Effect of Resistant Starch and Inulin on the Properties of Imitation Mozzarella Cheese

Abstract

The effect of two fat replacers (inulin and resistant starch) on functional properties and microstructure of imitation Mozzarella cheeses was evaluated. Three groups of imitation Mozzarella cheeses were manufactured by adding 0, 2.4, 4.8, 7.2, 9.6, and 12% (w/w) inulin or resistant starch instead of the fat, respectively. Inclusion of the inulin up to 7.2% in cheese, fat content of it was significantly (p ≤ 0.05) decreased and the moisture and pH values of it were significantly (p ≤ 0.05) different. Inclusion of resistant starch up to 9.6% in cheese, fat content of it was significantly (p ≤ 0.01) decreased and the moisture and pH values of it were significantly (p ≤ 0.05) different. The physical properties of these cheeses were significantly different (p ≤ 0.05) among them. The textural properties were determined by instrumental texture profile. With increased levels of inulin or resistant starch from 2.4 to 12% (w/w), the hardness of the resultant imitation cheeses were significantly increased (p ≤ 0.05), while the cohesiveness and springiness decreased and the meltability and stretch ability had reduced (p ≤ 0.05), compared with the control, especially containing resistant starch stretch ability had reduced more. The microstructure of imitation Mozzarella cheese was observed by scanning electron microscopy. The scanning electron microscopy results indicated that interactions between casein and inulin or resistant starch in imitation cheeses accounted for variance properties of imitation cheese. It is concluded that inulin or resistant starch could be used to replace up to 7.2% of the fat in imitation cheese and that the preferred substitution was inulin.

Keywords:

• Imitation Mozzarella cheese
• Resistant starch
• Inulin
• Properties
• Texture
• Microstructure

INTRODUCTION

Imitation Mozzarella cheese is often used on pizzas, lasagna, cordon bleu products, and so on as a cheaper alternative to natural Mozzarella cheese.^[1] Imitation cheese is a relatively high-fat product which contains 20–27% fat; thus there is potential for the formulation of low-fat versions of this product. In the 1990s, the cheese industry successfully introduced many new cheese products many of which were low in fat.^[2,3] A reduction in fat is known to affect the product’s texture and melting ability,^[4] so care is needed when selecting the type of fat replacer, especially since imitation cheese products are typically designed for molten cheese applications. Lots of food researchers have examined the effect of reducing the fat content of natural Mozzarella and process cheeses.^[3,5–7] Recently, Montesinos, Cottell, O’Riordan, and O’Sullivan^[4] sought to replace up to half the fat content of imitation cheeses with two types of resistant starch (RS; Novelose 240 or 330). However, it was found that reducing the fat caused the cheeses to become harder, with the lowest fat cheese (12%) having a hardness value considered undesirable from a sensory perspective. Hennelly, Dunne, O’Riordan, and O’Sullivan^[8] showed that increasing the moisture content of imitation cheese reduced the hardness problems associated with replacement of fat by a soluble dietary fiber (inulin). It is possible that a similar approach might be successful in reducing excessive hardness of reduced fat cheeses containing Novelose 240.

Inulin is a generic term composition of heterogeneous blends of fructose-oligosaccharides. Nutritionally, inulin is regarded as a soluble dietary fiber.^[9,10] RS as insoluble fiber, but has the physiological benefits of soluble fiber. Both inulin and RS are dietary fibers that offer many beneficial effects on the gastrointestinal system. These healthy effects are revealed by selectively stimulating the growth or activity of certain indigenous bacteria.^[11,12] Being metabolized by advantageous colonic microorganisms which are called probiotics is the important property of dietary fibers. This may lead to an enhanced bacterial growth and increased fecal bulk, therefore, fibrous materials have prebiotic effects. The health benefits of dietary fibers for consumers such as reducing glycaemic responses to carbohydrate ingestion, decreasing the risk of colorectal cancer, and enhancing mineral absorption makes it a valuable prebiotic. In the colon, RS lowered colonic pH and increased faucal bulk. The portion of the latter that was fermented by the intestinal microflora produced a wide range of short-chain fatty acids, which has a positive impact on bowel health, including a degree of protection against bowel cancer.^[13–15] Inulin has been successfully added, as a partial fat substitute, to imitation Mozzarella cheese. It didn’t affect the cheese meltability. The inulin-containing cheeses at 54 g moisture per 100 g had increased hardness but the 56 g moisture per 100 g had similar hardness to the control.^[8]

