Effect of Gellan, Alone and in Combination With High-Methoxy Pectin, On The

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Food Hydrocolloids 24 (2010) 744e754
Contents lists available at ScienceDirect
Food Hydrocolloids
journal homepage: www.elsevier.com/locate/foodhyd
Effect of gellan, alone and in combination with high-methoxy pectin, on the

structure and stability of doogh, a yogurt-based Iranian drink
H. Kiani a, M.E. Mousavi a, H. Razavi a, E.R. Morris b, *
a
b
Department of Food Science and Technology, Faculty of Biosystems Engineering, Campus of Agriculture and Natural Resources, University of Tehran, Karaj, Iran
Department of Food and Nutritional Sciences, University College Cork, Cork, Ireland
a r t i c l e i n f o
a b s t r a c t
Article history:
Received 7 August 2009
Accepted 29 March 2010
Doogh is a traditional Iranian drink prepared by fragmentation and dilution of yogurt, with addition of
salt and avouring. In the present work, we have used viscometry, microscopy, particle-size analysis and
measurements of serum separation to explore the effect of very low concentrations of gellan gum (0.01,
0.03 and 0.05 wt %), alone or in combination with 0.25 wt % high-methoxy pectin (HMP), on its structure
and stability. HMP is known to prevent association of casein particles in acidic milk drinks by steric
stabilisation, forming a protective layer bound electrostatically to the surface of the particles. Doogh
incorporating 0.25 wt % HMP alone showed satisfactory stability on storage for w10 days at 5 C, but
after 15 days there was obvious separation into a dense sediment and a much clearer upper layer that
occupied more than 80% of the total volume, which we attribute to progressive sedimentation of individual sterically-stabilised particles. Samples incorporating gellan (with or without HMP), by contrast,
showed rapid development of a clear serum phase, with little further separation at longer times (up to
w1 month), suggesting expression of uid by contraction of a gel network (i.e. syneresis rather than
sedimentation). Particle size increased dramatically (more than 10-fold) with increasing concentration of
gellan, and at the highest concentration studied (0.05 wt %) a continuous network of casein-rich strands
was observed by phase-contrast microscopy. The concentration of NaCl used in the doogh samples
(0.5 wt % z 85 mM) is known to be sufcient to maintain gellan in its ordered (double-helix) conformation, which has higher charge density than individual molecules of HMP. We suggest that network
structure is formed by electrostatic attachment of gellan to fragments of acid-casein gel, thus increasing
particle size and inhibiting surface-coverage by HMP, with weaker associations between gellan helices
allowing the samples to ow. Observed decrease in serum volume with increasing concentration of
gellan is attributed to formation of progressively stronger coupled networks with greater resistance to
syneresis. Stabilisation of doogh with 0.05 wt % gellan in combination with 0.25 wt % HMP had no
adverse effect on organoleptic acceptability, and reduced serum volume on protracted storage to w10%
(in comparison with over 80% for the same concentration of HMP alone), suggesting that gellan could be
of practical value in extending the shelf-life of doogh (and related acidic milk drinks).
2010 Elsevier Ltd. All rights reserved.
Keywords:
Acidic milk drinks
Doogh
Gellan
Pectin
Microscopy
Rheology
1. Introduction
Doogh is a nutritious and refreshing acidic milk drink native to
Iran. It is one of many acidied dairy beverages produced worldwide. These range from products acidied by bacterial fermentation to drinks made by direct acidication of milk with fruit juice
and/or acids (Nakamura, Yoshida, Maedab, & Corrediga, 2006) but
usually have viscosities similar to that of unprocessed milk (Laurent
& Boulenguer, 2003).
* Corresponding author. Tel: 44 1234 825523.

E-mail address: ed.morris@ucc.ie (E.R. Morris).
0268-005X/$ e see front matter 2010 Elsevier Ltd. All rights reserved.
doi:10.1016/j.foodhyd.2010.03.016
Traditionally, doogh was prepared by churning full-fat yogurt

after partial dilution with water, followed by removal of fat,
further fermentation to achieve satisfactory taste and acidity, and
nal dilution, with addition of salt and spices, to give a drink
ready for consumption. In current commercial production,
reduction in fat content is normally achieved by using skim milk
to prepare the yogurt, and the nal product is avoured with
essential oils, such as those from mint or zizifore (Ziziphora
tenuior), homogenised and packaged. Similar yogurt-based
beverages are popular in countries neighbouring Iran, and
include a Turkish product known as ayran (Kksoy & Kili, 2003;
Koksoy & Kilic, 2004). These drinks may differ from doogh in, for
example, dilution ratio, fat content, rheological characteristics
H. Kiani et al. / Food Hydrocolloids 24 (2010) 744e754
and taste (Kiani, Mousavi, & Emam-Djomeh, 2008). Like doogh,

