ARTICLE IN PRESS

International Dairy Journal 14 (2004) 161172

Effect of pH and calcium level on the biochemical, textural and functional properties of reduced-fat Mozzarella cheese
Jeremiah J. Sheehan, Timothy P. Guinee*
Dairy Products Research Centre, Teagasc, Moorepark, Fermoy, Co. Cork, Ireland Received 1 April 2003; accepted 14 July 2003

Abstract Reduced-fat Mozzarella cheeses were made using starter culture (control, CL), lactic acid (directly acidied, DA) or a combination of starter culture and lactic acid (DAS1 and DAS2) to reduce the pH during manufacture. The resultant cheeses differed in pH at 5 d and calcium content (mg g1 protein): CL, 5.42 and 28.58; DA, 5.89 and 19.38; DAS1, 5.64 and 18.54; and DAS2, 5.49 and 18.31. Compared to CL, the DA and DAS cheeses had higher levels of moisture, moisture-in-non-fat substances, non-expressible serum (g g1protein), and lower levels of protein. These changes were paralleled by higher mean values for stretchability and owability, and lower mean values for rmness in the DA and DAS cheeses over the 70-d ripening period. Reducing the pH from 5.89 in the DA cheese to p5.64 in the DAS cheeses, by adding starter culture and increasing the time between whey drainage and curd milling, increased primary proteolysis in the unheated cheese and the stretchability and owability (as measured using the modied Schreiber method) of the melted cheese. r 2003 Elsevier Ltd. All rights reserved.
Keywords: Reduced-fat Mozzarella; pH; Calcium; Texture; Proteolysis; Functionality of heated cheese

1. Introduction Reduction in the fat content of cheese, including lowmoisture part-skim Mozzarella (LMM), increases the hardness, springiness, viscosity and elasticity of the unheated cheese. Similarly, the quality of the heated cheese is impaired, as reected by a reduction in owability and increases in apparent viscosity and chewiness (Tunick et al., 1993a, b; Merrill, Oberg, & McMahon, 1994; Fife, McMahon, & Oberg, 1996; Rudan & Barbano, 1998; Rudan, Barbano, Yun, & Kindstedt, 1999; Metzger, Barbano, Kindstedt, & Guo, 2001b; Metzger, Barbano, & Kindstedt, 2001a). These defects are associated with concomitant reductions in moisture-in-non-fat substances (MNFS), proteolysis and amount of free oil, and increases in the proportion of intact casein. Various approaches have been used to counteract the adverse effects of fat reduction on heatinduced functional properties of Mozzarella cheese. These include homogenisation of cheesemilk to increase the surface area of the fat phase (Jana & Upadhyay,
*Corresponding author. Tel.: +353-25-42204; fax: +353-25-42340. E-mail address: tguinee@moorepark.teagasc.ie (T.P. Guinee). 0958-6946/$ - see front matter r 2003 Elsevier Ltd. All rights reserved. doi:10.1016/S0958-6946(03)00167-5

1991; Tunick et al., 1993b; Poudaval & Mistry, 1999); alterations of make-procedure (e.g., varying pasteurisation temperature, preacidication of milk, altering cut size and cook temperature) to increase the levels of MNFS and reduce the calcium content (Merrill et al., 1994; Metzger et al., 2001b); addition of fat replacers to the cheesemilk (Rudan, Barbano, Guo, & Kindstedt, 1998; McMahon, Alleyne, Fife, & Oberg, 1996); and/or addition of exopolysaccharide-producing cultures (Perry, McMahon, & Oberg, 1998). These methods have resulted in varying degrees of success. Metzger et al. (2001b) reported that preacidication of milk with organic acids to a pH of 5.85.6, in combination with a starter culture, resulted in a reduction in calcium level of low-fat (6%, w/w) LMM but did not signicantly affect the levels of moisture or MNFS; the experimental cheeses generally had a lower pH (p5.2) than the control (B5.45) at times X15 d. The reduction in calcium content resulted in an increase in protein hydration and a decrease in hardness of the unheated cheese, and an increase in owability of the melted cheeses, to an extent depending on the preacidication pH, the type of acid used and nal pH of the cheese. More recently, Guinee, Feeney, Auty, and

