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Partial replacement of fat by functional fibre in imitation cheese: Effects on rheology and microstructure
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International Dairy Journal 16 (2006) 910–919 www.elsevier.com/locate/idairyj
Partial replacement of fat by functional fibre in imitation cheese: Effects on rheology and microstructure Clara Montesinos-Herreroa, David C. Cottellb, E. Dolores O’Riordana,, Michael O’Sullivana a
b
Department of Food Science, University College Dublin, Belfield, Dublin 4, Ireland Electron 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 (G 00 ) increased with increasing contents of both fibres. The crossover temperature (G0 ¼ G 00 ) 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 nutritionally 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, & O’Dea, 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. Corresponding author. Fax: +353 1 7161147.
E-mail address: [email protected] (E. Dolores O’Riordan). 0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.08.008
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 & O’Riordan, 2001; Zallie & Chiu, 1989; Zwiercan, Lacrourse, & Lenchin, 1987). Mounsey and O’Riordan (2001) also studied the effect of different 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 & O’Riordan,
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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 ingredients 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 manufacturing 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 & O’Riordan, 2001). The influence of the added starch on the meltability, texture, rheology and microstructure of imitation 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 qualitative data are obtained from the latter sections by staining them histochemically for starch, fat and protein. In addition, the putative relationship between these microstructural 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 percentage): 42.99% water, 28.41% rennet casein (80% protein) (Kerry Ingredients, Listowel, Ireland), 16.65% hydrogenated palm and 8.2% rapeseed oil (Trilby Trading Ltd., Liverpool, England), 1.49% total emulsifying salts, containing 1.02% trisodium citrate, 0.47% disodium phosphate (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
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maintained—using the steam jacket—for 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 O’Riordan (1999). Six samples from each block of imitation cheese were assessed within one week of manufacture. The elastic/storage modulus (G 0 ) which reflects the ability of a material to store energy while maintaining its structural integrity was measured. The viscous/loss modulus (G 00 ), 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 G 00 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 (G 00 / G 0 ), 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 O’Riordan (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 corresponding RS powder. Specimens were transferred (under vacuum at 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 mm 3 mm 5 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. Cryosections, 8 mm in thickness, were taken from cubes of cheese approximately 10 mm 10 mm 8 mm using a cryo-microtome (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 (1 g 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, 1.0 g KI 100 mL1) 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 considered 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 ¼ 21.36N240+278.86, R2 ¼ 0:9327; Hard2 ness ¼ 34.81N330+282.02, R ¼ 0:9844) when the percentage 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 by N330, but was increased by increasing concentrations of N240 (Table 1). 3.3. Rheology For all cheeses the values of G 0 (Fig. 1) and G 00 (Fig. 2) decreased, while tan d values (Fig. 3) increased as the temperature was increased from 22 to 55 1C. In this temperature range (22–55 1C) the effect of the added RS was to increase both the values of G0 and G 00 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 G 0 and G 00 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 inclusion—particularly notable for the cheese containing 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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Table 1 Hardness, cohesiveness and crossover temperature values of control imitation cheese and imitation cheeses containing Novelose240 (N240) or Novelose330 (N330) Hardness (N)a
Novelose (%)
0 5 7.5 10 12.5
Cohesiveness
Crossover temperature (1C)
N240
N330
N240
N330
N240
N330
303a 367bx 400cx 508dx 564ex
433by 530cy 621dy 741ey
0.35a 0.40bx 0.40bx 0.42bcx 0.43cx
0.38ay 0.36ay 0.35ay 0.36ay
54.6a 54.8ax 57.9bx 55.3ax 54.8ax
59.5by 57.8bx 60.3bcy 63.0cy
a
For 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.
250
120 100 G" (KPa)
G' (KPa)
200
150
80 60 40
100
20
50
0 0 20
30
40
(A)
50 60 Temperature (°C)
70
80
90
20
30
20
30
40
(A)
50 60 Temperature (°C)
70
80
90
120
250
100
G" (KPa)
G' (KPa)
200
150
80 60 40
100
20 50
0 0 20
(B)
30
40
50 60 Temperature (°C)
70
80
90
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.
(B)
40
50 60 Temperature (°C)
70
80
90
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.
3.4. Microstructure 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).
3.4.1. 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 10 mm) and the size distribution more uniform than was observed for N330, for which particle size ranged from 2 to
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7 6 Tan_delta
5 4 3 2 1 0 20
30
20
30
40
(A)
50 60 Temperature (°C)
70
80
90
7 6 Tan_delta
5 4 3 2 1 0 (B)
40
50 60 Temperature (°C)
70
80
90
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.
