1221正式稿 Msc Thesis.Yiwei Chen - ku

U N I V E R S I T Y O F C O P E N H A G E N F A C U L T Y O F S C I E N C E

Master thesis Yiwei Chen

Stabilization of acidified milk drinks by high-methoxyl pectin and carboxymethyl cellulose

Supervisor: Richard Ipsen, University of Copenhagen

Co-supervisor: Claus Rolin, CP Kelco Aps

Submitted: 06/06/2017

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Preface The present study is a master thesis of 30 ECTS which is written as a part of the MSc Program in

Food Science and Technology with specialization in Dairy Science and Technology. The project has

been performed in collaboration with the Department of Food Science, University of Copenhagen

and CP Kelco Aps, Lille Skensved, Denmark. The study is based on experimental work performed at

the Department of Food Science, Section of Ingredient and Dairy Technology, University of

Copenhagen. The study has been carried out from September 2016 to June 2017 under the supervision

of Richard Ipsen, Department of Food Science, University of Copenhagen and Claus Rolin, CP Kelco

Aps, Lille Skensved.

I would like to acknowledge my supervisor Richard Ipsen and co-supervisor Claus Rolin, for giving

me the great opportunity to work with this project, for valuable guidance and discussions, for endless

help and support for my study and life throughout the entire period of my thesis.

A special thanks is given to Karen Christiansen, Process Technologist at CP Kelco, for helping me

with the practical performance of my experiments at CP Kelco. Thanks Rasmus Hilleke at CP Kelco

for helping me with the data treatment. I would also like to thank Birthe Kirstine Eriksen and other

employees for warm support and concerns during my stay at CP Kelco.

I would also like to thank Glykeria Koutina, Anni Bygvrå Hougaard, Guanchen Liu, Xiaolu Geng,

Pia Skjødt Pedersen at the Section of Ingredient and Dairy Technology for the guidance and

suggestions on my experiments in the lab. I also express my thanks to Zihan Qin and Chuantao Peng

for helping me with the data analysis and result interpretation.

Finally, I would like to thank my dear family and friends in Denmark and China, for always providing

great encouragement and support in all possible ways in my life.

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Abstract The objective of this study was to further investigate the stabilization mechanisms of acidified milk

drinks (AMD) by stabilizers, HM pectin (DE 75, DE 68) and CMC. Stabilizers in 3% MSNF acidified

milk drinks and model systems at pH 4.15 and pH 3.75 were prepared. The measurements of zeta

potential, intrinsic viscosity, hydrodynamic radius were conducted to study the hydrodynamic

structure of stabilizers. The sedimentation test, centrifugal stability analysis, particle size distribution

measurements were conducted to evaluate their stabilization functions for AMD. The stabilizer

concentration in the serum of AMD was measured to indicate the adsorption of stabilizers onto casein

micelles.

The zeta potential result showed that higher amount of free carboxyl groups and higher pH could

result in more negative charge of polymers. Intrinsic viscosity and hydrodynamic radius of stabilizers

in yoghurt serum at pH 4.15, pH 3.75 and LiAc buffer were measured. CMC showed lower

hydrodynamic size in both methods. From the intrinsic viscosity test, the result showed a trend that

pectin DE 75 > pectin DE 68 > CMC irrespective of the solvents. In yoghurt serum, pectin DE 75

and CMC showed higher intrinsic viscosity at pH 4.15 than at pH 3.75, however unexpectedly, pectin

DE 68 showed higher intrinsic viscosity at pH 3.75 than at pH 4.15. The two pectins showed lower

intrinsic viscosity in LiAc than in yoghurt serum while CMC showed lower intrinsic viscosity in

yoghurt serum.

The measurements of sedimentation test, instability index, transmission profiles and particle size

distribution showed that the stabilization of AMD could be influenced by stabilizer type, pH and

stabilizer concentration. The efficiency of stabilizing followed: pectin DE 75 > pectin DE 68 > CMC

under the same pH condition. The AMD showed better stability at pH 4.15 for all stabilizers. By

increasing the concentration of stabilizers, different stabilizers at pH 4.15 and pH 3.75 can achieve

similar stability level for AMD. At pH 3.75, stronger adsorption of stabilizers onto casein micelles

was found than at pH 4.15 and CMC showed stronger adsorption than pectins.

Generally, the molecular dimension of different stabilizers, as well as the pH, ionic strength, co-solute

(Ca2+) of the solvent, DE or DS of the stabilizer which influence the hydrodynamic volume and

negative charge of the stabilizer molecules, may play an important role in the steric and electrostatic

stabilization for the AMD system.

iii

Abbreviations and symbols AMD Acidified milk drinks CMC Carboxy methyl cellulose DE Degree of esterification DLS Dynamic Light Scattering DS Degree of substitution DVS Direct vat set GalA Galacturonic acid GDL Glucono-!-lactone HG Homogalacturonan HM High-methoxyl LiAc Llithium acetate LM Low-methoxyl MSNF Milk solid non fat NIR Near-infrared RCA Relative centrifugal acceleration RCF Relative centrifugal force RG Rhamnogalacturonan Rha Rhamnose ROI Region of interest SEC Size exclusion chromatography STEP Space and time resolved extinction profiles

D [4,3] Volume weighted mean diameter " Shear rate # Shear stress $% Viscosity of the pure solvent $& Viscosity of the dissolved polymer $' Relative viscosity $%& Specific viscosity $'() Reduced viscosity $*+, Inherent viscosity KH Huggins coefficient

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Contents

Preface.....................................................................................................................................iAbstract..................................................................................................................................iiAbbreviations and symbols...................................................................................................iii1. Introduction....................................................................................................................1

1.1. Acidified milk drinks ....................................................................................................... 11.2. Pectin ................................................................................................................................ 31.3. CMC ................................................................................................................................. 51.4. Stabilization mechanism .................................................................................................. 61.5. Objectives ........................................................................................................................ 7

2. Theory of methods..........................................................................................................92.1. Zeta potential ................................................................................................................... 92.2. Intrinsic viscosity ........................................................................................................... 102.3. Hydrodynamic radius ..................................................................................................... 132.4. Accelerated stability analysis ......................................................................................... 15

3. Materials and Methods.................................................................................................173.1. Materials ........................................................................................................................ 173.2. Preparation of acidified milk drinks .............................................................................. 173.3. Zeta potential ................................................................................................................. 193.4. Intrinsic viscosity ........................................................................................................... 193.5. Hydrodynamic radius ..................................................................................................... 213.6. Conductivity ................................................................................................................... 233.7. Determination of serum stabilizer concentration ........................................................... 233.8. Sedimentation test .......................................................................................................... 233.9. Accelerated stability analysis ......................................................................................... 243.10. Particle size distribution ................................................................................................. 243.11. Statistical analysis .......................................................................................................... 24

4. Results and Discussion..................................................................................................274.1. Zeta potential ................................................................................................................. 274.2. Intrinsic viscosity ........................................................................................................... 294.3. Hydrodynamic radius ..................................................................................................... 334.4. Serum stabilizer concentration ....................................................................................... 434.5. Sedimentation test .......................................................................................................... 444.6. Instability index ............................................................................................................. 464.7. Particle size distribution ................................................................................................. 474.8. Transmission profiles ..................................................................................................... 494.9. Summary ........................................................................................................................ 52

5. Conclusion....................................................................................................................546. Perspectives...................................................................................................................567. Reference......................................................................................................................57APPENDIX I. Calculation procedure for determination of [.]...........................................61APPENDIX II. Instability index and stabilizer concentration.............................................64APPENDIX III. Transmission profiles.................................................................................65

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1. Introduction

1.1. Acidified milk drinks

Acidified milk drinks (AMD) is a broad classification of popular products including yoghurt drinks,

blends of fruit-based ingredients with milk (Jensen, Rolin, & Ipsen, 2010). Figure 1 shows the

production flow of acidified milk drinks. The acidification method could be fermentation with starter

cultures, or direct acidification with acid, such as glucono-!-lactone (GDL) and citric acid, or juice

concentrate (Du et al., 2007; Jensen et al., 2010; Lucey, Tamehana, Singh, & Munro, 1999). The

fermented products may be available with live lactic acid bacteria in cold chain storage, or without if

there is heat treatment after the fermentation for a longer product shelf life at ambient temperature

(Du et al., 2007). Based on the market trend and customer demand, the AMD milk solid non fat

(MSNF) content varies from less than 1% to 8.5% (Laurent & Boulenguer, 2003). The pH of the

products usually ranges from 3.6-4.6 (Du et al., 2009; Wu, Liu, Dai, & Zhang, 2013).

Figure 1. The production flow of acidified milk drinks.

Heat treatments of milk at 90 °C will cause denaturation of whey proteins, which will aggregate with

casein micelles, involving k-casein, through hydrophobic interactions and forming intermolecular di-

sulphide bonds. The presence of β-lactoglobulin was necessary for any association of whey protein

with casein micelles to occur (Corredig & Dalgleish, 1999). Cross-linking or bridging by denatured

whey proteins produces a rigid gel network by casein micelles (Lucey, 2004; Lucey, Tamehana,

Singh, & Munro, 1998; Lucey, Tet Teo, Munro, & Singh, 1997). In the production of yoghurt drinks

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with a fermented base, whey proteins denatured by pasteurization tend to aggregate with the complex

of denatured whey proteins associated with casein micelles, during acidification (Lucey et al., 1998).

Yoghurt drinks are usually manufactured by homogenization of acid casein gels with fermented bases.

This means that they can be considered as suspensions of colloidal casein gel particles (Lucey et al.,

1999). It is presumed that the casein particles observed in stable beverages are aggregates or clusters

remaining after homogenization and dilution of the original gel.

In order to avoid protein aggregation and subsequent macroscopic whey separation due to the

instability of caseins at their isoelectric points, a stabilizer must be added. Anionic polysaccharides,

such as HM pectin and CMC are widely used to stabilize acidified milk drinks by preventing milk

protein flocculation that results in sedimentation (Du et al., 2009; Jensen et al., 2010; Laurent &

Boulenguer, 2003; Tromp, De Kruif, Van Eijk, & Rolin, 2004; Wu, Du, Li, & Zhang, 2014; Wu et

al., 2013).

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1.2. Pectin

Pectin is a worldwide used functional food ingredient as a gelling agent and stabilizer. According to

FAO and EU regulations, pectin (E440) is extracted from edible plant materials which are usually

citrus fruits and apples, it should contain no less than 65% of galaturonic acid on the ash-free and

anhydrous basis after washed with acid and alcohol (JECFA, 2017).

Figure 2. Conventional structure of pectin (Willats, Knox, & Mikkelsen, 2006).

The pectin structure contains three major building blocks: homogalacturonan (HG),

rhamnogalacturonan Ι (RGΙ), rhamnogalacturonan ΙΙ (RGΙΙ). Xylogalacturonan and apiogalacturonan

have also been mentioned as substitution galacturonans (Caffall & Mohnen, 2009; Christiaens et al.,

2015). HG is linear with 1, 4-linked α-D-GalA, RGΙ contains repeated disaccharide [→4)-α-D-GalA-

(1→2)- α-L-Rha-(1→] and side chains of mostly arabinan and galactan attached to the rhamnose

residues. RGΙΙ actually has a backbone of homogalacturonan with diverse side chains on galacturonic

acid residues (Willats et al., 2006). The figure above shows the conventional model of pectin structure,

however, the exact arrangement of these domains are still in debate among researchers in recent years

(Willats et al., 2006).

Pectins are anionic polyelectrolytes that contain a large number of ionizable carboxylic groups with

a strong affinity for small counter-ions (Lopes da Silva & Rao, 2006). Thus their conformational and

functional properties depend on their polyelectrolytic properties.

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In the HG domain, the C-6 carboxyl groups of some α-D-GalA residues can be methyl-esterified.

Depending on the degree of esterification (DE), pectin can be categorized as high-methoxyl (HM)

pectin or low-methoxyl (LM) pectin, which means DE > 50% and DE < 50% respectively (Hansen,

Nielsen & Rolin, 2008; Willats et al., 2006). The DE of pectin determines the linear charge density

of the macromolecule, and thus it is the most important factor influencing the ion binding properties

of pectins (Lopes da Silva & Rao, 2006). The higher the linear charge density, the stronger the

interaction of counter-ions with ionic groups (Kohn, 1972).

The degree of blockiness is the percentage of carboxyl groups inhabiting blocks of at least 5 among

galacturonic acid residues. They are calcium-reactive segments and can be increased by plant pectin

esterase (Willats et al., 2006).

Figure 3. α (1→4) linked D-galacturonic acid partly methyl esterified (CP Kelco, 2005)

Standardization is a current industrial practice to produce pectin samples with consistent properties.

As pectins are extracted from a diversity of natural raw materials, they are commonly characterized

by variability in structure and functionality. Thus pectins from different production batches are

diluted with sucrose or dextrose to attain a standard performance (Lopes da Silva & Rao, 2006).

OOCOOCH3

OH

OHO

OCOOCH3

OH

OH

O

O OCOOH

OH

OH

COOCH3

OH

OHO

O

COOH

OH

OH

O

O O

O

COOCH3

OH

OH4 1 41

4 1 41

4 1 41

αα

αα

αα

5

1.3. CMC

Carboxy methyl cellulose or cellulose gum (E466), which is abbreviated as CMC or NaCMC, is a

partial sodium salt of carboxymethyl ether of cellulose that is obtained from fibrous plant materials.

The molecular weight is approximately higher than 17000, with the degree of polymerization n to be

about 100 (JECFA, 2017).

The CMC polymers contain substituted units of β(1 → 4)-linked glucopyranose residues (Du et al.,

2009), with a general formula C6H7O2(OR1)(OR2)(OR3), where R = H, or CH2COOH, or CH2COONa

(JECFA, 2017).

Figure 4. Carboxy methyl cellulose with a DS of 1.0 (Coffey, Bell, & Henderson, 2006).

Cellulose is hygroscopic, insoluble but able to swell in water, dilute acid, and most solvents. It can

be soluble in concentrated acids but may undergo considerable degradation through acetal (glycosidic)

hydrolysis. At high temperature (> 150°C) cellulose can be hydrolysed in basic media, and oxidation

would occur (Coffey et al., 2006).

CMC is anionic, linear and water-soluble, the polymer can exist either as the free acid or its sodium

salt or as mixtures. The sodium salt is common for food use, because the free acid form is insoluble

in water (Coffey et al., 2006). The degree of substitution (DS), which is the average number of

carboxymethyl groups per repeated unit, is regulated to be 0.2 to 1.5. For food application DS is

usually narrowed down to 0.6-0.95 (Du et al., 2009).

