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A comparison of native state of casein micelles of buffalo and cow milk and its molecular changes under different
physico-chemical conditions
S. Ahmad, F.M. Anjum, J.F. Grognet, Frédéric Gaucheron
To cite this version:
S. Ahmad, F.M. Anjum, J.F. Grognet, Frédéric Gaucheron. A comparison of native state of casein micelles of buffalo and cow milk and its molecular changes under different physico-chemical conditions.
IDF International Symposium on Sheep, Goat and other non-Cow Milk, May 2011, Athènes, Greece.
�hal-01454321�
A comparison of native state of casein micelles of buffalo and cow milk and its molecular changes under different physico-
chemical conditions
Sarfraz Ahmad Sarfraz Ahmad, Faqir Muhammad Anjum, Jean- François Grongnet and Frederic Gaucheron
Contact: [email protected]
2
World production
(Billion L) 2009
Cow milk 580.
5 Buffalo milk 90.3
Others 25.8
Total 696.
Buffalo milk is the world’s second most produced milk (13%)6
> 92% of this production is from India (68% with 70.0 Billion L) and Pakistan (24% with 21.6 Billion L)
Buffalo milk is richer in all major components like protein, fat, lactose
& minerals than cow milk
Technological transformation into dairy products is very little in both major buffalo milk producing countries (Pakistan=3% and India=15%)
A vast knowledge exists on technological transformation of cow milk but a very limited information on the effects of processing on buffalo milk
Source: F.A.O, Food and Agriculture Organization
Introduction-Buffalo Introduction-Buffalo
milk milk
3
Microfiltratio
n Casein Gel
Acidificatio
n Alkalinization
To gain knowledge on casein micelles of buffalo milk:
- native state
- molecular changes under different physico-chemical conditions
Casein micelles of cow milk was used as a reference
Ionic strength
Heat treatment
Objective Objective
Casein micelles
Buffalo milk was richer in all major components
particularly casein contents than that of cow milk
pH= 6.76 Fat= 41 Lactose=
48.0 Ash=
7.7 TN=
33.5 NCN=
7.4 NPN= 1.6 CN= 26.1 TS= 136.7 g/k
g pH= 6.81
Fat= 70 Lactose=
52.1 Ash=
8.4 TN=
43.5 NCN=
8.9 NPN= 1.7 CN= 34.6 TS= 174.5 g/k
g
General General
Composition Composition
TN: Total nitrogen; NCN: Non casein nitrogen; NPN: Non protein nitrogen; CN:
Casein; TS: Total solids
Native Casein Native Casein
Micelles Micelles
Buffal o
Co w
Nanoclusters Association of α
s1-, α
s2& β - casein
Model of Carl Holt
κ -casein
Size: 200 nm Charge: -20 mV
Hydration:
1.9 g of H2O.g-
1 dry pellet
Size: 180 nm Charge: -20 mV
Hydration:
2.2 g of H2O.g-
1 dry pellet
κ-CN and αs2-CN
β-CN
β-Lg αs1-CN
Cow
α -LA
HPLC profiles are similar: 4 classes of caseins are present in both milks
κ-CN and αs2-CN
αs1-CN β-CN
β-Lg
Buffal o
Native Casein Native Casein
micelles micelles
Proteins fractions
Surface area
(µV*sec) Buffalo milk (%)
Surface area
(µV*sec) Cow milk
(%) Buffalo/Co w
κ-CN + αs2-CN 38077712 21 9134691 15 1.40
αs1-CN 56047939 31 18835282 30 1.03
β-CN 71787660 39 22010568 35 1.11
All caseins classes are more important in buffalo milk than that of cow milk
RP-HPLC
Profiles
Size of Particles and Aggregates
0 2 4 6 8 10
0,0 0,1 1,0 10,0 100,0 1000,0 10000,0 Size (µm)
Volume (%)
Cow milk
6.74
5.38
3.67
Acidification induces aggregation of casein
Identical process of protein aggregation during acidification in both milks
02 46 108
0.0 0.1 1.0 10.0 100.0 1000.0 10000.
0 Size (µm)
Volume (%)
6.76
4.89 3.31
Buffalo milk
02 46 108
0.0 0.1 1.0 10.0 100.0 1000.0 10000.
0 Size (µm)
Volume (%)
02 46 108
0.0 0.1 1.0 10.0 100.0 1000.0 10000.
