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LWT - Food Science and Technology 41 (2008) 1847e1856

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Structural and textural characteristics of reduced-fat cheese-like

products made from W1/O/W2 emulsions and skim milk
C. Lobato-Calleros a, A. Sosa-Perez a, J. Rodrguez-Tafoya a, O. Sandoval-Castilla b,
C. Perez-Alonso c, E.J. Vernon-Carter b,*
a
Departamento de Preparatoria Agrcola, Universidad Autonoma Chapingo, Km 38.5 Carretera Mexico-Texcoco, 56230 Texcoco, Mexico
b
Area de Ingeniera Qumica, Universidad Autonoma Metropolitana-Iztapalapa, San Rafael Atlixco 186, Col. Vicentina,
09340 Mexico, D.F., Mexico
c
Facultad de Qumica, Universidad Autonoma del estado de Mexico, Paseo Colon esq. Paseo Tollocan s/n, Col. Residencial Colon,
50120 Toluca, Estado de Mexico, Mexico
Received 9 September 2007; received in revised form 9 January 2008; accepted 13 January 2008

Abstract

The chemical composition, yield, structural arrangement, instrumental textural characteristics, and preference sensory evaluation of reduced-
fat cheese-like products manufactured from skim milk and different water-in-oil-in-water (W1/O/W2) emulsions were determined. A full-fat
white fresh cheese (WFC) was prepared from milk containing 27 g of milk-fat L1, and five reduced-fat white fresh cheese-like products
(EC) were made from skim milk added with 25 g of multiple emulsions L1 containing canola oil and stabilized/emulsified by amidated
low-methoxyl pectin (LMP), carboxymethylcellulose (CMC), gum Arabic (GA), and blends of GAeCMC or GAeLMP. The chemical compo-
sition, yield, structural arrangement and texture of the cheese-like products were affected by the biopolymers used as emulsifying/stabilizing
agents of the multiple emulsions. CMC produced an EC with similar textural behaviour than the WFC cheese. GA contributed to a higher yield
and fat content in the EC cheese in comparison with CMC and LMP cheese. GA and LMP contributed to increased values of hardness and
chewiness of the EC cheese. The cheese made with multiple emulsions incorporating GA and LMP emulated best the textural characteristics
of the WFC cheese. All of the EC cheese showed marked differences in microstructure.
2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.

Keywords: Cheese-like products; Textural characteristics; Microstructure; Multiple emulsions; Biopolymers

1. Introduction properties. The response of the cheese to external forces can

The development of cheese-like products containing emul-
sified vegetable oils in substitution of saturated milk-fat can
contribute to improve consumers health (Yu & Hammond,
2000).
Cheese is a complex, multiphase, multicomponent colloidal
system, whose microstructure arises from a balance of cooper-
ative and competitive molecular interactions between its
chemical components, and gives the cheese its characteristic
* Corresponding author. Tel.: 52 55 8044648; fax: 52 55 8044900.
E-mail address: jvc@xanum.uam.mx (E.J. Vernon-Carter).
reveal structural features directly related to texture (Bhaskara-
charya, Friedhuber, & Shah, 1998; Kalab, Allan-Wotjas, &
Shea Miller, 1995). Any changes in the composition will result
in different structural arrangements and different textural char-
acteristics (Bryant, Ustunol, & Steffe, 1995; Lobato-Calleros,
Ramrez-Santiago, Osorio-Santiago, & Vernon-Carter, 2002).
Consumers are demanding low- and reduced-fat products,
with a healthy saturatedeunsaturated fat balance, in order to
reduce the risk factor for heart disease, which is now the
worlds leading cause of death (Lobato-Calleros, Rodrguez,
Sandoval-Castilla, Vernon-Carter, & Alvarez-Ramirez, 2006).
In general terms, low- and reduced-fat cheese exhibit poor
texture (Drake, Boylston, & Swanson, 1996), due to significant