50 mm. The surface of N240 appeared smooth and the globular 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 2–19 mm for the control, 1–12 mm for 12.5% N240 and 0.5–7 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
Fig. 4. Scanning electron micrographs ( 1500) of (A) Novelose240 and (B) Novelose330 powders. C: coalesced particles.
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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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.
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 40 mm 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 & O’Riordan, 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. 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.
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 welldefined particles, e.g., starch, were better observed using specific staining with iodine and LM. SEM yielded highresolution topographical images that allowed the measurement 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 distribution 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 O’Riordan (2001) reported that incorporation 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. 7. Electron micrographs ( 7500) of imitation cheese containing 10% of either (A) Novelose240 or (B) Novelose330. S:starch; P: protein matrix.
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 distribution of fat in different ways. The increase in fat emulsification was noticeable even at the lowest concentrations 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 coalescence of fat and hence smaller fat globules.
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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 increase in cohesiveness of Mozzarella cheese when the fat content was decreased. On the other hand, (Mounsey & O’Riordan, 2001) observed that replacing protein by starch caused 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 starch–protein 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 temperature 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 G 00 values. The fact that N330 has a higher waterholding capacity (1.8–2.0 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 temperatures455 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 & O’Riordan 1999, 2001), which consistently 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 O’Riordan (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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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 protein dehydration. In the present study, the honeycomb structures 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, O’Sullivan, & O’Riordan, 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 cost savings. 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 elucidating 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 magnification 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 acknowledged. References Ahmed, R., Segal, I., & Hassan, H. (2000). Fermentation of dietary starch in humans. The American Journal of Gastroenterology, 95, 1018–1019. Auty, M. A. E., Fenelon, M. A., Guinee, T. P., Mullins, C., & Mulvihill, D. M. (1999). Dynamic confocal scanning laser microscopy methods for studying milk protein gelation and cheese melting. Scanning, 21, 299–304. Baghurst, P. A., Baghurst, K. I., & Record, S. J. (1996). Dietary fiber, non-starch polysaccharides and resistant starch—a review. Food Australia, 48, S3. Burkwall, M. P., Jr. (1973). Preparation of a high protein simulated cheese product. US Patent 3,741,774. Clark, G. (1980). Staining procedures. Baltimore, London, UK: Williams & Wilkins, p. 415. Drury, R. A. B., & Wallington, E. A. (1967). Carleton’s histological technique. London, UK: Oxford University Press. Ennis, M. P., & Mulvihill, D. M. (1997). Cheese analogues. In T. M. Cogan, P. F. Fox, & R. P. Ross (Eds.), Proceedings of the fifth cheese symposium, 11–13th March 1997 (pp. 1–14). Dublin, Ireland: Teagasc. Freck, J. A., & Kondrot, L. V. (1974). Process for making simulated feta and cheese products. US Patent 3,836,677. Guinee, T. P., Auty, M. A. E., & Mullins, C. (1999). Observation on the microstructure and heat-induced changes in the viscoelasticity of commercial cheeses. The Australian Journal of Dairy Technology, 54, 84–89. Haralampu, S. G. (2000). Resistant starch—a review of the physical properties and biological impact of RS3. Carbohydrate Polymers, 41, 285–292. Hennelly, P. J., Dunne, P. G., O’Sullivan, M., & O’Riordan, E. D. (2005). Increasing the moisture content of imitation cheese: Effects on texture, rheology and microstructure. European Food Research and Technology, 220, 415–420. IDF. (1958). Determination