0 Size (µm)
Volume (%)
Acidificati Acidificati
on on
Micellar Ca & Pi Solubilization
Diffusible Ca & Pi when pH
Solubilization of micellar calcium phosphate
♦: Buffalo milk
■: Cow milk
0 10 20 30 40 50
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 [Ca]
(mM)
pH Calcium in soluble phase
0 5 10 15 20 25 30
3.0 3.5 4.0 4.5 5.0 5.5 6.0 6.5 7.0 Pi
(mM)
pH
Inorganic phosphate in soluble phase
Acidificati Acidificati
on on
Micellar hydrations
micellar hydration with pH (corresponds to collapse of outer hairy layer of casein micelles and dissociation of Ca and Pi)
micellar hydration with pH (corresponds to of electrostatic interactions between casein and increase of volume of casein micelles)
micellar hydration with pH (corresponds to precipitation of casein micelles at pHi and general charge neutralization)
♦: Buffalo milk
■: Cow milk
0.0 0.5 1.0 1.5 2.0 2.5
3 3.5 4 4.5 5 5.5 6 6.5 7
H2O content of pellets of casein micelles (g H2O/g dry pellet)
pH
Acidificati Acidificati
on on
Size of the casein micelles and which approves their disruption at pH > 8.6
An in size is observed at pH > 10.8 may be due to reticulation of casein particles
-30 -28 -26 -24 -22 -20 -18 -16
6 7 8 9 10 11 12
Zeta (mV)
pH
0 50 100 150 200 250 300
6 7 8 9 10 11 12
Size of casein micelles (nm)
pH
A significant in the net negative charge of the casein micelles may be due to:
- changes in the ionizations of proteins and κ-casein-associated sugars - replacement of Na+ by Ca2+
Alkalinizatio Alkalinizatio
n n
Micellar size and charge
♦: Buffalo milk
■: Cow milk
Micellar hydration
Ionic cosmotropes (Ca & Pi ions) promote association of hydrophobic areas of protein by
binding water strongly and thus reducing the volume of water available to hydrate exposed
protein surfaces
0 1 2 3 4 5 6
6 7 8 9 10 11
Micellar hydration (g of water/g of dry pellet)
pH
Alkalinizatio Alkalinizatio
n n
♦: Buffalo milk
■: Cow milk
0.0 0.5 1.0 1.5 2.0 2.5
0 10 20 30 40 50 60
Time (m in)
AU
Supernatant of buffalo milk at pH ~6.7
0.0 0.5 1.0 1.5 2.0 2.5
0 10 20 30 40 50 60
Time (min)
AU
Supernatant of buffalo milk at pH ~9.7
0.0 0.5 1.0 1.5 2.0 2.5
0 10 20 30 40 50 60
Time (min)
AU
Supernatant of buffalo milk at pH ~10.8
0.0 0.5 1.0 1.5 2.0 2.5
0 10 20 30 40 50 60
Time (min)
AU
Cow milk at pH ~6.7
κ-CN & αs2-CN
αs1-CN β-CN
β-Lg
0.0 0.5 1.0 1.5 2.0 2.5
0 10 20 30 40 50 60
Time (min)
AU
κ-CN & αs2-CN
αs1-CN
β-CN
β-Lg Buffalo milk at pH ~6.7
0.0 0.5 1.0 1.5 2.0 2.5
0 10 20 30 40 50 60
Time (m in)
AU
Supernatant of cow milk at pH ~6.7
0.0 0.5 1.0 1.5 2.0 2.5
0 10 20 30 40 50 60
Time (min)
AU
Supernatant of cow milk at pH ~9.7
0.0 0.5 1.0 1.5 2.0 2.5
0 10 20 30 40 50 60
Time (min)
AU
Supernatant of cow milk at pH ~10.8
RP-HPLC
At pH 10.8 protein
molecules have been degraded
All caseins are increasing
in the
supernatants as a function of pH which approves the
increase of [TN]
Alkalinizati Alkalinizati
on on
Micellar Ca and Pi
Supernatan t
Ultrafilterate
0 2 4 6 8 10
6 7 8 9 10 11
pH
[Calcium] (mmol/kg)
0 5 10 15 20 25 30 35 40 45
6 7 8 9 10 11
pH
[Calcium] (mmol/kg)
0 2 4 6 8 10 12
6 7 8 9 10 11
pH
[Phosphate] (mmol/kg)
0 5 10 15 20 25 30 35
6 7 8 9 10 11
pH
[Phosphate] (mmol/kg)
[Ca] & [Pi] in supernatants and in ultrafiltrates of both milks
Association of calcium phosphate onto disrupted casein colloids???
