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International Dairy Journal 16 (2006) 910919
Partial replacement of fat by functional fibre in imitation cheese:
Effects on rheology and microstructure
Clara Montesinos-Herreroa, David C. Cottellb, E. Dolores ORiordana,, Michael OSullivana
aDepartment of Food Science, University College Dublin, Belfield, Dublin 4, IrelandbElectron Microscopy Laboratory, University College Dublin, Belfield, Dublin 4, Ireland
Received 18 May 2005; accepted 11 August 2005
Abstract
The rheology and microstructure of a control imitation cheese were compared with cheeses containing Novelose240 (N240, native
resistant starch) or Novelose330 (N330, retrograded resistant starch), as a source of fibre to replace fat. Hardness increased linearly with
fibre content and to a greater extent for N330 than for N240. Cohesiveness increased linearly with N240 content but was not influenced
by N330. The elastic modulus (G0) and the viscous modulus (G00) increased with increasing contents of both fibres. The crossover
temperature (G0 G00) was unaffected by N240, but was increased by N330. Over 50% of the fat content of imitation cheese was replaced
with resistant starches without impacting on meltability. The microstructure of imitation cheese was observed by scanning electron
microscopy and light microscopy. The latter, a cheaper and simpler technique than that normally used in microstructure studies,
facilitated the explanation of the effects of fibre on the rheology of imitation cheese.
r 2005 Elsevier Ltd. All rights reserved.
Keywords: Imitation cheese; Fibre; Resistant starch; Rheology; Microstructure
1. Introduction
Imitation cheese is increasingly used in the food industry
as an ingredient for prepared foods. The health attributes
of imitation cheese could be improved by adding nutri-
tionally beneficial ingredients such as fibre and lowering
the fat content. However, not every source of fibre can be
used since it is important that the textural properties of the
cheese are not negatively altered. Resistant starch (RS) is a
source of fibre that is available as a fine powder and its
benefits have been widely assessed (Haralampu, 2000;
Muir, Young, & ODea, 1994; Phillips et al., 1995;Silvester, Englyst, & Cummings, 1995). RS assays as
insoluble fibre, but has the physiological benefits of soluble
fibre. In the small intestine, RS may be slowly absorbed
and promote an increased malabsorption of starch, which
is important for the use of RS in food formulations for
people with certain forms of diabetes (Muir et al., 1994). In
the colon, RS lowers colonic pH and increases faecal bulk.
The portion of the latter that is fermented by the intestinal
microflora produces a wide range of short-chain fatty
acids, which has a positive impact on bowel health,
including a degree of protection against bowel cancer
(Ahmed, Segal, & Hassan, 2000; Baghurst, Baghurst, &
Record, 1996; Scheppach, Bartram, & Richter, 1995).
There are a number of published studies in which starch
has been incorporated into imitation cheese, mainly to
replace the more expensive casein (Burkwall, 1973; Freck &
Kondrot, 1974; Mounsey & ORiordan, 2001; Zallie &
Chiu, 1989; Zwiercan, Lacrourse, & Lenchin, 1987).
Mounsey and ORiordan (2001) also studied the effect ofdifferent native starches on the characteristics of imitation
cheese and found a reduced meltability and cohesiveness
with increasing starch concentration, while hardness was
increased by wheat, potato and maize, but reduced by
waxy-maize or rice starch.
In recent years much attention has been given to the
microstructure of natural and imitation cheese. Several
techniques have been used for this purpose, for example
scanning electron microscopy (SEM: Lee, Klostermeyer,
Schrader, & Buchheim, 1996; Mounsey & ORiordan,
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doi:10.1016/j.idairyj.2005.08.008
Corresponding author. Fax: +3531 7161147.
E-mail address: [email protected] (E. Dolores ORiordan).
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2001), confocal laser microscopy (Auty, Fenelon, Guinee,
Mullins, & Mulvihill, 1999: Guinee, Auty, & Mullins, 1999)
and transmission electron miscroscopy (Tunick, 2001). In
particular, the use of SEM has become the method of
choice in many investigations and it has proved to be an
efficacious method to identify the components when fat,
protein and moisture are the major constituents. However,there is a growing interest in incorporating other ingre-
dients in the imitation cheese that could be more difficult to
identify by SEM. In this regard, the tinctorial qualities of
light microscopy (LM) would offer a distinct advantage in
the explanation of the changes occurring in the structure of
imitation cheese.