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Fox (2002) found that reducing the calcium-to-casein ratio from B28 to B21 mg g1, by preacidication of milk prior to renneting, resulted in higher levels of moisture and MNFS, and a lower protein level in the resultant directly acidied LMM. These changes resulted in marked increases in protein hydration in the unheated cheese and in the owability and stretchability of the heated cheeses, even though the pH of the LMM from pre-acidied milk was relatively high, i.e., B5.8 compared to 5.4 in the control LMM made using starter culture (Guinee et al., 2002). In general, reducing the pH of cheese from 5.8 to 5.4 increases the ratio of solubleto-colloidal calcium (Guinee, Harrington, Corcoran, Mulholland, & Mullins, 2000b), an occurrence, which would be expected to increase the degree of casein hydration (Sood, Gaind, & Dewan, 1979; Kindstedt, Zielinski, Almena-Aliste, & Ge, 2001; Metzger et al., 2001b; Ge, Almena-Aliste, & Kindstedt, 2002). Moreover, the hydration of para-casein in dilute model systems increases with decreasing pH in the range 65.3 (Creamer, 1985). The objective of the current study was to evaluate the effects of lowering the calcium-to-casein ratio and altering the pH of reduced-fat LMM on its texture and heat-induced functionality.

2. Materials and methods 2.1. Cheese manufacture In each of three trials, four types of reduced-fat LMM were manufactured by alteration of the make-procedure. Acidication was achieved either by: a starter culture as in conventional production (control cheese, CL), addition of lactic acid (directly acidied cheese, DA), or by a combination of added lactic acid and starter culture (directly acidied cheeses with starter culture, DAS1 and DAS2). The four cheeses were further differentiated according to pH of milk at rennet addition (setting), pH of the curd at whey drainage, and pH of the curd at milling/plasticisation (Table 1). Mid-lactation milk from the autumn-calving Friesian herd at the Dairy Production Centre, Moorepark was collected on three separate occasions over a 4-week period. The raw milk (2200 L) was standardised to a protein-to-fat ratio of 3.4:1, by the addition of skim milk, held overnight at o10 C, pasteurised at 72 C 15 s, and pumped into cylindrical, jacketed, stainless steel, 500 L vats with automated variable-speed cutting and stirring (APV Schweiz AG, CH-3076 Worb 1, Switzerland). The CL cheese was manufactured as described previously (Guinee, Mulholland, Mullins, & Corcoran, 2000c) with certain modications, as described below. Milk (454 kg) was inoculated with a starter culture consisting of Streptococcus thermophilus

and Lactobacillus helveticus, added at levels of 1.0% and 0.5%, w/w, respectively. The cultures were grown separately overnight at 37 C in 10%, w/w, reconstituted skim milk powder that had been heat-treated at 90 C for 30 min. Chymosin (Single strength Chy-max plus, B50 Chymosin units mL1; Pzer Inc, Milwaukee, WI, USA) was added at a level of 0.18 mL kg1 milk and the milk allowed to set at 36 C. All gels were cut at a rmness that was equivalent to an elastic shear modulus (G 0 ) value of 60 Pa, as measured using low-amplitude strain oscillation rheometry (Guinee, Pudja, & Mulholland, 1994). After cutting (B30 min later), the curds whey mixture was allowed to heal for 10 min and then stirred continuously, with the speed of stirring being increased gradually from 11 to 25 rpm over 22 min. The curdswhey mixture was cooked to 38 C, at a rate of 0.2 C min1 and continually stirred until the pH of the curd decreased to 6.1. The mixture was then pumped to rectangular jacketed stainless 500 L nishing vats (warmed to 38 C by circulating water), where the curdwhey mixture was allowed to settle for 2 min prior to drainage of the whey. After whey drainage, the curds (B3639 kg) were cheddared in the nishing vat, and milled at pH 5.23, as measured at 38 C by inserting a pH probe (Radiometer Analytical S.A., Villeurbanne Cedex-Lyon, France), calibrated at 38 C with reference buffer solutions, directly into the curd. The milled curd was dry salted at a level of 4.1%, w/w, mellowed for 20 min, and transferred to the stretching unit (Automatic Stretching Machine, Model d; CMT, S. Lorenzo di Peveragno CN, Italy). The curd was heated to 58 C by dosing with hot water at 78 C (used at a rate of B1.4 kg kg1 curd and added over a period of 8 min) and batch plasticised mechanically. The molten plastic curd mass was then mechanically conveyed by auger to the moulding section where it was formed into a cylindrical mass which was automatically cut into 2.3 kg portions (24 cm long) that were lled into rectangular moulds. The resultant blocks were cooled in dilute brine (10%, w/w, NaCl; 0.2%, w/w, Ca; pH 5.1) at B4.0 C for 30 min to allow cooling to a surface temperature of 24 C and a core temperature of o45 C. The cheeses, approximately 14 loaves per vat, were then vacuum-wrapped and stored at 4 C. For the manufacture of DA, DAS1 and DAS2 cheeses, the milk (29 C) was acidied directly to pH 5.6 with dilute lactic acid (5%, w/w) (Wardle Chemicals, Calveley, Nr. Tarporley, Chesire, CW6 9JW). The cheesemaking procedure was otherwise similar to that for the CL cheese, apart from the differences as outlined in Table 1. While the DA milk was not inoculated with starter culture, the DAS1 and DAS2 cheesemilks were inoculated with the same type and level of starter culture as the CL cheesemilk. Owing to the high tendency of the DA and DAS curds to mat following cutting of the gel, the healing period (3 min) was shorter than that of the