Alkalinizatio Alkalinizatio
n n
♦: Buffalo milk
■: Cow milk
RP-HPLC
β-Lg disappeared
with of
temperatures
Modification of casein molecules particularly at 125°C
Disappearance of β-Lg was due to its denaruration and interaction with κ- CN
Modification of casein molecules may be due to:
-proteolysis -deamidation
-dephosphorylation -lactosylation
-formation and breakdown of s-s bridge
-intermolecular reactions
0 10 20 30 40 50
Time (min) 0.0
0.2 0.4 0.6 0.8 1.0 1.2
AU
0.0 0.2 0.4 0.6 0.8 1.0 1.2
AU
0.0 0.2 0.4 0.6 0.8 1.0 1.2
0 10 20 30 40 50
Time (min)
AU
0.0 0.2 0.4 0.6 0.8 1.0 1.2
AU
0.0 0.2 0.4 0.6 0.8 1.0 1.2
AU
NH
80°C
95°C
110°C
125°C
Buffalo Milk Cow Milk
κ-CN & αs2-CNαs1-CN β-CN
β-Lg κ-CN & αs2-CNαs1-CNβ-CN β-Lg
Heat Heat
Treatment
Treatment
Micellar Size and Charge
Overall size with temperature in both milks
100 140 180 220 260 300
70 80 90 100 110 120 130
Temperature (°C)
Size (nm)
Heat Heat
Treatment Treatment
-20.0 -18.0 -16.0 -14.0 -12.0 -10.0
70 80 90 100 110 120 130
Temperature (°C)
Zeta potential (mV)
♦: Buffalo milk
■: Cow milk
No effects of heat treatment on the zeta potential
around casein micelles of milks of both species
Micellar hydration
Micellar hydration remained constant
0.0 0.5 1.0 1.5 2.0 2.5 3.0
70 80 90 100 110 120 130
Temperature (°C) Micellar hydration (g H2O.g-1 dry pellet)
Heat Heat
Treatment Treatment
♦: Buffalo milk
■: Cow milk
17
Buffalo casein gel presents
- bigger particles than that of Cow casein gel with less
void spaces
Buffalo
Cow
Casein Casein
Gel Gel
Microstructure
0 1 2 3 4 5 6 7 8
1 1 0 1 0 0 1 0 0 0
Siz e ( µ m) CMLG
BMLG 71 ± 7
113 ± 7 µm
Volume (%)
♦: Buffalo milk
■: Cow milk
BMLG is firmer
0.0 0.5 1.0 1.5 2.0 2.5
BMLG CMLG
F max Fin (N)
BMLG has - higher visco-elastic
moduli (G´ & G˝) - more interactions or more building
material?
- Both have similar loss tangent (δ) = same structure, same
solid/liquid like properties
BMLG
100 1000 10000
0.2 0.3 0.4
CMLG
100 1000 10000
0.01 0.1 1 10
Frequency (Hz)
0.2 0.3
♦: G/ 0.4
■:G//
▲: Loss tangent (tan δ)
Loss tangent (tan δ) Storage (G') & elastic
(G") moduli (Pa)
Casein Casein
Gel Gel
Rheology
19
BMLG shows
- less syneresis with both methods - more water holding capacity
0 2 4 6 8 10 12 14 16
Surface syneresis Centrifugal syneresis
BMLG CMLG
%
Casein Casein
Gel Gel
Syneresis
The molecular changes were similar for both milks qualitatively
Precipitation/aggregation of casein suggested similar isoelectric pH
Minerals solubilized in the same manner (pH for total solubilization of Ca ~ 3.5 and of Pi ~ 4.7)
Micellar hydration (decrease and increase showing the similar behavior and also complete neutralization at pH 4.6)
Acidification process (already well established for cow milk) can not be directly extrapolated to buffalo milk so adaptations are necessary
Conclusio Conclusio
ns ns
Microfiltration-Native Casein micelles
Native casein micelles from buffalo milk were bigger in size, less hydrated, more mineralized with similar charge than that of cow milk
Similar classes of caseins in both milk but higher concentration in buffalo milk
Acidification
Effects of heat treat treatments were qualitatively similar for both milks but quantitatively different related to the compositional differences
The biochemical modifications were progressively
occurred with of intensity of heat treatments with major effects observed at 125°C in both milks
Conclusio Conclusio
ns ns
Heat treatments Alkalinization
Casein gel
Bigger size, undissolved Ca and lower hydration of casein micelles of buffalo milk resulted into a firmer gel
Ca & Pi in soluble phase due to possible precipitation onto casein
disruption & aggregation of casein molecules and size of micelles upto 120 nm
net - ive charge due to loss of net + ive charge ; micellar
hydration and solubility of casein micelles
Acknowledgeme Acknowledgeme
nt nt
08/02/13 23