0023-6438/$34.00 2008 Swiss Society of Food Science and Technology. Published by Elsevier Ltd. All rights reserved.
doi:10.1016/j.lwt.2008.01.006
1848 C. Lobato-Calleros et al. / LWT - Food Science and Technology 41 (2008) 1847e1856
changes occurring in the protein structural mesh (Gunasekaran
& Ding, 1999). The textural characteristics of the cheese are de-
termined by the combined structural properties of the protein
matrix and the fat droplets immersed in the former (Lobato-
Calleros et al., 2007). As fat content is reduced, more non-
interrupted protein zones compose the cheese structure. In
consequence, a high degree of cross-linking of protein mole-
cules occurs resulting in three-dimensional networks exhibiting
high resistance to the deformation (Lobato-Calleros et al.,
2006). When formulating low- and reduced-fat cheese one
must seek formulation ingredients that interact or embed in
the protein matrix avoiding its aggregation. The success of
the different ingredients used as milk-fat replacers depends
on one or more of the following abilities: (1) water binding
capacity, (2) potential for developing fat-like characteristics,
(3) capacity to interact with other ingredients, and (4) capacity
to modify the protein structural arrangement and thus mechan-
ical response to external factors (McMahon, Alleyne, Fife, &
Oberg, 1996; Tamime, Muir, Shenana, Kalab, & Dawood,
1999). In this way, it is desirable to secure optimal functionality
from each component in order to achieve the wished properties,
including texture (Dickinson & Stainsby, 1987).
In both natural and processed foods, fats are commonly
found as oil-in-water or water-in-oil emulsions (McClements,
1999), and more recently, water-in-oil-in-water (W1/O/W2)
multiple emulsions have been proposed as being suitable for
developing reduced-fat food products (Lobato-Calleros
et al., 2006), as these systems comprise an inner aqueous
phase that effectively reduce the fat mass fraction occurring
in an equivalent O/W emulsion. Morphology of W1/O/W2
emulsions deviates greatly from that of O/W emulsions, in
that they exhibit very small inner water droplets encased in
much larger oil droplets. The size and distribution of the
W1/O/W2 droplets is affected greatly by the choice of external
aqueous phase emulsifier/stabilizer employed. Both, droplet
size and the emulsifier/stabilizer utilized may modify the
rheological and textural properties of the cheese through their
interactions with the milk proteins (Lobato-Calleros et al.,
2006).
The aim of this work was to determine the chemical
composition, yield, microstructure, instrumental texture, and
preference sensory evaluation by consumers of reduced-fat
cheese-like products made from W1/O/W2 emulsions and
skim milk in comparison with a full-fat white fresh cheese.
2. Materials and methods
2.1. Materials
The hydrophilic emulsifier (WE) (Panodan SDK Datem,
esters of monoglycerides and diglycerides of diacetyltartaric
acid) and hydrophobic emulsifier (OE) (Grinsted PGPR 90,
esters of polyglycerol and polyricinoleate fatty acids) were
purchased from Danisco Mexico, S.A. de C.V. Canola oil
(CO) (Capullo, Unilever de Mexico, S.A. de C.V.) was
used as the oil phase of the W1/O/W2 emulsion. The biopoly-
mers used were gellan gum (GG) (K3B408, CP Kelco, S.A. de
C.V., Mexico), gum Arabic (GA) (Acacia senegal, Industrias
Ragar, S.A. de C.V., Mexico), carboxymethylcellulose
(CMC) (Cerol 50000, Grupo Dermet, S.A. de C.V., Mexico),
and amidated low-methoxyl pectin (LMP) (Genu pectin
LM-101 AS, CP Kelco, S.A. de C.V., Mexico). The water
used in all of the experiments was double-distilled.
2.2. Preparation of the W1/O/W2 emulsions
W1/O/W2 emulsions were prepared at room temperature
using a two-stage emulsification procedure (Lobato-Calleros
et al., 2006). In the first stage, a W1/O emulsion having 20 g/
100 g of dispersed aqueous phase (W1) was prepared. W1, con-
taining GG (0.1 wt%), and WE emulsifier (1.6 g/100 g) was
added drop-wise to the oil phase (O) made up by CO added
with OE emulsifier (6.4 g/100 g) using a high shear Silverson
L4R homogenizer (Silverson Machines, Ltd., Waterside, Che-
sham, Buckinghamshire, England) at 5800 rpm for 5 min. In
the second stage this primary W1/O emulsion (20 g/100 g)
was re-emulsified at 5200 rpm for 10 min using the Silverson
homogenizer into five different biopolymers aqueous solutions
(80 g/100 g): GA (10 wt%), CMC (0.5 g/100 g), LMP (2 g/
100 g), GA CMC (10 g/100 g 0.5 g/100 g), GA LMP
