PROTEIN MINERAL INTERACTIONS FACTORS AFFECTING MILK PROTEIN AND MINERAL INTERACTION

1. welcome
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2. Presentation on: Protein-mineral interaction
Presented by
M Archana
MDK1702
Dairy Chemistry
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3. Protein minerals interaction:
 In milk the protein fraction casein and the mineral fraction that is
milk salts particularly Calcium, magnesium, phosphates and
citrates plays an important role. So the interaction between these
salts with casein micelles is called as protein mineral
interaction.
 In milk casein constitutes around 80% of total protein, caseins
belongs to the family of phosphoproteins.(Kawaski and Weiss,
2003).
 Phosphorylation is a post translational modification of caseins
and it occurs at the serine groups of proteins and rarely threonine
group.
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4.  Due to the placing of serine residues along the molecular sequence
of caseins αs1, αs2 and β-casein most of the phosphorylated
residues are found in clusters these are called as colloidal calcium
phosphate clusters (CCP).
 CCP is an integrating factor in the casein micelle and its removal
results in drastic changes in the micellar properties including an
increase in heat stability.
 Calcium fortification is a growing trend in the food industry
especially for milk and dairy products, however fortifying milk
with calcium can be a challenge especially if the product has to be
heat treated.
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5.  When soluble calcium salts are added to milk, the ionic calcium
modifies the mineral equilibrium of milk consequently the physico
chemical characteristics of casein micelles are modified and heat
stability decreases.
 The insoluble calcium phosphate particles such as Hydroxy
appatite (HA) or calcium carbonates rather than soluble calcium
phosphates are often used for fortification of milk.
 When HA particles were added to cow milk it was reported that
they donot modify the ionic calcium content or the pH of milk but
the protein present in milk can interact with HA particles.
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6.  The negative sites are formed by oxygen atoms associated with
phosphate groups are called P-sites, positively charged groups
called C-sites formed by the calcium ion in the crystal lattice.
 C-sites can complex specifically with negatively charged groups
of proteins such as carboxyl or phosphoserine groups.
 As the milk proteins are negatively charged at the natural pH of
the milk, they can adsorb to the C-sites of Hydroxy appatite.
 These adsorbed hydroxy appatite to casein causes them to acquire
negatively charged particles providing an electrostatic stabilization
to the proteins particles.
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7.  Whey proteins from whey also able to bind to HA particles but to
a lesser extent as that of caseins.
 In milk the phosphoserine groups of the caseins are involved in
the formation casein micelle structure and are bound to colloidal
calcium phosphate.
 The salt composition of milk is likely to affect the adsorption of
proteins on to the HA particles as the binding of protein has been
shown to depend on the mineral composition of suspending
medium.
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8.  The calcium ions have been shown to give a positive charge to the
surface of HA and this enhances protein adsorption by increasing
the number of sites available for protein binding through their
carboxyl and phosphoserine groups.
 As the milk contains free calcium, phosphate and citrate ions it is
likely that these ions will adsorb on to the HA particles.
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10. Influence of physicochemical conditions on mineral
distribution and stability of casein micelles:
 The micellar calcium phosphate plays an important role in the
maintenance of the structure of the casein micelle.
 It is useful to modify the physicochemical condition because the
heat induced changes allow to have a better understanding of the
properties of minerals and especially between salts of calcium
phosphates and casein micelles.
 Commonly occuring physicochemical events during the
technological operations such as acidification, heat treatment,
cooling, addition of chelants, addition of NaCl, addition of
divalent ions.
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11. 1) Acidification:
 The different acido-basic group of mineral constituents (organic
and inorganic phosphates, citrates, carboxylic acid residues etc.)
become more and more protonated when pH of milk is reduced.
 Consequently micellar calcium phosphate and small amounts of
magnesium citrates associated with casein micelles are dissolved
and the caseins are liberated in to the diffusable fractions of milk.
 The milk at pH 5.2 one part of calcium and total inorganic
phosphates are solubalised so that micellar calcium phosphate is
completely destroyed at this pH value.
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12. Cont…
 Calcium is totally solubalised only at pH 3.5 .
 The compilation of this results of pH induced solubalisation
suggests that calcium ion is implicated in to two types of
association that is one is inorganic phosphate and other is organic
phosphate (phosphoserine residues of casein molecules).
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13. 2) Heat treatment:
 Casein micelles are stable against heat treatments, however
several changes depending on the intensity of heat treatment is
well determined.
 Calcium phosphate present in diffusable fraction becomes less
soluble during heat treatment and consequently decrease in
calcium and inorganic phosphate after heat treatment.(Wahlgren
etal, 2003).
 By using NMR technique of determining Calcium and phosphate
an association of these constituents to casein micelles during heat
treatment at 30-64ºC.
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14. Cont..
 If heat treatment is < 95ºC for few minutes the modifications of
salt equilibrium are considered as reversible. In case of severe
heating > 120ºC for 10 min causes irreversible changes in the
casein micelles and salt partition.
 Phosphoserine residues of casein micelles can be partially
hydrolysed and decrease in calcium and phosphate content in
diffusable fraction.
 This causes changes in the structure and composition of the
original micellar calcium phosphate into a more insoluble form
which are identified as crystalline β-Tricalcium phosphate or
hydroxyappatite.
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15. 3) Cooling:
 The solubility of micellar calcium phosphate increases when the
temperature decreases and consequently the cooling of milk
induce a dissociation of micellar calcium phosphate.(Ichilczylc-
leone etal, 2003).
 The cooled milk shows one part of micellar calcium phosphate is
transferred into the diffusable fraction within 24 hours.
 These changes are reversible and the previous partition may be
re-established on rewarming.
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16. 4) Addition of chelants:
 Chelants have high affinity for cations and are able to displace
them, especially calcium.
 Thus addition of calcium chelating agents(citrate, EDTA, oxalate
etc.) to milk induces an increase of calcium and inorganic
phosphate contents in the diffusable fractions of milk.
 Consequently the casein micelles are more or less destroyed by the
disruption of micellar calcium phosphate
 In industry these added chelants improves heat stability and
storage life of concentrated milk products and prevent deposit
formation on heat exchangers and various membrane surfaces. 16