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J.J. Sheehan, T.P. Guinee / International Dairy Journal 14 (2004) 161172 Table 1 Treatments and details of the make-procedures used for manufacture of experimental reduced-fat Mozzarella cheeses Cheese code CL pH at different stages of manufacture At set At whey drainage At curd milling Acidication procedurea pH adjustment before set pH decrease between setting and whey drainage pH decrease between whey drainage and salting Details of cheesemaking stepsb Temperature of milk on adding acid ( C) Set temperature ( C) Stirring speed, rate of increase (rpm/min) Temperature at scald ( C) pH at milling Times for cheesemaking stages (min)d Ripening period (starter addition to set) Set (rennet addition) to cut End of cut to stirring (healing) Start of stirring to scald Scald to start of whey drainage End of drain to milling 6.41 6.13 5.23 DA 5.60 5.60 5.61 DAS1 5.60 5.60 5.40 DAS2 5.64 5.54 5.20 163

Starter culture Starter culture Starter culture

Lactic acid None None

Lactic acid Starter culture Starter culture

Lactic acid Starter culture Starter culture

NAc 36 1.0 38 5.23

29 29 0.6 38 5.60

29 29 0.6 38 5.40

29 29 0.6 38 5.20

30 31 10 14 41 85

NA 14 3 22 7 56

10 13 3 22 9 77

10 13 3 22 11 165

a Acidication during cheese manufacture was achieved by addition of a starter culture (CL), lactic acid (DA), or a combination of lactic acid and a starter culture (DAS1 and DAS2). Dilute lactic acid solution (5%, w/v), prepared by diluting concentrated lactic acid (80%, w/v) with distilled water, was used. b Full details of cheese manufacture are given in text. c NA, not applicable. d The values given are the means of the three trials.

CL curd. The cooking rate (0.5 C min1) for the DA and DAS curds was similar and higher than that of the CL curds. After reaching the scald temperature, the DA and DAS curdwhey mixtures were held for a period of 711 min to allow the curd particles to attain the desired resilience. Following whey drainage, the DA curd was cut into slabs, which were trenched, piled and held for B60 min (Table 1). The DAS1 and DAS2 curds were treated similarly and held until the pH reached pH 5.40 (DAS1) or pH 5.20 (DAS2). The DA and DAS curds were treated as for CL from this stage of cheesemaking onwards. 2.2. Sampling of cheese Cheese was grated to yield particles of o1 mm, in a Krups Rotary 350 food processor tted with a universal blade (Robert Krups GmbH & Co, Postfach 190460, D5650 Solingen 19, Germany). Shredded cheese was prepared by cutting the block, stored at 4 C, into 25 mm cubes (Cheese Blocker; Bos Kaasgereedschap, Bodengraven, Postbus 2410 AC, Netherlands) and immediately shredding in a Hallde RG-350 machine (AB Hallde Maskiner, Kista, Sweden) using the raw

food grating disc (K) to yield shreds E25 mm long and E4 mm diameter. Cheese was cut with a Unika cutter (model WG-300; BOS Kaasgereedschap, Bodegraven, Netherlands) giving 6.5 mm thick slices from which discs (45.5 mm) were cut using a stainless steel borer. Cylindrical samples (weight, 1570.05 g; B13 mm diameter and 33.7 mm height) were procured using custommade stainless steel borers.