(10 g/100 g 2 g/100 g). The W1/O/W2 emulsions were
prepared by triplicate and codified as: ECGA, ECCMC, ECLMP,
ECGAeCMC, and ECGAeLMP, respectively.
The numberelength mean diameter (D1,0) of the W1/O/W2
emulsions and of the whole milk was determined using a Mal-
vern particle size analyzer series 2600 (Malvern Instruments,
Malvern, Worcestershire, UK). D1,0 of inner aqueous droplets
of the multiple emulsions was determined using an optical
microscope (Olympus BX45, Olympus Optical Co., Tokyo,
Japan) coupled to an image analyzer system (digital Olympus
camera C3030, Olympus America Inc., USA and Image Pro-
Plus version 4.5 software, Media Cybernetics, Inc., L.P., Silver
Spring, USA). Analysis was carried out within 1 h of initial
preparation.
2.3. Cheese-making process
A control white fresh cheese (WFC) was prepared from
milk containing 27  0.1 g of milk-fat L1 by blending whole
milk (38  0.1 g of milk-fat L1) with skim milk (0.1  0.05 g
of milk-fat L1). Five reduced-fat white fresh cheese-like
products (EC) were elaborated from skim milk added with
27 g of the W1/O/W2 emulsions L1, and were given the
same codes as those of the W1/O/W2 emulsions from which
they were prepared. Cheese were prepared from 15 L batches
of formulated milk by triplicate in a completely randomized
design. The formulated milks were vat-pasteurized at
63  0.1  C for 30 min, cooled to 36  0.5  C, and added
with 1.5 mL L1 of a calcium chloride solution at 10 g/100 g
and 0.25 mL L1 of microbial rennet (M. miehei, strength
1:10,000, Industrias Cuamex, S.A. de C.V., Mexico). After
coagulation time of approximately 30 min, the curd was cut
into 1 cm3 cubes. About 30% of the whey was drained, and
salting was carried out by adding 6.3 g of table salt L1 of
 C. Lobato-Calleros et al. / LWT - Food Science and Technology 41 (2008) 1847e1856
milk. The curds were transferred to 1 kg round polyvinylchlor-
ide containers, kept at room temperature (20  2  C) for 2 h,
and then placed in a cooling chamber (10  0.5  C, 85e90%
RH), for 24 h. Cheese were stored at 4  C.
After 3 days of preparation, the cheese were analyzed for
protein by the Kjeldahl method, fat by the Gerber method,
pH on warm macerates and moisture by oven drying
(AOAC, 1995), by triplicate. Composition results were
reported in wet basis.
2.4. Microstructure determination
Scanning electron microscopy (SEM) was performed on the
cheese after 3 days storage using cylinder samples 0.5 cm in
diameter by 0.5 cm in height that were fixed in 2.5 g/100 g
buffered glutaraldehyde (0.1 M phosphate buffer, pH 7.2) as
fixative to crosslink proteins at room temperature for 6 h.
The fixed samples were washed with phosphate buffer and
dehydrated a graded ethanol series (30, 50, 70, 80, 90 and
100 g/100 g, 30 min in each) and placed in acetone for 1 h
(Lobato-Calleros et al., 2002). Samples were critical point
dried in a CPA II Technics Critical Point Dryer (Tousimis,
Rockville, MD). Each sample was fragmented before mount-
ing on SEM stubs to reveal their internal microstructure and
coated with a thin layer of gold (Kalab, 1979) in a Fine
Coat Ion Sputter JFC 1100 (Jeol Ltd., Akishima, Japan).
A high vacuum JEOL Scanning Electron Microscope JMS-
6360LY (Jeol Ltd., Akishima, Japan), at 20 kV, was used to
view each sample at magnification of 500 and 3000. Rep-
resentative micrographs were selected for presentation.
Digitalized SEM micrographs (3000) were analyzed with
an Image Pro-Plus software version 4.5. The size of actual fat
droplets or of the voids they occupied was reported as the
numberelength mean diameter of an equivalent sphere (Agui-
lera & Stanley, 1999; Rawl, 2007) because frequently SEM
micrographs show uneven surface topography. Fat globules
or fat voids perimeters were manually highlighted (Crites,
Drake, & Swanson, 1997), and micrographs size bar was
used as reference scale (Kamath, Morr, & Schenz, 1998).
2.5. Instrumental texture characteristics
Texture profile analysis (TPA; Bourne, 2002) was per-
formed at room temperature (20  2  C) on cylinders, 1 cm
in diameter by 1 cm in height, after 3 and 15 days of cheese
manufacture, using a Stable Micro Systems Texturometer
model TA-XT2i (Texture Technologies Corp., White Plains,
NY, USA). Each sample was cut from the central part of
each cheese piece using a borer and a sharp knife, and held
at 20  2  C in closed glass containers. In all cases the sam-
ples were compressed by 50%, using two compression cycles
at a constant crosshead velocity of 2 mm s1. Mechanical pri-