however, they all contain aggregated casein.
Casein constitutes about 80% of the total protein content of
bovine milk, and comprises four main components, as1, as2, b and k
(Fox & McSweeney, 1998). The a and b caseins have a high content
of phosphate, attached by ester linkages to serine residues of the
protein chains. A distinctive feature of k-casein is an extensively
glycosylated macropeptide region at the C-terminal end of the
molecule. Virtually all (>95%) of the casein in milk exists as
micelles, which are roughly spherical and range in size from 50 to
300 nm, with an average diameter of w120 nm. The a- and
b-caseins are clustered in the centre of the micelle; the k-casein
molecules lie at the surface, with the glycosylated C-terminal
sequences protruding to form an expanded hairy layer which acts
as a barrier to aggregation (Holt, 1992; Holt & Horne, 1996; Walstra,
1990). In production of yogurt from fresh milk, the charge on the
macropeptide is suppressed by reduction in pH, which causes
collapse of the hairy layer and eliminates the steric, entropic and
electrostatic barrier to agglomeration.
In addition to casein, milk contains other proteins which remain
soluble at pH values low enough to cause agglomeration of casein;
these are known as whey proteins and contribute about 20% of the
total protein content. The principal whey protein is b-lactoglobulin,
which constitutes about 50% of the total content of whey protein in
bovine milk (Fox & McSweeney, 1998). In commercial production of
yogurt, the milk is normally pre-heated (typically for w10e20 min
at w90 C). This heat treatment causes association of denatured
b-lactoglobulin to the surface of the casein micelles, by disulphide
bridging to k-casein (Horne & Davidson, 1993; Mulvihill &
Grufferty, 1995), and the pH at the onset of acid-induced gelation
of pre-heated milk is close to the isoelectric point of the attached
whey protein (Horne & Davidson, 1993), indicating that the critical
requirement for aggregation is now suppression of electrostatic
repulsion between b-lactoglobulin appendages, rather than
collapse of the hairy layer, which occurs only at lower pH.
Acid-casein gels can be formed chemically, by direct addition of
mineral acid or by decomposition of glucono-d-lactone (GDL) into
gluconic acid (Arshad, Paulsson, & Dejmek, 1993; Cobos, Horne &
Muir, 1995; Lucey, van Vliet, Grolle, Geurts, & Walstra, 1997). In
production of yogurt, pH is reduced by fermentation with bacteria
that convert milk sugar (lactose) into lactic acid (Mulvihill &
Grufferty, 1995; Tamime & Marshall, 1997). Commercial yogurts
are produced in two different forms: set-style, where the milk is
fermented in the retail cartons, giving a continuous gelled structure, or stirred, where fermentation is carried out in large tanks,
and the acid gel is then disrupted by stirring and sieving to give
a more uid product.
In stirred yogurt, the particles of fragmented acid-casein gel
remain in contact with one another, giving a self-supporting
structure. On dilution to acidied beverages (such as doogh and
ayran), however, the particles are separated and free to sediment
under gravity, causing massive loss of stability, which becomes
more severe as the extent of dilution is increased (Glahn, 1982;
Lucey, Tamenaha, Singh, & Munro, 1999). This phenomenon,
known industrially as wheying off (Kravtchenko, Parker, &
Trespoey, 1995; Syrbe, Bauer, & Klostermeyer, 1998), leads to
separation into a casein-rich lower layer and an upper layer of clear
serum, which can often occupy more than half the total volume.
Doogh is particularly unstable, because the extent of dilution is
greater than in most other yogurt-based beverages, including
ayran. Hydrocolloid stabilisers are commonly used to retard separation in acidic milk drinks (Kiani et al., 2008; Koksoy & Kilic, 2004;
Laurent & Boulenguer, 2003; Nakamura et al., 2006).
One way in which they can act is by increasing viscosity, and
therefore reducing the rate of sedimentation. Viscosity increase can
745
also be achieved by using a ropy starter culture to produce

extracellular bacterial polysaccharides (EPS) in the yogurt base
(Kksoy & Kili, 2003). A related approach to reduction in sedimentation rate (Paraskevopoulou et al., 2003) is to incorporate
xanthan, which gives solutions with a pourable weak gel structure (Ross-Murphy, 1984) capable of holding particles in suspension over long periods of time. However, the stabiliser most
commonly used (Kravtchenko et al., 1995; Parker, Boulenguer, &
Kravtchenko, 1994; Towler, 1984) is high-methoxy pectin (HMP).
HMP is best known as the gelling agent in jams and marmalade,
but its use in acid milk drinks is now of at least comparable
commercial importance (Willats, Knox, & Mikkelsen, 2006). The
structure of pectin is complex (Ridley, ONeill & Mohnen, 2001; Visser
& Voragen, 1996; Voragen, Schols & Visser, 2003), but centres on
(1 / 4)-linked linear sequences of a-D-galacturonate. In commercial
HMP (Christensen, 1986; May, 1990; Rolin, 1993) w65e75% of the
galacturonate residues occur as the methyl ester. The ester content
can be reduced by pectin methylesterase enzymes, or by hydrolysis
with alkali or acid, to give low-methoxy pectin (LMP), which typically
has a degree of esterication (DE) of w30e35%.
Addition of pectin to milk at its natural pH of w6.5 (Fox &
McSweeney, 1998) can cause separation into two layers, or phases, one of which is casein-rich and the other pectin-rich (Antonov,
Grinberg, Zhuravskya, & Tolstoguzov, 1982; Maroziene, & de Kruif,
2000). Phase separation in mixtures of unlike biopolymers is
common; there is usually an enthalpic advantage from molecules
being surrounded by others of the same type, which can outweigh
the entropic advantage of intimate mixing (Grinberg & Tolstoguzov,
1997; Morawetz, 1965; Morris, 1990; Picullel, Bergfeldt, & Nilsson,
1995; Piculell, Nilsson, Falck, & Tjerneld, 1991; Suchkov, Grinberg
& Tolstoguzov, 1981; Tolstoguzov, 1986, 1991). In mixtures of
polymer molecules with larger particles, such as oil droplets or
casein micelles, there is an additional mechanism of segregation,
known as depletion occulation (Dickinson, 1992; Dickinson,
Semenova, Antipova, & Pelan, 1998; Hemar, Tamehana, Munro, &
Singh, 2001; Langendorff, Cuvelier, Launay, & Parker, 1997;
Maroziene & de Kruif, 2000). As particles approach one another,
it may become impossible for polymer molecules to penetrate the
gap between them, with consequent loss of entropy. Association of
the particles into ocs reduces the depleted volume, and is
therefore thermodynamically favourable. However, although
pectin may reduce the colloidal stability of micellar casein at
around neutral pH, it can confer stability under acidic conditions
where the micelles have passed through their isolelectric point of
w4.9 and their net charge has changed from negative to positive
(Tuinier, Rolin, & de Kruif, 2002).
The effectiveness of HMP in stabilising acid dairy drinks has
been known for over 50 years (Doesburg & de Vos, 1959), and the
underlying mechanism is now understood reasonably well (Parker
et al., 1994). A major source of instability in these products is the
tendency of fragmented acid-casein gels to re-associate into larger
particles that sediment more rapidly. Aggregation can be suppressed by electrostatic association of (negatively-charged) pectin
molecules to the surface of the (positively-charged) casein particles, to form a protective layer that maintains colloidal stability. A
useful analogy can be drawn (Parker et al., 1994) with stabilisation
of oil-in-water emulsions by adsorption of protein onto the surface
of the oil droplets (Dickinson, 1992, 2003; Dickinson et al., 1998). In
both cases, mechanical energy must be applied to the system to
create small particles (acid-casein fragments; oil droplets), and the
adsorbed polymer (dispersing agent) prevents re-aggregation.
Stabilisation of acid-casein dispersions by HMP, however, requires
enough pectin to cover the surface of the particles. Lower concentrations can cause instability (Maroziene & de Kruif, 2000; Syrbe
et al., 1998; Tuinier et al., 2002) by attachment of individual pectin
746
molecules to more than one particle, thus inducing formation of large

