Partial Replacement of Fat by Functional Fibre in Imitation Cheese-Effects on Rheology and Microestruc~1

7/30/2019 Partial Replacement of Fat by Functional Fibre in Imitation Cheese-Effects on Rheology and Microestruc~1

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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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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




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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


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

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Fig. 7. Electron micrographs ( 7500) of imitation cheese containing

10% of either (A) Novelose240 or (B) Novelose330. S:starch; P: protein

matrix.



9/10

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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