Journal of Food Engineering 84 (2008) 5764 www.elsevier.

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Inuence of pre-gelatinised maize starch on the rheology, microstructure and processing of imitation cheese
John S. Mounsey a,*, E. Dolores ORiordan b
b a Teagasc, Food Research Centre, Moorepark, Fermoy, Co.Cork, Ireland School of Agriculture, Food Science and Veterinary Medicine, College of Life Sciences, University College Dublin, Beleld, Dublin 4, Ireland

Received 12 January 2007; received in revised form 12 April 2007; accepted 17 April 2007 Available online 27 April 2007

Abstract Imitation cheeses were manufactured with increasing levels (09%, w/w) of pre-gelatinised maize starch in partial replacement of rennet casein. At increased starch levels, longer processing times (10 min at 78 C using 9%, w/w starch) were necessary compared to the control (5 min) because of the reduced protein present to emulsify/stabilise the fat droplets. Scanning electron and confocal microscopy revealed that increased starch addition resulted in a less homogeneous protein matrix, with a honeycomb appearance although the fat globules remained small and uniformly sized. With increased levels of starch up to 9% (w/w), the storage modulus, peak stress and stress relaxation times signicantly increased while hardness values remained unchanged. Results indicated that the inclusion of pre-gelatinised maize starch impaired the hydration of the casein as well as the thermoplastic properties of the imitation cheese and has most application in food products where ow resistance is required, particularly at increased temperatures. 2007 Elsevier Ltd. All rights reserved.
Keywords: Pre-gelatinised maize starch; Imitation cheese; Microscopy; Rheology; Processing conditions

1. Introduction Imitation cheese is widely used as an ingredient in prepared foods such as pizza, lasagne, cordon bleu products where its rheological properties, that dictate whether it melts or maintains its structure, are critical (Mounsey & ORiordan, 2001). Hence, determination of parameters such as textural hardness, dynamic rheology, empirical melt determination as well as microstructural evaluation are widely used to assess the critical physical properties of imitation cheese (Guinee, Caric, & Kalab, 2004; Montesinos-Herrero, Cottell, ORiordan, & OSullivan, 2006; Mounsey & ORiordan, 1999, 2001; Song & Park, 1986; Yang & Taranto, 1982). For cost and health reasons, there has been increased interest recently in incorporation starch into imitation cheese formulations where the interaction between the added starch and the protein source (usually
*

Corresponding author. Tel.: +353 25 42443; fax: +353 25 42340. E-mail address: john.mounsey@teagasc.ie (J.S. Mounsey).

casein) has been shown to inuence the rheological properties of the imitation cheese (Guinee et al., 2004; Montesinos-Herrero et al., 2006; Mounsey & ORiordan, 1999, 2001). Imitation cheese hardness and storage modulus (G0 ) have been increased by partially replacing fat with resistant maize starch-derived products such as maltodextrin and resistant starch (Montesinos-Herrero et al., 2006). Mounsey and ORiordan (2001) showed that replacing 15% of the protein rennet casein in imitation cheese with dierent native starch types resulted in reduced meltability of the imitation cheese but had variable eects on the texture and microstructure of rennet casein-based imitation cheese. It was evident during the manufacture of the imitation cheese that the oil of starch-containing products was emulsied more rapidly compared to the control product. Scanning electron microscopy (SEM) images of the imitation cheese showed a uniform distribution of spherical fat droplets in a smooth protein matrix. However it was evident that the starch remained as discrete entities in the protein

0260-8774/$ - see front matter 2007 Elsevier Ltd. All rights reserved. doi:10.1016/j.jfoodeng.2007.04.017