ocs. This process of bridging occulation (Dickinson, 1992) has its
maximum effect (Maroziene & de Kruif, 2000) when the concentration of HMP reaches the value required to give 50% coverage of the
surface of the particles. As the pectin concentration is raised beyond
this point, bridging is progressively replaced by attachment of individual polymer chains to individual particles, until their surface is
fully covered and the dispersion becomes colloidally stable. Further
increase in HMP concentration can cause loss of stability by depletion
occulation, but this occurs only at concentrations well above those
used in practical applications of pectin in dairy products.
The concentration of HMP required to ensure stability in acid milk
drinks is around 0.25 wt % (Willats et al., 2006). Most (up to w90%) of
the HMP is not adsorbed; the excess is needed to drive attachment of
pectin chains to the casein particles, but can then be removed
without loss of stability (Tromp, de Kruif, van Eijk, & Rolin, 2004).
The particular effectiveness of HMP in stabilising acid milk drinks
can be traced to its primary structure. Measurements of zeta potential
(Parker et al.,1994) have shown that the net negative charge of casein
particles coated with adsorbed HMP is insufcient for electrostatic
repulsion between the particles to prevent re-aggregation. However,
a more general way in which adsorbed polymers can prevent
aggregation is by forming a thick, expanded layer whose conformational entropy would be lost by particleeparticle association.
This mechanism, which is known as steric stabilisation, has two
conicting requirements (Dickinson, 2003; Parker et al., 1994). The
polymer chains must bind strongly, to keep them attached to the
particles, but they must also retain sufcient conformational freedom
to create an entropic barrier to aggregation. Structurally-regular
polymers cannot satisfy both requirements simultaneously, but block
copolymers can, if one type of block forms stable associations with
the surface of the particles and the other type remains unattached
and conformationally mobile.
Although pectin is not, of course, a block copolymer, the distribution of ester groups along the polymer chains can give regions of
high negative charge (low ester content) which bind tightly to casein
particles, and regions of lower charge (higher ester content) which
confer steric stabilisation. Because of its high content of unesteried
carboxyl groups, LMP binds strongly, but does not have sufcient
highly esteried sequences to form a thick, conformationally mobile
layer. HMP, however, gives the required combination of strong
attachment and residual exibility. As would be expected from this
interpretation, HMP fractions that include long sequences of unesteried polygalacturonate, known as calcium-sensitive fractions
because of their ability to form egg-box junctions (Grant, Morris,
Rees, Smith, & Thom, 1973) with Ca2, are more effective in stabilising acidic milk drinks than fractions with a more even distribution
of esteried and non-esteried residues (Glahn & Rolin, 1996;
Laurent & Boulenguer, 2003; Tuinier et al., 2002; Willats et al., 2006).
One polysaccharide that has received little attention as
a potential stabiliser of acidic milk drinks is gellan gum. Gellan
(Sworn, 2000) is an extracellular anionic polysaccharide produced
by the bacterium Sphingomonas elodea (ATCC 31461; formerly
known as Auromonas elodea or Pseudomonas elodea) on aerobic
fermentation. It has a linear tetrasaccharide repeating sequence
(Jansson, Lindberg, & Sandford, 1983; ONeill, Selvendran, &
Morris, 1983) of: /3)-b-D-Glcp-(1 / 4)-b-D-GlcpA-(1 / 4)-b-DGlcp-(1 / 4)-a-L-Rhap-(1/. The native polysaccharide, as biosynthesised, has an L-glyceryl substituent on O(2) of the 3-linked
glucose and about half the repeat units have an acetyl group on
O(6) of the same residue (Kuo, Mort, & Dell, 1986). In normal
commercial production of gellan gum, however, the polysaccharide is released from the fermentation broth by treatment
with hot alkali, resulting in essentially complete removal of both
types of acyl substituent by alkaline hydrolysis.
It has been shown by X-ray bre diffraction analysis