17. 5) Addition of NaCl:
 The addition of NaCl to milk leads to a slight decrease in the pH
and increase in the Ca2+ concentration in diffusable phase.
 These changes would corresponds to exchangers of divalent
cations or protons which were attached directly to phosphoseryl
residues of casein molecules by sodium(Na+)[9, 28, 30, 31]
 Increase in the ionic strength induces decrease in the activity
coefficients of the diffusable ions and consequently increase in
dissociation of ionic pairs.
 As a consequence of these exchanges, the hydration of casein
micelles is increases but their size and charge stay constant. 17

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19. 6) Addition of divalent cations(especially calcium):
 The addition of divalent cations induces important modifications
in the salt distribution between aqueous and micellar phases.
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20. Cont..
 The modifications depends on type of cations (calcium
magnessium iron, zinc, copper) and the concentration at which
they are added.
 The modifications of salt balance are controlled by the solubilities
of phosphates and citrates salts in diffusable fractions the
modification of salt balance of milk induced by calcium are most
important, thus after addition of 10mM of CaCl2 to milk about
80% of this ion was associated with casein micelles
 As the casein micelles are strongly modified their zeta potential
and hydration decreases with a consequence decrease in their heat
stability.
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21.  Refference:
Text book of advanced dairy chemistry vol:3 lactose,
water, salts and minor constituents 3rd edition
Edited by P L H McSweeney
P F Fox
LUCILE TERCINIER and co-workers 2014., J. Agric. Food
Chem.,“Interactions of Casein Micelles with Calcium
Phosphate Particles”
FREDERIC GUCHERON 2005 INRA, UMR Science et
Technologie du Lait et de l’OEuf, 65 rue de Saint-Brieuc, 35042
Rennes, France «The minerals of milk”
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22. thank you
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