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Zeta potential of sodium caseinate in water-ethanolsolutions

Samir Mezdour, J. Korolczuk

To cite this version:Samir Mezdour, J. Korolczuk. Zeta potential of sodium caseinate in water-ethanol solutions. Milch-wissenschaft Milk Science International, AVA-Verlag, 2010, 65 (4), pp.392-395. �hal-01173905�

Zeta potential of sodium caseinate in water-ethanol solutions

By S. M EZDOUR1 and J. KOROLCZUK

2"'

1UMR 1145 1ngenierie Precedes Aliment, A~roParisTech, Cnam, INRA, 1 Av .. 91300 Massycedex, France· 2 1NRA-Agrocampus Rennes, UMR1253 Science et Technologie du Lait et de I'OEuf, 65 rue de Saint-Bneuc, 35042 Rennes cedex, France. •E-mail: JOzef.korolcz:uk@gmail.com

The zeta (~) potential at pH 7.0 is -42, - 10 or -15 m V for an industrial sodium caseinate dissolved in water, in 50% or in 70% ethanol, respectively. The casein solubility as a function of U1e ~ potential can be represented by an equation de­scribing the Ionisation process. The half solubility-is at~ = - 13 m V and w1thin 2.3 m V from the inflection point the protein solubility changes from 50 to 90% or to 1 0%. The electrostatic surface potential is - 101 m V In water and it increases to -155 m V in 70% ethanol. The nominal thickness of the electric double layer decreases from 3.5 nm for water solution to 2.9 nm tor the 70% ethanol. The distance (dl) between the surface or shear and the surface of casein aggregate is 3.1. 7.8 and 6. 7 nm, respectively, for water, 50 0r 70% ethanol. The d~ mainly depends on the viscosity of the solvent.

Das Zeta-Potenziaf von Natriumcaseinat im W~sser-Ethanoi-Losungen

Oas Zeta- (~)Potential bei pH 7,0 betragt -42, ~1 0 oder -15 mV bei einem industriellen Natriumcaseinat nach Aufl6-sung in Wasser oder in 50 bzw. 70% Ethanol. Die Clilseinloslichkeit als eine Funktion des ~-Potential s kann durch eine Fennel charaktorisiert werden, die den lonisatrqfi.~fQ:Zess beschreibt. Die halftige Li:lslichkeit liegt bei 1; = -13 mV, und innerhalb von 2,3 f1V vom Flektionspunkt andertsleh die EiweiBioslichkeit von 50 aut 90% oder aur 10%. Das elektrosta-

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tische Oberflachenpotenzial betragt -101 mV in Wasser und erhoht sich aut -155 mV in 70% Ethanol. Die nominelle Starke der elektrischen Doppelschicht vermindert sich von 3,5 nm bei wassriger L6sung aut 2,9 nm bei 70% Ethanol. Die Distanz (d~) zwischen der Scheroberflache und der Oberflache des Caseinaggregats betragt 3,1, 7,8 und 6,7 nm bei Wasser und 50 bzw. 70% Ethanol. D~; ist wesentlich von der Viskositat des L6sungsmittels abhangig. 58 Caseinate (zeta potential in solutions) 58 Caselnat (Zeta-Potenzial in L6sungen)

1. Introduction The casein fractions in milk and in sodium caseinate

form aggregates called micelles. The zeta (s) potential of casein micelles in milk serum and in water-based buffers has already been studied (1-8). The aim of this work is to analyse the effect of the ethanol concentration on the t; potential, the electrostatic surface potential, the nominal thickness of the electric double layer, the posi­tion of the surface of shear and the relations between the t; potential and the sodium caseinate solubility in wa­ter-ethanol mixtures.

