NMR Study of the Water Diffusion in Casein Systems. Effect ...€¦ · Euro food’s water, March 2004 Materials and methods vThe micellar casein solutions were prepared : Øwith

Euro food’s water, March 2004

NMR Study of the Water Diffusion in NMR Study of the Water Diffusion in Casein Systems.Casein Systems.

Effect of the Effect of the MicellarMicellar Casein Casein ConcentrationConcentration

F. Mariette A. MétaisCEMAGREF, Food process engineering research unit,

Rennes


ObjectivesObjectives

Ø To study the effect of casein micelle concentration on the water diffusion coefficient

Ø To find a model to explain the water diffusion in casein systems

Ø To study the effect of coagulation by rennet on the water diffusion coefficient


How to measure the water diffusion by How to measure the water diffusion by NMR ?NMR ?

v To obtain a NMR signal, the sample is introduced into a magnetic field

if the magnetic field is homogenous, the NMR signal is insensitive to molecular diffusion.

But, if the magnetic field is inhomogeneous, then the NMR signal become sensitive to molecular diffusion.

So, a linear field gradient is applied in order to measure the Diffusion coefficient.

- +- +

mag

net

mag

net

sample


NMR signal with gradient


NMR signal without gradient (g=0)

90°x90°x 180°y180°yR

F P

uls

e

RF

Pu

lse

90°x90°x 180°y180°y

RF

Pu

lse

RF

Pu

lse

g



∆ NMR signal with gradient

90°x90°x

RF

Pu

lse

RF

Pu

lse

180°y180°y

g

∆= Dd 6

∆

90°x90°x 180°y180°yR

F P

uls

e

RF

Pu

lse

NMR signal with gradient

g

d

d


[ ]kDtgI

tgI−=

=exp

),0(),(

0

K = F(g ,∆)

g, the gradient strength and ∆, the diffusion time


g

Ln(I/I0) D



v by changing g with ∆ = cte

g

Ln(I/I0)

No restriction, D is the self-diffusion coefficient

Restriction D is an apparent diffusion coefficient

g

Ln(I/I0)

g

Ln(I/I0)

g

Ln(I/I0)

Heterogeneous diffusion processApparent diffusion coefficientHomogeneous diffusion process

Self diffusion coefficient

v by changing g for different ∆ values


Why using NMR to measure the water Why using NMR to measure the water diffusion coefficient ?diffusion coefficient ?

v Non invasive technique, Ø So the measurements could be done without any perturbation of the

diffusion process.v no tracer, Ø the measurements could be done without any specific chemical

preparation.v the diffusion could be measured in any directionØ If Dx=,Dy=Dz then the diffusion is isotropicØ If Dx≠,Dy ≠ Dz then the diffusion is anisotropicØ so diffusion anisotropy could be detected.

v the diffusion could be measured for different travelling distances,

Deff=D0 Deff< D0

d

2*r>> d

d

2*r≤ d


Why using NMR to measure the water Why using NMR to measure the water diffusion coefficient ?diffusion coefficient ?

v Non invasive technique, Ø So the measurements could be done without any perturbation of the

diffusion process.v no tracer, Ø the measurements could be done without any specific chemical

preparation.v the diffusion could be measured in any direction, Ø so diffusion anisotropy could be detected.

v the diffusion could be measured for different travelling distances, Ø so “restricted diffusion” from impermeable barrier could be observed

v as NMR is molecular specific Ø so the diffusion coefficient from several molecules could be

measured simultaneously.


Milk composition and structureMilk composition and structure

Water : 87%

Milk is an o/w emulsion : Fat 3-4%

protein : 3.2-3.4%

Lactose : 4.6%Ash : 0.7%

Casein micelle 80% Milk is a colloidal

suspension of caseinSoluble protein 20%

Casein micelle plays a major role in the functional properties of the milk


Casein micelles Casein micelles

Caseins are organised as "micelles"

roughly spherical

highly hydrated with about 4 – 6 g water/g

protein .

Largemean diameter: 300 nm

The integrity involves many interactions such as

hydrophobic bonding, hydrogen bonding,

electrostatic interaction, disulphide bonding and

calcium bonding.Schmidt’s model


Materials and methodsMaterials and methods

v The micellar casein solutions were prepared :Ø with a native micellar casein powder (INRA, Rennes)Ø Rehydrated in a water – NaCl solution (80 mM), Ø from 1 to 22 g/100g of water

vThe gel was obtained :Ø After renneting at 30°C during 120 minutes

vThe NMR measurements were performed at 20°CØ A low field NMR (0.47 Tesla) “The Minispec” (Bruker)Ø With a pulsed field gradient unit with a maximum strength of 4 T/m


Attenuation of the NMR signal according Attenuation of the NMR signal according to the pulsed field gradient strengthto the pulsed field gradient strength

A linear variation was observed for all the samples for both protein solutions and gels

So the diffusion coefficient is the self diffusion coefficient

-1,1

-0,9

-0,7

-0,5

-0,3

-0,1

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8

k (109 rad2 s m-2)

Ech

o a

tten

uat

ion

ln (

I g/I 0

)

(rel

ativ

e u

nit

s)

NPC solution DM=19%NPC solution DM=17%Linéaire (NPC solution DM=19%)

-1,2

-1,0

-0,8

-0,6

-0,4

-0,2

0,0

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8

k (109 rad2 s m-2)

Ech

o a

tten

uat

ion

ln (

I g/I 0

)

(rel

ativ

e u

nit

s)

NPC gel DM=19%NPC gel DM=17%

In solution In gel


Effect of the observation time D on the Effect of the observation time D on the water diffusion coefficientwater diffusion coefficient

Whatever the diffusion time ,

a straight variation of the echo attenuation was observed

The self-diffusion is constant, the diffusion mechanism is the same.