This work aims to investigate the feasibility of manu-
facturing an imitation cheese with reduced fat and that also
contains dietary fibre in the form of RS. The inclusion of
RS is not an attempt to reduce cost; rather it is to increase
the health benefits by replacing up 75% of the hardened fat
in imitation cheese. The starch is added at a later stage in
the manufacturing process, compared with previous studies,
to avoid dehydration of the protein matrix and some of the
imitation cheese manufactured has higher concentrations of
starch than those previously reported (Mounsey & ORior-
dan, 2001). The influence of the added starch on the
meltability, texture, rheology and microstructure of imita-
tion cheese are studied. A particular aim of the present
investigation is to use a correlative microscopy approach in
which imitation cheese is examined using cryo-SEM and
images thus obtained are assessed in conjunction with
images from light microscopy cryo-sections. Firm qualita-
tive data are obtained from the latter sections by staining
them histochemically for starch, fat and protein. Inaddition, the putative relationship between these micro-
structural findings and the physical properties of the
imitation cheeses containing different types of RS, e.g.,
hardness, melting temperature and elasticity is assessed.
2. Materials and methods
2.1. Manufacture of imitation cheese
A control imitation cheese was manufactured according
to the following formulation, (expressed as weight percen-
tage): 42.99% water, 28.41% rennet casein (80% protein)
(Kerry Ingredients, Listowel, Ireland), 16.65% hydroge-
nated palm and 8.2% rapeseed oil (Trilby Trading Ltd.,
Liverpool, England), 1.49% total emulsifying salts, con-
taining 1.02% trisodium citrate, 0.47% disodium phos-
phate (Ellis and Everard, Dublin, Ireland), 1.60% sodium
chloride (Salt Union, Cheshire, England), 0.58% citric acid
(Jungbunzlauer, Pernhofen, Austria), and 0.09% sorbic
acid (Hoechst Ireland Ltd., Dublin, Ireland). All the
ingredients except citric acid were mixed in a twin-screw
cooker (model CC-010, Blentech Corporation, CA, USA)
and were maintained at a temperature of 50 1C by injecting
steam into the jacket. The direct steam valve was opened to
heat the mixture to 80 1C, and this temperature was
maintainedusing the steam jacketfor 5 min, or until a
uniform mass was obtained. Citric acid was then added and
mixed for a further 1 min. Using a similar process,
imitation cheeses, containing 5, 7.5, 10, or 12.5%
Novelose240 (N240; 40% granular RS) or Novelose330
(N330; 30% retrograded RS) (National Starch and
Chemicals, Manchester, England) were manufactured.N240 or N330 were added in direct replacement (on a
weight basis) of hardened palm oil in the control
formulation. The Novelose was incorporated into its
respective batch after closing the steam valve once the
temperature of 80 1C had been reached. The rest of the
procedure was the same as for the control cheese. Three
batches of each cheese were manufactured.
2.2. Compositional analysis
Samples of imitation cheese were analysed for moisture by
the IDF (1958) method, fat by the Gerber method (National
Standards Authority of Ireland, 1955) and protein using the
semi-micro Kjeldahl method (IDF, 1993). The pH was
determined by placing the electrodes of a pH meter (model
9450, Unicam Ltd., Cambridge, England) directly into a
small block of imitation cheese, equilibrated at 22 1C. All
compositional analyses were performed in triplicate.
2.3. Dynamic rheology test
Dynamic oscillatory measurements were performed on
cheese samples using a controlled stress rheometer (model
SR 2000, Rheometrics Inc., Piscataway, NJ, USA) as
described by Mounsey and ORiordan (1999). Six samplesfrom each block of imitation cheese were assessed within
one week of manufacture. The elastic/storage modulus (G0)
which reflects the ability of a material to store energy while
maintaining its structural integrity was measured. The
viscous/loss modulus (G00), representing the ability of a
material to dissipate mechanical energy by converting it to
heat through molecular motion was also measured. The
temperature at which G0 and G00 are equal to each other is
referred to as the crossover temperature (CT) and this value
was used as an indication of cheese meltability. Tan d, the
loss angle (G00/ G0), an index of the viscoelasticiy of the
material, was also measured.
2.4. Texture profile analysis
The textural properties of the imitation cheese were
measured two days post manufacture as described in
Mounsey and ORiordan (2001); six samples were analysed
from each cheese batch.