2. Materials and methods Industrially produced, spray-dried sodium caseinate

(Armor Proteines, Saint Brice en Cogles, France) was reconstituted in deionised water as described previ­ously (9, 1 0). Ethanol at 20 "C was slowly added with continuous mixing, to reach the final volume fraction from 0.1 to 0.7. The solutions were kept for 1 hat 20"C and continuously stirred in a water bath. Then the pH of the solutions was adjusted to 7.0 with 0.5 M HCI or 0.5 M NaOH and allowed to re-equilibrate for 1 h at room tem­perature. The casein content in final solutions was 1 0 g·L-1

. Soluble caseins were separated from precipitate by centrifugation at 2000g for 30 min. The zeta potential of soluble casein aggregates in water and in up to 70% (v/v) ethanol solutions was measured by a Laser Zee Meter, Model 501 (Pen Kern Inc., NY, USA). The scat­tering light detector was situated at 90 o to the incidence beam. The relative dielectric constants and the viscosity of aqueous ethanol solutions were taken from (11 , 12).

3. Results and discussion At pH 7.0 the zeta potential of casein aggregates

soluble in water is about -42 m V (Fig .1). lt decreases progressively to about -10 m V in 50% ethanol solution and increases again to - 15 m V in 70% ethanol. Such behaviour is most probably caused by the dissociation of casein aggregates and the differences in the compo­sition of the soluble fraction (9, 10). The zeta potential as a function of the ethanol volume fraction (E) is quite well represented by a parabolic equation:

I;= -40.7(±1.3)+ 124(±7.8)-E-127(±11 )·E2; R2=0.899; Error=3.8; N=9

The zeta potential of casein micelles dissolved in milk serum and in different water-based buffers varies be­tween -30 and - 10 m V (1-8). lt decreases with the addi­tion of calcium, magnesium, copper and iron ions (1, 7) and with lowering pH (3, 5).

Sodium caseinate is totally soluble in up to 20% ethanol (Fig. 1 ); then the solubility decreases progres­sively to about 20% in 45% ethanol and increases again for higher ethanol concentrations. The smallest solubili­ty coincides with the lowest zeta potential. Ca

0

-10

l -20

>..J'

• -30

-40

-50 0 0 .1 0.2 0.3 0.4 0 5 0.6 0.7

E

80

~ oo~

~ :0

40 ~ (/)

• 20

0

Fig. 1: Zeta potential (~) and casein solubility evolution at pH 7.0 as a function or ethanol volume fraction (E).

sein micelles dissociation and the solubility increase in over 50% ethanol have been previously observed (9, 10, 13-19). The solubility (S) as a function of the ethanol volume fraction (E) can be represented by the Gaussian type curve:

S = Smax - (Smax - Sm;n)·exp[-(E-Em;n)2/(2·D2) ] [1]

with Smax = 100%, Smin =minimal solubility, Em;n = E for Sm;n. D =change in E causing the solubility shift between Smax and exp( -1 /2)·(Smru.-Sm;n). For the experimental da­ta (Fig. 1 ): Smin = 19.9%, Emin = 0.48, D = 0.123, R2 = 0.991, Error = 3.1, N = 9.

100 > E Ol

80 N

lea 11

J ~

~ 50% .g 40 > • E (/)

~ 20

11 <!

0 -20 -18 -16 -14 -12 -10

~ (m V)

Fig. 2: Casein solubility at pH 7.0 as a function of zeta po­tential (s).

The casein solubility as a function of the zeta poten­tial (Fig. 2) satisfactorily fits an equation representing the ionisation process (20):

S = Smax/{1 +exp[(l;-~)/.1]} [2] with I;; = inflection point zeta potential, being an intercept and .1 being the slope of the straight line in s versus LN[(Smax·S)/S) coordinates. For the experimental re­sults from Fig. 1: 1;1 = -12.9±0.06 m V, .1 = 1.06±0.05 mV, R2 = 0.991 , Error= 0. 16, N = 9. Within 2.2·.1 = 2.3 m V from the inflection point the solubility changes from 50 to 90 or to 1 0%. it confirms that around the inflection point the minor modification of the molecular electrostatic

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properties provokes important changes in protein solu­bility(9, 10, 14, 15, 17-19).

