Proteins

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Proteins

• Colloidal Stability

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Proteins

• 95 of nitrogen in milk
• 5 NPN
• genetic variants

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Casein

• Precipitation at pH 4.6
• Not globular
• Form micelles with CCP
• ?s1 ?s2 ? ? 4 1 4 1.6
• ?s1, ?s2, ?-caseins are phosphoproteins
• Precipitate with Ca2
• ?-casein glycosylated
• Rennet sensitive
• p- ?-casein and GMP
• Not heat sensitive, above 120C become insoluble,
  at low pH heat sensitive
• Hydrophobic
• High charge
• Phosphates esterified to serine residues, ionized
  at pH of milk, bind Ca2

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Casein

• ?s1-casein Highest charge and highest phosphate
  content
• ?s2-casein two cysteine residues, S-S bridge
• ?-casein most hydrophobic but also hydrophilic,
  large number of proline residues, like a soap, T
  and ionic strength affect association, below 5C
  no association, part goes into solution
• ?-casein degradation product of ?-casein by
  plasmin, hydrophobic portion, residue left is
  proteose-peptones
• ?-casein two cysteine residues, can form
  intermolecular disulfide bonds, oligomers (5-11),
  
• 2/3 of the molecule have a carbohydrate group
  which is esterified to threonines, galactose,
  galactosamine, N-acetylneuraminic acid residues,
  hydrophilic

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Serum proteins

• Globular proteins, high hydrophobicity
• Heat sensitive, denatured
• ?-lactalbumin coenzyme for lactose synthesis, pH
  and salt dependent
• ?-lactoglobulin hydrophobic, no phosphate and
  less proline
• Two S-S linkages, one free sulfhydryl group
• pH and ionic strength dependent dimer
• Serum albumin S-S linkages
• Immunoglobulins antibodies against antigens
• IgM contain cryoglobulin, agglutination
  flocculation of particles, fat globules, bacteria
• Heat treatment inactivate agglutinins
• Proteose-peptone not heat sensitive, not
  precipitated at pH 4.6, precipitated by 12 TCA,
  degradation products of ?-casein, glycoprotein
  and others
• Lysozyme enzyme
• Lactoferrin iron binding protein, inhibitor of
  some Bacillus

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Proteins in milk
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Colloidal structure of milk

• Casein micelles colloidal stability
• Spherical particles
• 40-300 nm in diameter
• 104 casein molecules
• 8 g calcium phosphate / 100 g casein
• Proteose-peptone, enzymes
• Voluminous, more water than dry matter
• Negative charge

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• Submicelles
• 12-15 nm
• 20-25 casein molecules
• Hydrophobic and salt bridges
• Hydrophobic parts are buried inside
• Charged hydrophilic groups at outer layers
• Two major types with or without k-casein
• Hydrophilic part of k-casein sticking out the
  submicelle surface
• Adding excess of calcium and phosphate results in
  aggregation of submicelles into larger units
  micelles
• Calcium phosphate lowers electric charge on the
  surface and make them more compact
• Aggregation
• Due to protruding hair of k-casein in the
  aggregate submicelles cannot come closer
• A spherical aggregate formed with hydrophilic
  part of k-casein protruding out
• Hydrodynamic thickness of the hairy layer is
  about 7 nm

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Model

• Submicelles contain small regions of calcium
  phosphate attached to a serine residue
• Micelles are loose
• Hairs of k-casein on the outside provide
  stability
• Possible protein-protein contacts between
  submicelles
• Dry casein 0.7 ml/g
• Without hairy layer 2-2.5 ml/g
• With hairy layer 4 ml/g

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• K-casein determines the micelle size
• Voluminosity increases with decreasing micelle
  size, thickness of hairy layer and low protein
  content
• Large micelles have a higher CCP
• T, pH, aCa2 dependent changes

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Dynamic equilibrium
Casein molecules
Casein micelle
Submicelles
Caphosphate
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Dynamic equilibria

• In 10-12 s, the micelle structure changes
• Exchange of components between micelles and
  surroundings
• Mineral compounds exchange the fastest
• The counterions present as free ions in the
  electrical double layer would exchange very
  rapidly
• Some of the components of colloidal phosphate
  also exchange
• Submicelles can diffuse in and out (1 min)
• Size distribution of micelles depends on
  aggregation and disintegration
• Casein micelles can be broken by intense
  homogenization, but then rapidly reaggregate into
  original size
• There are also free caseins , increase at low T
• Exchanges decrease at low T

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Low Temperature

• Dissolution of ?-casein due to weakening of
  hydrophobic bonds
• Other caseins dissolve at a lower extent
• Voluminosity increases
• Loose ?-casein also protrudes from micelle
• Disintegration of micelles
• Dissolution of CCP
• Viscosity increases
• Colloidal stability increases
• 24 h at 4?C
• Changes are reversible, heating to 50?C and
  cooling to 30?C restores original properties

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High temperature

• Micelles shrink
• CCP increases
• T above 70C, casein molecules become flexible
• Above 100C, dissolution of part of k-casein
• At pH 7.2 dissolution complete
• Lower than pH 6.2 no dissolution
• Serum proteins associate with micelles

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Acidity

• As the pH is lowered
• Colloidal phosphate goes into solution, complete
  at 5.25
• Removal of calcium continues until isoelectric
  point of casein
• ? potential (charge) decreases
• Association of H with acid and basic groups of
  protein
• Increase in Ca2 activity, associate with acid
  groups
• Further decrease in pH causes dissociation of
  Ca2 negative charge increases and decreases
  again due to association with H ions, and then
  positively charged
• First swelling of micelles and then shrinkage
• Part of casein goes into solution at pH 5.3

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Acidity

• Particles are different according to pH but size
  are similar
• At normal pH
• Colloidal phosphate keep micelles together
• At low pH
• Phosphate dissolves resulting in weaker bonds
• Swelling of micelles
• Dissolution of caseins
• Internal salt bridges between positive and
  negative groups on the protein molecules keep
  molecules together
• The attraction is strongest at pH 4.6
• At pH 5.25, the number and/or strength of the sum
  of all kinds of bonds is weakest

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• Disintegration
• Weakening of the bonds between submicelles or
  between protein molecules in the submicelles
• Dissolution of colloidal phosphate by adding
  excess of a Ca binder like citrate, EDTA or
  oxalate
• Sodium dodecyl sulfate or large quantities of
  urea which break hydrogen bonds and/or
  hydrophobic interactions

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Colloidal stability

• DLVO theory electrostatic repulsion and
  attractive van der Waals forces
• Micelles have ? potential
• Micelles move and encounter each other due to
  Brownian motion
• Energy needed to bring two micelles together from
  an infinite to a close distance
• Steric repulsion between hairy layers (volume
  restriction)
• Hairy layers overlap (mixing)
• If solvent quality is poor then attraction

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Model

• Hydrodynamic thickness of hairy layer for a
  micelle is 7 nm
• If hairy layer overlap then steric repulsion
  occurs (about 20 nm)
• At this distance electrostatic repulsions are
  negligible
• Electrostatic repulsion by itself not enough to
  prevent flocculation but determines the closest
  approach of the micelles
• Crosslinking, salt bridging, Ca bridging,
  formation of colloidal phosphate linkages, at
  high T covalent linkages between amino acids

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Instability

• Change in micelles and then aggregation
• By changing environmental conditions
• e.g. Ethanol results in lower solvent quality for
  the hairs, collapse, colloidal phosphate passes
  to another state, lower the pH lower the ethanol
  concentration

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Aggregation of casein micelles