2.5. Microstructural analysis using scanning electron
microscopy
Cryo-SEM (JEOL JSM-5410LV Scanning Microscope,
JEOL Instruments, Tokyo, Japan) was used to examine the
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microstructure of N240 and N330 powders before adding
them as an ingredient to the imitation cheese. The cryo unit
used was an Oxford Instruments Cryo Preparation System
CT 1500 (Oxford Instruments, Oxford, England). A
double-sided adhesive tape was adhered to the specimen
stub and then it was placed in contact with the correspond-
ing RS powder. Specimens were transferred (under vacuumat 180 1C) to the cryo chamber. The samples were
sublimed at 85 1C, sputter-coated with gold (3 mA,
2 min) at 180 1C under an atmosphere rich in Argon and
then introduced in the microscope chamber where it was
examined using an accelerating voltage of 10 KV, and a
spot size of about 9 nm. Micrographs of N240 and of N330
powders were taken at 500, 1500 and 5000 magnification.
The microstructure of the imitation cheese was also
examined. Blocks approximately 1 mm3 mm5 mm
were cut from the stored imitation cheese using a scalpel,
mounted on a specimen holder and cryofixed by rapid
plunging into nitrogen slush (210 1C). Specimens were
transferred (under vacuum at 180 1C) to the cryo
chamber and the interior exposed by slicing off the apex
of the block using a scalpel. The samples were then
sublimed, coated and subsequently introduced into the
microscope chamber for examination, as outlined above.
Several images were taken of each specimen at different
magnifications ranging from 500 up to 7500 fold. Two
samples were analysed from each type of imitation cheese.
2.6. Microstructural analysis using light microscopy
Samples of imitation cheese made with 12.5% (w/w)
N240 or 12.5% (w/w) N330 were examined by LM. Cryo-sections, 8 mm in thickness, were taken from cubes of cheese
approximately 10 mm10mm 8 mm using a cryo-micro-
tome (model Starlet 2212 Bright Instrument Company
Ltd., Huntingdon, England). The sections were placed on
glass slides, labelled and then dried in an incubator at 30 1C
for 24 h.
Following drying, three components of the imitation
cheese specimens, protein, fat and starch, were sequentially
stained greenish blue, red and blue/black, using Fast Green
(Clark, 1980), Sudan III (Drury & Wallington, 1967) and
Iodine, respectively.
The fixed specimens were flooded with aqueous Fast
Green (0.5 g 100 mL1) for 1 min, to stain protein and then
rinsed with distilled water. The samples were subsequently
well rinsed in aqueous triethyl phosphate (60 g 100 mL1)
(TEP), then immersed in a solution of Sudan III (1g
100 mL1 TEP) for 11 min to stain the fat. Stained samples
were then covered in DPX (gum/xylene) mountant prior to
examination. Finally, to allow the examination of starch,
some stained sections were flooded with an aqueous iodine
solution (0.3 g I2, 1.0g KI 100mL1) for 1 min and then
rinsed with distilled water; these latter sections were not
mounted prior to examination.
Images of the stained sections were recorded using a LM,
model Nikon Eclipse E600 fitted with a digital camera
(Nikon DXM1200, Nikon UK, Kingston, Surrey). Three
samples of each block of cheese were analysed in this way
within a week of manufacture.
2.7. Statistical analysis
PROC GLM of SAS (SAS Institute, Cary, NC, USA)was used to determine differences between treatment means
and correlation coefficients. Treatment means were con-
sidered significantly different at Pp0:05 unless stated
differently.
3. Results
3.1. Composition of cheese
The mean water, protein, ash contents and the pH of the
imitation cheese were 52.270.4%, 20.870.4%, 4.170.3%,
and 6.170.06%, respectively. The fat content in the
control cheese was 22.470.2% and was reduced to
17.870.6%, 16.170.2%, 13.970.3% and 11.270.1% as
the hydrogenated palm oil was replaced with 5%, 7.5%,
10% or 12.5% N240 or N330, respectively.
3.2. TPA results
The hardness of the imitation cheeses increased linearly
(Hardness 21.36N240+278.86, R2 0:9327; Hard-
ness 34.81N330+282.02, R2 0:9844) when the percen-
tage of RS was increased, to a greater extent in the case of
N330 than for N240 (Table 1). The cohesiveness of
the imitation cheese was not significantly affected byN330, but was increased by increasing concentrations of
N240 (Table 1).