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matrix. This may have been due to starch insolubility arising from the poor availability of water and/or the processing temperaturetime combination being insucient to fully gelatinise and solubilise the starch (Mounsey & ORiordan, 2001). Guinee et al. (2004) concluded that starches with high ratio of amylose to amylopectin tend to retrograde and undergo gelation more readily than those with a lower level of amylose during imitation cheese storage. This starch gelation within the imitation cheese structure with storage impedes imitation cheese melting ability, possibly because of its irreversibility at temperatures reached in the product during heating (<100 C) (Guinee et al., 2004). Previous research by Mounsey and ORiordan (1999) showed that the inclusion of increasing levels of pre-gelatinised maize starch in partial replacement of the protein in imitation cheese greatly reduced the meltability and peak tan d values of imitation cheese compared to a control, which had no starch. It was apparent that imitation cheeses containing pre-gelatinised starches would only be useful in specialist applications such as when low meltability in a heated system is required, e.g., Welsh rarebits, deep fried cheese or in a cold application, e.g., salad bar. Against this background, the objective of this work was to examine the eects of pre-gelatinised maize starch on the rheology, microstructure and processing of imitation cheese and use the ndings to attempt to elucidate why pre-gelatinised maize starch had such dramatic eects on the rheology of imitation cheese on heating. 2. Materials and methods 2.1. Manufacture of imitation cheeses A control imitation cheese was manufactured with the following formulation: 48.8% water, 24.5% rennet casein (82% protein) (Kerry Ingredients, Listowel, Ireland), 26% vegetable fat (Trilby Trading Ltd., Liverpool, England), 2.18% emulsifying salts, [1.08% trisodium citrate, 0.62% citric acid (Jungbunzlauer, Pernhofen, Austria), 0.48% disodium phosphate (Ellis and Everard, Ireland)], 1.67% sodium chloride (Salt Union, Cheshire, England) and 0.1% sorbic acid (Hoechst Ireland Ltd., Dublin, Ireland). All ingredients (except citric acid) were blended in a twinscrew cooker (model CC-010, Blentech Corporation, CA) at 35 C and heated to 78 C using direct steam. Citric acid was added. After 5 min of mixing at 100 rpm the product was packaged, cooled to 4 C and vacuum packed (model C 10H, Webomatic, Bochum, Germany) 24 h later. During cooking the solid screw agitators of the cooker turned in opposite directions causing the product to be folded into the centre and moved around the cooker in a counter clockwise direction resulting in a well-emulsied homogeneous cheese mass. Using a similar manufacturing process, a series of imitation cheeses were prepared with 3%, 5%, 7% or 9% (w/w) pre-gelatinised maize starch (National Starch and Chemical, Manchester, England) by replacing

15%, 25%, 35% or 45%, respectively of the casein protein in the control and reducing the concentration of emulsifying salts used to solubilise the casein accordingly. 2.2. Compositional analysis of imitation cheeses Compositional analysis of imitation cheeses were determined as previously described by Mounsey and ORiordan (2001). 2.3. Microstructural analysis using cryo-scanning electron microscopy (SEM) The microstructure of the imitation cheeses was examined using SEM as previously by Mounsey and ORiordan (2001). 2.4. Microstructural analysis using confocal microscopy The control imitation cheese and the product containing 9% (w/w) pre-gelatinised maize starch were examined using confocal microscopy by a modication of the method used by Guinee, Auty, and Mullins (1999). Slices of imitation cheese, measuring %10 mm 10 mm 3 mm, were cut using a razor blade from a sample of imitation cheese at 4 C. One drop (%50 lL) of a staining solution [0.02% w/ v Nile Red (Sigma Chemical Co., Poole, Dorset, UK) and 0.0005% (w/v) Nile Blue (sulphate salt; Sigma Chemical Co., Poole, Dorset, UK) was added to the at horizontal surface of the cheese slice and a coverslip placed on top. Nile Red stains liquid fat when excited at 488 nm and Nile Blue stains protein when excited at 633 nm. After 10 min at 24 C, the sample was examined using a Zeiss LSM310 confocal scanning laser microscope (Carl Zeiss Ltd., Welyn Garden City, Herts, UK). Images of a layer 20 lm below the sample surface were acquired at both 488 nm (using an Argon laser) and 633 nm (using a HeliumNeon laser) to show the distributions and microstructures of the fat and protein phases. 2.5. Texture prole analysis (TPA) Textural properties were measured with an Instron Universal Testing Machine (Instron Model 4301, Instron Corp., Canton, MA) as described by Mounsey and ORiordan (2001). The textural parameters; hardness and cohesiveness were calculated as described by Bourne (2002). 2.6. Stress relaxation Samples for stress relaxation measurements were prepared as described for TPA measurements (see Section 2.5). Each specimen was lubricated with rapeseed oil at the parallel surfaces in contact with the cross-head and compressed to 80% of its initial height at a cross-head speed of 25 mm/min with the Instron. A 100 N loadcell was used. Relaxation curves were recorded for 8 min