mary characteristics of hardness, springiness and cohesiveness
were selected from the two successive uniaxial compression
cycles of TPA. Also the secondary characteristic of chewiness
(hardness  cohesiveness  springiness) was determined. Fif-
teen measurements were carried out on each cheese variation.
1849
2.6. Sensory evaluation
After 3 and 15 days of storage, the cheese were evaluated
by a consumer panel of 80 (32 males and 48 females) recruited
among faculty, staff and students of the Universidad Auton-
oma Chapingo (Texcoco, State of Mexico) who stated that
they were cheese lovers and users.
Each of the six cheese prepared in this work was cut in
1 cm3 cubes samples, were coded with three-digit random
numbers, and randomly presented to the panel. The con-
sumers preference for the cheese was scored on a five-point
hedonic scale (1 dislike extremely; 2 dislike moderately;
3 neither like or dislike; 4 like moderately; 5 like
extremely) (Farkye, Prasad, Rossi, & Noyes, 1995).
2.7. Data analysis
Simple classification variance analysis was applied and
whenever it was adequate Tukeys test was used in order to
determine differences between the composition, textural and
sensory data of the cheese. The significance was established
at p  0.05. Data analysis was done using Statgraphics Plus
software (Statistical Graphics Corp., Manugistics, Inc., Cam-
bridge, MA).
3. Results and discussion
3.1. Droplet diameter of the multiple emulsions
All of the multiple emulsions were made up by spherical oil
droplets containing within them a large number of water drop-
lets (0.1  0.0 mm) (Fig. 1). The multiple emulsion morphol-
ogy showed in Fig. 1 is classified as type C (Garti, 1997).
The D1,0 of the EC emulsions was affected by the type of bio-
polymer(s) used as stabilizers in the external aqueous phase
(W2). D1,0 varied from 2.5 to 5.5 mm (Table 1). D1,0 of the
ECCMC emulsion was not-significantly different from that of
milk-fat globules in fluid milk used to prepare the WFC
Fig. 1. Morphology of the multiple emulsion droplets used for manufacturing
the low-methoxyl pectin cheese (ECLMP).
1850 C. Lobato-Calleros et al. / LWT - Food Science and Technology 41 (2008) 1847e1856
Table 1
Chemical composition, numberelength mean diameter of the fat droplets in the full-fat white fresh cheese (WFC) and reduced-fat cheese-like products (EC), and
of the fresh W/O/W emulsions or fat in milk used in the cheese formulation (mean  SD, n 9)
Cheese D1,0a of milk or W/O/W Fatb (g/100 g) Proteinb (g/100 g)
code emulsions (mm)
WFC 2.6  0.0a 15.3  0.8d 17.0  0.4ab
ECGA 5.5  0.1d 13.0  0.0c 17.6  0.5bc
ECCMC 2.5  0.2a 11.3  0.3a 18.9  0.4d
ECLMP 4.4  0.3c 11.5  0.5ab 18.2  0.5cd
ECGAeCMC 3.7  0.0b 12.3  0.6abc 16.0  0.3a
ECGAeLMP 3.7  0.3b 12.8  0.3bc 16.8  0.2ab
GA: gum Arabic; CMC: carboxymethylcellulose; LMP: amidated low-methoxyl pectin; D1,0: mean arithmetic diameter of milk-fat globules W1/O/W2 emulsion
droplets used in the manufacture of cheese; Deq: mean equivalent diameter of the fat droplets in the cheese; a,b,c,d,e: different superscripts within the same column
indicate that the means differ significantly ( p  0.05).
a
Determined after 1 h of manufacture.
b
Determined after 3 days of manufacture.
cheese. The D1,0 values of the rest of the EC emulsions (ECGA,
ECLMP, ECGAeCMC, and ECGAeLMP) were significantly higher
( p  0.05) than those of the milk-fat globules and the ECCMC
emulsion. GA contributed to the largest emulsion droplets for-
mation, followed by LMP and CMC, respectively. Blending of
GA with CMC or LMP produced emulsion droplets which
were non-significantly different ( p > 0.05) between them-
selves, but were intermediate in size and significantly different
( p  0.05) to those observed for ECMC and ECLMP emulsions.
The effect of CMC on the ECMC emulsion droplet size is
probably due the formation of a barrier of hydrated macromol-
ecules around each emulsion droplet (Coffey, Bell, & Hender-
son, 1995) which retards collision and aggregation of the
droplets, i.e., the action of CMC is that of a stabilizing agent
rather than emulsifying agent. Furthermore, CMC acts at rel-
atively low concentrations as an effective macromolecular
thickening agent, increasing the viscosity of the aqueous phase
and modifying its rheology (Coffey et al., 1995), with the lat-
ter factor controlling the movement of dispersed emulsion
droplets and impeding incipient droplet coalescence (Lobato-
Calleros et al., 2007). LMP formed coarser emulsion droplets