3.3. Rheology
For all cheeses the values of G0 (Fig. 1) and G00 (Fig. 2)
decreased, while tand values (Fig. 3) increased as the
temperature was increased from 22 to $55 1C. In this
temperature range (2255 1C) the effect of the added RS
was to increase both the values of G0 and G00 of the
imitation cheese. This effect was always greater in the case
of N330 (Figs. 1B and 2B) than for N240 (Figs. 1A and
2A); tan d was little affected by the added starch in this
temperature range.
At temperatures 455 1C, both G0 and G00 decreased with
increasing temperature for the control cheese and the
cheeses with the lower concentrations (5% or 7.5% w/w) of
RS. However, for cheeses containing higher concentrations
of RS the elastic and viscous modulii of the cheeses
increased, and the increase was greater at higher levels of
fibre inclusionparticularly notable for the cheese contain-
ing 12.5% N240. In the case of tan d, at temperatures
4551C the values for the control imitation cheese
increased but those of the RS containing cheeses (with
the exception of 5% N240) tended to level off or decrease
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to an extent dependant on the level of starch inclusion.
Results showed no significant effect of N240 on CT whereas
N330 increased CT at the highest level of addition. For
the most part the cheeses containing N330 had a higher
CT than those containing corresponding levels of N240
(Table 1).
3.4. Microstructure
3.4.1. Microstructure of N240 and N330
Electron micrographs showed clear differences between
N240 and N330 powders (Fig. 4). The average size of the
particles of N240 was noticeably smaller (between 2 and
10mm) and the size distribution more uniform than was
observed for N330, for which particle size ranged from 2 to
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0
50
100
150
200
250
20 30 40 50 60 70 80 90
Temperature (C)
G'(KPa)
0
50
100
150
200
250
20 30 40 50 60 70 80 90
Temperature (C)
G'(KPa)
(B)
(A)
Fig. 1. Effect of increasing temperature on the storage modulus (G0) of
imitation cheese containing 0% (B), 5% (&), 7.5% (n), 10% (), 12.5%
(m) of Novelose240 (A) or Novelose330 (B). Each point represents the
mean of three replicate trials.
Table 1
Hardness, cohesiveness and crossover temperature values of control imitation cheese and imitation cheeses containing Novelose240 (N240) or
Novelose330 (N330)
Novelose (%) Hardness (N)a Cohesiveness Crossover temperature (1C)
N240 N330 N240 N330 N240 N330
0 303a 0.35a 54.6a
5 367bx 433by 0.40bx 0.38ay 54.8ax 59.5by
7.5 400cx 530cy 0.40bx 0.36ay 57.9bx 57.8bx
10 508dx 621dy 0.42bcx 0.35ay 55.3ax 60.3bcy
12.5 564ex 741ey 0.43cx 0.36ay 54.8ax 63.0cy
aFor each column, means having the same superscript letter a, b, c, d, or e did not differ significantly. For each physical property, means having the same
superscript letter x or y did not differ significantly between cheeses containing equivalent levels of N240 or N330.
0
20
40
60
80
100
120
Temperature (C)
G"(KPa)
0
20
40
60
80
100
120
20 30 40 50 60 70 80 90
20 30 40 50 60 70 80 90
Temperature (C)
G"(KPa)
(A)
(B)
Fig. 2. Effect of increasing temperature on the loss modulus (G00) of
imitation cheese containing 0% (B), 5% (&), 7.5% (n), 10% (), 12.5%(m) of Novelose240 (A) or Novelose330 (B). Each point represents the
mean of three replicate trials.
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50mm. The surface of N240 appeared smooth and theglobular particles tended to aggregate into clumps and in
some cases coalesced to form elongated structures. N330
had a creased surface, and the conformation of the
particles was characterised by heterogeneous protrusions
and cavities.