(Chandrasekaran, Millane, Arnott, & Atkins, 1988) that gellan exists
in the solid state as a 3-fold coaxial double helix, similar to that of
iota carrageenan (Arnott, Scott, Rees, & McNab, 1974). Adoption of
double-helix structure when solutions of gellan are cooled from the
disordered state at high temperature can be seen as a sharp
conformational transition, which has been monitored by a variety
of physical techniques, including optical rotation (Crescenzi &
Dentini, 1988; Crescenzi, Dentini, Coviello, & Rizzo, 1986;
Grasdalen & Smidsrd, 1987; Milas, Shi, & Rinaudo, 1990;
Robinson, Manning, & Morris, 1991), circular dichroism (Crescenzi
et al., 1986; Matsukawa & Watanabe, 2007), solution viscosity
(Grasdalen & Smidsrd, 1987; Milas et al., 1990) and differential
scanning calorimetry (Robinson et al., 1991). As found for other
charged polysaccharides, the disordereorder transition moves to
progressively higher temperature with increasing concentration of
salt. The accompanying changes in rheology, and in loss of
conformational order on heating, however, are more complex.
On addition of low concentrations of salt (NaCl) to solutions of
gellan in water, the solutions remain uid (Morris, Richardson, &
Whittaker, 1999), and the orderedisorder transition on heating
follows the same temperature-course as the disordereorder transition on cooling (Robinson et al., 1991). The onset of gel formation
(by 1.0 wt % gellan) occurs when the concentration of NaCl reaches
w65 mM (w0.4 wt %), and is accompanied by development of two
orderedisorder processes. As the salt concentration is increased
beyond the threshold value for gelation, the rst orderedisorder
process decreases in magnitude and continues to follow the same
temperature-course as the single transition observed on cooling.
The second orderedisorder process, however, moves steeply to
higher temperature (with accompanying increase in thermal
hysteresis between the onset of gelation on cooling and completion
of gel melting on heating), and increases in magnitude, paralleling
the progressive increase in gel strength with increasing concentration of NaCl.
A proposed interpretation (Morris, Gothard, Hember, Manning,
& Robinson, 1996; Robinson et al., 1991) is that formation of
a cohesive gel network requires association of gellan helices into
stable aggregates, and that Na cations promote the aggregation
process by site-binding to the helices, thus suppressing the electrostatic repulsion between them. In terms of this interpretation,
the rst orderedisorder process observed on heating under gelling
conditions can be attributed to dissociation of residual unaggregated helices, and the second to dissociation of aggregated
helices at higher temperature. Qualitatively similar behaviour is
observed with other Group I metal ions, but their effectiveness in
promoting gel formation decreases with ionic radius, in the order
Cs > K > Na > Li (Grasdalen & Smidsrd, 1987). At high
concentrations of salt (above w300 mM NaCl) gel strength
decreases, which can be attributed (Morris et al., 1999) to excessive
helixehelix association into very large aggregates, with consequent
reduction in the total number of junction zones in the gel network.
A maximum in gel strength, which can be explained in the same
way, is also observed with increasing concentration of Group II
cations, but it occurs at much lower cation concentrations, around
stoichiometric equivalence to the carboxyl groups of the gellan, and
there is no indication that the increase in gel strength up to the
maximum begins only once the cation concentration has reached
a threshold value. A likely interpretation (Morris et al., 1996) is that
divalent cations promote aggregation by site-binding between pairs
of carboxyl groups on neighbouring helices, to give structures
analogous to the egg-box junctions proposed (Grant et al., 1973) for
calcium-induced gelation of alginate or pectin. Computer modelling
(Chandrasekaran & Thailambal, 1990) has shown that a packing
arrangement of this type is sterically feasible for Ca2 gellan.
At concentrations of polymer and/or cations below those

required to give true (self-supporting) gels, gellan, in its ordered
(double-helix) conformation, can form uid (pourable) weak gel
networks similar to those of ordered xanthan. For example, Morris
et al. (1999) observed weak gel rheology for 1.0 wt % gellan at
NaCl concentrations above w25 mM but below the threshold
concentration required to give true gels. In a more recent study by
Rodrguez-Hernndez, Durand, Garnier, Tecante, & Doublier (2003),
where a xed concentration of CaCl2 (10 mM) was incorporated in
very dilute solutions of gellan (0.05 wt % and less), weak gel
properties were observed down to slightly below 0.005 wt %. The
window of polymer/salt concentrations at which weak gels are
formed can be extended by shearing gellan solutions as they are
cooled through the temperature-range of the disordereorder
transition. Using this approach, Sworn, Sanderson, and Gibson
(1995) obtained uid gellan networks, capable of suspending
particles, at a polymer concentration (0.125 wt %) where ordered
xanthan shows only the predominantly liquid-like properties of
a normal polymer solution.
In the present work, we have explored the effect of low concentrations of ordered gellan, alone and in combination with a higher
concentration of HMP, on the structure and stability of doogh.
2. Materials and methods
2.1. Materials
Fresh skim milk (fat content by Gerber analysis z 0.2 wt %) was
obtained from the dairy plant at the University of Tehran. A direct
vat-set starter culture (YF-3331 from Chr. Hansen, Denmark) was
used for yoghurt production. Gellan gum (Kelcogel) was from CP
Kelco, San Diego, CA, USA, and high-methoxy (HM) citrus pectin
from Provisco, Switzerland (PROVladd PEC 1902; DE > 72%).
Sodium chloride and lactic acid were from Merck. Deionised water,
produced by reverse osmosis, was used throughout.
2.2. Sample preparation
Fresh skim milk was heated for 15 min at 90 C, inoculated with
the starter culture at 40 - 42 C, fermented for 6 h to pH 4.0e4.2,
and cooled to 5 C to stop the fermentation. The total solids (TS)
content of the resulting yoghurt was determined by evaporation of
w1 g sample on a boiling bath, followed by oven-drying at 102 C
until constant weight was attained, yielding a value of TS 9.6 wt
%. A diluted stock solution was then prepared by addition of water
and salt (NaCl) to an NaCl concentration of 0.75 wt % and total
solids content (including the added salt) of TS 7.5 wt %, followed
by thorough mixing (Janke & Kunel RW20 DZM mixer), and
homogenisation at a pressure of 150 bar (APV 1000 laboratory
homogenizer). Doogh samples were prepared from aliquots of the
stock solution by addition of water and/or polysaccharide solutions
to a nal salt concentration of 0.5 wt % NaCl and nal total solids
content (including salt and polysaccharides) of 5.0 wt %, with
gentle mixing at room temperature.
Three fermentation batches, identied as Batch 1, Batch 2 and
Batch 3, were studied. For each of these, pectin (where used) was
incorporated at a xed concentration of 0.25 wt % in the resulting
samples of doogh, and gellan was used at concentrations of 0.00, 0.01,
0.03 and 0.05 wt % (see Table 1). Polysaccharide solutions for use in
doogh were prepared by dispersing the powder in cold water and
dissolving on a shaking water bath for 20 min at high temperature.
Three temperatures of dissolution were evaluated: 70, 80 and 90 C.
All preparations were stored at 5 C until measurements were made.
For characterisation of the viscosity of polysaccharide solutions
under conditions of pH and ionic environment matched to those in
747
doogh, the following method of sample preparation was used.