2.3. Analysis of cheese 2.3.1. Composition and proteolysis Grated cheese was analysed in triplicate, at 5 d, for moisture, fat, protein, salt, calcium, and phosphorous using standard IDF methods (Guinee, Auty, & Fenelon, 2000a). The pH was measured on a cheese slurry prepared from 20 g cheese and 12 g distilled water (British Standards Institution, 1976). The levels of total nitrogen soluble at pH 4.6 (pH4.6SN) and in 5% phosphotungstic acid soluble nitrogen (PTAN) were measured, as described previously (Fenelon, Guinee, Delahunty, Murray, & Crowe, 1999).

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All cheeses were analysed by ureapolyacrylamide gel electrophoresis (PAGE) at 1, 35, and 70 d. PAGE was performed on a Protean II xi vertical slab gel unit (BIORAD Laboratories Ltd., Watford, Herts, UK), using a separating and a stacking gel, as described by Feeney, Fox, and Guinee (2001). Cheese samples (equivalent to 2.5 mg cheese protein) were dissolved in 1 mL of sample buffer and incubated at 55 C for B10 min. The gels (1 mm thick) were pre-run at 280 V for B40 min prior to sample application and 9 mL samples were loaded using a micro-syringe (Hamilton, CH-7402, Bonaduz, Switzerland). The samples were run through the stacking gel and separating gel at 280 and 300 V, respectively. The gels were stained overnight in Coomassie brilliant blue G250 and de-stained in repeated changes of distilled water. 2.3.2. Cheese rheology All cheeses were analysed using compression on a TAHDi Texture Prole analyser (Stable Micro Systems, Godalming, Surrey GU7 1YL, England). Six samples (25 25 25 mm cubes) were obtained from each cheese, placed in an airtight plastic bag and held at 8 C overnight. Samples were withdrawn from 8 C and immediately compressed to 30% of original height at a rate of 60 mm min1 at room temperature. Fracture stress was dened as the force per unit area to induce fracture, and rmness as the force required to compress the cheese to 30% of its original height. 2.3.3. Non-expressible serum The levels of cheese serum expressed by centrifugation at 12 500g at 20 C, was measured as described by Guinee et al. (2002). The levels of non-expressible serum (NES) were then calculated from data on moisture content and expressible serum. 2.3.4. Evaluation of cheese functionality on cooking Flowability was measured by (i) a modied Schreiber method, dened as the percentage increase in the diameter of a disc of cheese (45.5 mm diameter, 6.5 mm thick) on melting at 280 C for 4 min in an electric fan oven (Guinee et al., 2000b) and (ii) a modied Olson & Price method (Olson & Price, 1958), dened as the percentage increase in the length of a 15 g cylinder of cheese (13 mm diameter and 33.7 mm height), enclosed in a graduated glass cylindrical tube tted with a holed rubber bung, on melting at 180 C for 7.5 min in an electric fan oven. The stretchability of the molten cheese on a pizza base was measured by uniaxial extension at a velocity of 0.066 m s1 (Guinee & OCallaghan, 1997). Prior to heating, the shredded cheese was distributed uniformly at a xed loading (2.5 kg m2) onto a pizza base, which was pre-cut in half, with the two halves aligned to form a ush interface. The base with cheese was then baked at 280 C for 4 min in an electric fan oven. After baking,

the pizza was placed on the platform unit of a custombuilt stretch apparatus, which consisted of xed and rolling elements. The pizza was positioned so that the interface between the two halves of the base coincided with the junction between the xed and rolling elements. The pizza was clamped, one-half to the xed element, the other to the rolling element. The rolling element was drawn along a rail system at a constant speed of 0.066 m s2 by a motor-driven winch system, resulting in stretching of the molten cheese mass. Stretch was dened as the distance travelled 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. 2.4. Statistical analysis Three replicate cheesemaking trials were undertaken; in each, four different cheeses (CL, DA, DAS1 and DAS2) were produced using different make-procedures designed to vary the calcium level and pH in the cheese (Table 1). A randomised complete block design which incorporated the four make-procedures (treatments), and three replicate trials (blocks) was used for analysis of the response variables relating to cheese composition (Table 2). Analysis of variance (ANOVA) was carried out using a SAS procedure (SAS, 1995), where the effect of makeprocedure and replicates were estimated for all response variables. Duncans multiple-comparison test was used as a guide for pair comparisons of the make-procedure means. The level of signicance was determined at Po0:05: A split-plot design was used to determine the effects of make-procedure, storage time and their interaction on the response variables measured at regular intervals during storage, i.e., pH, pH4.6SN, PTAN, NESP, owability and stretchability (Tables 3 and 4). ANOVA for the split-plot design was carried out using a general linear model (GLM) procedure of SAS (1995). Statistically signicant differences (Po0:05) between means were determined by Fishers least signicant difference. The four cheeses from each trial were analysed at various times for fracture stress and for proteolysis, by ureaPAGE. None of the cheeses, apart from CL, fractured over the 70-d storage period. The data for both PAGE and fracture stress (for CL) are presented as supportive data but were not analysed statistically.