of dry matter in cheese and processed cheese. Standard FIL-IDF 4. Brussels, Belgium: International Dairy Federation. IDF. (1993). Milk: Determination of nitrogen content, Part 3: Block digestion method (semi-micro rapid routine method), Annex-Modified procedure for milk products. Standard FIL-IDF 20B. Brussels, Belgium: International Dairy Federation. Lee, S. K., Klostermeyer, H., Schrader, K., & Buchheim, W. (1996). Rheological properties and microstructure of model process cheese containing low molecular weight emulsifiers. Nahrung, 40, 189–194. Mounsey, J. S. (2000). Characteristics of imitation cheese containing starch. Ph.D. Thesis. Dublin, Ireland: NUI. Mounsey, J. S., & O0 Riordan, E. D. (1999). Empirical and dynamic rheological data correlation to characterize melt characteristics of imitation cheese. Journal of Food Science, 64, 701–703. Mounsey, J. S., & O’Riordan, E. D. (2001). Characteristics of imitation cheese containing native starches. Journal of Food Science, 66, 586–591. Muir, J. G., Young, G. P., & O’Dea, K. (1994). Resistant starchimplications for health. Proceedings of the Nutritional Society of Australia, 18, 23–32. Murphy, K. J. (1999). Properties of imitation cheese containing non-casein proteins. M. Agr. Sc. Thesis. Dublin, Ireland: University College. National Standards Authority of Ireland. (1955). Determination of percentage of fat in cheese (IS69). Dublin, Ireland: N.S.A.I. National Starch and Chemicals (2001). N240 and N330. In http:// www.foodstarch.com/products_services/product. Nielsen, L. E., & Landel, R. F. (1994). Particulate-filled polymers. Mechanical Properties of Polymers and Composites II (2nd ed). New York, USA: Marcel Dekker inc. pp. 379–442. Noel, T. R., Ring, S. G., & Whittam, M. A. (1993). Physical properties of starch products: Structure and function. In E. Dickinson, & P. Walstra
ARTICLE IN PRESS C. Montesinos-Herrero et al. / International Dairy Journal 16 (2006) 910–919 (Eds.), Food colloids and polymers: Stability and mechanical properties (pp. 126–137). Cambridge, UK: Royal Society of Chemistry. Phillips, J., Muir, J. G., Birkett, A., Lu, Z. X., Jones, G. P., O0 Dea, K., et al. (1995). Effect of resistant starch on fecal bulk and fermentation-dependent events in humans. American Journal of Clinical Nutrition, 62, 121–130. Rayan, A. A., Kalab, M., & Ernstrom, C. A. (1980). Microstructure and rheology of process cheese. Scanning Electron Microscopy, 3, 635–644. Rudan, M. A., Barbano, D. M., Yun, J. J., & Kindstedt, P. S. (1999). Effect of fat reduction on chemical composition, proteolysis, functionality, and yield of Mozzarella cheese. Journal of Dairy Science, 82, 661–672. Savello, P. A., Ernstrom, C. A., & Kalab, M. (1989). Microstructure and meltability of model process cheese made with rennet and acid casein. Journal of Dairy Science, 72(1) 1–1. Scheppach, W., Bartram, H. P., & Richter, F. (1995). Role of short-chain fatty acids in the prevention of cholorectal cancer. European Journal of Cancer, 31a, 1077–1080.
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Silvester, K. R., Englyst, H. N., & Cummings, L. H. (1995). Ileal recovery of starch from whole diets containing resistant starch measured in vitro and fermentation of ileal effluent. American Journal of Clinical Nutrition, 62, 403–411. Taranto, M. V., & Yang, C. S. T. (1981). Morphological and textural characterisation of soybean Mozzarella cheese analogs. Scanning Electron Microscopy, 3, 483–492. Tunick, M. H. (2001). Transmission electron microscopy of Mozzarella cheese made from microfluidized milk. In TEKTRAN Agricultural Research Services. www.nal.usda.gov/ttic/tektran/data Zallie, J. P., & Chiu, C. -W. (1989). Imitation cheese containing enzymatically debranched starches in lieu of caseinates. European Patent 0,363,741,A2. Zwiercan, G. A., Lacrourse, N. L., & Lenchin, J. M. (1987). Imitation cheese products containing high amylose starch as total caseinate replacement. US Patent 4,695,475.
International Dairy Journal 16 (2006) 910–919 www.elsevier.com/locate/idairyj
Partial replacement of fat by functional fibre in imitation cheese: Effects on rheology and microstructure Clara Montesinos-Herreroa, David C. Cottellb, E. Dolores O’Riordana,, Michael O’Sullivana a
b
Department of Food Science, University College Dublin, Belfield, Dublin 4, Ireland Electron 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 (G 00 ) increased with increasing contents of both fibres. The crossover temperature (G0 ¼ G 00 ) 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 nutritionally 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, & O’Dea, 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. Corresponding author. Fax: +353 1 7161147.