There are several studies about inulin or starch as replacing partial fat. They had been incorporated into imitation cheese and studied the effect on the characteristics of imitation cheese. In the past studies, the main source of protein was milk rennet casein in imitation cheese, but this study is yak milk rennet casein made from yak milk which is richer in almost all the main nutritional components and is regarded as natural concentrated milk compared with cows’ milk.^[16] The concentrations of micellar minerals in yak milk were higher than that of cows’ milk. The concentrations of calcium and phosphate in the serum phase of yak milk are much higher than those in cows’ milk. The hydration of casein micelles in yak milk was higher than that of cows’ milk. The physico-chemical properties of casein micelles in yak milk differ greatly from that of cows’ milk. The incorporation of inulin and RS, as a partial fat replacer, specifically in yak milk imitation cheese, has not been previously reported. Therefore, the objectives of this study is to investigate the level of inulin and RS incorporation on the textural and microstructural properties of imitation cheese.

MATERIALS AND METHODS

Manufacture of Imitation Mozzarella Cheeses

A control imitation Mozzarella cheese was manufactured with the following formulation: 49.3% water, 22% yak milk rennet casein (90.7% protein; Hua Ling Ltd., Gan Su, China), 24% vegetable fat (Lu Hua Ltd., Shan Dong, China), 2.1% emulsifying salts (1.4% trisodium citrate, 0.7% disodium phosphate [Sinopharm Chemical Reagenent Co., Ltd., Shang Hai, China]), 1.9% sodium chloride (Sinopharm Chemical Reagenent Co., Ltd., Shang Hai, China), 0.6% citric acid (Yao Hua Ltd., Tian Jin, China) and 0.1% Sorbic acid (Hoechst Ireland Ltd., Dublin, Ireland). All dry ingredients (casein, emulsifying salts, inulin/RS) were mixed for 1 min in a twin-screw cooker (model CC-010, Blentech Corporation, CA) at room temperature. After 1 min, the vegetable fat was added and all ingredients were heated to 85°C using direct steam and sheared continuously. Citric acid was added. After 5 min of mixing at 100 rpm the product was packaged, cooled to 4°C and vacuum packed (model C10 H, 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 center and moved around the cooker in a counter clock-wise direction resulting in a well-emulsified homogeneous cheese mass. Using a similar manufacturing process, a series of imitation cheeses were prepared with 2.4, 4.8, 7.2, 9.6, and 12% (w/w) inulin (Energave, Mexico) by replacing 10, 20, 30, 40, and 50%, respectively, of the vegetable fat in the control and reducing the concentration of emulsifying salts used to solubilize vegetable fat accordingly. An additional product was also manufactured in which the vegetable fat was replaced with rice RS (Xiwang Ltd., Shan Dong, China), and added the same emulsifying salt levels as the control.

Compositional Analysis

Samples of imitation cheese were analyzed 1 day after manufacture for protein,^[17] moisture,^[4] and fat (National Standards Authority of China, 2003) content. The pH was determined by the electrodes of a pH meter (model 9450, Unicam Ltd., CA, England) directly into a small block of imitation cheese, equilibrated at 22°C. All compositional analyses were performed in triplicate.

Instrumental Texture Profile Analysis (TPA)

Textural profile analysis of imitation Mozzarella cheese was determined after 48 h manufacture using an Instron Universal Testing Machine (Instron Model 4301, Instron Corp., Canton, MA, USA). The textural parameters were calculated according to the method of Mounsey.^[18] Six samples were analyzed from each cheese batch. The determination parameters were that pretest speed was 2.0 mm/s, test speed was 1.0 mm/s, post speed was 2.0 mm/s, two-bite time intervals was 5.0 s and the probe is P/5.