3. Results and discussion 3.1. Cheesemaking Both DA and DAS milk coagulated rapidly, forming gels that were sufciently rm (60 Pa) to cut in B14 min,

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J.J. Sheehan, T.P. Guinee / International Dairy Journal 14 (2004) 161172 Table 2 Composition of reduced-fat Mozzarella cheeses made using different make-procedures and with different pH and calcium levelsa,b Composition Cheese code CL Moisture (%, w/w) Fat (%, w/w) Protein (%, w/w) MNFS (%, w/w)d FDM (%, w/w)d S/M (%, w/w)d Ash (% w/w) Ca (mg g1 protein) P (mg g1 protein) pH at 1 d 51.21b 10.33a 33.15a 57.12b 21.18a 2.82a 4.08a 28.58a 19.38a 5.42c DA 57.62a 9.01b 29.19b 63.32a 21.25a 2.71a 3.25b 19.38b 15.68b 5.89a DAS1 57.47a 8.80b 29.72b 63.01a 20.72ab 2.60a 3.07b 18.54b 15.48b 5.64b DAS2 56.63a 8.84b 30.62b 62.12a 20.39b 2.63a 3.09b 18.31b 16.02b 5.49c SEDc 1.13 0.35 0.67 1.03 0.31 0.23 0.19 0.90 0.68 0.03 165

The values within row not followed by common alphabet differ (Po0:05). a The values presented are the means of three replicates. b Acication during cheese manufacture was achieved by addition of a starter culture (CL), lactic acid (DA), or a combination of lactic acid and a starter culture (DAS1 and DAS2). Details are described in Section 2 and in Table 1. c SED=standard error of difference. d MNFS=Moisture-in-non-fat-substances; FDM=fat-in-dry matter; S/M=salt in moisture. Table 3 Mean squares (MS) and probabilities (P) for aggregated changes in pH, proteolysis as measured by pH 4.6-soluble N (pH4.6SN) and 5% phosphotungstic acid-soluble N (PTAN), rmness and non-expressible serum per gram protein (NESP) in reduced-fat Mozzarella cheese made using different make-procedures and with different pH and calcium levels, and stored for 70 d at 4 Ca,b Factor pH MS Main plot Make-procedure Error Subplot Storage time Interaction (make-procedure time) Error 0.581 0.007 P o0.0001 pH4.6SN MS 15.263 0.449 P 0.0004 PTAN MS 0.167 0.011 P 0.0036 Firmness MS 257493 7372 P 0.0003 NESP MS 0.610 0.066 P 0.011

0.021 0.003 0.002

o0.0001 0.253

27.175 0.646 0.443

o0.0001 0.200

0.076 0.015 0.003

o0.0001 0.0002

10269 2342 1808

0.0015 0.27

0.040 0.006 0.001

o0.0001 o0.0001

a The make-procedures differed with respect to type of milk and curd acidication, and pH at set, whey drainage and curd milling, as described in Table 1. b For both the response variables pH and pH4.6SN, the df for the main plot and the subplot were 6 and 27, respectively; for both PTAN and rmness were 6 and 31, respectively; and for non-expressible serum were 6 and 32, respectively.

Table 4 Mean squares (MS) and probabilities (P) for aggregated changes in ow, as measured by both the Schreiber or modied Olson & Price methods, and stretch of heated reduced-fat Mozzarella cheese made using different make-procedures and with different pH and calcium levels, and stored for 70 d at 4 Ca,b Factor Flow: Schreiber MS Main plot Make-procedure Error Subplot Storage time Interaction (make-procedure time) Error 697 51 P 0.004 Flow: Olson & Price MS 32164 4414 P 0.02 Stretch MS 2907 274 P 0.0082

452 22 36

o0.0001 0.766

19071 683 811

o0.0001 0.6088

530 114 131

0.0093 0.5801

a The make-procedures differed with respect to type of milk and curd acidication, and pH at set, whey drainage and curd milling, as described in Table 1. b For the response variable Schreiber ow the df for the main plot and the subplot were 6 and 27, respectively; and for both Olson & Price ow and stretch the corresponding values were 6 and 31, respectively.