Fresh skim milk and NaCl were mixed in the same proportions as
in the stock solutions of diluted yogurt, the mixture was gradually
acidied to pH 4.1 by addition of 0.1 M lactic acid, and water was
added to give concentrations of NaCl and milk solids 50% higher
than those in plain doogh (i.e. without added polysaccharides).
Particles of agglomerated protein formed on acidication were
removed by centrifugation (15 min at 2500 rpm, then two 20 min
periods at 4000 rpm) followed by vacuum ltration (0.22 mm
Millipore lter), which also removed whey proteins. Polysaccharide solutions were prepared in water at concentrations
three times those used in the doogh samples (i.e. 0.75 wt % pectin
and/or 0.03, 0.09 or 0.15 wt % gellan), and the nal solutions for
rheological characterisation were obtained by mixing 15 mL of
polysaccharide solution with 30 mL of protein-free ltrate from
the acidied mixture of milk and NaCl.
2.3. Methods of investigation
Viscosity measurements were made at 20 C on a Haake Rotovisco RV12 viscometer (Gebruder Haake GmbH, Karlsruhe,
Germany), using the NV geometry (doubled-gap coaxial-cylinder;
German Institute of Standards, DIN 54 453) recommended by
Schramm (1981). Analyses were performed in triplicate at least
24 h after production of the samples.
For characterisation of phase separation and sedimentation,
10 mL samples of doogh were held for 15 days at 5 C in tubes of
diameter 1.5 cm and height 18 cm and the volume of separated
serum was measured during the storage time.
Table 1
Parameters derived from double-logarithmic plots of specic viscosity (hsp) versus
shear rate g_ : slope; intercept at g_ 1 s1 ; correlation coefcient (r2).
Doogh
batch
[Pectin]
(wt %)
[Gellan]
(wt %)
Slope
(s)
Intercept
r2
0.00
0.00
0.00
0.00
0.00
0.01
0.03
0.05
0.488
0.552
0.549
0.618
29.0
51.9
102.6
169.9
0.970
0.979
0.978
0.988
0.25
0.25
0.25
0.25
0.00
0.01
0.03
0.05
0.444
0.505
0.517
0.596
29.0
61.8
135.8
234.3
0.940
0.993
0.993
0.995
0.00
0.00
0.00
0.00
0.00
0.01
0.03
0.05
0.344
0.463
0.577
0.622
2.7
14.4
56.2
125.9
0.849
0.959
0.991
0.990
0.25
0.25
0.25
0.25
0.00
0.01
0.03
0.05
0.418
0.465
0.563
0.603
11.6
33.1
106.9
184.1
0.867
0.965
0.988
0.990
0.00
0.00
0.00
0.00
0.00
0.01
0.03
0.05
0.517
0.555
0.584
0.615
13.5
30.1
79.5
138.8
0.974
0.986
0.995
0.997
0.25
0.25
0.25
0.25
0.00
0.01
0.03
0.05
0.411
0.491
0.554
0.601
16.7
44.3
119.4
201.5
0.930
0.981
0.992
0.995
No
doogh
0.00
0.00
0.00
0.01
0.03
0.05
0.483
0.443
0.408
12.5
14.8
17.8
0.998
0.964
0.995
No
doogh
0.25
0.25
0.25
0.25
0.00
0.01
0.03
0.05
0.347
0.373
0.401
0.395
8.2
13.4
21.2
31.2
0.985
0.981
0.960
0.995
748
Particle size was determined by light scattering, using a Mastersizer 2000S (Malvern Instruments Ltd., UK). Analysis was performed 24 h after preparation of the samples, and was based on the
principle of Fraunhofer (Huang, Kakuda, & Cui, 2001). Size distribution was characterised by volumetric percentage and mean
particle diameter, obtained from the Mastersizer 2000 software.
Microstructure was visualised by phase-contrast light microscopy
(Leica Galen III, Germany), and recorded using a digital camera
mounted on the microscope. Protein particles were stained noncovalently by Rhodamine B dye (Merck), giving bright regions in the
micrographs; 0.5 mL of a 0.05% w/v Rhodamine B solution was added
to each 9.5 mL of doogh, and the samples were then diluted 10-fold.
Observations were made immediately after preparation of the slides.
Sensory analysis was carried out after storage for 1 day at 5 C,
using 20 panellists familiar with the product. Samples were evaluated for aroma, taste, homogeneity, mouthfeel and overall
acceptability, on a 5-point hedonic scale. Results were analysed
using SAS 9.1 software (SAS Institute Inc., Cary, NC, USA). The effects
of the stabilisers were characterised by ANOVA and, if necessary,
the means were compared by Duncans multiple comparison test.
3.1. Viscosity measurements

A recent power law analysis of the variation of shear stress (s)
with shear rate g_ for mixtures of doogh with gellan gum (Kiani,
Ebrahimzadeh-Mousavi, Emam-Djomeh, & Yarmand, 2008) indicated small changes in consistency index (K) and ow behaviour
index (n) on varying the temperature at which the gellan was dissolved (across the range 70e90 C). As illustrated in Fig. 1, however,
the effect of dissolution temperature is small in comparison with
the experimental scatter of the viscosity measurements, particularly at low shear rates. In the present analysis, the values of s for all
three temperatures at which gellan and pectin solutions were
prepared (70, 80 and 90 C) were therefore averaged (with omission of any obvious outliers), and used to construct ow curves of
viscosity h s=g_ versus g_ (at 20 C).
The plots obtained are illustrated in Fig. 2a for doogh from Batch
3, with no added polysaccharide (plain doogh) and with gellan at
1000
10000
Viscosity (mPa s)
Stress (mPa)
3. Results
1000
100
10
100
10
0.1
1
0.1
1
10
100
1000
10
100
1000
100
1000
-1
Shear rate (s )
-1
Shear rate (s )
10000
1000
100
1000
Specific viscosity
Stress (mPa)
100
10
10
0.1
10
100
1000
-1
Shear rate (s )
Fig. 1. Flow curves (20 C) of shear stress versus shear rate for illustrative samples of
doogh (Batch 3) incorporating (a) 0.03 wt % gellan or (b) 0.03 wt % gellan plus 0.25 wt
% HMP, after the polysaccharides had been dissolved at temperatures of 70 (B), 80 (6)
or 90 (,) C.
0.1
0.1
10
-1
Shear rate (s )
Fig. 2. Shear-rate dependence of (a) viscosity and (b) specic viscosity for doogh
(Batch 3) alone (B), and with gellan at concentrations (wt %) of 0.01 (C), 0.03 (6) and
0.05 (:).
the three concentrations studied (0.01, 0.03 and 0.05 wt %). The
curves show substantial shear-thinning (pseudoplasticity), but
level out at high shear rate, towards the viscosity of water (1 mPa s
at 20 C). To remove the direct contribution of water to the overall
viscosity, experimental values of h were converted to specic
viscosity (hsp):
hsp h hs =hs
(1)
where hs is the viscosity of the solvent (Bohdaneck & Kovr, 1982).