Meltability Test

The meltability was determined in triplicate using a modification method of Mounsey and O’Riordan.^[19] Cylinders (25 mm diameter, 20 mm height, 10 ± 0.05 g weight) were cut from blocks of imitation cheese, wrapped in aluminium foil and tempered to 10°C. The foil was then removed and the cylinders were individually placed into one end of a Pyrex glass tube (250 mm length, 30 mm diameter). The end containing the cheese was closed with a solid rubber stopper and the opposite end was plugged with a stopper pierced with a hole to allow gas to escape. The tubes were placed horizontally in a conventional oven at 180°C for 10 min. The tubes were removed from the oven and, after 5 min at room temperature, the horizontal distance flowed (length) from a reference line was measured in millimeters using vernier caliper.^[19,20]

Stretch Ability

The stretch ability of the molten imitation Mozzarella cheese on a pizza base was measured by uniaxial extension at a velocity of 0.066 m/s.^[21] Before heating, the shredded cheese was distributed uniformly at a fixed loading (2.5 kgm^–2) onto a pizza base, The base with cheese was then baked at 232°C for 5 min in an electric fan oven. After baking the pizza was placed on the platform unit of a custom-built stretch apparatus, which consisted of fixed and rolling elements. The pizza was positioned so that the interface between the two halves of the base coincided with the junction between the fixed and rolling elements. The pizza was clamped, one-half to the fixed element, the other to the rolling element. The rolling element was drawn along a rail system at a constant speed of 0.066 ms^–2 by a winch, resulting in stretching of the molten cheese mass. Stretch was delimited as the distance traveled by the mobile element to the point where all extended strings and/or sheets of molten cheese between the two halves of the pizza base had broken.^[22]

Microstructure Analysis Using Scanning Electron Microscopy (SEM)

Microstructure analysis of imitation cheese was performed as described by Mounsey and O’Riordan.^[18] Blocks approximately 1 × 3 × 5 mm were cut from the stored imitation cheese using a scalpel, mounted on a specimen holder, and dehydration. The samples were then sublimed, coated, and subsequently introduced into the microscope chamber for examination. Several images were taken of each specimen at different magnifications ranging from 500 up to 2000 fold. Two samples were analyzed from each type of imitation cheese.

Statistical Analysis

The imitation Mozzarella cheeses were manufactured in triplicate. All tests were replicated four times. Procedure (PROC) general linear model (GLM) of SAS (SAS Institute, Cary, NC, USA) was used to determine differences between treatment means. Treatment means were considered significantly different at p < 0.05.

RESULTS AND DISCUSSION

Mounsey^[18] and Noronha^[20] reported that imitation cheese was typically used in pizzas, cheese sauces, and other related foods which required different functional properties for both the melted and unmelted states. Unmelted cheese must have good shred ability and texture, while melted cheese should flow well without browning/blistering.

Compositional Analysis of Imitation Mozzarella Cheeses

The compositional analyses of the imitation Mozzarella cheeses containing various levels of inulin or RS were respectively shown in Tables 1 and 2. From Table 1 we can see, when the replacement of the fat with inulin, no significant (p > 0.05) differences was obtained for protein. But all products had significant (p ≤ 0.05) differences in moisture and pH values, when the inulin was added up to 7.2%. Fat content of imitation cheeses was significantly (p ≤ 0.05) decreased as the inulin content increased.

TABLE 1 Compositional analysis of imitation cheese containing inulin

Inulin (%)	Protein (%)	Fat (%)	Moisture (%)	pH
0(control)	19.20 ± 0.15^a	23.21 ± 0.14^a	47.58 ± 0.43^b	5.52 ± 0.03^c
2.4	19.26 ± 0.14^a	20.83 ± 0.29^b	47.67 ± 0.31^b	5.54 ± 0.03^c
4.8	19.29 ± 0.14^a	18.29 ± 0.14^c	47.74 ± 0.48^b	5.54 ± 0.03^c
7.2	19.31 ± 0.39^a	16.01 ± 0.22^d	48.02 ± 0.58^a	5.60 ± 0.02^b
9.6	19.36 ± 0.18^a	13.41 ± 0.24^e	48.07 ± 0.27^a	5.63 ± 0.03^b
12	19.34 ± 0.31^a	11.02 ± 0.30^f	48.16 ± 0.67^a	5.71 ± 0.05^a

Data represents the mean ± standard deviation of three batches of cheese; triplicate analysis was performed on each batch;

Numbers with the different letter were significantly different (p < 0.05) in the same row, otherwise it was not significantly different.