than CMC. The role of pectin as emulsifier and/or stabilizer is
yet not clearly understood. Leroux, Langendorff, Schick,
Vaishnav, and Mazoyer (2003) reported that only a limited
quantity of pectin is adsorbed on the oil surface (attributed
to protein residues present within the pectin molecules) with
the rest remaining in the bulk phase. In contrast, Dickinson
(2003) mentioned that commercial pectin extracted from citrus
peel or apple pomace is not effective as emulsifying agent
irrespective of its degree of esterification. Pectin dispersions
have relatively low viscosities compared with other biopoly-
mers (Voragen, Pilnik, Thibault, Axelos, & Renard, 1995),
among them CMC. GA is a commercially important emulsify-
ing agent that is unique in that it can be used at very high con-
centrations exhibiting very low viscosity. Its emulsifying
ability is due to a small amount of protein which is covalently
bound to a highly branched polysaccharide structure. GA
forms strong viscoelastic films around oil droplets providing
stability to against oil droplet coalescence through strong
steric repulsion forces (Garti & Reichman, 1993). GA has
a very high molecular weight so that its diffusion to the newly
Yieldb (g/100 g) Moisture after 3 days (moisture Deqb of fat droplets
after 15 days) (g/100 g) in cheese (mm)
17.8  0.1d 60.4  0.1a (53.3  0.1a) 3.7  0.3b
15.1  0.2b 62.8  0.1c (58.0  0.2d) 6.9  0.2e
13.6  0.1a 60.2  0.2a (53.5  0.1a) 3.2  0.2a
13.1  0.1a 63.2  0.1d (56.6  0.1c) 5.5  0.1d
15.8  0.2c 61.9  0.1b (55.4  0.1b) 4.5  0.1c
14.8  0.1b 63.9  0.2e (57.2  0.3b) 4.8  0.1c
formed interface during the emulsifying process is relatively
slow, which coupled with the fact that only a small fraction
of the gum contained in the continuous phase is adsorbed at
the interface, results in relatively large droplet sizes formation,
unless very high concentrations of the gum are employed in
the emulsification process (Phillips & Williams, 2000). It is
logical to assume that when using GA blends with CMC or
LMP both emulsifying and stabilizing mechanisms are
involved.
3.2. Cheese composition
The WFC cheese had a fat content that was significantly
higher ( p  0.05) than that of the EC cheese (Table 1), which
it is expected, as it was elaborated from milk standardized to
27 g L1, whereas the EC cheese were made from skim milk
(0.1 g L1) added with 27 g L1 of multiple emulsion contain-
ing 16.2 g of canola oil L1. The fat content in the EC cheese
varied depending on the biopolymer used in the respective
multiple emulsion preparation (Table 1). The ECGA cheese
had a higher fat content ( p  0.05) than ECLMP and ECCMC
cheese, but similar fat content ( p > 0.05) to the ECGAeLMP
and ECGAeCMC cheese. This seems to indicate that when the
emulsion stabilizing mechanism was due mainly to the forma-
tion of an adsorbed biopolymer layer around the oil droplets,
these were more efficiently retained in the protein matrix
because of the interactions occurring between the adsorbed
biopolymer molecules and the protein chains of the cheese
network (Lobato-Calleros et al., 2006). WFC cheese had sig-
nificantly lower protein content ( p  0.05) than the ECLMP
and ECCMC cheese, but was non-significantly different
( p > 0.05) the ECGA, ECGAeLMP and ECGAeCMC cheese
(Table 1). Protein content in the cheese was inversely propor-
tional to their fat content. Thus the ECLMP and ECCMC cheese
had significant higher ( p  0.05) protein content than the rest
of the EC cheese. The moisture content of WFC cheese was
60.4 g/100 g after 3 days of manufactured, while that of the
EC cheese varied between 60.2e63.9 g/100 g. All of the
cheese showed a substantial decrease in moisture content
(7.64e11.75 g/100 g) with storage time. Moisture loss during
the 15-day period was less for all the EC cheese than for the
 C. Lobato-Calleros et al. / LWT - Food Science and Technology 41 (2008) 1847e1856
WFC cheese (Table 1), which can be explained due to the
water holding ability of the hydrocolloids (Clark, 1994).
The WFC and EC cheese that had higher fat contents ex-
hibited higher yields (Table 1). Milk-fat, one of the major com-
ponents in milk, is trapped in the casein matrix during cheese
making, so it is not surprising that the cheese yield decreased
as the fat content of the milk was reduced, and so did the total
solids content. These results are in accordance to those reported
for cheese-like products prepared from skim milk added with
O/W emulsions (Lobato-Calleros et al., 2007).