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following deformation, with a sampling rate of 5 point/s. The peak stress (Pa) and stress relaxation time [the time (s) required for the initial stress to relax to 1/e (i.e., 1/the exponential) of its initial value (van Vliet, 1991) were determined. Four samples from each block of imitation cheese were assessed. 2.7. Dynamic rheology evaluation of imitation cheeses Rheological characterisation of the imitation cheeses was undertaken as previously described by Mounsey and ORiordan (2001). Parameters included the storage modulus (G0 ) and the loss modulus (G00 ). 2.8. Statistical analysis The imitation cheeses were manufactured in triplicate. All tests were replicated 4 times. PROC GLM of SAS (SAS Institute, Cary, NC, USA) was used to determine differences between treatment means. Treatment means were considered signicantly dierent at P 6 0.05. 3. Results and discussion 3.1. Processing conditions In the case of the control product a smooth homogeneous product was formed after heating the ingredients in the cheese cooker at 78 C for 5 min. During manufacture, the complete emulsication of oil was assessed visually by noting the time taken for the disappearance of free oil. In the case of pre-gelatinised maize starch-containing products, extra cooking time was necessary at the high levels of casein replacement because of the reduced protein present to emulsify and stabilise the fat droplets. Imitation cheeses containing 5%, 7% or 9% (w/w) pregelatinised maize starch required cooking times of 6.5, 8 and 10 min, respectively. Because it is dispersible in cold water, the pre-gelatinised starch would have hydrated more rapidly than the casein and inhibited the action of the calcium chelating (emulsifying) salts, and consequently reduced the conversion of rennet casein to more soluble sodium paracaseinate. Additionally, the pre-gelatinised starch brought about an immediate viscosity increase in the early stages of imitation cheese manufacture, which would have further decreased the proteins ability to diffuse to the fat interface resulting in poorer fat emulsication. 3.2. Compositional analysis of imitation cheeses The imitation cheese products had similar fat (25.9 0.19%, w/w), moisture (48.9 0.17%, w/w) and pH values (5.79 0.05). Protein content of imitation cheeses decreased from 20.5 0.3% (w/w) at 0% (w/w) starch levels to 11.0 0.1% (w/w) at 9% (w/w) levels of starch inclusion.

3.3. Microstructure The rheological properties of imitation cheese are related to the molecular interactions and spatial arrangements of its component ingredients. In this study, two techniques, cryogenic scanning electron microscopy (SEM) and confocal microscopy were used to examine the imitation cheese microstructures. The SEM image of the control imitation cheese (Fig. 1) is in agreement with earlier ndings (Guinee et al., 1999; Mounsey & ORiordan, 1999, 2001; Song & Park, 1986) in having a smooth protein matrix interspersed regularly with spherical fat globules. The confocal micrograph of the control (Fig. 2) also shows uniformly distributed fat globules up to 20 lm in diameter, in a continuous protein phase. This smooth continuous appearance of the protein matrix arises from the conversion of the insoluble calcium paracasein using emulsifying salts, temperatures of $80 C and mixing to form hydrated sodium paracaseinate. Images (using SEM) of imitation cheeses where pre-gelatinised maize starch was incorporated at levels of 3% to 9% (w/w) in partial casein replacement are shown in Figs. 37. A confocal micrograph of the product containing 7% (w/w) pre-gelatinised maize starch is shown in Fig. 8. The product containing 3% (w/w) pre-gelatinised maize starch had a similar relatively smooth protein matrix compared to the control but had slightly larger fat globules (Fig. 3). The addition of 5%, 7% or 9% (w/w) pre-gelatinised maize starch (Fig. 46, respectively) resulted in smaller more numerous fat globules and a less homogeneous protein matrix than the control or the product containing 3% (w/ w) pre-gelatinised maize starch. At these higher levels of starch inclusion, irregular shaped starch particles became more numerous and disruptive of the protein structure. Previous researchers found that as the fat content of imitation cheese was partially replaced with resistant starch and

Fig. 1. Scanning electron microscopic image of the control (0%, w/w starch) imitation cheese at 500 magnication (F: fat globule; P: protein matrix).