The viscosity of the solution is a function of the polymer molecular weight and is also influenced by

the pH. Below pH 4.0 the free acid is substantially produced, which can cause precipitation of the

polymer, above pH 10 viscosity decreases and cellulose degradation becomes critical (Coffey et al.,

2006). The effect of solutes on the viscosity can also be significant, as divalent cations can generally

6

form less soluble salts of CMC, and decrease the solution quality, such as haze would occur. However,

monovalent salts form soluble salts of CMC, the viscosity and clarity are relatively unaffected at low

to moderate salt levels (Coffey et al., 2006).

1.4. Stabilization mechanism

Figure 5. Illustration of the collapse of k-casein and adsorption of pectin with the decrease of pH (Tromp et al., 2004).

As shown in Figure 5, casein micelles in milk at pH 6.7 maintain in a stable status because of the

steric repulsive interactions (Tromp et al., 2004; Tuinier, Rolin, & de Kruif, 2002). The κ-casein

chains protruding from the surface of the casein micelle into the surroundings contributes to the native

stabilization mechanism of casein micelles (Tuinier et al., 2002). The extended κ-casein chains

increase their entropy, and in order to prevent the decrease of their entropy from overlapping the κ-

casein chains, a mutual repulsion is formed between casein micelles (Tromp et al., 2004). When the

pH drops close to the isoelectric point (pH 4.6) of caseins during acidification, the protruded κ-casein

chains collapse, thus the failed steric stabilization leaves the casein micelles unstable and tend to

aggregate (de Kruif, 1998).

Casein micelles become positively charged when the pH gets lower than their isoelectric point pH4.6,

the negatively charged pectin chain would adsorb onto the micelle surface with the charged blocks,

while the uncharged chains stretch out into the solution forming loops causing repulsive interaction

between casein micelles (Tromp et al., 2004). It has been studied that in diluted acidified milk systems,

pectin stabilizes casein micelle particles by adsorbing onto their surface through electrostatic

interactions (Tuinier et al., 2002), and the adsorption started to occur at and below pH 5.0. Different

7

explanations have been proposed for the stabilization mechanisms. Pereyra, Schmidt, and Wicker

(1997) proposed that steric stabilization was found to be responsible, with one part of the pectin

adsorbing to the casein aggregate surface and another protruding from the surface forming loops and

dangling tails. The entropy of the protruding pectin chains contributes to the stability due to

introduction of steric repulsion between the casein aggregates (as cited in Jensen et al., 2010). It is

reported that casein aggregates are stabilized by multilayer of pectin, where the interior part is

adsorbed more tightly to the casein aggregates than the exterior layer (Tuinier et al., 2002). The

concentration and the type of pectin, the concentration of the casein and the ionic strength and pH

influence the stability of AMD (Glahn & Rolin, 1994).

CMC is commonly used as a stabilizing agent for its low cost in acidified milk drinks instead of pectin

in China (Du et al., 2009). The electrosorption of CMC onto casein micelles takes place below pH

5.2 and the adsorption could prevent flocculation of casein micelles by steric repulsion. Besides, the

non-adsorbed CMC could increase the viscosity of serum and slow down the sedimentation of casein

particles. At low pH, The adsorbed CMC layer could result in repulsion between the casein micelles

as k-caseins do at neutral pH (Du et al., 2007).

The quality of the solvent will affect the ability of adsorbed polymers to stabilize casein micelles

(Everett & McLeod, 2005). In good solvents, the polymer will dissolve in the solvent phase and casein

micelles will be prevented from aggregating; in poor solvents, the adsorbed polymer will lie compact

on the surface of the micelle. In this case, the steric repulsive mechanism is minimized (Everett &

McLeod, 2005).

1.5. Objectives

Stabilization of acidified milk drinks works optimally within a narrow range between pH 3.8 - pH

4.2 (Lucey et al., 1999). At the ends of this interval a larger stabilizer dosage is needed, and outside

of the interval stability becomes poorer. When the pH increases to the upper pH limit, the positive

charge of protein would decrease and the adsorption between protein and stabilizers could get

weakened. However, at the lower pH limit, the reason remains uncertain. There are two proposed

hypotheses:

• The attraction force between protein and stabilizer becomes weaker at low pH because the

negative charge of the dissociated carboxyl groups decreases, thus weaker adsorption could

be expected.

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• Another possibility is: the stabilizers adsorb strongly onto casein micelle particles even at

such low pH, but the stabilizer layer around the protein tends to aggregate with other

stabilizer chains because the stabilizers become less soluble at lower pH than the optimal pH

range.

Thus a further investigation of the stabilization mechanisms of acidified milk drinks (AMD) by

stabilizers was carried out with HM pectin (DE 75, DE 68) and CMC. Stabilizers in 3% MSNF

acidified milk drinks and model systems at pH 4.15 and pH 3.75 were prepared. The measurements

of zeta potential, intrinsic viscosity, hydrodynamic radius were conducted to study the hydrodynamic

structure of stabilizers. The sedimentation test, centrifugal stability analysis, particle size distribution

measurements were conducted to evaluate their stabilization functions for AMD. The serum from the

acidified milk drinks were taken to measure the serum stabilizer concentration for studying the

adsorption of stabilizers onto casein micelles.

A number of questions were listed to be investigated:

• At lower pH, is the adsorption between protein and stabilizers stronger or weaker?

• How does the pH influence the stabilizers and the acidified milk drinks?

• How does the solvent of the system influence the behavior of stabilizers?

• What is the difference of the stabilization functionalities between different stabilizers

investigated?

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2. Theory of methods

2.1. Zeta potential

The liquid layer surrounding the particle exists as two parts, an inner region, stern layer, where

the ions are strongly bound and an outer region, diffuse layer, where they are less firmly

associated. The ions and particles form a stable entity inside the diffuse layer. When a particle

moves, ions within the boundary move with it. Those ions beyond the boundary stay with the

bulk dispersant. The potential at this boundary, which is the surface of hydrodynamic shear,

is the zeta potential (Malvern Instruments, 2017).

Figure 6. Schematic representation of zeta potential (Malvern Instruments, 2017).

In general, the ζ-potential provides an estimate of the charge of a particle measured at the

slipping plane, which depends on the charge on the actual particle plus the charge associated

with any ions that move along with the particle in the electric field.

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2.2. Intrinsic viscosity

The intrinsic viscosity is a measure of the hydrodynamic volume occupied by a macromolecule,

which is closely related to the size and conformation of the macromolecular chains and independent

of the concentration in the solution. In dilute solutions, by definition, the polymer chains are separated

and there is negligible interaction between them (Lopes da Silva & Rao, 2006). Therefore, intrinsic

viscosity of a polymer in solution depends only on the dimension and the molecular weight of the

polymer chain (Kulicke & Clasen, 2004).

Viscosity The viscosity $ (Pa⋅s) is defined as the ratio of the shear stress #(Pa) and the shear rate " (s-1):

$ = #"(1)

The shear stress #(Pa) as can be seen in Fig 7, is the force F (N) that is exerted on the surface

element A (m2) parallel to the direction of the flow:

# =5

6 (2)

Figure 7. Schematic representation of a flow field between two parallel plates. The force F acts on the area A, resulting in the shear stress. The shear rate is the velocity difference Δν between two fluid layers separated by the distance Δh (Kulicke & Clasen, 2004).

The shear rate " (s-1) is the ratio of the velocity difference Δν (m/s) of two fluid layers and the distance

Δh (m) between them. It describes how fast the fluid layers move in respect to one another in a laminar

flow profile:

" = ∆8

9, (3)

The relative viscosity $' is the ratio of the viscosity $ of a solution to that of the pure solvent $%

$' =$$%(4)

11

When the viscosity of the solution is considered as the sum of the viscosity of the dissolved polymer

$&and the solvent viscosity $%:

$ = $& + $%(5)

then the specific viscosity $%& of the polymer in the solvent is the ratio of the dissolved polymer

viscosity to the solvent viscosity:

$%& =$&$%=$ − $%$%

= $' − 1(6)

According to the Huggins equation, the reduced viscosity $'()is the ratio of the specific viscosity

$%& to the concentrations of the solutions c:

$'() =$%&?= $ + @A ∙ $ C ∙ ?(7)

The intrinsic viscosity $ of a polymer is the reduced viscosity extrapolated to c→0

$ = limI→J

$'() (8)

The unit for the reduced viscosity can be expressed as ml/g or dl/g. Thus the intrinsic viscosity $ has

the same unit. The intrinsic viscosity $ is determined from the y-axis intercept of a plot of $%&/?

against the concentration c and an extrapolation of the linear fit of the data to c→0.

Usually a double graphic extrapolation of both the Huggins and the Kraemer equations combined are

used to estimate the value of $ more accurately.

The Kraemer equation utilizes the inherent viscosity $*+,:

$*+, =ln($')?

= $ + @ ∙ $ C ∙ ?(9)

The Huggins coefficient KH is constant for a given polymer-solvent system. Strictly speaking, the

Kraemer equation is only valid for a Huggins constant KH =1/3 (Kulicke & Clasen, 2004). If the KH

does not meet the requirement, a linear fit can be obtained initially from the Huggins equation,

because it’s more accurate over a broader range than the Kraemer equation (Kulicke & Clasen, 2004).

The Huggins coefficient KH is specific for a given polymer-solvent system. The slope of the curves

also depends on the intrinsic viscosity squared. Whereas for polymer samples with low molar masses

and low intrinsic viscosities the curves almost seem to be independent of the concentration, steep

12

slopes are observed for polymer samples with high intrinsic viscosities and high molar masses

(Kulicke & Clasen, 2004).

Capillary viscosimeter Capillary viscosimeters are the most widely used type of viscosimeters. They cannot be used to

determine an exact viscosity, but are commonly used in quality control and management. The

capillary viscosimeter used in this study is shown in Fig 8. The flow of the examined solution is

achieved through gravity, thus the sample flows under its weight through a known capillary length l

with a defined radius R. The times of a certain sample volume running between two measurement

points (M1 and M2) are measured. During the measurement, the entire closed capillary should be

immersed in a water bath that keeps the temperature constant within ± 0.1 °C, in order to get accurate

measurement.

Figure 8. The Cannon-Fenske Routine Viscometer consists of 2 tubes, the tube with capillary-1 and the venting tube-2, the reservoir-3, the upper timing mark M1-4, the lower timing mark M2-5, the pre-run sphere-6, the capillary-7, the measuring sphere-8 and the tube expanding-9.

Calculation of relative viscosity OP

According to Hagenbach-Couette correction for the Hagen-Poiseuille Law:

QR=S ∙ TU ∙ ∆V ∙ R8 ∙ Q ∙ W

−X ∙ Y ∙ Q8 ∙ S ∙ W ∙ R

(10)

with ∆V = Y ∙ [ ∙ ℎ(11)

The fluid of volume V that flows through the capillary per time unit t is determined by factors like

the radius R and the length l of the capillary, the mean filling height ℎ and the liquid viscosity $ and

13

density Y. The correction factor m comes from the Hagenbach - Couette correction term that will be

provided in a table together with commercially available capillaries.

With only the running time t and the density of the solution Y being variables, the constant values are

merged to a capillary constant K, which is a specific factor for a particular capillary and is determined

and provided by the manufacturer:

$ = @ ∙ Y ∙ R(12)

According to Hagenbach-Couette correction the viscosity can be determined via the (corrected)

running times. correction times ^ are listed by the manufacturer for the particular capillary and have

to be subtracted from the measured running times t.

RI_'' = R − ^(13)

Thus for relative viscosity $', it’s only necessary to measure the running times of the pure solvent

and the sample solution. It is assumed that the density of the dilute polymer solution Y%_abc*_+ equals

the density of the pure solventY%_a8(+c, because of the very small polymer concentration c:

$' =$$%=@ ∙ Y%_abc*_+ ∙ R%_abc*_+@ ∙ Y%_a8(+c ∙ R%_a8(+c

=R%_abc*_+R%_a8(+c

(14)

In a summary, the relative viscosity $' can be calculated as (CP Kelco control method):

$' =R%_abc*_+ −

@R%_abc*_+

R%_a8(+c −@

R%_a8(+c

(15)

2.3. Hydrodynamic radius

Hydrodynamic radius is measured by Dynamic Light Scattering (DLS), which is a technique

classically used for measuring the size of particles typically in the sub-micron region, dispersed in a

liquid. DLS measures Brownian motion and relates this to the size of the particles. The larger the

particle or molecule, the slower the Brownian motion will be (Malvern Instruments, 2017).

The size of a particle is calculated from the translational diffusion coefficient by using the Stokes-

Einstein equation:

d e = fg3S$h

(16)

where d (H) is hydrodynamic diameter, D is translational diffusion coefficient, k is Boltzmann's

constant, T is absolute temperature, $ is viscosity.

14

The diameter obtained by this technique is the diameter of a sphere that has the same translational

diffusion coefficient as the particle. The hydrodynamic diameter of a non-spherical particle is the

diameter of a sphere that has the same translational diffusion speed as the particle.

In dynamic light scattering, the speed at which the particles diffuse due to Brownian motion is

measured by determining the rate at which the intensity of the scattered light fluctuates when detected

by a suitable optical arrangement.

A typical dynamic light scattering system comprises of six main components. Firstly, a laser provides

a light source to illuminate the sample contained in a cell. An attenuator adjusts the intensity of the

laser source onto the sample. For dilute materials, most of the laser beam passes through the sample,

but some light will be scattered by the particles within the sample at all angles. A detector is used to

measure the intensity of the scattered light. In the Zetasizer Nano series, the detector position will be

at either 173° or 90°, depending upon the particular model. Then a correlator compares the scattering

intensity at successive time intervals to obtain the rate at which the intensity is varying. The

information is then sent to a computer with Nano software to analyse the data for size information.

Figure 9. Optical configurations of the Zetasizer Nano series for dynamic light scattering measurements (Malvern Instruments, 2017).

It is a good practice to report the size of the peak based on an intensity analysis because the first order

result from a DLS experiment is an intensity distribution of particle sizes. The intensity distribution

is naturally weighted according to the scattering intensity of each particle fraction.

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2.4. Accelerated stability analysis

An accelerated characterization of the stability of a dispersion system can be obtained from multi-

sample analytical centrifugation by LUMiSizer (LUM GmbH, Berlin, Germany) with STEP (space

and time resolved extinction profiles) technology, which provides measurement of the transmission

rate of NIR light as function of time and position over the entire sample length simultaneously

(Sobisch & Lerche, 2008).