-1,40

-1,20

-1,00

-0,80

-0,60

-0,40

-0,20

0,00

0,0 0,1 0,2 0,3 0,4 0,5 0,6 0,7 0,8 0,9

k (109)

Ln(I/

I 0)

∆ = 60 ms

∆ = 210 ms∆ = 160 ms

∆ = 110 ms


v From the diffusion time, the distance over which the water molecules diffused could be deduced from :

(6D∆)1/2

v So the distance probed by the water molecules was between Ø 8 µm (∆ = 7.5 ms)Ø 43 µm (∆ = 210 ms)

v so the self-diffusion is measured on a micrometer length-scale “micro-diffusion”

Effect of the observation time D on the Effect of the observation time D on the water diffusion coefficientwater diffusion coefficient


Effect Effect of of the casein the casein concentration on concentration on the the water water selfself--diffusion coefficientdiffusion coefficient

0,60

0,65

0,70

0,75

0,80

0,85

0,90

0,95

1,00

0 5 10 15 20 25casein / water (g / 100 g)

D /

D0(

10-9

m2 s-1

)In solution

More the casein concentration increase more the water diffusion decrease.


0,60

0,65

0,70

0,75

0,80

0,85

0,90

0,95

1,00

0 5 10 15 20 25

casein / water (g / 100 g)

D /

D0(

10-9

m2 s-1

)

Effect Effect of of the casein the casein concentration on concentration on the the water water selfself--diffusion coefficientdiffusion coefficient

In gel

� In solution

p In renneted gel

The water diffusion is not modified after the aggregation of the casein induced by the rennet.


How to explain the water mobility reduction in How to explain the water mobility reduction in casein dispersion ?casein dispersion ?

First case : we suppose an obstruction effect induced by the impenetrable slow moving casein micelles.



First case : we suppose an obstruction effect induced by the impenetrable slow moving casein micelles.

+=

21

10 φ

DDeff

The self-diffusion can be described by :

Effective self-diffusion coefficient

Pure water self-diffusion coefficient

Volume fraction occupied by the micelles


V = 0,62 cm3/g

0,60

0,70

0,80

0,90

1,00

0 0,02 0,04 0,06 0,08 0,1 0,12 0,14 0,16

φ (%)

D/D

0

Obstruction effect induced by Obstruction effect induced by impermeable micelle ?impermeable micelle ?

The model do not explained the experimental results

So another effect should be considered



Second case : we suppose two fluxes, one around the micelle and one through the micelle


Obstruction model included two water fluxesObstruction model included two water fluxes

C2,D2

C1,D1

D1 is the water self-diffusion inside the micelleC1 is the water content inside the micelle

D2 is the water self-diffusion outside the micelleC2 is the water content outside the micelle

According to the model proposed by Jönsson et al*, the self-diffusion can be decomposed in :

*B. Jönsson, H. Wennerstrom, P. G. Nilsson, P. Linse , Self-diffusion of small molecules in colloidal systems, Colloid & Polymer Sci 264 (1986) 77-88.


⋅⋅+⋅+

⋅−⋅+

⋅

⋅+⋅=

water

cas

water

cascas

water

cas

water

cascas

water

cascaswaterwater

eff

mm

Kmm

mmK

mm

mm

DD5.01

110

υ

υυ

Obstruction effect induced by permeable Obstruction effect induced by permeable micelle ?micelle ?

The water diffusion according to the cell-model is given by :


+×= cas

water

cas

VKυυ

β

1122

1122

5.0 CDCDCDCD

+−

=β

where

C1, D1

C2, D2

C1, D1

C2, D2


with

• The water diffusion inside the casein micelle D1 ?• The micelle hydration C1 ?• The water diffusion outside the micelle D2• The water concentration outside the micelle C2


0,60

0,70

0,80

0,90

1,00

0 0,05 0,1 0,15 0,2 0,25

casein/water (g/100g)

D/D

0


The best fit is obtained with K = 1,60 ± 0,01


From k and if a casein voluminosity of V = 5 g water/g casein was assumed *, the water self-diffusion inside the micelle D1 could be estimated

++

−+×=

kV

kV

CCD

Dcas

eau

cas

caseau

cas

5.01

221

υυ

υυ


with C2 = 1 g.cm-3, D2 = DO

= 0.75 cm3.g-1,

= 1 cm3.g-1

caseinυwaterυ


The water diffusion coefficient inside the miceIle was 1.45 10-9 m2 S-1


If we compare to the self-diffusion of pure water (1.99 10-9 m2 S-1) • the water diffusion is slightly reduced • the reduction could be explained by the obstruction induced by the protein molecules

If we compare to the self-diffusion of protein (3 10-12 m2 S-1) • the water diffusion is very high, • the water diffusion reduction could not be explained by strong water-protein interaction


ConclusionConclusion

Ø The self-diffusion coefficient could be measured and the water molecules are free to diffuse for large distances (micrometer scale)

Øin solutions ØIn gels

Ø No restriction from impermeable barriers was involved in the hindrance of the water diffusion

Ø The water self-diffusion coefficients were not modified by the coagulation with rennet D sol = D gel.

Ø The water diffusion in casein micelle could be explained by two water fluxes

ØOne around the casein micellesØOne through the casein micelles


PerspectivePerspective

v Could we generalized this approach to other protein systems (globular protein) ?v Could we include in the model other effects ?Ø Amount of soluble compounds Ø Amount of fat

v Could we describe the water diffusion in real product (cheese…) ?v Could we relate the water diffusion in micro-length scale to macroscopic length scale ?

Work in progress….

Documents

NMR Study of the Water Diffusion in Casein Systems. Effect ...€¦ · Euro food’s water, March 2004 Materials and methods vThe micellar casein solutions were prepared : Øwith