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Fig. 2. Confocal scanning microscopic image of the control (0%, w/w starch) imitation cheese at 500 magnication (F: fat globule; P: protein matrix).

Fig. 5. Scanning electron microscopic image of the imitation cheese containing 7% (w/w) pre-gelatinised maize starch at 500x magnication.

Fig. 3. Scanning electron microscopic image of the imitation cheese containing 3% (w/w) pre-gelatinised maize starch at 500 magnication.

Fig. 6. Scanning electron microscopic image of the imitation cheese containing 9% (w/w) pre-gelatinised maize starch at 500 magnication.

Fig. 4. Scanning electron microscopic image of the imitation cheese containing 5% (w/w) pre-gelatinised maize starch at 500 magnication.

Fig. 7. Scanning electron microscopic image of the imitation cheese containing 5% (w/w) pre-gelatinised maize starch at 3500 magnication (S: starch particle).

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tion cheese. Montesinos-Herrero et al. (2006) also showed noticeable phase boundaries separating the starch from the protein matrix. This exclusion eect increases the eective concentrations of each component and can lead to separation of the system producing a multi-textured gel (Morris, 1991; Tolstoguzov, 1991). 3.4. Compressional analysis Peak stress (measured at 20% compression) and hardness (measured at 80% compression) were used as indices of products strength, while stress relaxation time was used to indicate the viscoelastic behaviour and strength of internal bonding of the imitation cheeses, respectively. The eects of increasing levels of pre-gelatinised maize starch on hardness, peak stress and stress relaxation time of imitation cheese are presented in Fig. 9. Increasing levels of starch had no signicant eect on imitation cheese hardness. In contrast, peak stress values of imitation cheeses increased signicantly (P 6 0.05) from 425 41 kPa at 0% (w/w) starch levels up to 659 34 kPa at 9% (w/w) levels of starch inclusion. The control imitation cheese (0%, w/ w starch) had a stress relaxation time of 35.3 1.6 s. The stress relaxation times increased signicantly (P 6 0.05) to 140 7.5 s with increasing levels of pre-gelatinised maize starch to 9% (w/w), possibly indicating reinforcement of the product through amylose and amylopectin association. Previous authors suggested that the inclusion of starch resulted in an increase in interactions such as salt linkages and hydrogen bonds due to association of amylose and amylopectin molecules (Mounsey & ORiordan, 2001). With the increased addition of pre-gelatinised maize starch in the present work, these forces probably contributed to increased strength in the imitation cheese when subjected to small-scale deformation resulting in the higher peak

Fig. 8. Confocal scanning microscopic image of the imitation cheese containing 7% (w/w) pre-gelatinised maize starch at 500 magnication (F: fat globule; P: protein matrix; S: starch particle).

maltodextrin, the fat globules also became smaller (Montesinos-Herrero et al., 2006). The micrograph of the product containing 5% (w/w) pre-gelatinised maize starch (Fig. 7) at a high magnication (3500) shows the honeycomb nature of the protein matrix surrounding an irregularly shaped object (presumably starch). The honeycomb network may be indicative of protein dehydration. In the product containing 7% (w/w) pregelatinised maize starch, these irregular objects disrupting the protein matrix are particularly evident (Fig. 5). The clarity of the particles in this SEM may be due to more extensive sublimation of moisture exposing more surface detail than in the other products. Montesinos-Herrero et al. (2006) also obtained honeycomb-like structures in imitation cheese containing maltodextrin (Novolose330). They suggested the high water-binding capacity of the maltodextrin may have pulled the water from the protein matrix and promoted its dehydration. The confocal micrograph of the product containing 7% (w/w) pre-gelatinised maize starch (Fig. 8) shows dark stained irregular-shaped regions distributed throughout the protein matrix between the fat globules. These dark regions corresponded to similar areas positively stained for starch (results not shown). Guinee et al. (1999) using confocal microscopy identied similar non-uorescing dark regions as starch particles in cheese analogues containing pre-gelatinised starch. The increased replacement of casein with pre-gelatinised maize starch led to longer processing time to emulsify the oil. This may have been as a result of the poorer emulsifying properties of casein arising from poor hydration due to competition for water by the starch. The SEM and confocal images in the present work provide evidence that interactions between casein, and the pre-gelatinised starch components are probably unfavourable leading to mutual exclusion of each component from the polymer domains of the others in the imita-