Figure 10. Measurement scheme of the multi-sample analytical centrifuge with photometric detection (Sobisch & Lerche, 2008).

The measurement scheme is shown in Figure 10. The light source sends out parallel NIR-light that

illuminates and passes through the entire sample cell on the rotor, the transmitted light is detected by

thousands of sensors with a micro-scale resolution arranged linearly across the whole sample length

(Sobisch & Lerche, 2008). The distribution of local transmission rate is recorded at preset time

intervals (from every second up to hours) over the entire sample length by the detector (Detloff,

Sobisch, & Lerche, 2007).

Figure 11. Illustration of transmission profile evolution of polydisperse sedimentation (LUM, 2017).

Figure 11 shows an evolution of the transmission profile of polydisperse sedimentation (LUM, 2017).

The transmission rate (%) is plotted as a function of the radial position (mm) from the center of

16

rotation, the transmission profiles are displayed as a sequence (Sobisch & Lerche, 2008). The

evolution of the transmission profiles contains the information on the kinetics of separation behavior,

which is based on concentration changes due to creaming, sedimentation, flocculation, coalescence

or phase separation, and allows particle characterizations and evaluation of particle interactions

(Detloff et al., 2007; Lerche, 2002; Sobisch & Lerche, 2008; Sobisch, Lerche, Detloff, Beiser, & Erk,

2006).

Instability index The Instability Index has been used for quick product ranking and quality control regarding phase

separation and sample destabilization.

The Instability Index is quantified by the clarification at a given separation time, divided by the

maximum clarification. The clarification quantifies the increase in transmission (decrease of particle

concentration) due to phase separation by sedimentation or creaming, flotation (Detloff, Sobisch, &

Lerche, 2013).

The Instability Index is a dimensionless number between 0 and 1. Zero indicates the sample is very

stable with no changes of particle concentration and 1 means that sample is very unstable that

dispersion has completely phase separated.

The evaluation or ranking of samples can be obtained by comparing the Instability Index value, for

the same ROI (region of interest - range of the sample analysed), RCA (relative centrifugal

acceleration) and time of separation.

17

3. Materials and Methods

3.1. Materials

Two kinds of HM Pectin with different degree of esterification (DE): pectin of 75.29% DE and pectin

of 68.61% DE, and CMC were supplied by CP Kelco, Lille Skensved, Denmark. Water used in this

study was ultrapure Mili-Q water (Millipore, Bedford, MA. USA) with resistivity of 18.2 MΩ•cm at

25 °C.

Table 1. Material information of pectin DE 75, pectin DE 68, CMC provided by CP Kelco.

Pectin DE 75 Pectin DE 68 CMC

Gum % 67,7 Gum % 78,2 NaCMC % 99,6

DE (%) 75,29 DE (%) 68,61 2% solution viscosity (mPa.s) 180

Degree of Blockiness (%) 16 Degree of

Blockiness (%) 30,7 Degree of Substitution 0,93

1% pH 3,84 1% pH 3,75 1% pH 7

3.2. Preparation of acidified milk drinks

3% MSNF sweetened acidified milk drinks with a series of concentrations of the stabilizer (pectin

DE 75, pectin DE 68 and CMC, respectively) at pH4.15 and pH3.75 were prepared. These samples

were used for measurements of stability evaluation by LumiSizer, particle size by MasterSizer,

sedimentation test and serum stabilizer concentration.

Preparation of diluted and sweetened 6% MSNF yoghurt drink

Yoghurt of 17% milk solid non fat (MSNF) was made by reconstitution of medium-heat skim milk

powder (Arla Foods amba, Denmark) at CP Kelco. The medium-heat skim milk powder was

dissolved in de-ionized water at 46 °C. Then the reconstituted skim milk was pasteurized at 85 °C for

12 min and then fermented at 42 °C with Chr. Hansen YC-350 DVS freeze dried starter culture. The

mixture was fermented to pH 4.2, and then cooled to 5°C for storage.

18

The diluted and sweetened 6% MSNF yoghurt drink was made of 35.3% of the above prepared 17%

MSNF yoghurt, 15.0% sucrose and 49.7% de-ionized water. Firstly, the 17% MSNF yoghurt, sucrose

and only part of the de-ionized water were weighed till 88% of the aimed total amount. And then the

mixture was stirred by laboratory high shear mixer (Silverson, MA. USA) for 4 min to fully dissolve

the sucrose. Before the weight reach the set value, the pH of the mixture was adjusted to 3.75 or 4.15

with 50% w/w citric acid solution or 1M Na2CO3. Finally the rest part of de-ionized water was added

to reach the aimed weight.

Stock solutions of stabilizers with adjusted pH 1% pectin (DE75 and DE68) solutions and 3% CMC solution were prepared with different procedures.

For preparing 1% pectin stock solutions, pectin was dissolved in 85% total weight of de-ionized water

under high shear mixing with Silverson mixer for 5 min. The undissolved pectin on the mixer was

also flushed into the solution, then the weight was adjusted to 90% total amount. Then the solution

was heated at 75°C for 30 min. After the solution was cooled to room temperature, it was adjusted to

pH4.15 or pH3.75 with 50% w/w citric acid solution or 1M Na2CO3. Finally the weight was reached

to the aimed amount by adding de-ionized water.

For preparing 3% CMC stock solution, CMC was added in 85% total weight of de-ionized water with

Jiffy mixer (Jiffy Mixer Co. Inc. CA. USA) at 13 000 g for 20 min (Sorvall RC 5B, DuPont

Instruments, Wilmington, USA). It was stirred at room temperature for one hour and then stirred

overnight with magnetic stirrer. After the solution was adjusted to pH4.15 or pH3.75 with 50% w/w

citric acid solution or 1M Na2CO3, the final weight was reached with de-ionized water.

Sample systems build-up

The pH adjusted 6% MSNF sweetened yoghurt drink, with the corresponding pH adjusted stabilizer

stock solution and water were pumped respectively into the inlet of the PandaPLUS homogenizer

(GEA Niro Soavi, Denmark), and the mix was homogenized at 180-200 bar. The intake of the three

pumps were programmed to generate ten different concentrations of one stabilizer in 3% MSNF

yoghurt drink system at pH 4.15 or pH 3.75. The final stabilizer concentrations (% w/w) for each

sample system were listed in Table 2.

19

Heat treatment

Samples were heated at 75°C for 15 min in the water bath and cooled to room temperature gradually.

Table 2. Concentrations% w/w of different stabilizers in 3% MSNF yoghurt drink systems at pH 4.15 and pH 3.75.

PectinDE75 pH 4.15

PectinDE75 pH 3.75

Pectin DE68 pH 4.15

Pectin DE68 pH 3.75

CMC pH 4.15

CMC pH 3.75

0,267 0,267 0,308 0,308 0,865 0,864 0,201 0,201 0,232 0,232 0,704 0,692 0,162 0,162 0,188 0,188 0,596 0,578 0,141 0,141 0,163 0,163 0,519 0,509 0,124 0,124 0,143 0,143 0,471 0,466 0,100 0,100 0,116 0,116 0,383 0,375 0,081 0,081 0,094 0,094 0,324 0,320 0,060 0,060 0,069 0,069 0,246 0,243 0,037 0,037 0,043 0,043 0,159 0,159 0,011 0,011 0,013 0,013 0,048 0,048

3.3. Zeta potential

0.1% w/w pectin and CMC in MiliQ water were prepared, adjusted to pH 4.15 and pH 3.75 with 1M

NaCO3 and 50% citric acid.

The zeta potential of pectin and CMC solutions were determined by Zetasizer ZSP (Malvern

Instruments, Malvern, UK).

Samples were filled in DTS1070 disposable folded capillary cells and then equilibrated at 25°C for

60 s. 3 measurements with 15 runs for each measurement were conducted at 25°C.


Sample preparation In order to measure the intrinsic viscosity of three stabilizers in the serum of yoghurt at pH3.75,

pH4.15 and LiAc buffer, solutions of serum from 3% MSNF yoghurt with a series of concentrations

(0.00~0.05 % w/w) of each stabilizer were prepared. For the preparation of stabilizers in 0.3M LiAc

buffer, 0.1% w/w stabilizer in LiAc stock solutions were prepared and then diluted to the

concentration of 0.00 % ~ 0.05 % w/w.

20

The serum of 6% MSNF sweetened yoghurt was used to dilute into 3% later. The yoghurt was heat

treated at 76 °C for 30 min, after cooling to room temperature, it was centrifuged at 10,000 rpm for

20 min, then the supernatant was transferred to bottles. The serum was frozen for storage.

0.1% w/w stabilizer stock solutions were prepared under agitation of the magnetic stirrer, after heat

treatment at 75 °C water bath for 30 min, the solutions were cooled to room temperature, then adjusted

to the desired pH and concentration.

The frozen serum of 6% MSNF sweetened yoghurt was also heat treated in the same water bath and

then pH adjusted. Both the 0.1% w/w stabilizer stock solutions and serum were filtered through

Whatman Grade 41 filter paper (pore size 20µm) with a Buchner funnel connecting to the vacuum

pump, in order to prevent small particles or hair to block the capillary viscosimeter. Finally, the pH

adjusted 0.1% stabilizer solutions, water and 6% MSNF serum were mixed as Table 3 shows, to

obtain samples with each stabilizer concentrations 0.00~0.05% w/w in 3% MSNF serum at pH3.75

and pH4.15, respectively.

Table 3. Intrinsic viscosity- Sample preparation for each stabilizer and pH condition.

Stabilizer concentration in samples (% w/w)

0.00 0.01 0.02 0.03 0.04 0.05

0.1% stabilizer stock solution (g) 0 4 8 12 16 20 Water (g) 20 16 12 8 4 0 Serum 6% MSNF (g) 20 20 20 20 20 20

Measurement

Two capillary viscosimeter, which were Cannon-Fenske-Routine viscometer, type no.513 03/50,

apparatus no. 907766 (K=0.003738 mm2/s2) and no. 907768 (K=0.003799 mm2/s2), were used to

measure the intrinsic viscosity.

For each prepared solution, the same solution was filled in two capillary viscosimeters. The enclosed

part of the two capillary viscosimeters were merged under the thermostatic water bath and the

measurements were conducted at 25 ± 0.1°C. The time of the liquid running pass the mark M1 and

M2 was recorded. For each solution, the time was measured for three times.

21


Concentrations of 0.1% and 0.01% w/w stabilizer in MiliQ water, 3% yoghurt serum, 3% simulating

serum buffer and 0.3M lithium acetate buffer solutions were prepared.

0.1% and 0.01% w/w stabilizer in MiliQ water

Firstly, 0.4% w/w stock solution of pectin DE75, pectin DE68 and CMC were prepared with MiliQ

water. Further it was diluted to 0.1% and 0.01% w/w with MiliQ water for measurements. All the

solutions were adjusted to pH4.15 or pH3.75 before weighed up finally.

0.1% and 0.01% w/w stabilizer in 3% yoghurt serum

The frozen yoghurt serum of 6% MSNF yoghurt drink was heated at 75 °C water bath for 30 min.

Then it was clarified with different methods, such as filtration with 0.22µm syringe filter (Q-Max RR,

diameter 25 mm, membrane-cellulose acetate, Fresenette, Denmark), centrifugation at 3500rpm,

2600×[ 25min (SORVALL RTA6000D with H-1000B Swinging bucket rotor, radius of rotor

R=19cm, Du Pont, USA), and centrifugation at 5000rpm, 2800×[ 20min (Thermo Scientific SL16R,

radius of rotor R=10cm, Germany). The relative centrifugal force (RCF) is converted from centrifuge

rotor speed (rpm) according to the equation below:

Tjk = 1.118×10mn ToC(17)

where RCF is relative centrifugal force in unit of gravity (×[), R is the radius of the rotor in

centimeters, S is the speed of the centrifuge in revolutions per minute.

60 mL 0.1% w/w stabilizer in 3% MSNF yoghurt serum was made with 30 mL 6% MSNF yoghurt

serum, 15 mL 0.4% w/w stabilizer stock solution, 15 mL MiliQ water. Before all the MiliQ water

was added in the solution, the pH was adjusted to pH4.15 or pH3.75 with 20% w/w citric acid solution

or 0.3M Na2CO3. Then 0.01% w/w stabilizer in 3% MSNF yoghurt serum was made from dilution of

the forementioned 0.1% w/w solution. The pH was also adjusted before the final weighing up.

Simulated serum buffer for 3% MSNF sweetened yoghurt drinks

A 1L simulated serum buffer of pH4.15 and pH3.75 were prepared respectively from the following

listed compositions, according to Hansen, Nielsen and Rolin, (2008). And regarding the preservative

to prevent bacteria growth and deterioration of the buffer, the sodium azide concentration was set to

be 0.20 g/L (Du et al., 2009). The calcium lactate (C6H10O6Ca, 5H2O), di-potassium hydrogen

22

phosphate (K2HPO4), potassium di-hydrogen phosphate (KH2PO4), sodium azide (NaN3), lactose

(C12H22O11) and sucrose (C12H22O11) were dissolved in MiliQ water firstly. Then the lactic acid 90%

was added with a beaker, and the water for rinsing the beaker was also transferred into the solution.

And next sodium hydroxide (NaOH) was added. Finally, before the system was diluted till 1000 mL,

the buffer solution was adjusted pH 4.15 or 3.75 with 20% w/w citric acid and 0.3M Na2CO3. The

simulated serum buffer was filtered with 0.22µm syringe filter (Q-Max RR, diameter 25 mm,

membrane-cellulose acetate, Fresenette, Denmark) before use.

Similar to the way of making the stabilizer in yoghurt serum system, for 100 mL 0.1% w/w stabilizer

in simulated serum buffer for 3% MSNF sweetened yoghurt drinks, 50 mL simulated serum buffer,

25 mL 0.4% w/w stabilizer stock solution, 25 mL MiliQ water were mixed and pH adjusted. Then

the 0.01% w/w stabilizer in SSB was prepared from diluting the 0.1% w/w solution for 10 folds. And

the pH was also adjusted before the final weighing up.

Table 4. Composition of simulated serum buffer (Hansen, Nielsen & Rolin, 2008).

Chemicals g/L Ca-lactate 6.52 Di-potassium hydrogen phosphate (K2HPO4) 1.40 Potassium di-hydrogen phosphate (KH2PO4) 1.02 Sodium azide (NaN3) 0.20 Lactose (C12H22O11) 20.00 Sucrose (C12H22O11) 150.00 Lactic acid (90%, C3H6O3) 3.62 Sodium hydroxide (NaOH) 0.22

0.1% and 0.01% w/w stabilizer in LiAc

As for the sample of 0.1% and 0.01% w/w stabilizer in 0.3M lithium acetate buffer, the prepared

solutions from intrinsic viscosity measurement were share used.