3.4.2. Fat globule size and distribution in the cheese
SEM of the control imitation cheese (Fig. 5A) showed a
continuous protein matrix (P) containing fat globules (F)
with a size range from 2 to 16 mm, with larger globules
predominant. The particles of fat were smooth surfaced
and were mostly spherical in shape though some of the
globules presented a degree of deformation. Honeycomb
structures (H) were commonly observed in an otherwise
uniform protein matrix. As the amount of starch in the
imitation cheese was increased, the fat globules became
smaller and the particle size distribution more uniform (not
shown). The ranges of the diameters of the fat globules
were 219 mm for the control, 112 mm for 12.5% N240 and
0.57 mm for 12.5% N330 (Fig. 5). Thus, the observed
average diameters of the fat particles were approximately
9 mm in the control cheese, 5 mm in the 12.5% N240 and
3 mm in the 12.5% N330 imitation cheeses. LM sections of
imitation cheese containing 12.5% (w/w) N240 stained for
lipid (red) and protein (blue) (Fig. 6A) showed an uneven
distribution of fat. Fat globules were seen clustered in
the protein matrix with relatively large areas bereft of fat.
In contrast, sections of imitation cheese containing 12.5%
(w/w) N330 stained for lipid, protein and starch (Fig. 6B)
showed an even distribution of fat droplets compared with
the clustering of fat particles observed in sections of cheese
containing N240.
3.4.3. Resistant starches in the cheese
When N240 RS was incorporated into an imitation
cheese formulation, its presence could be seen in sections
stained with iodine (Fig. 6C). The granules of RS had a
relatively uniform size and homogeneous spherical shape
and showed little diffusion of the granule boundaries into
the protein matrix (stained blue). In fact the clusters of
granules were found over a pale background rather than
blue, suggesting that the granules of starch were isolated
from the protein matrix. The specific staining of the starch
by iodine revealed pale linearities in some areas, these
possibly represented zones of coalescence of the granules;
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0
1
2
3
4
5
6
7
20 30 40 50 60 70 80 90
Temperature (C)
Temperature (C)
Tan_
delta
0
1
2
3
4
5
6
7
20 30 40 50 60 70 80 90
Tan_
delta
(A)
(B)
Fig. 3. Effect of increasing temperature on the loss angle (tan delta) of
imitation cheese containing 0% (B), 5% (&), 7.5% (n), 10% (), 12.5%
(m) of Novelose240 (A) or Novelose330 (B). Each point represents the
mean of three replicate trials.
Fig. 4. Scanning electron micrographs ( 1500) of (A) Novelose240 and
(B) Novelose330 powders. C: coalesced particles.
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such zones were seen in varying degrees of completeness.
In contrast, the particles of N330 (Fig. 6D) had a broad
size distribution although they were generally bigger in
comparison with the particles of N240 and their shape was
remarkably different, presenting an irregular appearance.
The contour of the particles of N330 was diffused into the
protein matrix and these particles were not grouped
together but in general were well distributed throughout
the protein matrix. The identification of the particles of RS
using SEM was possible, although not always certain (S in
Fig. 5B and C). Whenever these particles were clearly seen,
the maximum diameters were around 15 mm for N240 and
40mm for N330.
3.4.4. Interaction between resistant starches and protein
matrix
Closer observation of the Novelose particles at higher
magnifications by SEM revealed some details of
its appearance and interaction with the protein matrix
(Fig. 7). In the case of N240, noticeable phase boundaries
separating starch granules from the protein matrix were
evident by the presence of a vacancy surrounding the starch
granule (S in Fig. 7A). Granules of N240 maintained, to
some extent, their original shape, although the protein
matrix around them made them appear slightly irregular.
In the case of N330 the shape was very irregular and the
size of the starch particles (S in Fig. 5C) was generally
bigger than those of N240. In addition, the manner in
which the particles of N330 were incorporated in the
protein matrix (P in Fig. 7B) was different, with a less
distinctive phase boundary. Particles of N330 (S in Figs. 5C
and 7B) appeared irregularly shaped and could be
distinguished from the protein matrix by the flatness of
their surface and the fact that no other structures (fat
globules, air holes, honeycomb structures) were present in
the area occupied by the retrograded starch. In fact, the
presence of honeycomb structures around the N330
particles usually helped to highlight it from the protein
matrix.
4. Discussion
SEM has been used extensively to characterise imitation
cheese (Mounsey, 2000; Mounsey & ORiordan, 2001;
Murphy, 1999; Rayan, Kalab, & Ernstrom, 1980; Savello,
Ernstrom, & Kalab, 1989; Taranto & Yang, 1981).