3.3. Cheese microstructure
Electron micrographs of the cheese showed different micro-
structure arrangements (Figs. 2e7). The progressive aggrega-
tion of the casein throughout cheese making to form a matrix
containing entrapped fat droplets was affected by substituting
the milk-fat globules by multiple emulsion droplets. The milk-
fat globules in the WFC cheese are stabilized by natural sur-
factants e the milk proteins e while in the EC cheese the
emulsion droplets were stabilized by different hydrocolloids
or their blends. Thus, different structural arrangements took
place in the EC cheese compared to that of the WFC cheese.
The formulated milks made with different hydrocolloids ex-
hibited different emulsion droplet sizes, which on turn affected
their spatial distribution and interactions with the protein ma-
trix, and eventually, cheese microstructure (Lobato-Calleros
et al., 2007). At this point it is important to mention that the
D1,0 droplet sizes observed in the cheese were slightly higher
than to those found in the formulated milks (Table 1), so that it
may be inferred that the W1/O/W2 emulsions maintained a rel-
atively high stability against droplet coalescence during the
cheese-making process. The microstructure of the WFC
cheese consisted of a relatively compact structure (Fig. 2a)
interrupted by numerous milk-fat globules of various sizes,
as indicated by the void spaces that were originally occupied
by the milk-fat globules (Fig. 2b). The ECCMC cheese was
characterized by a highly interrupted protein network, where
the void spaces were very small and uniform (Fig. 3a and
b), and were smaller than those exhibited by the WFC and
the rest of the EC cheese (Table 1). Embedded emulsion drop-
lets into the protein matrix can be observed for the ECCMC
a b
Fig. 2. SEM micrographs of the protein network (p) of the milk-fat control cheese (WFC) at low (a) and high (b) magnifications.
1851
cheese (Fig. 3c and d). Although in this work the SEM micros-
copy samples were not fixed in OsO4, we hypothesize that
probably CMCecasein interactions (Coffey et al., 1995)
took place, and that some of these complexes were fixed dur-
ing the sample preparation with glutaraldehyde (Lobato-Call-
eros et al., 2007). The ECGA cheese showed the least
interrupted protein network containing the largest emulsion
droplets sizes (Table 1), providing a lace-like appearance
(Fig. 4a and b). The casein matrix of ECGA cheese was coarser
than that of WFC and ECMC cheese (Figs. 2a and 3a) as more
numerous links among casein strands were formed on the pro-
tein dominating areas. Some relatively large and other smaller
spherical emulsion droplets covered by presumably casein
strands can be observed (Fig. 4b and c). These findings indi-
cate that the hydrophobic polypeptide chains of gum Arabic
adsorbed at the oil droplets surface (Phillips & Williams,
2000) could interact with casein strands of the protein matrix.
ECLMP cheese was characterized by a granular protein matrix,
containing amorphous and slightly elongated shaped structures
(Fig. 5a and b), probably composed by calcium pectate aggre-
gates. The surface of these amorphous structures was distinc-
tively smooth as compared with that granular texture of the
protein matrix. Furthermore, emulsion droplets that exhibited
variable sizes appeared within the amorphous structures
(Fig. 5c and d). Most polysaccharides are added to emulsions
to modify the properties of the continuous phase (Gancz,
Alexander, & Corredig, 2003) although a few have the ability
to form insoluble gel structures. LMP forms insoluble com-
plexes with calcium ions present in milk (Lobato-Calleros,
Robles-Martnez, Caballero-Perez, Aguirre-Mandujano, &
Vernon-Carter, 2001) by bridging carboxyl groups between
adjacent pectin chains (Leroux et al., 2003), creating junction
zones that entrapped emulsion droplets. When GA was
blended with CMC or LMP spherical oil droplets with inter-
mediate sizes to those produced by the individual polysaccha-
rides were observed (Table 1). Thus, the ECGAeCMC cheese
structure showed larger casein inter-chain voids (Fig. 6a and
b) than the ECCMC cheese (Fig. 3a and b), while the ECGAe
LMP cheese structure was finer (Fig. 6c and d) than that of
the ECGA cheese (Fig. 4a and b). An emulsion droplet in the
ECGAeCMC cheese (Fig. 7a) can be seen linked to the protein
network and covered by a surface layer conformed by smooth
p
1852 C. Lobato-Calleros et al. / LWT - Food Science and Technology 41 (2008) 1847e1856