Measurement

The hydrodynamic radius of stabilizer particles were measured by dynamic light scattering technique,

carried out with a Zetasizer ZSP (Malvern Instruments, Malvern, UK), equipped with a max output

10mW He-Ne laser at λ of 633nm. The measurement angle is 173° Non-Invasive Backscattering.

Disposable cuvettes DTS0012 were used for samples and equilibration was at 25°C for 60 s. Then 3

measurements of 10 runs each with 10 s run time were performed per sample.

23

3.6. Conductivity

Conductivity of the solvents of yoghurt serum at pH 4.15, pH 3.75, simulated serum buffer at pH

4.15, pH 3.75, LiAc buffer, were measured by a conductivity meter (Radiometer, Meterlab ION450,

Hach, USA) with four-pole electrode at 25°C.

3.7. Determination of serum stabilizer concentration

The AMD samples, with stabilizer concentration shown in Table 2, were centrifuged at 18 000 g for

20 min (Sorvall RC 5B, DuPont Instruments, Wilmington, USA). The supernatant after centrifugation

was thus considered to contain the non-adsorbed stabilizer in serum and the concentration was

determined by SEC (Size Exclusion Chromatography) at CP Kelco. The principle of SEC is that the

molecules are separated on basis of size, the larger molecules eludes first then the smaller molecules,

then salts. The effluent (0.3M LiAc) from the chromatography column passes four detectors,

Refractive Index (RI), Right and Low Angle Laser Light Scattering (RALLS/LALLS) and a viscosity

detector. Intrinsic viscosity can be determined from the output of the viscometer detector in

combination with the Refractive Index detector. Concentration was determined from the output of

the Refractive Index detector.

As control standard, dextran with the molecular weight approx.64,000 daltons (concentration about

2.0 mg/mL) and a control sample, pectin with a known IV (concentration 1 mg/mL) were used. All

samples must be analysed in duplicate. Since some supernatant samples (with higher stabilizer

concentration in the series) turned out to be opaque, might be because the samples were very stable

and casein micelles were present, therefore those samples were discarded.

3.8. Sedimentation test

The AMD samples in tubes were weighed, and centrifuged at 13 000 g for 20 min (Sorvall RC 5B,

DuPont Instruments, Wilmington, USA), then the tubes were turned upside down for 30 min to drain

out the upper layer of liquid. Afterwards the sediment in tubes were weighed again. The sediment

rate was expressed as the ratio of sediment weight to the total weight of the sample. Duplicate was

done for each sample.

24

3.9. Accelerated stability analysis

After the AMD samples were prepared according to the concentration in Table 2 for each stabilizer

at pH 4.15 and pH 3.75, all the samples were centrifuged in LumiSizer (LUM GmbH, Berlin,

Germany) for 15 min at 1000g. The parameters were set as follows: speed 2000 rpm, temperature

25°C, wave length 865 nm. The transmission profiles were recorded every 5 seconds for a total

duration of 15 min, thus 180 profiles were generated. When the data was extracted from the software,

every 5 of the files were selected, to gain a clear “diluted” version of the evolution in the curves.

3.10. Particle size distribution

The particle size distribution of the selected representative stable and unstable AMD samples were

measured using a Malvern Mastersizer 2000 (Malvern instruments Ltd, Worcs., England). The

instrument was set up according to the following parameters: Refractive index of the particles of 1.50,

a sample adsorption of 0.01 and a refractive index of the solvent 1.330. Measurements were

performed 1 day after production. Samples were introduced into a flow of degassed, demineralized

water until the obscuration was between 9% and 12%.

3.11. Statistical analysis

Statistical analysis was performed using the software IBM SPSS Statistics Version 24 (IBM Corp.,

Armonk, NY, USA). The results of triplicate analyses were used to calculate averages and standard

deviations. The data were analyzed by one-way analysis of variance (ANOVA) and two-way analysis

of variance (ANOVA) to determine statistical significance of the difference between the mean values

of independent variables.

Statistical analysis for zeta potential results

Two-way ANOVA

Two-way ANOVA was conducted to evaluate the significance of effects of pH, stabilizer and their

interaction effect on the zeta potential of solutions.

• One of the independent variable, pH, was set as fixed factor with two levels: pH4.15, pH 3.75.

25

• Another independent variable, stabilizer, was set as fixed factor with three levels: pectin with

DE 75, pectin with DE 68, CMC.

• The dependent variable was set as zeta potential

The results from the tests of between-subject effects showed that, the pH (PpH < 0.05), stabilizer

(Pstabilizer < 0.05), pH and stabilizer interaction (PpH * stabilizer < 0.05), have significant effects on the zeta

potential measured by ZetaSizer.

One-way ANOVA

One-way ANOVA was conducted to evaluate if there was significant difference between the three

mean value of zeta potential of each stabilizer at pH 4.15, pH 3.75 and unadjusted pH of their original

solutions.

The result showed the differences between the three mean value of zeta potential of each stabilizer at

pH 4.15, pH 3.75 and unadjusted pH of their original solutions were significant (P < 0.05).

Statistical analysis for intrinsic viscosity results

Paired T-test

Since two capillary viscosimeters were used, thus the paired T-test was performed in excel to evaluate

if the intrinsic viscosity results from the two capillary viscometers are significantly different.

Two-way ANOVA

Two-way ANOVA was conducted to evaluate the significance of effects of solvent, stabilizer and

their interaction effect on the intrinsic viscosity of solutions.

• One of the independent variable, solvent, was set as fixed factor with three levels: yoghurt

serum pH4.15, yoghurt serum pH 3.75, 0.3M lithium acetate buffer.

• Another independent variable, stabilizer, was set as fixed factor with three levels: pectin with

DE 75, pectin with DE 68, CMC.

• The dependent variable was set respectively, as intrinsic viscosity measured by capillary

viscometer 766, intrinsic viscosity measured by capillary viscometer 768.

26

Thus two-way ANOVA analysis were run two times respectively for the above mentioned

independent variables and dependent variables.

The results from the tests of between-subject effects showed that, for both four analyses, the solvent

(Psolvent < 0.05), stabilizer (Pstabilizer < 0.05), solvent and stabilizer interaction (Psolvent* stabilizer < 0.05),

have significant effects on the intrinsic viscosity measured by capillary viscometer 766 and capillary

viscometer 768.

One-way ANOVA

One-way ANOVA was conducted to evaluate if there is significant difference between the three

mean value of intrinsic viscosity or K value of each stabilizer in three different solvents. The

analyses were run for results from both capillary viscometer 766 and 768. They can be seen in the

Table 5 and 7, with layout in columns with 3 rows under each stabilizer. The significant differences

were marked with a superscript.

27

4. Results and Discussion

4.1. Zeta potential

Figure 12. Zeta potential of 0.1% w/w pectin DE 75, pectin DE 68 and CMC solutions at adjusted pH 3.75, pH 4.15 and unadjusted pH: pectin DE 75 at pH 3.20, pectin DE 68 at pH 3.16, CMC at pH 6.60. The error bars represent the standard deviation of three measurements of each sample.

Figure 12 shows the zeta potential of 0.1% w/w pectin DE 75, pectin DE 68 and CMC solutions at

pH 3.75 and pH 4.15 as adjusted, and original 0.1% w/w solutions without adjusted pH, which was

pH 3.16 for pectin DE68, pH 3.20 for pectin DE 75 and pH 6.60 for CMC. As shown by two-way

ANOVA, pH and stabilizer interaction had a significant effect (PpH*stabilizer < 0.05) on the zeta potential

of samples.

The pH of solutions had a significant effect (PpH < 0.05) on the zeta potential of the three stabilizers.

As seen from the figure, when the pH of the solution increased, specifically for pectin DE 68 from

pH 3.16 to pH 4.15, for pectin DE 75 from pH 3.20 to pH 4.15, for CMC from pH 3.75 to pH 6.60,

an increase in the negative zeta potential was observed. This corresponds to an increase of negative

charge on the polymer molecules, because more carboxyl groups dissociated at higher pH.

A significant difference was found between the zeta potential of pectin DE 75, pectin DE 68 and

CMC at both pH 3.75 and pH 4.15 (Pstabilizer < 0.05). The CMC showed a much lower zeta potential

than pectins with a mean value of -45.1 mV at pH 3.75, and a mean value of -50.1 mV at pH 4.15.

This indicated that CMC possessed more free carboxyl groups than pectin samples, hence they

-70

-60

-50

-40

-30

-20

-10

03,16 3,20 3,75 4,15 6,60

ZetaPoten

tial/mV

pH

PectinDE75 PectinDE68 CMC

28

resulted in more negative charges when dissociated in the solution at the pH investigated. It might be

attributed to the DS of CMC as 0.93, which is relatively high within food grade CMC DS range of

0.60 - 0.95 (Coffey et al., 2006).

The DE of pectin also showed a significant effect on the zeta potential of pectin samples in solutions

(P < 0.05). At pH 3.75 and pH 4.15, the pectin with a higher DE showed a less negative zeta potential

value than the pectin with a lower DE. This was because at a certain pH, pectin molecules of lower

DE had larger amount of free carboxyl group to dissociate hydrogen and thus to be more negatively

charged than the pectin with higher DE.

Another interesting result was found with the extent of the zeta potential of the three stabilizers

decreased when the pH increased from 3.75 to 4.15. It was noticed that, from pH 3.75 to pH 4.15, the

zeta potential of pectin DE 75 decreased from -29.1 mV to -30.9 mV, the zeta potential of pectin DE

68 decreased from -31.4 mV to -34.0 mV, the zeta potential of CMC decreased from -45.1 mV to -

50.1 mV. Their decreasing rate of zeta potential, which was calculated as 6% for pectin DE 75, 8%

for pectin DE 68 and 11% for CMC, followed an order as: CMC > pectin DE 68 > pectin DE 75. This

corresponded to the trend of the free carboxyl group amount contained by these polymer molecules:

CMC > pectin DE 68 > pectin DE 75. Thus it can be inferred that pH had a more pronounced effect

on the zeta potential of polymers with more free carboxyl groups.

The effects of pH and DE of pectins on zeta potential were in accordance with the results reported in

previous study (Schmidt & Schuchmann, 2016). Schmidt and Schuchmann (2016) have also found a

decrease of zeta potential of pectin of different DE with the pH increased from 2 to 4. At certain pH

investigated, a decrease of zeta potential with the decrease of DE of pectin DE 84, pectin DE 70 and

pectin DE 55 was observed (Schmidt & Schuchmann, 2016). The pectin with the lowest DE 55 was

noticed to reduce the zeta potential with the highest value when the pH was increased, while the

pectin of highest DE 84 reduced the least amount of zeta potential (Schmidt & Schuchmann, 2016).

In summary, the zeta potential of stabilizers in solutions are influenced by the amount of dissociated

carboxyl groups. This amount is influenced by the DE of the pectin or the DS of CMC, and the pH

of the solution. As a result, low DE of pectin, or high DS of CMC, which provide more amount of

free carboxyl groups, with high pH could lead to an increased negative charge, thus a more negative

zeta potential.

29

It could be inferred that, at higher pH, the negative charge of polymers would increase due to more

dissociation of carboxyl group, thus result in stronger intra- and intermolecular repulsion, then the

expanded molecule chain together with increased charge would make contributions to the steric and

electrostatic stabilization of the casein particles.


In dilute solutions, where interactions between polymer chains are negligible, the intrinsic viscosity

$ , as a good indication of the hydrodynamic volume of the biopolymer, depends only on the

dimensions of the polymer chain (Lopes da Silva & Rao, 2006).

Two capillary viscometers were used to measure the intrinsic viscosity. The paired T-test showed a

significant difference between the two capillary viscometers (P < 0.05). The pairs of data from two

viscometers for samples of pectin DE 75 in yoghurt serum pH 4.15, pectin DE68 in yoghurt serum

pH 4.15 and pH 3.75 showed significant difference. Thus the data were listed out separately in the

Table 5.

Table 5. Intrinsic viscosity of pectin DE 75, pectin DE 68, CMC in serum of 3% MSNF yoghurt pH 4.15, pH 3.75, 0.3 M LiAc measured by capillary viscometer 766, 768; the intrinsic viscosity of the stabilizers measured by size exclusion chromatography (SEC), provided by CPkelco. The significance of difference between the three mean value of intrinsic viscosity obtained from each capillary viscometer for each stabilizer in three different solvents are marked with a superscript, shown in columns with 3 rows under each stabilizer.

Table 5 shows the mean value of intrinsic viscosity, from three measurements of the same solution

Methods Solvent

Intrinsic viscosity dl/g

Pectin DE75

Pectin DE68 CMC

Capillary viscometer

766

Yog serum pH4.15 8,44 ±0,07

b 6,13 ± 0,05a 2,52 ± 0,17

a

Yog serum pH3.75 7,76 ± 0,15

a 6,49 ± 0,07b 2,20 ± 0,24

a

0.3M LiAc 7,50 ± 0,18a 5,25 ± 0,04

c 3,62 ± 0,07b


768

Yog serum pH4.15 8,83 ±0,08

a 5,95 ± 0,04a 2,57 ± 0,07

a

Yog serum pH3.75 7,85 ±0,11

b 7,09 ± 0,03b 2,23 ± 0,08

b

0.3M LiAc 7,47 ± 0,10c 5,27 ± 0,08

c 3,63 ± 0,03c

SEC 0.3M LiAc 7,18 5,24 ---

30

by each capillary viscometer, for pectin DE 75, pectin DE 68, CMC in serum of 3% MSNF yoghurt

at pH 4.15, pH 3.75 and 0.3 M LiAc measured by capillary viscometer 766, 768; the intrinsic viscosity

of the stabilizers measured by size exclusion chromatography provided by CP Kelco were also listed.

The significance of difference between the mean intrinsic viscosity values obtained from each

capillary viscometer for each stabilizer in three different solvents were marked with a superscript,

shown in columns with 3 rows under each stabilizer.

It should be noted that the intrinsic viscosity results were obtained from Huggins equation with the

concentration of the solution extrapolated to zero. The double graphic extrapolation of both the

Huggins and the Kraemer equations was not used. Since the double extrapolation of Huggins and

Kraemer equations requires strictly that KH =1/3 (Kulicke & Clasen, 2004), however, the K values of

stabilizers in yoghurt serum shown in Table 7 were not close to 0.33. Thus the linear fit was obtained

from the Huggins equation according to the suggestion (Kulicke & Clasen, 2004).