The average electrostatic surface potential (so) can be calculated by the equation (20):

[3]

where: z =electrical charge net in mol per mol of protein, e = elementary electronic charge = 1.602·1 0'

1 C, n =

3.14, eo = 8.854·10-12 ·J-1·C2·m·1 = dielectric vacuum permittivity, e =relative dielectric constant, r = radius of the molecule.

The net electrical charge (z) at the surface of the indi­vidual casein molecules at pH 7, calculated as de­scribed previously (9), decreases slightly with the rise in the ethanol concentration. lt is the highest, in absolute values, for a51 -casein and the smallest forK-casein. For N=9 experimental points, the following equations satis­factorily represent the relation between the electrical charge net (z) at pH 7.0 and the relative dielectric con­stant of the solvent (c):

ZCLs 1 = -26.3(±0.16) + 316(±9.6} I£ R2 = 0.994; Error = 0.09

z~ = -16.4(±0.1 2) + 167(±7.1} I£; R2 = 0.988; Error= 0.07

~2 = -18.4(±0.42) + 449(±25) I c:; R2 = 0.978; Error = 0.25

zK = -2.3(±0.18) + 13(±11) I e R2 = 0.175; Error= 0.10

The average charge, calculated from the composi­tion of the soluble fraction (1 0), is -16 for water solution, decreases to - 15 at E=0.3, increases again to - 16.3 at E=0.5 and drops to - 13.5 at E=0.7. These variations are small in absolute value but their effect on protein solubil­ity seems to be of great importance. The experimental net electrical charge level at the shear surface is -7 for water and it decreases to about --1 in over 40% ethanol. The experimental potential at the shear surface seems to be much closer to the x--casein potential than to the average charge. lt is believed that K-casein covers the surface of casein micelles (21-23}.

For different genetic variants, the net electrical charge (z) of the individual casein molecules dissolved in water at pH 6.6 is between -21 .0 and -23.5 for a51 -

casein, -12.2 and -17.1 for ag2-casein , -9.2 and - 13.8 for ~-casein and -2.0 and -3.0 for x--casein (24). The electrical charge of an average individual casein mole­cule in sodium caseinate at pH 7.0 in water can be esti­mated to be between -1 6.3 and - 18.7 (25-28).

In a dry state, an average radius of an individual ca­sein molecule would be 1.9 nm, it would be 2.9 nm for a casein molecule inside the micelles or 4.5 nm for free casein molecules in a random coil configuration (24). The radius of casein micelles in milk varies between 1 0 and 150 nm (5, 6, 29, 30). The radius of sodium casein­ate particles in water solutions varies between 20 nm and 171lm (31 -35). For the calculations of so potential we have taken r=2.9 nm.

For the assumptions taken above, the so would be-101 m V in water, increasing to -1 55 m V in 70% ethanol (Fig. 3} . The experimental s potential decreases from-42 mV for water solution to -11 mV at E=0.48 and in­creases to -15 m V at E=O. 7. The s and the so potentials show opposing trends where the effect of

• • •

• • •

• • •

• • •

•

leTl ~

•

Fig. 3: Experimental ~ potential and electrostatic surface potential (so) as a function of the ethanol volume fraction (E).

0

-20

> -40 .§.

..]i' -60

-80

-100 1;0 =-101 mV

0 2 d (nm)

-0

"' 0

~ ::> (/)

3 4

Fig. 4: Electrostatic potential (sd) evolution as a function of the distance (d) from the surface of casein particles.

the ethanol concentration is concerned. This might be caused either by the differences in the composition of the external layer of casein aggregates or by the dis­tance of the shear surface (d!) from the surface of ca­sein aggregates.

The electrostatic potential (sd) decreases progres­sively to zero (Fig. 4) with the increasing distance (d) according to the relation (20):

Sd =so· exp( -d/A.) [ 4]

where: 1;0 - is an electrostatic surface potential, A.- De­bye length or the distance from the surface where the electrostatic potential is equal to sole.