o
p

a b

p
o p

o
o o

p
c d
Fig. 3. SEM micrographs of the carboxymethylcellulose cheese (ECCMC) at low (a) and high (bed) magnifications. Some oil droplets (o) embedded in the protein
matrix (p) can be observed coated with a smooth and homogenous film presumably composed by CMC-protein.

o o

p
o

o o
a b

c
Fig. 4. SEM micrographs of the gum Arabic cheese (ECGA) at low (a) and high (b and c) magnifications. Some oil droplets (o) integrated to the protein matrix (p)
and enveloped fused casein micelles can be observed.
 C. Lobato-Calleros et al. / LWT - Food Science and Technology 41 (2008) 1847e1856 1853

p
p
p
p

a b

p
p
p

c d
Fig. 5. SEM micrographs of the low-methoxyl pectin cheese (ECLMP) at low (a) and high (bed) magnifications. Void spaces that were originally occupied by oil
droplets can be observed within ovoid structures of calcium pectate (pe), which on turn were dispersed into the protein matrix (p).

p
o
o p

a b

o
p

c d
Fig. 6. SEM micrographs of the gum Arabicecarboxymethylcellulose cheese (ECGAeCMC) at low (a) and high (b) magnifications, and gum Arabicelow-methoxyl
pectin cheese (ECGAeLMP) at low (c) and high (d) magnifications. Spherical oil droplets (o) were individually dispersed into the cheese protein matrix (p).
1854 C. Lobato-Calleros et al. / LWT - Food Science and Technology 41 (2008) 1847e1856

o
o

p
o
a b
Fig. 7. SEM micrographs of oil droplets (o) imbedded into the protein matrix (p) of the gum Arabicecarboxymethylcellulose (ECGAeCMC) (a) and gum
Arabicelow-methoxyl pectin (ECGAeLMP) (b) cheese.

areas (presumably formed by whey proteins and casein sub-
units) and granular structures of casein micelles. In contrast
an emulsion droplet of ECGAeLMP cheese (Fig. 7b) was
immersed in the protein matrix and it was covered by a pre-
dominantly smooth layer.
3.4. Cheese instrumental textural characteristics
The variation in textural characteristics of the WFC and EC
cheese with storage time are shown in Fig. 8. After 3 days of
storage the WFC cheese exhibited lower hardness than the
ECLMP and ECGAeCMC cheese ( p  0.05) but was non-signifi-
cantly different ( p > 0.05) from the rest of the EC cheese.
Chewiness and springiness of the EC were non-significantly
different ( p > 0.05) from that of the WFC cheese. The only
EC cheese that showed significantly lower cohesiveness
( p  0.05) from the WFC cheese was the ECGA cheese. It has
been reported that milk-fat globules substitution by oil-in-water
emulsion droplets in cheese-like products produced cheese with
lower hardness, springiness and chewiness, but with higher
cohesiveness (Lobato-Calleros et al., 2002). These authors
claimed that the low melting point and the more numerous ca-
nola oil droplets in comparison to the fewer and relatively high
melting point of milk-fat globules were responsible for the cre-
ation of a less dense and weaker protein matrix. In this work the
emulsion droplets were composed by canola oil but also con-
tained biopolymers in the external aqueous phase (W2), which
in general terms reinforced the protein matrix of the EC cheese.
As storage time increased the hardness and chewiness
increased substantially (Fig. 8), whereas the springiness and

a 10 b 4,7

8
4,3
mm

6
N

3,9
4

2 3,5
P C GA P C C P C GA P C C
A-L
M -CM LM CM WF -LM -CM LM CM WF
G GA GA GA

c 105 d 3,3
dimensionless

85
Nmm

65 2,9

45

25 2,5
P C GA P C C P C GA P C C
A-L
M
A-C
M LM CM WF -LM -CM LM CM WF
G G GA GA

Fig. 8. Texture characteristics of full-fat white fresh cheese (WFC) and reduced-fat cheese-like products (EC) after 3 and 15 days of manufacture: hardness (a),
springiness (b), chewiness (c), and cohesiveness (d).
 C. Lobato-Calleros et al. / LWT - Food Science and Technology 41 (2008) 1847e1856 1855