The calculation procedure of intrinsic viscosity and K value can be found in Appendix I.

Generally, the intrinsic viscosities of stabilizers were significantly different, irrespective of the

capillary viscometers used. The trend of intrinsic viscosity of stabilizers can be listed as: pectin DE

75 > pectin DE 68 > CMC. The intrinsic viscosities of the two kinds of pectin in LiAc buffer showed

similarity to the value provided by CP Kelco, which were measured by size exclusion

chromatography. However, the intrinsic viscosity regarding CMC could not be provided. The result

of pectin was in agreement with the study that reported a decrease of intrinsic viscosity of pectin with

decreasing DE (Lopes da Silva & Rao, 2006). Other studies have also shown a general decrease in

the hydrodynamic volume of the pectin molecule with decreasing DE (Deckers, Olieman, Rombouts,

& Pilnik, 1986), with both steric and electrostatic interactions playing an important role in these

conformational changes. On the contrary, Schmidt et al. (2017) an increased hydrodynamic radius

with decreasing DE (Schmidt, Schütz, & Schuchmann, 2017).

With regard to the effects of solvents on intrinsic viscosity, pectin DE 75 and CMC showed higher

intrinsic viscosity in yoghurt serum at pH 4.15 than at pH 3.75. This might be attributed to the

expansion of the polymer chain resulted from the electrostatic repulsions between the dissociated

carboxyl groups on the same molecule (Lopes da Silva & Rao, 2006). Previous studies have shown

that the intrinsic viscosity of citrus pectin increases with the pH increasing from 3 to 7 (Lopes da

Silva & Rao, 2006). In contrast to the behavior of pectin DE 75 and CMC in yoghurt serum, pectin

31

DE 68 showed increased intrinsic viscosity with decreased pH in yoghurt serum. And this was

observed in both capillary viscometers. This could not be explained based on existing research.

Pectin DE 75 and DE 68 in LiAc buffer presented lowest intrinsic viscosity, which might be related

to the ionic strength of LiAc buffer. As shown in Table 6, LiAc showed higher conductivity which

indicates a strong ionic strength. The increased ionic strength reduced the charge effects of the

dissociated carboxyl groups, which resulted in suppression of intramolecular electrostatic repulsion,

thus a decreased volume of the polymer chain (Lopes da Silva & Rao, 2006). Schmidt et al. (2017)

have also found decreased hydrodynamic radius of pectin under increased ionic strength with NaCl

present in the solution (Schmidt et al., 2017).

Table 6. Conductivity (ms/cm), of yoghurt serum at pH 4.15, pH 3.75, simulated serum buffer at pH 4.15, pH 3.75, LiAc buffer, measured at 25°C.

Solvent Conductivity (ms/cm), 25°C Yoghurt serum pH 4.15 2,908 ± 0,004 Yoghurt serum pH 3.75 2,882 ± 0,004 Simulated serum buffer pH 4.15 2,104 ± 0,013 Simulated serum buffer pH 3.75 2,006 ± 0,008 0.3 M LiAc 14,415 ± 0,290

Another contrary phenomenon was noticed with CMC, showing smaller intrinsic viscosities in

yoghurt serum than in LiAc buffer with higher ionic strength, for both capillary viscometers.

Considering the yoghurt serum contains calcium and magnesium ions (Jenness & Koops, 1962), a

possible reason might be that the salts of CMC formed with these divalent cations are generally less

soluble (Coffey et al., 2006). For the same polymer with different solvents, the intrinsic viscosity

decreases with the solvent quality decreasing (Kulicke & Clasen, 2004).

32

Table 7. K value of pectin DE 75, pectin DE 68, CMC in serum of 3% MSNF yoghurt at pH 4.15, pH 3.75, 0.3 M LiAc measured by capillary viscometer 766, 768. The significance of difference between the three mean K value obtained from each capillary viscometer of each stabilizer in three different solvents were marked with a superscript, shown in columns with 3 rows under each stabilizer.

Table 7 shows the mean K value of pectin DE 75, pectin DE 68, CMC in serum of 3% MSNF yoghurt

at pH 4.15, pH 3.75, 0.3 M LiAc measured by capillary viscometer 766, 768. The significance of

difference between the three mean K value obtained from each capillary viscometer of each stabilizer

in three different solvents were marked with a superscript, shown in columns with 3 rows under each

stabilizer.

The Huggins coefficient KH is specific for a given polymer-solvent system. Although the K

values of stabilizers in yoghurt serum were not close to 0.33, it could be noticed that the K values

with LiAc buffer were close to 0.33. This might be related to that LiAc was reported to be a good

solvent (Hansen, Nielsen & Rolin, 2008). However, in a good solvent, there are strong excluded

volume interactions which force the chain to open up and assume a very extended configuration

(Bohidar, 2015). This “good solvent” did not show higher intrinsic viscosity with the two pectins than

their intrinsic viscosity in yoghurt serum.

In summary, irrespective of the solvent system these stabilizers dissolved in, pectin DE 75 showed

highest intrinsic viscosity and CMC showed the lowest intrinsic viscosity. For pectin DE 75 and CMC,

they also showed higher intrinsic viscosity at pH 4.15 than at pH 3.75. Considering intrinsic viscosity

is an indication of hydrodynamic dimension of polymers, this trend may be correlated to their steric

stabilization functions. Thus a higher intrinsic viscosity may contribute to a better stability of AMD

because of more steric repulsion.

Methods Solvent K value

Pectin DE75

Pectin DE68 CMC


766

Yog serum pH4.15 0,57 ± 0,04

a 0,52 ± 0,04

a 6,53 ± 1,45

a

Yog serum pH3.75 0,77 ± 0,07

b 0,87 ± 0,06

b 10,64 ± 2,80

a

0.3M LiAc 0,36 ± 0,08c

0,40 ± 0,04a

0,67 ± 0,09b


768

Yog serum pH4.15 0,38 ± 0,02

a 0,65 ± 0,02a 6,05 ± 0,49

a

Yog serum pH3.75 0,78 ± 0,05

b 0,54 ± 0,02b 9,41 ± 0,99

b

0.3M LiAc 0,40 ± 0,05a 0,40 ± 0,06

c 0,70 ± 0,08c

33


The hydrodynamic radius of pectins and CMC of two concentrations (0.1% and 0.01% w/w) in four

different solvent systems (MiliQ water, yoghurt serum of 3% MSNF, simulated serum buffer, LiAc

buffer) at two different pH conditions (pH 4.15 and pH 3.75, excluding LiAc buffer) were

investigated.

In general, the size distribution by intensity of all samples showed large polydispersity. Thus, the

peak mode of the size distribution by intensity, instead of the Z-average size, was used to discuss the

hydrodynamic radius of stabilizer particles in different solvent systems.

Firstly, the hydrodynamic radius distribution of some solvents alone without dissolved stabilizers, as

well as stabilizers in each solvent system were described separately. Further, an overview of different

solvent system with stabilizers was displayed and the effects of solvents on the size distribution of

different stabilizers were discussed.

4.3.1 Size distribution of stabilizers in MiliQ water

Figure 13. Size distribution by intensity of 0.1% w/w pectin DE 75, pectin DE 68, CMC in MiliQ water at pH 4.15 and pH 3.75.

0

2

4

6

8

10

12

14

16

0,1 1 10 100 1000 10000

Intensity

/%

Size/r.nm

0.1%stabilizerinMiliQwater

0.1%PectinDE75inMiliQpH4.150.1%PectinDE75inMiliQpH3.750.1%PectinDE68inMiliQpH4.150.1%PectinDE68inMiliQpH3.750.1%CMCinMiliQpH4.150.1%CMCinMiliQpH3.75

34

Figure 14. Size distribution by intensity of 0.01% w/w pectin DE 75, pectin DE 68, CMC in MiliQ water at pH 4.15 and pH 3.75.

Figure 13 shows the size distribution of 0.1% w/w stabilizer concentration in MiliQ water. Pectin DE

75 showed peak modes at around 150 nm and 750 nm at pH 4.15, a peak mode of around 650 nm at

pH 3.75. A bimodal distribution was apparent for pectin DE 68, with one peak mode of around 200

nm and another peak near 850 nm. The size distributions of CMC samples were wide spread, with

size ranging from 1-10 nm, 10-100 nm, 170 nm for both pH conditions. For each stabilizer, the size

distribution of lower pH shifted slightly towards lower particle sizes. However, this trend was not

evident for the two pectins in Figure 14. At 0.01% w/w concentration, all four pectin samples

presented monomodal distribution with high intensity %. The pectin DE 68 had peak modes of around

270 nm, while pectin DE 75 had peak modes at 230 nm irrespective of pH conditions. CMC at pH

4.15 showed particle size ranging from 70-110 nm while the CMC at pH 3.75 had peaks of 40 and

70 nm. Small peaks ranging between 1-10 nm were observed for both CMC samples.

0

5

10

15

20

25

30

35

40

0,1 1 10 100 1000 10000

Intensity

/%

Size/r.nm

0.01%stabilizerinMiliQwater0.01%PectinDE75inMiliQpH4.15

0.01%PectinDE75inMiliQpH3.75



0.01%CMCinMiliQpH4.15

0.01%CMCinMiliQpH3.75

35

4.3.2 Size distribution of stabilizers in yoghurt serum (with and without stabilizer)

Figure 15. Size distribution by intensity of clarified serum of 3% MSNF yoghurt by 0.22 µm filter, relative centrifugal force (RCF) 2600×g for 20 min, clarified serum of 6% MSNF yoghurt by RCF 2800×g for 20 min, clarified serum of 3% MSNF yoghurt by RCF 2800×g for 20 min at pH 4.15 and pH 3.75.

Figure 15 shows the size distribution of clarified yoghurt serum of 3% MSNF yoghurt by 0.22 µm

filter, relative centrifugal force (RCF) 2600×g for 20 min, clarified serum of 6% MSNF yoghurt by

RCF 2800×g for 20 min, clarified serum of 3% MSNF yoghurt by RCF 2800×g for 20 min at pH

4.15 and pH 3.75.

The yoghurt serum was obtained from decanting the supernatant of centrifuged 6% MSNF sweetened

yoghurt drinks. This operation had brought in visible sediment fragments or large particles of protein

aggregates into the yoghurt serum. Thus, in order to further clarify the serum, different methods of

clarification (0.22pX filter, centrifuge at 2600×g and 2800×g for 20 min) were conducted.

All samples contained particles with a peak mode of around 0.6 nm, and another two peaks ranging

between 15-25 nm and 80-150 nm. Casein micelle has a particle diameter range of 10-300 nm, whey

protein has a particle diameter of 3-6 nm (Glantz et al., 2010; Hristov, Mitkov, Sirakova,

Mehandgiiski, & Radoslavov, 2016). Thus it can be inferred that the clarified serum samples contain

casein micelles. Although no peak was shown between 1-10 nm, it could be assumed that the whey

protein denatured and aggregated with casein micelles under the heat treatment during powder

production and acidification of yoghurt production (Lucey et al., 1999; Martin, Williams, & Dunstan,

2007).

0

2

4

6

8

10

12

14

16

0,1 1 10 100 1000 10000

Intensity

/%

Size/r.nm

Yoghurtserum3%YogSerum0.22umfiltered3%YogSerum2600gcentrifuged6%YogSerum2800gcentrifuged3%YogSerum2800gcentrifugedpH4.153%YogSerum2800gcentrifugedpH3.75

36

Figure 16. Size distribution by intensity of 0.1% w/w pectin DE 75, pectin DE 68, CMC in clarified (RCF 2800×g 20 min) serum of 3% MSNF yoghurt at pH 4.15 and pH 3.75.

Figure 17. Size distribution by intensity of 0.01% w/w pectin DE 75, pectin DE 68, CMC in clarified (RCF 2800×g 20 min) serum of 3% MSNF yoghurt at pH 4.15 and pH 3.75.

Figure 16 shows 0.1% w/w concentration of stabilizers in yoghurt serum. Pectin DE 75 had a peak

near 550 nm, pectin DE 68 showed size mode at around 300 nm, irrespective of pH conditions. CMC

at pH 4.15 had peak mode at 200nm while CMC at pH 3.75 had a peak at 230nm.

In figure 17, the concentration of stabilizers was diluted to 0.01%, the peaks of two pectins shifted

towards smaller sizes, ranging from 170-200 nm. However, the CMC showed unexpectedly larger

size, around 350 nm at pH 4.15 and 400 nm at pH 3.75.

The picture of the CMC sample was illustrated below. For all CMC samples, they appeared to be

more opaque or turbid, compared to pectin samples in yoghurt serum or the rest samples in different

solvent systems which were clear and transparent. The imaginable reason of microbial contamination

could be excluded because the measurements were repeated with newly prepared samples, however,

they presented the same phenomenon as described above. This might be related to the presence of

0

2

4

6

8

10

12

14

16

0,1 1 10 100 1000 10000

Intensity

/%

Size / r.nm

0.1%stabilizer in 3% YogSerum

0.1%PectinDE75pH4.15




0.1%CMCpH4.15

0.1%CMCpH3.75

0

5

10

15

20

25

30

35

40

0,1 1 10 100 1000 10000

Intensity

/%

Size/r.nm

0.01% stabilizer in 3% YogSerum0.01%PectinDE75in3%YogSerumpH4.150.01%PectinDE75in3%YogSerumpH3.750.01%PectinDE68in3%YogSerumpH4.150.01%PectinDE68in3%YogSerumpH3.750.01%CMCin3%YogSerumpH4.150.01%CMCin3%YogSerumpH3.75

37

casein micelles and calcium ions in the solution. The adsorption of CMC onto casein micelle can

happen below pH 5.2 (Du et al., 2007). Divalent cations can generally form less soluble salts of CMC,

and the decrease the solution quality, thus haze may occur (Coffey et al., 2006).

Figure 18. A representative sample of 0.01% w/w pectin in yoghurt serum (left), a representative sample of 0.01% w/w CMC in yoghurt serum (right).

4.3.3 Size distribution of stabilizers in simulated serum buffer (with and without stabilizer)

Figure19. Size distribution by intensity of simulated serum buffer (SSB) for 6% MSNF yoghurt, SSB for 3% MSNF yoghurt at pH 4.15 and pH 3.75, 0.22 µm filtered for all samples before measurement.

Figure 19 shows the size distribution of simulated serum buffer for 6% MSNF yoghurt drinks and the

diluted 3% MSNF simulated serum buffer with pH adjusted to 4.15 and 3.75, which were filtered

through 0.22pX filter prior to measurement. The size distribution of simulated serum buffers showed

a large population of particles with a peak mode of around 0.8 nm.