TABLE 2 Compositional analysis of imitation cheese containing RS

Resistant starch (%)	Protein (%)	Fat (%)	Moisture (%)	pH
0(control)	19.20 ± 0.15^a	23.21 ± 0.14^a	47.58 ± 0.43^c	5.52 ± 0.03^b
2.4	19.23 ± 0.29^a	20.97 ± 0.38^b	47.65 ± 0.49^c	5.53 ± 0.05^b
4.8	19.19 ± 0.25^a	18.32 ± 0.21^c	47.71 ± 0.34^c	5.54 ± 0.03^b
7.2	19.13 ± 0.19^a	16.08 ± 0.16^d	47.74 ± 0.50^c	5.54 ± 0.03^b
9.6	19.27 ± 0.24^a	13.37 ± 0.19^e	47.88 ± 0.30^b	5.56 ± 0.04^b
12	19.15 ± 0.28^a	11.13 ± 0.22^f	48.15 ± 0.25^a	5.64 ± 0.03^a

Data represents the mean ± standard deviation of three batches of cheese; triplicate analysis was performed on each batch;

Numbers with the different letter were significantly different (p < 0.05) in the same row, otherwise it was not significantly different.

When the replacement of the fat with RS, from Table 2 we can see no significant (p > 0.05) differences was obtained for protein. But all products had significant (p ≤ 0.05) differences in moisture and pH values, when the RS was added up to 9.6%. Fat content of imitation cheeses was significantly (p ≤ 0.01) decreased as the RS content increased. Thus, using different fat replaces will have different effective on imitation cheeses. Compared with inulin, RS had less effective on imitation cheeses.

With inulin or RS content increased, the moisture of imitation cheeses increased. The reason was that inulin or RS can improve water retention. It may result from the highly water absorption and water-holding ability of RS or inulin.^[23] The pH of imitation cheeses also increased, the reason may be caused by the increase of moisture. The possible cause of pH change was that the levels of lactic acid was diluted with water. When compared with the reported imitation Mozzarella cheese,^[24] the differences in moisture and pH values chiefly accounted for fat replacer.

Instrumental Textural Properties Analysis of Imitation Cheeses

The effects of the different inulin or RS on textural of imitation Mozzarella cheese were presented in Fig. 1. According to the Fig. 1a, the inulin or RS significantly (p ≤ 0.05) increased the imitation cheese hardness, moreover the RS increased more. The control imitation cheese (0% inulin or RS) had a textural hardness of 295 ± 11 (g). Increasing levels of RS or inulin to 5% levels significantly (p ≤ 0.05) increased the hardness to 381 ± 9 (g) or 365 ± 7 (g) respectively, when increasing levels of RS or inulin to 12% increased the hardness to 585 ± 10 (g) or 379 ± 14 (g), respectively. The control imitation cheese (0% inulin or RS) had a textural cohesiveness of 0.72 ± 0.006. After increasing levels of RS to 12% it decreased to 0.68 ± 0.005. When increasing levels of inulin to 9.6% it significantly (p ≤ 0.05) decreased to 0.64 ± 0.005.

FIGURE 1 The textural of imitation cheeses containing inulin or RS; (a) Hardness; (b) Cohesiveness; (c) Springiness.

According to the Fig. 1c, the inulin or RS significantly (p ≤ 0.05) decreased the imitation cheese springiness. The control imitation cheese (0% inulin or RS) had a textural springiness of 0.78 ± 0.002. Increasing levels of RS or inulin to 5% it significantly (p ≤ 0.05) decreased to 0.76 ± 0.004 or 0.76 ± 0.002 respectively, when increasing levels of RS or inulin to 12% it decreased to 0.68 ± 0.006 or 0.66 ± 0.004, respectively.