cohesiveness remain fairly constant in all of the cheese. The Table 2

changes in hardness and chewiness are probably due to the Preference evaluation results (mean  SD, n 80) of the full-fat white fresh
cheese (WFC) and reduced-fat cheese-like products (EC) after 3 and 15
decrease in the moisture content of the cheese (Fig. 8). It is days of manufacture
generally established that the greater the moisture content,
Cheese code Mean overall preference Mean overall preference
the softer the cheese (Gunasekaran & Ak, 2003). In our cheese (3 days) (15 days)
the hardening effect due to moisture loss is greater than the
WFC 3.7a (1.0) 3.2b (0.7)
softening effect of proteolysis. Singh and Verma (1996) ECGA 3.2a (0.9) 2.6ab (0.7)
reported that the hardness of cheddar cheese increased up to ECCMC 3.0a (0.7) 2.2a (0.4)
120 days of ripening, due to moisture losses, and slow lipoly- ECLMP 3.9a (1.1) 3.1ab (0.9)
sis and proteolysis. ECGAeCMC 2.9a (0.9) 2.3ab (0.6)
Differences in the textural behaviour among the EC cheese ECGAeLMP 3.2a (0.9) 2.7ab (0.7)
with time can be attributed to the effect of the biopolymers on Different superscripts within the same column indicate that the means differ
the size and distribution of the emulsion droplets (Table 1) in significantly ( p  0.05). 1 dislike extremely; 5 like extremely; GA: gum
Arabic; CMC: carboxymethylcellulose; LMP: amidated low-methoxyl pectin.
the protein matrix and the interactions taking place among the
biopolymers molecules and cheese components (i.e., milk pro-
teins and water). For example in the case of the ECLMP cheese, grinding sensation that could possibly be related to the low
the highest hardness and chewiness can probably be associated values of hardness and chewiness and high values of springi-
to the formation of relatively large aggregates of calcium pec- ness that it exhibited (Fig. 8) at the end of the storage period.
tate (Fig. 5aed). So it seems that the stress resistance of the
protein matrix was improved due to this factor. 4. Conclusions
The lowest cohesiveness ( p > 0.05) of the ECGA, indicated
that this cheese had a lesser capacity to deform before ruptur- The textural and microstructure characteristics of white
ing, probably due to the openness of the cheese structure fresh cheese were altered by the substitution of the milk fat
(Fig. 4a and b). Compact cheese tend to break less easily (Bry- globules by multiple emulsion droplets. The emulsifiers/stabi-
ant et al., 1995). lizers used in the formulation of the multiple emulsions af-
While all of textural characteristics of the ECCMC cheese fected the size of the fat droplets, which in turn spatially
were non-significantly different ( p > 0.05) to those of the distributed differently within the protein matrix, giving rise
WFC cheese after 3 days, the textural characteristics of hard- to different microstructure and textural characteristics. In
ness, cohesiveness and chewiness showed significant differ- this work, it was established that reduced-fat cheese-like prod-
ences ( p  0.05) to those of the WFC cheese after 15 days ucts manufactured from skim milk and water-in-oil-in-water
(Fig. 8). The ECCMC cheese showed less variation in its tex- emulsions stabilized with hydrocolloids closely emulated
tural characteristics with storage time than the WFC cheese some of the textural characteristics of its full milk-fat counter-
(Fig. 8), so that this relatively long-term stability seems to part, despite their completely different structural arrange-
be closely related to the functional properties of the non- ments. Additionally, reduced-fat cheese-like products had
adsorbing CMC at the oilewater interface. similar preference scores than that corresponding to its full
When GA was blended with CMC, the textural characteris- milk-fat counterpart.
tic of hardness tended to increase significantly ( p  0.05)
compared to the ECCMC cheese after 15 days of storage. Acknowledgements
When GA was blended with LMP the cohesiveness of the
ECGAeLMP cheese was significantly higher, but the chewiness The authors thank the Consejo Nacional de Ciencia y Tec-
was significantly lower ( p  0.05) than that of the ECLMP nologa (CONACyT) for grants 52317-Z and U45992-Z, and
cheese. The overall behaviour of the polysaccharides blends to Biol. Yolanda Hornelas-Uribe of the Instituto de Ciencias
is determined by a balance between competing thermody- del Mar y Limnologa of the Universidad Nacional Autonoma
namic and rheological factors that are beyond the scope of de Mexico for the expertise provided for obtaining the SEM
this study. micrographs.
3.5. Preference sensory evaluation
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