aggregates mistakenly called “Casein Micelles” in
ca. 1930s and the name has persisted.
• They do not have a classical micelle structure of surfactants.
• Many models developed during past 40 years.
• Internal Structure models
• Framework structure formed by linear and branched
polymerization of caseins.
• Coat-Core Models
• Different structure in interior of particle than on the particle
surface.
• Sub-unit Models
• Colliodal particle consists of many smaller protein aggregates
8"
Framework
Model
• Branched Copolymers
• No role assigned to calcium
phosphate
• No proposal on how growth of
polymer network is limited to
colloidal size.
J. Garnier and B. Ribadeau-Dumas
J. Dairy Res. 1970, 37:493-504
9"
Submicelle Model
• Subunits have hydrophobic domains in center and hydrophilic
domains on surface.
• Subunits have differing amounts of κ-casein
• Subunits with large amount of κ-casein limit growth of the
particle
• Subunits held together via calcium phosphate clusters
D. G. Schmidt
1982
10"
Revised Submicelle
Model
• Casein subunits about 10 to
15 nm diameter.
• Clusters of calcium
phosphate located with
subunits
• Hydrophilic peptide chains
protrude from subunits
• Hairy layer removed by
renneting
• Hairy layer collapses if
ethanol added to milk
P. Walstra
Int. Dairy J., 1999, 9:189-192.
11"
Entangled
Framework Model
• Open structure of entangled chains
of casein molecules
• Crosslinked by small clusters of
calcium phosphate
• Segment density decreases from
interior to exterior of particle.
• Hairy layer on particle surface. C. Holt and D. S. Horne
Neth. Milk Dairy J., 1996, 50:85-111
12"
Dual Binding
Framework Model
• Caseins aggregate via
hydrophobic domains
• Phosphoserine domains bind
to calcium phosphate
clusters.
• κ-casein acts as a chain
terminator
D. S. Horne
Int. Dairy J., 1998, 8:171-177
13"
Investigating Supramolecular
Structure of Casein Micelles
Ph. D. research by Dr. Bonney Oommen
• Used Freeze drying Technique
• Investigated
McMahon, D. J. and B. S. Oommen. 2008. Supramolecular structure of the
casein micelle. J. Dairy Sci. 91:1709-1721.
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Copper Grid Parlodion film coat
Poly-L-lysine Casein micelles
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Wash
Uranyl stain
Wash
Flash freezing in (l)N2
cooled Freon
Freeze Drying
Electron Beam
EM Freeze Drying Technique
developed by Bill McManus
15"
Bovine Casein Micelles
• Casein micelles come in
various sizes.
• Casein micelle structure
varies between colloidal
particles.
• Need a model that
satisfies all possible
combinations of the
proteins.
16"
What About Other Species?
• Variation in casein
types
• Variation in casein
concentration.
• Variations in casein
micelle structure.
• Need a model that
satisfies all possible
combinations of the
proteins.
Human Pigmy Goat
Horse
17"
How did we examine it’s internal structure?
Stereopairs of images
can be obtained
18"
How do Caseins link together?
• Each cluster of phosphate groups attached to serine groups can link together
with calcium
• forming a protective layer around a calcium phosphate nanocluster.
• Each hydrophobic regions can attach to other hydrophobic regions on other
proteins.
• forming chains of caseins
• Functionality (f ) is the number of other entities with which they can
simultaneously interact.
κ: P-serine clusters = 0; H’phobic regions =1; f = 1
β: P-serine clusters = 1; H’phobic regions =1; f = 2
αs1: P-serine clusters = 2; H’phobic regions =2; f = 3 or 4
αs2: P-serine clusters = 3; H’phobic regions =1; f = 4
19"
(a) Using a stereo-pair view a single plane
on the periphery of a casein supramolecule
and look at how many entities attached
either in plane, or above and below it
(b) Assign a functionality (f) to
each electron dense region
f = 1 f = 2 f = 3 f ≥ 4
(c) Assign calcium phosphate
nanoclusters to be at f ≥4
(d) Assign casein to remaining electron dense areas
20"
Developed a model of the casein micelle
that involves protein aggregation and
stabilization of calcium phosphate
• Nanoclusters of calcium phosphate
act as nodes that tie together the
chains of aggregating proteins.
• Caseins prevent calcium phosphate
from nucleating into crystals.
• Hairs on periphery consist of
multiple protein molecules
• Aggregation occurs in various
forms to give an irregular structure
21"
Do All Casein powders form
Supramolecules?
• Calcium caseinate forms colloidal particles that are “similar” to casein
micelles in milk,
• Sodium caseinate can be converted into colloidal particles by adding
calcium.
100
nm
100
nm
Skim
Milk
Sodium
Caseinate
Calcium
Caseinate
100
nm
22"
Casein Micelle Summary
! Irregular lattice supramolecular structure of casein based on
flexible open conformation of individual proteins which can
prevent pathological calcification of the mammary gland
! Satisfies the concepts of self aggregating, interdependent,
and diversity that are found in natural systems.
! Internal chains grow until encounter with chain terminating
protein or bond with another chain
! Calcium phosphate clusters form because of low solubility
and casein polymerization because of calcium sensitivity
and hydrophobicity.
23"
How do you Concentrate the Casein
Micelles in Milk?
• Concentrate skim milk (3.2% protein, 2.6% casein)
using microfiltration
• Further concentrate using evaporation to 18% casein
• Manufactured by
Prof. Lloyd Metzger
at South Dakota
State University
• PhD research by Lu
Ying at Utah State
University
Further concentration by
evaporation
24"
What happens
when you cool a
micellar casein
concentrate?
• HC-MCC forms a gel
when cold and is liquid
at 50°C.
• Gelation temperature
depends on % protein
R² = 0.97
0
5
10
15
20
25
30
35
40
45
15.0 17.0 19.0 21.0 23.0 25.0
!"#$%&'##()&%*'+,'-.*/-'%01!2%
3-"*'()%#'4'#%052%
25"
Microstructure
of Cold Gelled
• Transmission electron
microscopy
• Casein micelles are
close-packed together
What causes cold
gelation?
• Sphere of hydration
around casein
micelles includes
“hairy” layer of
protein chains.
• Protein tendrils in
hairy layer restrict
movement of
particles
Casein micelles move
closer together as you
concentrate them
27"
Micellar Casein Concentrate
Summary
• MCC can be made with up to 23% protein
• No cold gelation if protein <16%
• Cold gelation is reversible.
• Need to warm MCC gel to disperse casein
micelles.
• Cold gelation of MCC is caused by steric
interference between protein tendrils on the
casein micelles
28"
Thank You
• Donald.McMahon@usu.edu
• www.usu.edu/westcent