Montesinos et al.^[4] had shown that it was possible to reduce up to half the fat of imitation cheese with RS while still maintaining reasonable functional properties. One of the disadvantages of such fat replacement was a substantial increase in hardness, which other studies^[8,25] had also observed. Replacing the fat content by inulin or RS significantly (p < 0.05) increased the hardness and decreased the springiness (p < 0.05), similar to the observations of Haralampu^[26] and Noronha.^[20] When increased levels of RS or inulin from 2.4 to 9.6%, it did not significantly affect the cohesiveness (p < 0.05) of the imitation cheese (Fig. 1). Increasing inulin or RS content of imitation cheese caused an increase in cheese hardness and this increase may partly be attributed to the reduction of fat content. Zisu et al.^[27] showed cheese texture was related to the microstructure, with a reduction of fat resulting in the protein matrix becoming more compact, with the spaces that were once occupied by fat globules decreasing. In addition to fat reduction, the presence of RS also contributed to increase hardness. But Hennelly et al.^[8] had shown that the effect of fat replacement on cheese texture was dependent on the nature of the fat being replaced. The cohesiveness values of imitation cheese did not change with increasing moisture content, a finding consistent with those by Hennelly et al.^[8] Noronha et al. observed that cheese cohesiveness was not significantly affected when the fat content decreased, which may be partly explained by the microstructure.^[20] The light microscopy (LM) and SEM images showed there was little interaction between starch particles and the protein matrix. Thus, these trends were among the most difficulty to explain in the present study as they seem to result from a complex interaction of a number of variables.

Meltability and Stretch Ability of Imitation Mozzarella Cheese

All inulin or RS significantly (p < 0.05) decreased meltability of imitation Mozzarella cheese, products relative to the control with RS causing the greatest reduction (Fig. 2). With the addition of inulin or RS increasing, the meltability significantly decreased (p < 0.05). The meltability of inulin or RS containing products differed significantly from each other, with the exception of the product containing 7.2% (w/w) RS, which had no significant (p < 0.05) difference. RS generally caused a greater reduction in imitation Mozzarella cheese meltability, compared to the equivalent inulin.

FIGURE 2 Meltability of imitation cheeses with or without various inulin or resistant starch measured 48 h after manufacture.

The meltability of imitation Mozzarella cheese was one of its most important characteristics, as one of the main applications of the product was as an ingredient in cooked foods. Mounsey had shown that liquid fat contributes to the melting properties of imitation cheese.^[18] El-Bakry observed that water played an important role in cheese due to its hydration and solubilization of both protein and salts within the matrix. Protein hydration allowed greater interaction of the protein with the fat phase ensuring good emulsification and this led to good meltability in the cheese.^[28] Increasing inulin or RS content of imitation cheese, the moisture was significantly different (p ≤ 0.05), compared with the control (Tables 1 and 2). When adding inulin or RS, the water of imitation cheese increased, but the effect of decreased fat content should be more intensity.

The stretch ability of the heated cheese showed a trend similar to that noted for meltability (Fig. 3). All cheeses adding inulin or RS significantly (p < 0.05) decreased stretch ability. With the addition of RS increasing, the stretch ability significantly decreased (p < 0.05). When added of 12% RS, imitation cheese hardly had stretch ability. The stretch ability of inulin or RS containing products differed significantly from each other. RS generally caused a greater reduction in imitation cheese stretch ability, compared to the equivalent inulin. Adding inulin from 4.8 to 9.6%, the stretch ability of imitation cheese had not significantly decreased (p < 0.05). But when adding 12%, the stretch ability significantly decreased (p < 0.05) compared with others.

FIGURE 3 Stretch ability of imitation cheeses with or without various inulin or resistant starch measured 48 h after manufacture.