When an electric field is applied, the charged parti­cles migrate together with a certain volume of the sur­rounding liquid and the counter ions dissolved in it. The boundary at which the relative motion sets in, between the immobil ised layer and the mobile particle is often called the surface of shear or the slip plain and the elec­trostatic potential at this surface is called the zeta poten­tial (t;). The precise position of the surface of shear is not known.

The Debye length (A.) or the nominal thickness of the electric double layer can be calculated by the equation (20):

A, = £o·£·ks·T

2·lr ·N A·e2

[5]

where: k8 = 1.381 ·1 0'23 JK1 is the Boltzmann constant, T is the absolute temperature, lr is the total ionic strength and NA = 6.022·1 023 is the Avogadro number.

The Debye length outside casein aggregates de-

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creases from 3.5 nm for water solution to 2.9 nm for the ethanol volume fraction E=0.7 (9). The following linear equation satisfactorily describes the relation between (A.) and the ethanol volume fraction (E):

A= 3.52(±0.01)- 0.883(±0.02)·E ; R2 = 995; Error= 0.015 ; N = 9

The distance (d~) between the surface of casein mo­lecule and the surface of shear can be calculated from the transformed equation (4): d~ = -A.·LN(~/~0). For r=2.9 nm d!;(in nm) = 3.0(±0.11) + 14.8(±1.5)·E - 1.85 (±5. 1 ) ·E2-16.8(±4. 7) ·E3

; R2 = 0.997; Error= 0.12; N = 9

8 3

7

E' (ij'

.ss ~ >oft 2s " 5 ~ • • 4

~--~--~~~~--~--~--~ 1 0.1 0.2 0.3 0.4 0.5 0.6 0.7

E

Fig. 5: Distance of the surface of shear (dt) from the sur­face of casein aggregates and viscosity (11) of etha­nol/water mixtures taken from (1 1) as a function of the ethanol volume fraction (E).

(Fig. 5). A sim ilar tendency is also observed for the vis­cosity (11) evolution of the ethanol/water mixtures: 11 (in mPa·s) = 0.98(±0.04) + 3.98(±0.54)·E + 5.5 (±1.8)·E2 -11 .7(±1 .7)·E3

; R2 = 0.998; Error = 0.04; N = 9. A linear relation can be observed between the d~; and the sol­vents viscosity (11):

d( = 0.89(±0.27) + 2.43(±0.22)·T]; R2 = 0.984; Error= 0.23; N = 9

This means that the position of the surface of shear de­pends mainly on the physical cohesion of the solvent. lt also suggests that the 1; potential only indirectly repre­sents the so potential, and that the surface composi tion of casein aggregates cannot be deduced from the t; po­tential.

The method used in this study does not enable indi­vidual casein molecules to be detected, but the casein aggregates of r>50 nm. For the samples analyzed, the modal volume radius of sodium caseinate aggregates dissolved in water is about 200 nm, decreasing pro­gressively to 60 nm for E=0.6 (36). The aggregates of the modal volume radius are composed of 3.3·1 05 ca­sein molecules for water solution and 1.0·1 04 molecules for 60% ethanol. They are respectively composed of about 70 layers of casein molecules for water solution and about 20 layers for 60% ethanol. The casein mole­cules in the aggregates contain 66% water with the counter ions dissolved in it. Because of the presence of the counter ions, the electrostatic charge of the mole­cules is almost totally neutralised. The electrostatic charge attenuation coefficient for the molecules situ­ated in the centre of the aggregates would be about 1 o· 100 and that for the molecules situated in the second layer from the surface between 1 o·3 and 1 o·4. Thus, only the external layer of the molecules is to be taken into account for the estimation of the electrostatic surface

potential. This signifies that the surface potential for the individual casein molecules and that of casein aggre­gates would be similar.

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