However, in the present study, combining the results of
SEM and LM facilitated an improved interpretation of the
effects of RS on the texture and rheology of imitation
cheese and was particularly useful in explaining differences
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Fig. 5. Electron micrographs of (A) control 52% moisture imitation cheese ( 500), (B) imitation cheese containing 12.5% Novelose240 ( 500) and
(C) imitation cheese containing 12.5% Novelose330 ( 800). F: fat globule; P: Protein matrix; H: honeycomb structure; S: starch.
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between the effects of the two starches. Well-defined
structures, e.g., globules of fat were reliably imaged by
SEM, making their identification certain. Other less well-
defined particles, e.g., starch, were better observed using
specific staining with iodine and LM. SEM yielded high-
resolution topographical images that allowed the measure-
ment of the size of the fat and starch particles. However,
the distribution of the latter particles in the protein matrix
was better determined using specific staining with Sudan
III and iodine and examination by LM. Using SEM, only
the surface of the cheese could be seen, while LM showed
the components of the cheese in all the focal planes of the
section, giving an in-depth representation of the distribu-
tion of particles throughout the cheese matrix. LM was
found to be an unequivocal way to differentiate the
components of the imitation cheese analysed in this study.
Mounsey and ORiordan (2001) reported that incorpora-
tion of high amylose starches increased the hardness of the
imitation cheese, which they attributed to hydrogen
bonding of amylose leached from the starch particles
during the cheese cooking. The RS used in the present
study have a high concentration of amylose and probably
increased the hardness of the cheese by the same
mechanism. A notable finding was the large difference
between the effects of N330 and N240 on hardness for
which there are a number of possible explanations. Firstly,
the diffuse character of the N330/Protein boundary, as
observed by LM, suggests that the leaching of amylose
mentioned above may have occurred to a greater extent for
N330 than for N240. Secondly, Nielsen and Landel (1994),
in their studies relating to particulate-filled polymers,
suggested that the reinforcing effect of a filler, such as
starch, is greater when spheres are replaced by more
elongated or flat particles.
In the present study the particles of N240 were observed
to be more spherical than those of N330, both in the dry
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Fig. 6. Light microscopy images of imitation cheese containing 12.5% of either Novelose240 (A, C) or Novelose330 (B, D). Red: fat globules; blue:
protein matrix; purple: starch.
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state and in the cheese. Thus, when the spherical fat
globules are replaced by N240 a lower hardening effect is
expected than when they are replaced with N330. In
addition, as the amount of RS was increased in the
imitation cheeses, SEM images showed a higher degree of
emulsification, and the different starches affected distribu-
tion of fat in different ways. The increase in fat
emulsification was noticeable even at the lowest concentra-
tions of N330 (not shown) and the effect increased with the
RS concentration. In contrast, only at the highest
concentrations of N240 was the emulsification improved,
and always to a lower extent than N330.
Rayan et al. (1980) found a direct correlation between
the degree of emulsification (fineness of fat particles) and
hardness of cheese, thus the effects of amount and type of
starch on hardness may also partly arise through indirect
effects on fat emulsification. Ennis and Mulvihill (1997)
suggested that increasing the viscosity of the aqueous phase
reduced the frequency of oil droplet collisions and
stabilised the oil in water emulsion in cheese. Particles of
N330 bound more water than N240 and the cheese
containing the former had greater viscosity than that
containing N240 which may have led to reduced coales-
cence of fat and hence smaller fat globules.
The reduction of fat content and the increase of starch in
the formulation of imitation cheese may have opposite
effects on the cohesiveness of the cheese matrix. Rudan,
Barbano, Yun, and Kindstedt (1999) reported an in-
crease in cohesiveness of Mozzarella cheese when the fat
content was decreased. On the other hand, (Mounsey &
ORiordan, 2001) observed that replacing protein by starchcaused a reduction in the cohesiveness of imitation cheese.
The role of starch in the reduction of cohesiveness could be
due to structural failure on deformation due to stress
localisation at the starchprotein matrix interface, as found
by Noel, Ring, and Whittam (1993) in starch products.
When the stress applied to the cheese cannot be dissipated
by deformation the matrix breaks more easily. N330 has
bigger and less spherical particles that appear (by LM and
SEM) to be better integrated into the matrix than those of
N240, in the case of the former this may restrain the
deformation of the matrix and thus reduce the cohesiveness
of imitation cheese to a greater extent. The cohesiveness
results obtained in the current study probably reflect a
combined effect of decreased fat and increased starch
content. In the case of N240 the influence of reduced fat
content may be dominant.