160 140

800

700

Hardneess (N)/Stress relaxation time (s)

100 80 60

600

500

400 40 20 0 3 6 9 300

Starch level (%, w/w)

Fig. 9. Textural hardness ( ) at 80% compression, peak stress (h) and stress relaxation time (j) at 20% compression of imitation cheese containing various levels of added pre-gelatinised maize starch. Measurements were undertaken at 22 C. Each curve represents the mean of triplicate trials.

Peak stress (kPa)

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stress values and the longer stress relaxation times with 20% deformation. Zhou and Mulvaney (1998) found that casein gels with higher ratios of casein to water had longer stress relaxation times compared to gels with lower casein to water ratios. These ndings might indicate that the increased stress relaxation times of the imitation cheeses containing increasing levels of pre-gelatinised maize starch are indicative of a less hydrated casein matrix in the presence of starch. The exclusion eect between the protein and starch, which was evident in the micrographs in section 3.3, and its generation of a multi-textured gel probably contributed to increased proteinprotein interactions in the casein matrix resulting in enhanced structural properties when subjected to low deformation. However on large-scale (destructive) deformation, these forces did not contribute to increased imitation cheese strength as measured by textural hardness. With higher levels of starch and the consequent reduction in the protein content there would likely have been a reduction in the corresponding number of bonds capable of reformation after destructive strain. Also, relatively weak adhesion and concentration of stress between the ller (granules or associated molecules of amylose or amylopectin) and the protein matrix probably caused fracture in the system at high deformation. Previous researchers showed the eect of starch in causing structural failure on deformation due to stress localisation at the starchprotein matrix interface (Noel, Ring, & Whittam, 1993). Mounsey and ORiordan (2001) showed that imitation cheeses containing high amylose (2528% amylose) wheat, potato or maize starches, which were added in native granular form in partial replacement of protein, had increased hardness values compared to a control. These authors suggested that the granular shape of the starch as well as hydrogen bond formation following leaching of amylose during processing of the imitation cheese contributed to the increased textural hardness. Imitation cheese hardness and storage modulus (G0 ) have also been increased by partially replacing fat with resistant maize starch-derived products such as maltodextrin and resistant starch (Montesinos-Herrero et al., 2006). In the present work, the lack of granular structure in the added pre-gelatinised starch probably meant it had reduced strength when subjected to large deformation compared to granular starch. 3.5. Dynamic rheological properties of imitation cheeses as a function of temperature Dynamic rheology provides useful information relating to the heat-induced changes in the viscoelasticity of imitation cheese (Mounsey & ORiordan, 2001). The eects of increasing levels of pre-gelatinised maize starch on the rheological parameters; storage modulus (G0 ) and loss modulus (G00 ) of imitation cheese measured as a function of temperature are shown in Figs. 10 and 11, respectively. At 22 C the control product had G0 and G00 values of 71.7 16 kPa and 29.4 4.7 kPa, respectively. The addi-

1000

100

G' (kPa)

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0.1 20 30 40 50 60 70 80 90

Temperature (oC)
Fig. 10. Eect of heating temperature on the storage modulus (G0 ) of imitation cheese containing 0 (j), 3 (h), 5 (M), 7 (), 9 (s) % (w/w) added pre-gelatinised maize starch. Each curve represents the mean of triplicate trials.