Microstructure Analysis of Imitation Mozzarella Cheese

The protein and fat components were responsible for the resulting texture and functionality of cheeses. In this study, SEM was used to examine the imitation cheese microstructures.^[29] The SEM image of the control imitation cheese (Figs. 4a and 4e) was in agreement with earlier research in having a smooth protein matrix interspersed regularly with spherical fat globules. Images (SEM) of imitation cheeses where inulin or RS was incorporated at levels of 2.4 to 12% (w/w) in partial fat replacement were shown in Figs. 4b–4d or Figs. 4f–4h, respectively.

FIGURE 4 Scanning electron microscopic image of the imitation Mozzarella cheese control (a, e), containing inulin; (b: 2.4%, c: 7.2%, d: 12%) or RS (f: 12%, g: 12%, h: 7.2%) at 500, 1000, 2000× magnification.

The product containing 2.4% (w/w) inulin had a similar relatively smooth protein matrix, compared to the control but had slightly larger fat globules (Fig. 4b). The addition of 7.2 and 12% (w/w) inulin (Figs. 4c and 4d, respectively) resulted in lesser numerous fat globules and had more porosity in protein matrix, especially Fig. 4d, than the control or the product containing 2.4% (w/w) inulin. The diverseness properties of imitation Mozzarella cheese were related to the molecular interactions and spatial arrangements of its component ingredients. Lesser fat globule numerous and size had been associated with reduced meltability, there was relationship between fat globule numerous and cheese meltability in the present study (Fig. 2), but objected to the observations of Mounsey and Riordan.^[18] The protein matrix for all the inulin-incorporated cheeses appeared to be much more porosity, with apparent honeycombing suggesting that the added inulin had bound any loose water, thus preventing the formation of honeycomb structures during sample preparation.

In addition to the fat replacement imbibing water, the porosity of the protein matrix of containing inulin cheeses may had partially contributed to greater moisture retention over cheeses make with containing RS that exhibited a dense protein network.^[24] According to McMahon et al., fat replacement may interfere with shrinkage of the protein matrix and lower the force involved in expelling water from curd particles.^[25] They showed that a low-fat Mozzarella cheese made with a particular type of fat replacement increased the openness of cheeses and led to an increase in moisture content.

FIGURE 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.^[4]

FIGURE 6 Electron micrographs (×7500) of imitation cheese containing10% of either (A) Novelose240 or (B) Novelose330. S: starch; P: protein matrix.^[4]

SEM showed clear differences between the control and containing RS. When RS was incorporated into an imitation cheese formulation, its presence could be seen in Figs. 4f–4h. The granules of RS had a relatively uniform size and similar spherical shape and show diffusion of the granule boundaries into the protein matrix. In addition, the manner in which the particles of RS were incorporated in the protein matrix was different, with a less distinctive phase boundary. Similarly to the reports that particles of N330 (S in Figs. 5c and 6b) 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.^[4] Mounsey and Riordan^[18] 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, similar to the observations of this study. The RS used in imitation Mozzarella cheese had a higher concentration of amylose and probably increased the hardness of the cheese by the same mechanism.

CONCLUSION

The replacement of fat with increasing concentrations of inulin or RS resulted that they conspicuously affected properties of imitation cheese. From the present work, it can be concluded that containing inulin in cheese had significantly (p ≤ 0.05) differences in moisture and pH values. With increased levels of inulin or RS to 12% (w/w), the resultant imitation cheeses were harder, less cohesive and springiness, and also reduced meltability and stretch ability, especially containing RS had reduced more stretch ability, compared to the control. Microscopic results indicated that interactions between casein and inulin or RS in imitation cheese were unfavorable leading to mutual exclusion of inulin or RS phase, especially RS. During the casein phase it resulted that many honeycomb appearance at high magnification which may affect properties of imitation cheese.

FUNDING

This work was supported by the project Heilongjiang Province Educational Department General Project and Heilongjiang Province Colleges and Universities in the Industrialization of Scientific and Technological Early Stage of Research and Breeding Project.

ACKNOWLEDGMENTS

The authors thank Mr. Li Xiao-Dong and Dai Xian-Qi for his technical assistance, Shanghai Sangon Biotech Ltd., Merck China, and Heilongjiang Academy of Agricultural Sciences.