It can be noticed that in Figure 20-22, most of the samples also showed peaks below 1 nm,

corresponding to the size distribution of the simulated serum buffer.

0

2

4

6

8

10

12

14

16

0,1 1 10 100 1000 10000

Intensity

/%

Size/r.nm

Simulatedserumbuffer

6%SSB0.22umfiltered

3%SSB0.22umfilteredpH4.15

3%SSB0.22umfilteredpH3.75

38

Figure 20. Size distribution by intensity of 0.1% w/w pectin DE 75, pectin DE 68, CMC in simulated serum buffer (SSB) for 3% MSNF yoghurt at pH 4.15 and pH 3.75.

Figure 21. Size distribution by intensity of 0.01% w/w pectin DE 75, pectin DE 68, CMC in simulated serum buffer (SSB) for 3% MSNF yoghurt at pH 4.15 and pH 3.75.

Figure 22. Size distribution by intensity of 0.01% w/w pectin DE 75, pectin DE 68, CMC in simulated serum buffer (SSB) for 5.7% MSNF yoghurt at pH 4.15 and pH 3.75.

0

2

4

6

8

10

12

14

16

0,1 1 10 100 1000 10000

Intensity

/%

Size/r.nm

0.1%stabilizerin3%SSB0.1%PectinDE75inSSBpH4.150.1%PectinDE75inSSBpH3.750.1%PectinDE68inSSBpH4.150.1%PectinDE68inSSBpH3.750,1%CMCinSSBpH4.150,1%CMCinSSBpH3.75

0

5

10

15

20

25

30

35

40

0,1 1 10 100 1000 10000

Intensity

/%

Size/r.nm

0.01%stablizerin3%SSB0.01%PectinDE75inSSBpH4.15

0.01%PectinDE75inSSBpH3.75



0.01%CMCinSSBpH4.15

0.01%CMCinSSBpH3.75

0

5

10

15

20

25

30

35

40

0,1 1 10 100 1000 10000

Intensity

/%

Size/r.nm

0.01%stablizerin5.7%SSB0.01%PectinDE75in5.7%SSBpH4.150.01%PectinDE75in5.7%SSBpH3.750.01%PectinDE68in5.7%SSBpH4.150.01%PectinDE68in5.7%SSBpH3.750.01%CMCin5.7%SSBpH4.150.01%CMCin5.7%SSBpH3.75

39

Figure 20 shows 0.1% w/w stabilizers present in the simulated serum buffer system, pectin DE 75

exhibited main peak mode of 600 nm for both pH, and pectin DE 68 at pH 4.15 showed size near 400

nm while the pH 3.75 showed size mode at 350 nm and 1500 nm, CMC had distribution between 10-

100 nm. 0.01% w/w pectins in Figure 21, showed similar peak modes near 200 nm, and CMC

exhibited size distribution of 10-100 nm.

Figure 22 shows 0.01% w/w stabilizers in the simulated buffer for 5.7% MSNF AMD serum. These

samples were prepared when 0.1% w/w stabilizer in 3% SSB samples were diluted mistakenly with

SSB of 6% MSNF, instead of 3% MSNF. However, these samples did not show smaller sizes under

a stronger ionic strength, which contradicted the previous findings that the hydrodynamic radius of

polymers decreased with increased ionic strength due to the shielding effect of the charges on the

polymer chains (Schmidt et al., 2017).

4.3.4 Size distribution of stabilizers in LiAc

Figure 23. Size distribution by intensity of 0.1% w/w pectin DE 75, pectin DE 68, CMC in 0.3 M lithium acetate (LiAc) buffer.

Figure 24. Size distribution by intensity of 0.01% w/w pectin DE 75, pectin DE 68, CMC in 0.3 M lithium acetate (LiAc) buffer.

Figure 23 and 24 display the size distribution of 0.1% and 0.01% w/w stabilizers in 0.3 M LiAc

buffer, respectively. The sample of 0.3M LiAc buffer without stabilizer was not measurable by

ZetaSizer. The difference of size distribution between the two concentrations was not evident.

0

5

10

15

20

25

0,1 1 10 100 1000 10000

Intensity

/%

Size/r.nm

0.1%stabilizerin0.3MLiAc

0.1%PectinDE75inLiAc0.1%PectinDE68inLiAc0.1%CMCinLiAc

0

10

20

30

40

0,1 1 10 100 1000 10000

Intensity

/%

Size/r.nm

0.01%stabilizerin0.3MLiAc

0.01%PectinDE75inLiAc0.01%PectinDE68inLiAc0.01%CMCinLiAc

40

4.3.5 Overview

Figure 25. An overview of size distribution by intensity of 0.1% and 0.01% w/w pectin DE 75, pectin DE 68, CMC in MiliQ water – A, B; yoghurt serum of 3% MSNF – C, 0.1% and 0.01% w/w pectin DE 75, pectin DE 68, CMC in yoghurt serum of 3% MSNF – D, E; simulated serum buffer (SSB) for 3% MSNF yoghurt – F, 0.1% and 0.01% w/w pectin DE 75, pectin DE 68, CMC in simulated serum buffer (SSB) for 3% MSNF yoghurt – G, H; 0.1% and 0.01% w/w pectin DE 75, pectin DE 68, CMC in 0.3 M lithium acetate (LiAc) buffer – I, J.

Figure 25 shows an overview of size distribution by intensity of 0.1% and 0.01% w/w pectin DE 75,

pectin DE 68, CMC in different solvent systems investigated, which were MiliQ water (A, B),

yoghurt serum (C, D, E), simulated serum buffer (F, G, H), 0.3M LiAc buffer (I, J).

An increase of hydrodynamic radius of polymer particles with the increase of the concentration was

found, when comparing A and B, D and E for pectin samples, G and H, I and J for pectin samples.

The influence of pH of solutions on the hydrodynamic radius of stabilizers can not be distinguished

from A, B, D, E, G, H. When comparing the size distribution in LiAc buffer with yoghurt serum and

SSB, the suppression effect of increased ionic strength from LiAc buffer on the hydrodynamic radius

of stabilizers was not noticeable as found in previous study (Schmidt et al., 2017).

Observing from C to E and to D, where yoghurt serum was used as the solvent, the size distribution

shifted to be larger, which indicated that the adsorption of stabilizers onto casein micelles occurred,

41

and the particle size increased as the concentration of stabilizers increased. This finding was in

accordance with the results of previous studies (Du et al., 2007; Tuinier et al., 2002). They reported

that the electrostatic adsorption between positive charged casein micelles and negatively charged

pectin and CMC could happen below pH 5.0 and pH 5.2, respectively; an increase of hydrodynamic

radius of casein micelle with the present of pectin and CMC, as well as a further increase of particle

size with higher concentration of these stabilizers were reported (Du et al., 2007; Tuinier et al., 2002).

The peak appeared below 1 nm as observed in C, F, G, H was assumed to indicate the presence of

sucrose and lactose. The acidified milk drinks were sweetened with added sucrose, and the

preparation of simulated serum buffer also contained sucrose and lactose. It has been studied before

using dynamic light scattering measurement that, the peak means obtained from the intensity size

distribution data of a series of sucrose concentrations, ranging from 5 to 35% w/v, were measured as

0.82 ± 0.11 nm (Kaszuba, McKnight, Connah, McNeil-Watson, & Nobbmann, 2008). Malvern

Instruments has provided a measurement of sucrose at a concentration of 30%w/v by Zetasizer Nano,

showing the peak had a mode of 0.6 nm by volume distribution (Malvern Instruments, 2016).

According to Schultz and Solomon (1961), calculated effective hydrodynamic radius of lactose was

0.54 nm, sucrose was 0.52 nm; the radii obtained is a hypothetical sphere that shares the same

hydrodynamic behavior of the solute molecule including the water of hydration which is too firmly

bound to participate in the viscous shearing process (Schultz & Solomon, 1961).

The size distribution of CMC demonstrated peaks with modes between 1-10 nm and 10-100 nm, as

seen from A, B, G, H, I. It was presumed that CMC degradation has occurred under the low pH

condition of the sample solutions. It has been discussed that at temperature higher than 70 °C with

presence of 0.2 - 0.5 M citric acid, hydrolysis of CMC could occur (Takigami, et al., 2012), but no

evidence has been provided as a definite proof for the discussion. It has been reported that the

degradation of CMC at low pH (4.0) resulted in the decrease in viscosity of the serum CMC during

storage (Wu et al., 2014). However, the reference cited studied the hydrolysis of carboxy methyl

pullulan by 2.4 M perchloric acid at 65 °C (Glinel, Paul Sauvage, Oulyadi, & Huguet, 2000), which

may not be sufficient to explain the acid hydrolysis of CMC.

It seems that CMC is not easy to be hydrolyzed by dilute acid. Experiments of various CMC samples

with DP ranging from less than 100 to 200 and DS from 0.52 to 1.2, autoclaved at 121 °C with 0.5 N

H2SO4 for 15 minutes, as well as kept at 50 °C for 3 hours at pH 2.1 to 6.7 both showed no increase

42

in reducing value (Reese, Siu, & Levinson, 1950). Another study used 1 N H2SO4 for 5% CMC at

ambient temperature and 50-80°C, after two weeks, the viscosity of the solution remained unchanged,

which indicated that no hydrolysis occurred (Gautier & Lecourtier, 1991). In addition, according to

Andries Hanzen (personal communication, May 9, 2017), CMC innovation manager from CP Kelco,

CMC solution can be used at a wide range of pH 4-11 roughly, and there was also a case from their

customers where CMC was used below pH 4 without an issue; besides, experiments done for acid

degradation of CMC required rather harsh environment compared to the acid condition investigated

in this study.

The size distributions of A, B, D, G, H, I, J, all show CMC with the smallest hydrodynamic radius,

this result corresponded to the intrinsic viscosity result that CMC exhibited the lowest value in the

solution of yoghurt serum at pH 4.15, pH 3.75 and LiAc buffer, compared to pectin DE 75 and pectin

DE 68. Although the molecular weight and degree of polymerization of the CMC sample are

unknown, it could be assumed that CMC contains small fractions of polymer chains which attributed

to a lower hydrodynamic volume in the solution, in addition to the influence of the solvent system

with different pH, ionic strength and presence of divalent cations.

Such a correlation of intrinsic viscosity and hydrodynamic radius could also be found within the

sucrose. Mathlouthi and Reiser (1995) have reported the intrinsic viscosity of sucrose measured at

25°C to be 2.41 ml/g, which equals 0.0241 dl/g (Mathlouthi & Reiser, 1995). Compared with the

intrinsic viscosities of pectin and CMC samples, such a small hydrodynamic volume was also

corresponded with a size distribution down to < 1 nm.

The size distributions of D, G, I show that the hydrodynamic radius of the stabilizers followed a trend:

pectin DE 75 > pectin DE 68 > CMC, which was in accordance with the trend found in the intrinsic

viscosity data. However, in E, H, J, such a trend became undetectable. On the contrary to the

forementioned results, Schmidt and Schuchmann (2016) found an higher hydrodynamic radius of

pectin with lower DE, because of its higher degree of dissociation of carboxyl groups led to increased

intramolecular repulsion, thus a molecule expansion (Schmidt & Schuchmann, 2016).

43

4.4. Serum stabilizer concentration

Figure 26 shows the stabilizer concentration in the serum of each sample with pectin DE 75, pectin

DE 68 and CMC at pH 4.15 and pH 3.75, after centrifugation of the 3% MSNF AMD samples, plotted

against the original stabilizer concentration added in the AMD samples. For the AMD samples with

very low stabilizer concentrations, their serum stabilizer concentrations were not detectable by the

size exclusion chromatography, thus their serum concentrations were marked as 0 % w/w.

Figure 26. Stabilizer concentration in serum as a function of stabilizer concentration in acidified milk drinks.

Firstly, it can be observed that, for each sample, the stabilizer concentration in serum was smaller

than the original added concentration, which indicates that some part of the stabilizer was adsorbed

by the protein particles and became part of the sediment. Pectin can be considered as not adsorbed to

the casein aggregates when it did not follow the protein gel phase and sediment during centrifugation

(Jensen et al., 2010). Thus it can be inferred that, with the same added stabilizer concentration, the

lower the serum stabilizer concentration of that sample, the larger amount of certain stabilizer was

adsorbed onto the casein particles and centrifuged down in the sediment.

The CMC showed distinct lower serum concentrations compared with pectin samples at both pH

values investigated. In other words, CMC showed more adsorption onto casein particles than pectins.

However, under the same pH conditions, the difference of serum concentrations between pectin DE

75 and pectin DE 68 can not be distinguished.

0

0,02

0,04

0,06

0,08

0,1

0,12

0,14

0,16

0,18

0,2

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

Stabilizercon

centratio

ninserum

%w/w

Stabilizerconcentrationinacidifiedmilkdrinks%w/w

PectinDE75pH4.15PectinDE75pH3.75PectinDE68pH4.15PectinDE68pH3.75CMCpH4.15CMCpH3.75

44

For each stabilizer, at lower pH condition, samples showed decreased serum concentration, thus

higher adsorption amount. It has also been reported that the adsorption amount increased with

decreasing pH, when the pH was lowered form 4.8 to 3.5, because the positive charge of casein

micelles increased when the pH decreased, thus more pectin was needed to compensate for the charge

of the protein (Tuinier et al., 2002). Another study carried out by van der Wielen et al. (2008) showed

that above pH 3, the adsorbed amount of CMC decreased as the molecules became more dissociated

thus less dosage was needed to compensate for the positive charge of the protein (van der Wielen,

van de Heijning, & Brouwer, 2008). Du et al. (2007) have also discussed that, with pH dropped from

4.3 to 3.0, there was continuous adsorption of CMC onto the increased positively charged casein

micelles, plus the decrease of negative charge of CMC resulted in a collapse of adsorbed CMC layer,

more CMC was needed to build up a thick layer that could contribute to maintain the stability of the

system (Du et al., 2007).

4.5. Sedimentation test

Figure 27. Sedimentation rate (%) of 3% MSNF acidified milk drinks after centrifugation as a function of stabilizer concentration (% w/w) for pectin DE 75, pectin DE 68 and CMC at pH 4.15 and pH 3.75.

The figure 27 shows the sedimentation rate after centrifugation as a function of stabilizer dosage for

pectin DE 75, pectin DE 68 and CMC at pH 4.15 and pH 3.75 in 3% MSNF sweetened yoghurt drinks.