The increase in storage and loss moduli consequent to
higher levels of starch, observed throughout the tempera-
ture range in the current work, was most likely due to the
binding of water by the starch. Such binding reduces the
water available to plasticise the matrix resulting in elevated
G0 and G00 values. The fact that N330 has a higher water-
holding capacity (1.82.0 g water per g sample) than N240
(1.4 g water per g sample) (National Starch and Chemicals,
2001) may explain the different effects of the two starches.The increase observed in both G0 and G00 of the cheeses
containing starch, at temperatures4$55 1C, may possibly
be due to an increase in the level of starch hydration or
gelatinisation at these temperatures.
The fact that N240 had no significant effect on CT(Table 1) is not in agreement with the results from previous
studies (Mounsey & ORiordan 1999, 2001), which con-
sistently show that substituting casein with increasing levels
of starch lead to a decrease in the melting properties of
imitation cheese. This apparent inconsistency could be due
to the fact that in the present study RS was used to replace
fat and not casein. In addition, in the present study starch
was incorporated into the imitation cheese at the latest
stage of the manufacture in order to avoid the dehydration
of the protein matrix, which was identified by Mounsey
and ORiordan (2001) as an important factor influencing
the meltability of imitation cheese. The fact that N330 did
appear to elevate CT may simply reflect its better capacity
to bind water. Alternatively, the interaction between the
particles of N330 and the protein matrix may have
increased the extent of bonding between these constituents
and it would be reasonable to suggest that more energy
would be required to break such bonds. It is tempting also
to suggest that since the particles of N240 remained quite
separate from the protein matrix they exerted little
ARTICLE IN PRESS
Fig. 7. Electron micrographs ( 7500) of imitation cheese containing
10% of either (A) Novelose240 or (B) Novelose330. S:starch; P: protein
matrix.
C. Montesinos-Herrero et al. / International Dairy Journal 16 (2006) 910919 917
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influence on the amount of energy required to melt the
matrix.
Honeycomb structures were found in the SEM images of
control cheese and around the granules of N330 but not in
the cheese containing N240. Mounsey (2000) found
honeycomb structures around the granules of native starch
and suggested that these structures were a sign of proteindehydration. In the present study, the honeycomb struc-
tures in the control imitation cheese may be due to the
presence of pools of free water within the protein matrix
and their subsequent sublimation during the preparation of
the samples for SEM (Hennelly, Dunne, OSullivan, &
ORiordan, 2005). In the case of the honeycomb structures
found around N330 particles, it is possible that the high
water-binding capacity of N330 pulled the water from the
protein matrix and promoted its dehydration to some
extent. The lower water-binding capacity of the N240
granules was not apparently enough to exert a noticeable
dehydration. When the diameters of the particles of both
RS were compared before and after the addition to the
cheese it was found that the particles of N330 had generally
swelled while N240 particles had not.
5. Conclusions
High levels of fibre inclusion were achieved without
impairing the functional properties of imitation cheese,
possibly due to the novel approach of incorporating
resistant starch almost at the end of the manufacture.
The hardening effect and poorer meltability in the case of
N330 could probably be overcome by increasing the
moisture content, which would concomitantly lead to costsavings. The viable incorporation of fibre into imitation
cheese provides opportunities to change the perception of
this product from a mere cheap ingredient to a health
promoting and more nutritious food.
In this study, LM proved to be of great benefit as a
technique for examining the microstructure of imitation
cheese, mostly for its capacity to specifically distinguish the
different components of the cheese. In addition, while the
high magnification of SEM images was useful in elucidat-
ing some features of the microstructure (e.g., fat particle
size) the small sample size reduces the sampling accuracy.
However, using the broad sampling technique of LM in
conjunction with SEM, the sampling accuracy is secured
and full advantage of high resolution and high magnifica-
tion is obtained. Thus, using a combination of SEM and
LM, the understanding of the influence of functional fibre
on the physical properties of imitation cheese was greatly
improved.
Acknowledgements
This work was funded through the Food Institutional
Research Measure (FIRM) administered by the Irish
Department of Agriculture, Food and Rural Development.
The technical assistance of Mr. Barry Cregg from the
Electron Microscopy Department is gratefully acknowl-
edged.
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