100

10

G'' (kPa)
1 0.1 20 30 40 50 60 70 80 90

Temperature (oC)
Fig. 11. Eect of heating temperature on the loss modulus (G00 ) of imitation cheese containing 0 (j), 3 (h), 5 (M), 7 (), 9 (s) % (w/w) added pre-gelatinised maize starch. Each curve represents the mean of triplicate trials.

tion of pre-gelatinised maize starch type to imitation cheese at all the levels examined (with one exception), signicantly increased the G0 and G00 values at 22 C compared to the control. The only exception was the product containing 9% (w/w) pre-gelatinised maize starch that at 22 C had the same G00 as the control. It is noteworthy that despite having similar textural hardness values to the control under destructive deformation, the products containing pre-gelatinised maize starch had signicantly (P 6 0.05) higher G0

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values than the control at 22 C which agrees with higher peak stress values and stress relaxation times obtained also at non-destructive deformation. The G0 of all products decreased signicantly (P 6 0.05) with increasing measuring temperature from 22 C to 85 C, due to melting of the vegetable fat, weakening of proteinprotein interactions and softening of the protein matrix (Mounsey & ORiordan, 1999, 2001; Zhou & Mulvaney, 1998). With heating to $50 C, the elastic (G0 ) and viscous G00 components of the products containing starch decreased in a largely similar manner to the control. However at temperatures from 50 C to 80 C the G0 of the control became lower than the G00 . It has previously been shown that at temperatures above 60 C, the G0 of imitation cheese became lower than the G00 (tan d > 1) with the result of ow and melting behaviour in imitation cheese at these higher temperatures (Hennelly, Dunne, OSullivan, & ORiordan, 2005; Mounsey & ORiordan, 1999, 2001; Zhou & Mulvaney, 1998). Magnitudes of G0 were higher than G00 throughout the temperature range for imitation cheese containing pre-gelatinised maize starch at all levels, indicating that products containing starch retained their elasticity on heating more than the control, particularly at temperatures above 50 C. The eect of increasing pre-gelatinised maize starch in maintaining higher elasticity (G0 ) while showing relatively greater reductions in the viscous component (G00 ) compared to the control on heating is not surprising since the meltable casein matrix was replaced with the thermoplastic starch matrix. Mounsey and ORiordan (1999) showed that increasing levels of added pre-gelatinised starch impaired the meltability of imitation cheese and reduced the tan d values (G00 /G0 ) on heating. Guinee et al. (2004) concluded that starches with high ratio of amylose to amylopectin tend to retrograde and undergo gelation more readily than those with a lower level of amylose during imitation cheese storage. This starch gelation within the imitation cheese structure with storage would probably impede imitation cheese melting ability (Guinee et al., 2004) because of its irreversibility at temperatures reached in the product during heating (<100 C). Montesinos-Herrero et al. (2006) also showed increases in the G0 and G00 of imitation cheese throughout a similar temperature range with resistant starch/maltodextrin inclusion, particularly above 60 C, which they attributed to binding of water by starch. Such binding reduces the water available to plasticise the matrix resulting in elevated G0 and G00 values (Montesinos-Herrero et al., 2006). In the present work, the eects of pre-gelatinised starch on the dynamic moduli of imitation cheese were probably as a result of both dehydration of the protein matrix at higher temperatures where moisture bound by starch was probably unavailable to contribute to the mobility of the associated protein molecules at temperatures above 60 C as well as the lack of thermoplasticity of the starch phase in the imitation cheese. The imitation cheese products produced by the partial replacement of casein with pre-gelatinised starch

may have most application in deep-fried type applications such as breaded sticks, burgers with cheese inserts or cheese products where ow resistance on deep-frying is required. 4. Conclusions The replacement of casein with increasing concentrations of pre-gelatinised maize starch resulted in increased processing time required to form a well emulsied imitation cheese structure. Competition between the starch and the casein for water resulted in a less homogeneous protein matrix, with a honeycomb appearance at high magnication, which may have been an indication of protein dehydration. Microscopic results indicated that interactions between casein and the starch in imitation cheese were unfavourable leading to mutual exclusion of starch phase from the casein phase resulting in the formation of a multi-textured gel with modied textural and rheological properties. With increased levels of pre-gelatinised maize starch up to 9% (w/w), the textural properties such as peak stress and stress relaxation time increased while the hardness under large deformation remained constant. The inclusion of pre-gelatinised maize starch at high levels dehydrated the protein matrix and impaired the thermoplastic properties of the imitation cheese at temperatures above 60 C. Acknowledgement This research has been funded by the Irish Department of Agriculture and Food under the Food Institutional Research Measure (National Development Plan). References
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