The trend followed the characteristics of dose-response curves studied previously (Glahn & Rolin,

1994; Jensen et al., 2010; Laurent & Boulenguer, 2003).

0,00

2,00

4,00

6,00

8,00

10,00

12,00

14,00

16,00

18,00

20,00

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

Sedimen

tatio

nrate%

Stabilizerconcentration%w/w

SedimentationTest

PectinDE75pH4.15

PectinDE75pH3.75

PectinDE68pH4.15

PectinDE68pH3.75

CMCpH4.15

CMCpH3.75

45

It was observed that, for each stabilizer, there was an increase in the sedimentation rate as the

stabilizer concentration increased in the beginning. In this initial phase, the negatively charged

stabilizer particles were not sufficient to cover the positively charged casein micelles in the AMD

system, casein particles quickly reassembled into larger aggregates, thus bridging flocculation

occurred (Laurent & Boulenguer, 2003). Then the sedimentation rate decreased sharply, indicating

that an increasing amount of stabilizers were getting adsorbed onto casein micelles and more casein

micelles were fully covered with stabilizers around their surface. Finally, the steric and electrostatic

stabilization was reached at the bottom of the curve. When the concentration further increased, the

curves maintained at very low level of sedimentation rate.

It can be noticed that at a low sedimentation rate, for example at 2%, the concentration needed for

stabilizers varied. Pectin DE 75 showed the least dosage while CMC showed the highest. The sample

at pH 3.75 required higher concentration to reach a same stability as the sample at pH 4.15.

This result together with the serum stabilizer concentration result show that, increased adsorption of

stabilizers onto casein micelles can not guarantee better stability for the AMD system. This is also

agreed by former study that increased adsorption did not lead to improved stability but the opposite

was observed, instead (Jensen et al., 2010).

46

4.6. Instability index

Figure 28. Instability index of 3% MSNF acidified milk drinks measured by LumiSizer as a function of stabilizer concentration (% w/w) for pectin DE 75, pectin DE 68 and CMC at pH 4.15 and pH 3.75.

Figure 28 shows the instability index of 3% MSNF acidified milk drinks stabilized by a series of

concentrations (% w/w) of pectin DE 75, pectin DE 68 and CMC at pH 4.15 and pH 3.75. A higher

value of instability index indicates a less stable status where flocculation and sedimentation could

occur, while a lower value means a more stable profile exhibited by the AMD sample tested.

The instability index profiles resembled the characteristics of sedimentation rate curves in Figure 27.

In the initial stage with rather low stabilizer concentrations, the AMD samples all showed high

instability. Then with the increase of the stabilizer dosage, a sharp increase of instability index was

seen, indicating the samples were reaching a more stable phase. Finally, the instability index

decreased slowly with the continuing increased stabilizer concentration, samples maintained in a

stage of high-level stability.

In order to reach a certain stable status, for example instability index of 0.2, the dosage required for

stabilizers followed a trend: pectin DE 75 < pectin DE 68 < CMC, and at pH 3.75 the dosage needed

was higher than that of pH 4.15. This result in other words indicated that pectin DE 75 was an efficient

stabilizer for stabilizing the 3% MSNF AMD system investigated, the performance of pectin DE 68

was slightly less efficient, and CMC appeared to be a comparatively low efficient stabilizer; these

stabilizers displayed higher efficiency at pH 4.15 than at pH 3.75 in 3% MSNF AMD systems.

0

0,1

0,2

0,3

0,4

0,5

0,6

0,7

0,8

0,9

1

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

InstabilityInde

x

Stabilizer concentration%w/w

Stability profile of 3% MSNF acidified milk drinks

PectinDE75pH4.15

PectinDE75pH3.75

PectinDE68pH4.15

PectinDE68pH3.75

CMCpH4.15

CMCpH3.75

47

4.7. Particle size distribution

In order to further illustrate the particle size distribution of AMD samples, a representative stable

sample with an instability index of around 0.13 and a representative unstable sample with an

instability index of around 0.95 were picked out from each set of 10 AMD samples stabilized by each

stabilizer at each pH condition, then the particle size distribution of these samples were measured by

MasterSizer. The volume weighted mean diameter D [4,3], concentration of stabilizer, instability

index of these samples were listed in Table 8.

Table 8. Stabilizer concentration (% w/w), D [4,3]-volume weighted mean diameter (µm), instability index of representative stable and unstable samples picked out from AMD samples stabilized with a series of concentration of pectin DE 75, pectin DE 68, CMC at pH 4.15 and pH 3,75, respectively.

Stable AMD samples Concentration (% w/w)

D [4, 3]-Volume

weighted mean (µm)

Instability Index

Pectin DE 75 pH 4.15 0,08 0,5 0,13 Pectin DE 75 pH 3.75 0,12 0,6 0,13 Pectin DE 68 pH 4.15 0,12 0,5 0,15 Pectin DE 68 pH 3.75 0,16 0,6 0,14 CMC pH 4.15 0,47 0,6 0,14 CMC pH 3.75 0,69 0,7 0,11 Unstable AMD samples

Pectin DE 75 pH 4.15 0,04 2,1 0,94 Pectin DE 75 pH 3.75 0,06 1,0 0,94 Pectin DE 68 pH 4.15 0,04 2,3 0,92 Pectin DE 68 pH 3.75 0,07 2,2 0,93 CMC pH 4.15 0,16 39,0 0,97 CMC pH 3.75 0,24 5,6 0,95

48

Figure 29. Particle size distribution of representative stable AMD stabilized with pectin DE 75, pectin DE 68 and CMC at pH 4.15 and pH 3,75.

Figure 29 shows that stable AMD samples shared similar size distributions ranging between 0.05 - 5

µm, with the volume weighted mean diameter D [4, 3] as 0.5 – 0.7 µm. It could be noticed from table

8 that, upon achieving a similar stable status among the stable AMD samples, the required

concentration of stabilizers increased with the decrease of pH for each stabilizer, and the dosage

needed also followed the trend that pectin DE 75 < pectin DE 68 < CMC irrespective of pH conditions,

which were in accordance with the result of instability index and sedimentation rate.

Figure 30. Particle size distribution of representative unstable AMD with pectin DE 75, pectin DE 68 and CMC at pH 4.15 and pH 3,75.

In figure 30, the selected unstable AMD samples displayed polydisperse size distribution, with the

volume weighted mean diameter D [4, 3] > 1 µm. The samples with pectin DE 75 at pH 4.15, pectin

DE 68 at pH 4.15 and pH 3.75 showed similar size distribution with peaks ranging from 0.05 – 10

µm and 10 – 70 µm. The sample with pectin DE 75 at pH 3.75 showed a wide size distribution ranging

from 0.05 – 20 µm. The sample with CMC at pH 3.75 had a peak ranging from 0.05 – 5 µm, and

another peak from 15 – 100 µm, while the sample with CMC at pH 4.15 shifted towards a larger size

0

1

2

3

4

5

6

7

0,01 0,1 1 10 100 1000

Volume/%

Size/um

Particlesizeofstable3%MSNFAMD

PectinDE75pH4.15

PectinDE75pH3.75

PectinDE68pH4.15

PectinDE68pH3.75

CMCpH4.15

CMCpH3.75

0

1

2

3

4

5

6

7

0,01 0,1 1 10 100 1000

Volume/%

Size/um

Particlesizeofunstable3%MSNFAMDPectinDE75pH4.15PectinDE75pH3.75PectinDE68pH4.15PectinDE68pH3.75CMCpH4.15CMCpH3.75

49

distribution with peaks ranging from 0.2 – 5 µm and 15 – 400 µm. Compared with the size distribution

of stable samples in Figure 29, the larger particles shown in unstable AMD samples with D [4, 3] >

5 µm could be attributed to the bridging flocculation of casein particles induced by insufficient

stabilizer dosage.

The particle size results were in agreement with the former study which showed that stabilized

acidified milk drinks had casein particles smaller than 2 µm (Glahn & Rolin, 1994; Lucey et al.,

1999), and unstabilized beverages had a much larger particle size (Lucey et al., 1999).

4.8. Transmission profiles

The transmission profile of these unstable samples can be found in Figure 32, as A2 - pectin DE 75

pH 4.15, B3 - pectin DE 75 pH 3.75, C2 - pectin DE 68 pH 4.15, D3 - pectin DE 68 pH 3.75, E2 –

CMC pH 4.15, F3 – CMC pH 3.75. As for stable AMD samples, the one with pectin DE 75 pH 4.15

was shown as A4 in Figure 32, other samples can be found in Appendix III, B6 – pectin DE 75 pH

3.75, C6 – pectin DE 68 pH 4.15, D9 – pectin DE 68 pH 3.75, E5 – CMC pH 4.15, F7 – CMC pH

3.75.

Figure 31. Evolution of transmission profiles of an acidified milk drinks stabilized with 0.04% w/w pectin DE 75 at pH 4.15.

An example evolution of transmission profiles from LUMiSizer for unstable AMD sample with 0.04%

w/w pectin DE 75 at pH 4.15 is shown in Figure 31. The light transmission rate at each spot along

the sample cell was plotted against its radial position and was recorded every 5 seconds.

50

The insection in the transmission profiles at about 107 mm corresponded to the filling height of the

sample, the position of the cell bottom was at 130 mm. The transmission profiles represented the

variation of particle concentration in the sample, with high transmission rate indicating low particle

concentration and low transmission rate meaning high particle concentration (Sobisch & Lerche,

2008).

The first profile showed low transmission along the sample length except for the front part (107 –

112 mm), which indicated the formation of a supernatant layer already after 5 seconds. From the

second profile, a sharp front moved towards the cell bottom. This is presumed to reflect the fast

movement of aggregated casein particles due to the bridging flocculation resulted from the low

concentration of pectin DE 75. The distance between consecutive profiles was decreasing because

the resistance against compaction was increasing steadily (Sobisch & Lerche, 2008). Finally, the layer

of sediment was formed from around 126.5 mm to 130 mm.

Figure 32. An overview of transmission profiles of 3% MSNF acidified milk drinks stabilized by pectin DE 75 at pH 4.15 and pH 3.75 – A, B, pectin DE 68 at pH 4.15 and pH 3.75 – C, D, CMC at 4.15 and pH 3.75 – E, F. The concentration of stabilizers and instability index of the above samples are listed in Table 9.

51

Table 9. The concentration % w/w of stabilizers and instability index of AMD samples shown in Figure 32.

Stabilizers in AMD Concentration % w/w Instability index 1 2 3 4 1 2 3 4

A - Pectin DE 75 pH 4.15 0,011 0,037 0,060 0,081 0,940 0,936 0,184 0,128 B - Pectin DE 75 pH 3.75 0,011 0,037 0,060 0,081 0,915 0,932 0,943 0,425 C - Pectin DE 68 pH 4.15 0,013 0,043 0,069 0,094 0,957 0,921 0,335 0,182 D - Pectin DE 68 pH 3.75 0,013 0,043 0,069 0,094 0,873 0,936 0,929 0,448 E - CMC pH 4.15 0,048 0,159 0,246 0,324 0,969 0,965 0,539 0,302 F - CMC pH 3.75 0,048 0,159 0,243 0,320 0,879 0,937 0,951 0,741

Figure 32 shows the overview of the transmission profiles obtained from LUMiSizer for 3% MSNF

acidified milk drinks stabilized by pectin DE 75 at pH 4.15 and pH 3.75 – A, B, pectin DE 68 at pH

4.15 and pH 3.75 – C, D, CMC at 4.15 and pH 3.75 – E, F. The concentration of stabilizers increased

from 1 to 4. Table 9 shows the stabilizer concentration % w/w and instability index of AMD samples

shown in Figure 32. The complete version of transmission profiles and instability index for the 10

concentration series of each system can be seen in the Appendix II, III.

When comparing A1 and B1, C1 and D1, E1 and F1, AMD samples at pH 4.15 showed less stability

than at pH 3.75. In A1, C1, E1, the first transmission profile (the bottom red line) has already showed

sedimentation occurred with an increased transmission rate, then followed with a fast sedimentation

as the transmission rate decreased sharply reflecting the formation of an initial pack of sediments.

This was presumed to reflect the quick sedimentation of aggregated casein particles caused by ridging

flocculation. B1, D1, F1 showed polydisperse sedimentation. When the stabilizer concentration

increased in B2, D2, F2, sedimentation induced by bridging flocculation started to occur. In other

words, it was found that the bridging flocculation occurred at a lower dosage for AMD samples at

pH 4.15 than at pH 3,75.

It was also noticed that the AMD samples required less dosage to reach a relatively stable status at

pH 4.15 than at pH 3.75, as seen from A3 and B4, C3 and D4, E3 and F4. On the other hand, each

stabilizer with the same concentration showed better stabilizing efficiency at pH 4.15 than at pH 3.75,

when comparing A4 and B4, C4 and D4, E4 and F4. This was in agreement with the result of

instability index and sedimentation test.

Furthermore, the efficiency of stabilizers also followed the trend observed in the result of instability

index and sedimentation test: pectin DE 75 > pectin DE 68 > CMC, as seen from A4, C4, E4.

52

4.9. Summary

When combining the stability evaluation result from sedimentation test, instability index,

transmission profiles and particle size distribution, it can be found that the stabilization status of AMD

is influenced by stabilizer type, pH, stabilizer concentration. The stabilization functionality of pectin

DE 75, pectin DE 68 and CMC at pH 4.15 and pH 3.75 at different dosage varied, and the reasons

might be correlated with the characteristics of these polymers shown from the results of intrinsic

viscosity, hydrodynamic radius, zeta potential and serum stabilizer concentration measurements.

The dosage required for stabilizers to reach a same level of stability followed a trend: pectin DE 75

< pectin DE 68 < CMC. In general, it indicated that pectin DE 75 stabilized the 3% MSNF AMD

efficiently, the performance of pectin DE 68 was slightly less efficient, and CMC appeared to be a

comparatively low efficient stabilizer. This could be correlated with the result from intrinsic viscosity

and hydrodynamic radius and zeta potential. Among the three stabilizers investigated, under the same

pH condition, CMC had small hydrodynamic volume and large amount of negative charge, which

indicates a higher density of negative charge, thus it could be presumed that the adsorbed layer onto

casein particles might be thinner or flatter than pectin. Although CMC chains could also form loops

that extend out causing repulsive interaction, compared to the larger dimension of pectin, the weaker

steric repulsion to prevent aggregation might be the reason that CMC displayed less stabilizing

efficiency. On the other hand, by increasing the dosage, CMC was still able to reach the same stability

level as the pectin did, since more adsorption promoted to develop a thick layer for stabilization.