With values of h and hs expressed in units of mPa s, and with water
as solvent at 20 C (h 1 mPa s), Eq. (1) reduces to:
hsp h 1
(2)
As illustrated in Fig. 2b for the same samples of doogh as in Fig. 2a,

the resulting double-logarithmic plots of hsp versus g_ are acceptably linear. The slopes, intercepts (hsp at 1 s1, where log g_ 0)
and r2 values obtained by regression analysis (using the trendline
function in Microsoft Excel) are listed in Table 1 for all samples
1000
Viscosity (mPa s) at 1 s
-1
100
10
without pectin
1
0.00
0.02
0.04
0.06
Gellan concentration (wt %)
100
Viscosity (mPa s) with pectin
Viscosity (mPa s) at 1 s
studied (including the protein-free polysaccharide solutions, which

are indicated in the table as no doogh). In addition to the
expected increase in intercept with increasing concentration of
gellan, all samples of doogh (from Batches 1, 2 and 3, with or
without incorporation of 0.25 wt % pectin) show an accompanying
systematic increase in (absolute) slope of log hsp versus log g_ .
The values of h (mPa s) at g_ 1 s1 were obtained (Eq. (2)) by
addition of 1.0 to the intercepts listed in Table 1, and are shown in
Fig. 3 for samples prepared with (Fig. 3b) and without (Fig. 3a)
incorporation of 0.25 wt % pectin. In both cases, samples from
Batch 1 have substantially higher viscosities than those from Batch
2, with intermediate values for Batch 3. These differences (which
may have arisen from slight differences in fermentation temperature, pH at the end of the 6 h fermentation period, and/or the
subsequent dilution and homogenisation procedures) decreased
progressively as the concentration of gellan was increased.
However, this effect cannot be attributed to the overall viscosity
becoming dominated by that of the gellan component, because at
the highest gellan concentration used (0.05 wt %) the viscosity
values for the doogh samples (with or without pectin) are about
an order of magnitude higher than the corresponding values for
the protein-free polysaccharide solutions under the same conditions of pH and ionic environment. As discussed in Section 4,
a likely interpretation is enhanced association of casein by electrostatic coupling to gellan.
Fig. 4 shows the viscosities of the three batches of plain doogh
(with no added polysaccharides) in comparison with the corresponding values for preparations incorporating 0.25 wt % HMP (but
no gellan), and with the viscosity of 0.25 wt % HMP alone. Incorporation of pectin in the weakest batch of doogh (Batch 2) raised
the viscosity to a value only slightly above that of HMP alone. For
the strongest batch (Batch 1), the viscosity of the preparation with
added pectin was virtually identical to that of the corresponding
sample of plain doogh (with no pectin). Incorporation of HMP in the
intermediate batch (Batch 3) caused only a slight increase in
viscosity. The overall viscosity therefore seems to be dominated by
pectin when the doogh to which it is added is substantially less
viscous than the pectin alone, with the doogh becoming the
dominant component when it is substantially more viscous than
the pectin.
1000
-1
100
10
1
3
2
10
pectin alone
no gellan
with pectin
1
0.00
749
0.02
0.04
0.06

Fig. 3. Variation of viscosity (1 s1; 20 C) with gellan concentration in preparations
(a) with no added pectin and (b) with 0.25 wt % HMP, for polymer solutions alone (B)
and in combination with doogh from Batches 1 (:), 2 (C) and 3 (6).
10
Viscosity (mPa s) without pectin
100
Fig. 4. Comparison of viscosity (1 s1; 20 C) for doogh samples 1 (:), 2 (C) and 3
(6) with (vertical axis) and without (horizontal axis) 0.25 wt % HMP. The dashed line
corresponds to equal viscosities in the presence and absence of pectin; the horizontal
solid line shows the viscosity of 0.25 wt % HMP alone.
750
Fig. 5. Microstructure of doogh samples (Batch 2) visualised by phase-contrast microscopy. Proteins are stained by Rhodamine B and appear as bright zones in the micrographs.
Magnication is the same in all images (scale bars denote 250 mm). Top row: samples with no added pectin; bottom row: samples incorporating 0.25 wt % HMP; gellan
concentrations (from left to right within each row) of 0, 0.01, 0.03 and 0.05 wt %.
3.2. Microscopy
3.3. Particle-size analysis
Figs. 5 and 6 show micrographs obtained for samples of doogh

(Batch 2) with and without gellan and/or HMP. As would be
expected from the method of preparation, the microstructure of
plain doogh (i.e. with no added polysaccharides) at high magnication (Fig. 6) shows large, irregular fragments of aggregated
casein, separated from one another, and differing widely in size and
shape. At lower magnication (Fig. 5), the particles appear evenly
distributed, and incorporation of 0.25 wt % HMP has no discernable
effect on this homogeneous distribution.
On incorporation of gellan (0.01, 0.03 and 0.05 wt %), with or
without HMP, the distribution becomes less homogeneous, and at
the highest concentration of gellan (0.05 wt %) has the appearance
of a continuous crosslinked network, with the thickness of some
strands approaching w0.3 mm. Even larger assemblies (with
dimensions up to w1 mm) can be seen at the lower concentrations
of gellan (0.01 and 0.03 wt %), but these do not appear to form
a continuous network, and are less evident in the samples that also
include 0.25 wt % HMP.
As shown in Fig. 7, the particles detected in plain doogh by the