E-mail address: [email protected] (E. Dolores O’Riordan). 0958-6946/$ - see front matter r 2005 Elsevier Ltd. All rights reserved. doi:10.1016/j.idairyj.2005.08.008
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 & O’Riordan, 2001; Zallie & Chiu, 1989; Zwiercan, Lacrourse, & Lenchin, 1987). Mounsey and O’Riordan (2001) also studied the effect of different 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 & O’Riordan,
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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 ingredients 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 manufacturing 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 & O’Riordan, 2001). The influence of the added starch on the meltability, texture, rheology and microstructure of imitation 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 qualitative data are obtained from the latter sections by staining them histochemically for starch, fat and protein. In addition, the putative relationship between these microstructural 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 percentage): 42.99% water, 28.41% rennet casein (80% protein) (Kerry Ingredients, Listowel, Ireland), 16.65% hydrogenated palm and 8.2% rapeseed oil (Trilby Trading Ltd., Liverpool, England), 1.49% total emulsifying salts, containing 1.02% trisodium citrate, 0.47% disodium phosphate (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
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maintained—using the steam jacket—for 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 O’Riordan (1999). Six samples from each block of imitation cheese were assessed within one week of manufacture. The elastic/storage modulus (G 0 ) which reflects the ability of a material to store energy while maintaining its structural integrity was measured. The viscous/loss modulus (G 00 ), 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 G 00 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 (G 00 / G 0 ), 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 O’Riordan (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 corresponding RS powder. Specimens were transferred (under vacuum at 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 mm 3 mm 5 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. Cryosections, 8 mm in thickness, were taken from cubes of cheese approximately 10 mm 10 mm 8 mm using a cryo-microtome (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 (1 g 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, 1.0 g KI 100 mL1) 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 considered 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 ¼ 21.36N240+278.86, R2 ¼ 0:9327; Hard2 ness ¼ 34.81N330+282.02, R ¼ 0:9844) when the percentage 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 by N330, but was increased by increasing concentrations of N240 (Table 1). 3.3. Rheology For all cheeses the values of G 0 (Fig. 1) and G 00 (Fig. 2) decreased, while tan d values (Fig. 3) increased as the temperature was increased from 22 to 55 1C. In this temperature range (22–55 1C) the effect of the added RS was to increase both the values of G0 and G 00 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 G 0 and G 00 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 inclusion—particularly notable for the cheese containing 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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Table 1 Hardness, cohesiveness and crossover temperature values of control imitation cheese and imitation cheeses containing Novelose240 (N240) or Novelose330 (N330) Hardness (N)a
Novelose (%)
0 5 7.5 10 12.5
Cohesiveness
Crossover temperature (1C)
N240
N330
N240
N330
N240
N330
303a 367bx 400cx 508dx 564ex
433by 530cy 621dy 741ey
0.35a 0.40bx 0.40bx 0.42bcx 0.43cx
0.38ay 0.36ay 0.35ay 0.36ay
54.6a 54.8ax 57.9bx 55.3ax 54.8ax
59.5by 57.8bx 60.3bcy 63.0cy
a
For 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.
250
120 100 G" (KPa)
G' (KPa)
200
150
80 60 40
100
20
50
0 0 20
30
40
(A)
50 60 Temperature (°C)
70
80
90
20
30
20
30
40
(A)
50 60 Temperature (°C)
70
80
90
120
250
100
G" (KPa)
G' (KPa)
200
150
80 60 40
100
20 50
0 0 20
(B)
30
40
50 60 Temperature (°C)
70
80
90
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.
(B)
40
50 60 Temperature (°C)
70
80
90
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.
3.4. Microstructure 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).
3.4.1. 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 10 mm) and the size distribution more uniform than was observed for N330, for which particle size ranged from 2 to
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7 6 Tan_delta
5 4 3 2 1 0 20
30
20
30
40
(A)
50 60 Temperature (°C)
70
80
90
7 6 Tan_delta
5 4 3 2 1 0 (B)
40
50 60 Temperature (°C)
70
80
90
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.
50 mm. The surface of N240 appeared smooth and the globular 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 2–19 mm for the control, 1–12 mm for 12.5% N240 and 0.5–7 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
Fig. 4. Scanning electron micrographs ( 1500) of (A) Novelose240 and (B) Novelose330 powders. C: coalesced particles.
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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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.
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 40 mm 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 & O’Riordan, 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. 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.
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 welldefined particles, e.g., starch, were better observed using specific staining with iodine and LM. SEM yielded highresolution topographical images that allowed the measurement 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 distribution 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 O’Riordan (2001) reported that incorporation 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. 7. Electron micrographs ( 7500) of imitation cheese containing 10% of either (A) Novelose240 or (B) Novelose330. S:starch; P: protein matrix.
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 distribution of fat in different ways. The increase in fat emulsification was noticeable even at the lowest concentrations 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 coalescence of fat and hence smaller fat globules.
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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 increase in cohesiveness of Mozzarella cheese when the fat content was decreased. On the other hand, (Mounsey & O’Riordan, 2001) observed that replacing protein by starch caused 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 starch–protein 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 temperature 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 G 00 values. The fact that N330 has a higher waterholding capacity (1.8–2.0 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 temperatures455 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 & O’Riordan 1999, 2001), which consistently 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 O’Riordan (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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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 protein dehydration. In the present study, the honeycomb structures 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, O’Sullivan, & O’Riordan, 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 cost savings. 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 elucidating 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 magnification 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
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