The different stabilization effect of the two pectins may be also related to their intrinsic viscosity and

negative charge. Pectin DE 68 showed higher negative charge and lower intrinsic viscosity (smaller

hydrodynamic dimension) than pectin DE 75, plus pectin DE 68 has higher degree of blockiness

(more binding positions onto the casein particle surface), therefore the adsorption layer might be

flatter. On the other hand, pectin DE 75 with higher intrinsic viscosity and lower degree of blockiness

may have a more freely dangling loop contributing to the steric stabilization of the AMD system.

Besides, higher intrinsic viscosity of the polymers could also contribute to the higher viscosity of the

serum phase, which may also assist to maintain the stability.

At pH 3.75 the dosage needed was higher than that of pH 4.15 for the same stability, which indicated

that these stabilizers displayed higher efficiency at pH 4.15 than at pH 3.75 in 3% MSNF AMD

systems. It could be because at pH 4.15, the more negatively charged and expanded polymer chains

53

that adsorbed on the casein micelle contributed to the electrostatic and steric stabilization of the casein

particles, with the protruding chains maintaining a repulsion between the protected casein micelles.

Such repulsive interaction may also prevent more adsorptions of polymers as inferred by a higher

serum stabilizer concentration. However, at pH 3.75, the polymer chains are less negatively charged,

the molecules have less intra- and intermolecular repulsions that result in a more compact structure

(in accordance with the intrinsic viscosity of pectin DE 75 and CMC) and higher tendency to

aggregate. At such lower pH, the more positively charged casein particles also attracts more

negatively charged polymers to be adsorbed, which would increase the adsorption rate at lower pH

(lower serum stabilizer concentration).

Furthermore, it was found that in the initial phase of a dosage response curve, where stabilizer

concentration was very low (< 0.05% w/w), bridging flocculation happened at a lower dosage at pH

4.15, while the AMD at pH 3.75 had bridging flocculation at a slightly higher concentration of

stabilizer. It might be explained that at pH 4.15, the polymer chains are more expanded, due to a

higher degree of dissociation of free carboxyl groups that resulted in an intra-repulsion of the

molecules, and also the more negatively charged polymers have a stronger attraction onto positively

charged casein particles, therefore there is a higher tendency to form a “bridge” between the

neighboring particles.

54

5. Conclusion In this study, three stabilizers, pectin DE 75, pectin DE 68 and CMC were used to investigate the

stabilization mechanism for 3% MSNF acidified milk drinks at pH 4.15 and pH 3.75.

The zeta potential of stabilizers in solutions were influenced by the amount of dissociated carboxyl

groups. Low DE of pectin, or high DS of CMC, which provide more amount of free carboxyl groups,

with high pH could lead to an increased negative charge, thus a stronger intra- and intermolecular

interaction.

From the result of the intrinsic viscosity test, it was found that the hydrodynamic volume of the

stabilizers in different solvent environment could be influenced by pH, ionic strength and co-solute.

A significant difference was found between the intrinsic viscosity of the three stabilizers: pectin DE

75 > pectin DE 68 > CMC. For pectin DE 75 and CMC, the intrinsic viscosity in yoghurt serum pH

4.15 > yoghurt serum pH 3.75. However, pectin DE 68 showed the opposite. This was contrary to the

expectation and more study need to be done for an explanation. The intrinsic viscosity of pectin in

yoghurt serum showed higher value than that in LiAc buffer which is a solvent with higher ionic

strength, might be attributed to the shielding effect of charges by high ionic strength. However, CMC

had lower intrinsic viscosity in yoghurt serum than in LiAc, probably due to the presence of calcium

ions rendered CMC less soluble.

The hydrodynamic radius of CMC showed the smallest size distribution, however the difference

between pectin DE 75 and DE 68 were generally inconclusive. The effect of pH on the size

distribution of stabilizers were not evident. The hydrodynamic radius showed aggregation of

polymers with larger size distribution at higher stabilizer concentration. This method could be

optimized in the future study to avoid the influence of solution concentration.

The serum stabilizer concentration indicated stronger adsorption of stabilizers onto casein micelles

at pH 3.75 than at pH 4.15. This might be attributed to that at lower pH the decrease of negative

charge of stabilizers resulted in less intra- and intermolecular repulsions which would lead to a more

compact structure and higher tendency to aggregate, At such lower pH, the more positively charged

casein particles would also attract more negatively charged polymers to be adsorbed. This can support

the second hypothesis mentioned in the objectives. CMC showed stronger adsorption than pectins,

55

which might be related to its lower hydrodynamic volume that resulted in less steric repulsion

between molecules to aggregate.

The measurements of sedimentation test, instability index, transmission profiles and particle size

distribution showed that the stabilization of AMD is influenced by stabilizer type, pH and stabilizer

concentration. The efficiency of stabilizing followed: pectin DE 75 > pectin DE 68 > CMC at the

same pH. The AMD showed better stability at pH 4.15 for each stabilizer. By increasing the

concentration of stabilizers, different stabilizers at pH 4.15 and pH 3.75 can achieve similar stability

level for AMD. Within the concentration range and pH range investigated, higher concentration,

higher pH, stabilizer with larger hydrodynamic volume would promote stability of AMD.

Generally, the pH, ionic strength, co-solute (Ca2+) of the solvent, DE or DS of the stabilizer which

influence the hydrodynamic volume and negative charge of the stabilizer molecules, as well as the

molecular dimension of different stabilizers, may play an important role in the steric and electrostatic

stabilization for the AMD system.

56

6. Perspectives In this study, it was found that CMC is a less efficient stabilizer compared with the stabilization

performance of pectins. However, by increasing the dosage of CMC, it is still able to achieve the

same stability for the AMD systems, as illustrated by the particle size distribution of the stable AMD

samples. Considering the low cost of CMC, it is actually a cost-efficient ingredient for stabilizing the

AMD products. Understanding the stabilization mechanism of CMC with casein micelles helps to

further discover more functionalities for product application.

Regarding the evaluation of intrinsic viscosity measurements, the relative viscosities of a dilution

series, should lie between 1.2 and 2.5. The data points below the critical value of the relative viscosity

of 1.2 show deviations from the linear fit and should not be included in the extrapolation to the y-axis

for the determination of the intrinsic viscosity (Kulicke & Clasen, 2004). However, the data generated

in this study did not all fall into this recommended range. This could be taken into account in the

future measurements.

The yoghurt serum used for intrinsic viscosity measurements could have been properly clarified, to

avoid adsorption of polymers with casein micelles in the solution, which would result in deviation of

intrinsic viscosity results.

The hydrodynamic radius results showed influence from the concentration of the polymers. At higher

concentration (0.1% w/w) polymers aggregate together, thus the effective hydrodynamic radius of

the polymer molecules could not be obtained. In order to avoid influence of concentration on the size

distribution of particles, a series of concentration of polymer solutions could be prepared and their

sizes measured, then the hydrodynamic radius can be obtained by extrapolating the intensity peak

mode data to zero concentrations (Kaszuba et al., 2008; Schmidt et al., 2017).

In addition, the polymer solutions prepared before measurements of hydrodynamic radius may not

have been fully hydrated, since the waiting time before the measurement was not strictly controlled,

there might be aggregates of stabilizer molecules detected. For an optimization, all solution samples

could be prepared 12 h before measurements; both for the preparation of stabilizer stock solutions

and further dilutions of the stock solution (Schmidt & Schuchmann, 2016; Schmidt et al., 2017).

57

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APPENDIX I. Calculation procedure for determination of [.] As an example, the calculation procedure for determining [$] for pectin DE 75 in 3% MSNF serum at pH 4.15 by capillary viscometer 766 (K= 0,003738 mm2/s2) is listed below. The same procedure was used for all samples. Table 1. The efflux times for solvent (yoghurt serum of 3% MSNF) and various concentrations of pectin DE 75.

SampleConc.

[%]

0.00 0.01 0.02 0.03 0.04 0.05

Time[sec.] 303,69 330,78 360,20 389,45 425,81 463,53

303,63 330,88 360,26 389,34 425,43 463,95

303,72 330,46 360,16 390,62 425,31 464,46

The efflux time of each solution was measured for three times and recorded in Table 1. Then the of $', $%&, $%&/? were calculated for three replicates of efflux times, calculations were listed in Table 2-4. Then the $%&/? data were plotted against concentration, and the obtained linear fits were shown in Figure 1-3.

$' =R%_abc*_+ −

@R%_abc*_+

R%_a8(+c −@

R%_a8(+c

$%& = $' − 1

Table 2. The calculation of $', $%&, $%&/? according to the efflux time of the first records in table 1 for each solution.

Sampleconc.[%]

RunTime[sec.]

relativeŋ

specificŋ

specificŋ/C

0 303,69

0,01 330,78 1,0892 0,0892 8,9203

0,02 360,20 1,1861 0,1861 9,3039

0,03 389,45 1,2824 0,2824 9,4131

0,04 425,81 1,4021 0,4021 10,0530

0,05 463,53 1,5263 0,5263 10,5265

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Table 3. The calculation of $', $%&, $%&/? according to the efflux time of the second records in table 1 for each solution.

Sampleconc.[%]

RunTime[sec.]

relativeŋ

specificŋ

Specificŋ/C

0 303,63

0,01 330,88 1,0897 0,0897 8,97470,02 360,26 1,1865 0,1865 9,32550,03 389,34 1,2823 0,2823 9,40950,04 425,43 1,4011 0,4011 10,02870,05 463,95 1,5280 0,5280 10,5602

Table 4. The calculation of $', $%&, $%&/? according to the efflux time of the third records in table 1 for each solution.

Sampleconc.[%]

RunTime[sec.]

relativeŋ

specificŋ

specificŋ/C

0 303,72

0,01 330,46 1,0880 0,0880 8,80420,02 360,16 1,1858 0,1858 9,29150,03 390,62 1,2861 0,2861 9,53730,04 425,31 1,4003 0,4003 10,00840,05 464,46 1,5292 0,5292 10,5847

Figure 1. Huggins plot for pectin DE 75 in serum of 3% MSNF yoghurt at pH 4.15, according to the first records of efflux time.

y=39,616x+8,4549R²=0,95762

8,0

8,5

9,0

9,5

10,0

10,5

11,0

0 0,01 0,02 0,03 0,04 0,05 0,06

Concentrationg/dl

$ %&/I

63

Figure 2. Huggins plot for pectin DE 75 in serum of 3% MSNF yoghurt at pH 4.15 according to the second records of efflux time.

Figure 3. Huggins plot for pectin DE 75 in serum of 3% MSNF yoghurt at pH 4.15, according to the third records of efflux time. The linear equations generated from the Huggins plots shown in Figure 1-3 were listed in table 5 below. According to Huggins equation, [$] is the Y-axis intercept, KH is the quotient of the slope divided by the square of [$]. The average value and standard deviation were calculated for three measurements.

$'() =$%&?= $ + @A ∙ $ C ∙ ?

Table 5. The Huggins linear equations, [$] and KH of pectin DE 75 in serum of 3% MSNF yoghurt at pH 4.15 by capillary viscometer 766.

Capillaryviscometer

Solvent PectinDE75

766

YogSerumpH4.15

y=39,616x+8,4549R²=0,95762 8,45 0,55y=38,741x+8,4975R²=0,94363 8,50 0,54y=42,781x+8,3618R²=0,98463 8,36 0,61

Average 8,44 0,57StandardDeviation 0,07 0,04

y=38,741x+8,4975R²=0,94363

8,0

8,5

9,0

9,5

10,0

10,5

11,0

0 0,01 0,02 0,03 0,04 0,05 0,06

Concentratoing/dl

$ %&/I

y=42,781x+8,3618R²=0,98463

8,0

8,5

9,0

9,5

10,0

10,5

11,0

0 0,01 0,02 0,03 0,04 0,05 0,06

Concentrationg/dl

$ %&/I

[$] @A

64

APPENDIX II. Instability index and stabilizer concentration Table 1. Instability index of 3% MSNF acidified milk drinks stabilized by pectin DE 75 at pH 4.15 and pH 3.75 – A, B, pectin DE 68 at pH 4.15 and pH 3.75 – C, D, CMC at 4.15 and pH 3.75 – E, F, with a series of concentrations 1-10. The concentration is shown in Table 2.

Table 2. Stabilizer concentration % w/w of 3% MSNF acidified milk drinks stabilized by pectin DE 75 at pH 4.15 and pH 3.75 – A, B, pectin DE 68 at pH 4.15 and pH 3.75 – C, D, CMC at 4.15 and pH 3.75 – E, F.

A B C D E F

PectinDE75pH4.15

PectinDE75pH3.75

PectinDE68pH4.15

PectinDE68pH3.75

CMCpH4.15

CMCpH3.75

1 0,011 0,011 0,013 0,013 0,048 0,0482 0,037 0,037 0,043 0,043 0,159 0,1593 0,060 0,060 0,069 0,069 0,246 0,2434 0,081 0,081 0,094 0,094 0,324 0,3205 0,100 0,100 0,116 0,116 0,383 0,3756 0,124 0,124 0,143 0,143 0,471 0,4667 0,141 0,141 0,163 0,163 0,519 0,5098 0,162 0,162 0,188 0,188 0,596 0,5789 0,201 0,201 0,232 0,232 0,704 0,69210 0,267 0,267 0,308 0,308 0,865 0,864

A B C D E F

PectinDE75pH4.15

PectinDE75pH3.75

PectinDE68pH4.15

PectinDE68pH3.75

CMCpH4.15

CMCpH3.75

1 0,940 0,915 0,957 0,873 0,969 0,8792 0,936 0,932 0,921 0,936 0,965 0,9373 0,184 0,943 0,335 0,929 0,539 0,9514 0,128 0,425 0,182 0,448 0,302 0,7415 0,103 0,171 0,149 0,223 0,219 0,4986 0,091 0,125 0,121 0,163 0,144 0,3177 0,070 0,107 0,100 0,140 0,115 0,2928 0,071 0,092 0,087 0,109 0,083 0,2379 0,048 0,061 0,063 0,080 0,035 0,11310 0,021 0,033 0,030 0,032 0,018 0,019

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APPENDIX III. Transmission profiles

Figure 1. An overview of transmission profiles of 3% MSNF acidified milk drinks stabilized by pectin DE 75 at pH 4.15 and pH 3.75 – A, B, pectin DE 68 at pH 4.15 and pH 3.75 – C, D, CMC at 4.15 and pH 3.75 – E, F, with a series of concentrations 1-10. The concentration of stabilizers and instability index of the above samples are listed in Appendix II.

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