Malvern Mastersizer have a broad size-distribution, extending to
w25 mm (comparable to the dimensions of the particles in the
micrograph shown as Fig. 6) and centred at w6e7 mm. A peak at
12
Volume (%)
10
8
6
4
2
0
0.1
10
100
1000
10000
1000
10000
Particle size (m)
12
Volume (%)
10
8
6
4
2
0
0.1
10
100
Particle size (m)
Fig. 6. Microstructure of plain doogh, with no added gellan or pectin (top left-hand
image in Fig. 5), at higher magnication (scale bar denotes 60 mm).
Fig. 7. Particle-size distribution for doogh samples (Batch 2) prepared (a) without
pectin and (b) with 0.25 wt % HMP at gellan concentrations (wt %) of 0 (B), 0.01 (C),
0.03 (6) and 0.05 (:).
roughly the same position (w5 mm) was seen by Parker et al. (1994)
for model milk drinks prepared by 2:1 dilution of commercial
yogurt, and was attributed to fat globules. In view of the very low
content of fat (w0.2%) in the skim milk used to prepare the doogh
samples studied in the present work, however, it seems unlikely
that the peak seen (Fig. 7) for plain doogh can be explained in the
same way.
As was also indicated by microscopy (Fig. 5), incorporation of
0.25 wt % HMP has little effect on particle size (Fig. 7), but
progressive incorporation of gellan shifts the peak from the Mastersizer to progressively larger sizes (which again indicates that the
peak seen for plain doogh cannot be attributed to fat). At the
highest concentration of gellan studied (0.05 wt %) the size distribution extends to w1 mm, comparable to the size of the network
strands visualised by light microscopy (Fig. 5).
The peaks from the Mastersizer, however, show no indication of
the large (w1 mm) assemblies seen by microscopy at gellan
concentrations of 0.01 and 0.03 wt % (particularly in the absence of
HMP). Similar large assemblies were observed by Bourriot, Garnier,
and Doublier (1999) for mixtures of micellar casein with guar gum,
and were attributed to depletion occulation of the casein. The
weak ocs formed by the depletion mechanism are readily disrupted by shear (Bourriot et al., 1999). A likely explanation of the
apparent discrepancy between our evidence from microscopy and
particle-size analysis is therefore that the large assemblies visualised under quiescent conditions (Fig. 5) at 0.01 and 0.03 wt % gellan
arise from depletion occulation of acid-casein particles (Fig. 6) in
response to the presence of gellan, but that the resulting ocs are
broken down by ow through the Mastersizer.
As shown in Fig. 8, the average size (D3,2) of the stable particles
that resist disruption in the Mastersizer increases progressively
with increasing concentration of gellan, but is systematically higher
for the doogh samples incorporating 0.25 wt % HMP than for those
with gellan alone. The values, however, converge at the upper end
of the range of gellan concentrations studied.
3.4. Sensory analysis
Four preparations of doogh from Batch 2 were assessed: plain
doogh, and samples incorporating 0.05 wt % gellan and/or 0.25 wt %
HMP. The samples were rated for aroma, taste, homogeneity,
mouthfeel and overall acceptability, using a 5-point hedonic scale.
751
Fig. 9. Separation of serum from doogh samples (Batch 2) after storage for 15 days at
5 C, without pectin (left-hand frame) and with 0.25 wt % HMP (right-hand frame). The
concentrations of gellan used (from left to right within each frame) were 0, 0.01, 0.03
and 0.05 wt %.
The mean score for homogeneity was lower for doogh incorporating 0.05 wt % gellan alone than for the other three samples.
The panellists detected some slight graininess, which may correspond to the large brillar structures seen by light microscopy
(Fig. 5). However, no signicant differences in aroma, taste or
mouthfeel were observed, and the scores for overall acceptability
were also not signicantly different (p > 0.05).
3.5. Separation of serum on storage
Fig. 9 shows photographs of doogh samples (Batch 2) after
storage in cylindrical tubes for 15 days at 5 C. Little further change
was observed when the storage time was extended to 1 month. All
samples show separation into two layers, but the extent of separation decreases with increasing concentration of gellan, alone or in
combination with 0.25 wt % HMP.
The time-course of separation is shown in Fig. 10. Development
of an upper serum layer and a dense, opaque lower layer occurs
100
Serum volume (ml/100 ml)
50
D3,2 (m)
40
30
with pectin
without pectin
20
80
60
40
20
10
0
0
0
0
0.02
0.04
0.06
Fig. 8. Variation of mean particle diameter with gellan concentration for doogh with
no added pectin (B) and with 0.25 wt % HMP (C).
6
8
10
12
Storage time (days)
14
16
Fig. 10. Separation of serum layer (Fig. 9) from doogh (Batch 2) on storage at 5 C,
alone ()) and in combination with gellan at concentrations (wt %) of 0.01 (circles), 0.03
(triangles) or 0.05 (squares) in the presence (lled symbols) or absence (open symbols)
of 0.25 wt % HMP.
100
a
predominantly within the rst day of storage, with slower resolution at longer times. Throughout the 15 day storage period, the
volume of the serum layer at each concentration of gellan (0.01,
0.03 and 0.05 wt %) is consistently smaller for samples incorporating 0.25 wt % HMP than for those with gellan alone, but the
dominant effect is reduction in serum volume as the gellan
concentration is increased.
The serum volumes observed after storage for 1 day at 5 C are
shown in Fig. 11, plotted against the logarithm of the viscosity (h at
1 s1) of the same samples (obtained, as described in Section 3.1, by
adding 1.0 to the intercepts listed in Table 1). The plot shows good
linearity (r2 0.98), and there is no obvious systematic difference
between samples incorporating HMP and those prepared with
gellan alone. These observations suggest that the initial rapid
increase in serum volume early in the storage period (Fig. 10) may
arise predominantly from sedimentation of aggregated casein, with
the rate of sedimentation decreasing with increasing viscosity.
When the same comparison is made (Fig. 12) after storage for 5
days (Fig. 12a) or 15 days (Fig. 12b), however, there is no such direct
dependence of serum volume on log h, suggesting that the slow,
progressive increases observed (Fig. 10) after the rst day of storage
may involve a different mechanism of separation.
The serum volume measured after 15 days of storage at 5 C for
doogh incorporating 0.25 wt % HMP with no added gellan appears
in Fig. 12b as the rst point on the curve for samples with pectin.
It should be noted that there is no corresponding point in Fig. 11
(storage for 1 day) or Fig. 12a (storage for 5 days). The behaviour
of this preparation was qualitatively different from that of the
samples incorporating gellan (with or without HMP). No separation
was observed during the rst 5 days of storage. After w10 days,
however, it was possible to detect some slight sedimentation of
particles into a dense lower layer, although the supernatant
remained highly turbid. By the end of the 15 day storage period, the
upper (supernatant) layer was still slightly turbid (Fig. 9), but
obviously substantially depleted in casein particles, and occupied
more than 80% of the total volume (Fig. 12b). This behaviour seems
entirely consistent with progressive sedimentation of individual
fragments of acid-casein gel, sterically-stabilised by adsorbed HMP.
Development of a serum layer by samples incorporating gellan
(or by plain doogh), by contrast, resembled expression of uid from
a contracting gel network (i.e. syneresis rather than sedimentation).
100
5 days
80
60
without pectin
40
with pectin
20
0
1
10
100
Viscosity (mP s) at 1 s
1000
-1
100
15 days
752
80
60
without pectin
40
with pectin
20
0
1
10
100
1000
-1
Fig. 12. Correlation between viscosity and amount of serum formed after storage for
(a) 5 days and (b) 15 days at 5 C (Fig. 10) for doogh samples prepared with (C) and
without (B) 0.25 wt % HMP at gellan concentrations of 0, 0.01, 0.03 and 0.05 wt %.
Filled symbols - with pectin

Open symbols - without pectin
80
Throughout the time-course of separation, the upper (serum) layer

was clear, indicating that it contained no signicant amount of
aggregated casein.
60
2
r = 0.98
4. Discussion and conclusions
40
20
0
1
10
100
1000
-1
Fig. 11. Correlation between viscosity and amount of serum formed after storage for 1
day at 5 C (Fig. 10) for doogh samples prepared with (C) and without (B) 0.25 wt %
HMP at gellan concentrations of 0, 0.01, 0.03 and 0.05 wt %.
We propose, tentatively, the following unied interpretation of

our experimental results.
After initial rapid sedimentation of plain doogh (Fig. 10), the
acid-casein particles in the dense lower layer re-associate to give
a crosslinked network which then expresses uid by contracting as
more particleeparticle junctions are formed. In samples incorporating gellan, with no HMP, this structure is augmented by electrostatic attachment of gellan to the surface of the casein particles,
giving a coupled network. The concentration of NaCl in the doogh
samples (0.5 wt % z 85 mM) is sufcient (Milas et al., 1990;
Robinson et al., 1991) to stabilise the ordered (double-helix)
conformation of gellan at temperatures below w35 C. We
therefore envisage three types of junction in samples of doogh with

added gellan: (i) association of acid-casein particles, as proposed
for plain doogh; (ii) gellanecasein associations, giving bridging
between the casein particles, and (iii) tenuous association of gellan
helices, as in weak gels of gellan alone, allowing the coupled
network to ow in response to small stresses, with overall resistance to ow (viscosity) and network contraction (syneresis)
increasing with increasing concentration of gellan. Convergence of
viscosity values for different batches of doogh as the concentration
of gellan is increased (Fig. 3) can be explained by progressive
development of coupled-network structure, swamping batch-tobatch variations in the initial dispersions of fragmented acid-casein
gel. The accompanying increase in the size of particles resistant to
disruption by shear in the Mastersizer (Figs. 7 and 8) can be
attributed to increase in the number of stable electrostatic associations between gellan and acid-casein fragments.
Since gellan has one negative charge per tetrasaccharide repeat
unit, the linear charge density in the double-helix structure is twice
that (i.e. effectively 0.5 per residue). The corresponding value for
individual chains of HMP with DE z 70% is substantially lower
(w0.3 per residue). It seems likely, therefore, that in mixtures of
doogh with HMP and ordered gellan, the gellan component will
bind preferentially to the casein particles, inhibiting complete
surface-coverage by HMP. At low concentrations of gellan, however,
there may still be sufcient exposed surface on the casein fragments to allow HMP to participate in bridging occulation, which
could explain why the values of particle diameter observed (Fig. 8)
for mixtures of doogh with 0.01 and 0.03 wt % gellan are substantially higher when the mixtures also include 0.25 wt % HMP. The
convergence seen (Fig. 8) at the highest concentration of gellan
studied (0.05 wt %) can then be explained by almost complete
exclusion of HMP from the surface of the casein particles.
Irrespective of whether or not the above interpretation is
correct, our results indicate that gellan could be of practical value in
stabilising doogh (and similar acidic milk drinks). Because of its low
pH, doogh is microbiologically stable, and the factor dictating its
shelf-life is physical stability. As described in Section 3.5, 0.25 wt %
HMP abolishes visible separation at storage times up to w10 days at
5 C. After storage for 15 days, however, there is obvious massive
separation into a dense sediment and a much larger and much
clearer upper layer (Fig. 9).
At shorter storage times, incorporation of gellan has a detrimental effect, giving a layer of clear serum on the surface of the
product. In contrast to the progressive sedimentation seen with
HMP as stabiliser, however, there is little increase in the volume of
this serum layer at storage times greater than w5 days (Fig. 10).
Using 0.05 wt % gellan alone, the serum volume after protracted
storage is w12.5%. On incorporation of 0.25 wt % HMP, this value is
reduced (Fig. 10) to w10%, in comparison with over 80% (Fig. 12b)
for the same concentration of HMP alone. Gellan might therefore be
useful in extending the shelf-life of doogh, without seriously
compromising acceptability to the consumer.
In preparing the experimental samples studied in the present
work, polysaccharides were incorporated (Section 2.2) after the
doogh had been homogenised. In commercial production of acidic
milk drinks stabilised by HMP, however, it is more usual to add the
polysaccharide before homogenisation, to stabilise acid-casein
fragments as they are formed. This has the advantage of blocking
re-aggregation, so that the total surface area available for attachment of polysaccharide chains increases progressively throughout
the homogenisation process (Parker et al., 1994), rather than
reaching an equilibrium between fragmentation and re-aggregation. It seems likely, therefore, that even greater extension of shelflife could be achieved by applying the same procedure to doogh
with gellan as stabiliser, alone or in combination with HMP.
753
Acknowledgements
We thank Professor D.M. Mulvihill (University College Cork) and
Dr. C. Rolin (CP Kelco) for helpful discussions. We also thank Dr. Z.
Emam-Djomeh (University of Tehran) for her help in the investigation, and ZamZam Iran Co. for nancial and technical support.
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Food Hydrocolloids 24 (2010) 744e754

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