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compared with B31 min for the CL gel. The rapid coagulation of rennet-treated pre-acidied milk (at pH values o6.0) is in agreement with the results of previous studies (Keller, Olson, & Richardson, 1974; Kindstedt & Guo, 1997a; Metzger, Barbano, Rudan, & Kindstedt, 2000; Guinee et al., 2002) and may be attributed to the increased level of [Ca2+ ] and the reduction in the net negative charge of the casein (van Hooydonk, Hagedoorn, & Boerrigter, 1986). However, the coagulation times of the DA and DAS milks could be lengthened, if required, by reducing the milk temperature at set to p29 C (Guinee et al., 1994). The DA and DAS curds had a relatively high tendency to mat following cutting. This tendency reects the reduced level of calcium in the curd, owing to the low pH of these curds at whey drainage, and the higher proportion of total curd calcium that was soluble (see Guinee et al., 2000b). Both of these factors are expected to increase casein hydration (Sood et al., 1979), and hence, the uidity of curd particles and their tendency to ow and coalesce (knit) together, especially when allowed to settle or collide at low speed, e.g., during healing and the initial stages of stirring. The heated DA and DAS curds appeared more pliable, smooth, owable and uid than the CL curd during the cooking and stretching (plasticisation) of the salted curd in hot water. This trend, which was also noted previously (Kindstedt & Guo, 1997a; Guinee et al., 2002), again suggests a greater degree of casein hydration in the DA and DAS curds. 3.2. Cheese composition

in which the soluble Ca is removed from the curds at whey drainage (Czulak, Conochie, Sutherland, & van Leeuwen, 1969). The similar concentrations of calcium in the DA and DAS cheeses, despite the differences in plasticisation pH and nal pH, reect the similar pH values at setting and at whey drainage, factors which are the major determinants of the total Ca content of cheese (Lawrence, Heap, & Gilles, 1984).

3.3. Non-expressible serum The level of NES, given as g g1 protein (NESP), has been used as an index of the water holding capacity (WHC) of cheese, higher levels indicating higher WHC (Guinee et al., 2002). There was a signicant increase in the mean level of NESP during storage (Fig. 1), with the magnitude of the increase being most pronounced for CL. An increase in WHC during storage of LMM is one of the factors that assists in the conversion of a nonfunctional Mozzarella cheese to one which has the desired melt, ow and stretch properties (Kindstedt, 1995; Kindstedt & Guo, 1997b; McMahon, Fife, & Oberg, 1999). An adequate WHC prevents excessive dehydration at the temperature (X98 C) normally reached during pizza baking, and thereby minimises the risk of crusting and associated defects. The mean level of NESP over the 70-d storage period was signicantly affected by storage time, makeprocedure and their interaction (Table 3). The mean

2.0

The gross composition of the cheeses is summarised in Table 2. The composition of CL was similar to that reported for reduced-fat LMM in previous studies (Tunick et al., 1993b; Rudan et al., 1999; Poudaval & Mistry, 1999). Compared to CL, the DA and DAS cheeses had higher levels of moisture and MNFS, and lower levels of protein, ash and calcium. The increase in the levels of moisture and MNFS in the DA and DAS cheeses, as the set pH was lowered, is in agreement with earlier studies (Shehata, Iyer, Olson, & Richardson, 1967; Keller et al., 1974; Guinee et al., 2002) and is consistent with the concomitant reduction in calcium content (Sood et al., 1979; Creamer, Lawrence, & Gilles, 1985). The lower calcium content of the DA and DAS cheeses concurs with the results of previous studies (Keller et al., 1974; Metzger et al., 2000; Guinee et al., 2002). Factors contributing to the low calcium level in the latter cheeses included the low pH at setting and at whey drainage, which resulted in a high degree of solubilisation of micellar calcium phosphate (van Hooydonk et al., 1986) within the curd particles while still in contact with the whey. The whey acts as a vehicle

Nonexpressible serum, NESP (g g protein)

-1

1.8

1.5

1.3 0 10 20 30 40 50 60 70 Storage time (d)

Fig. 1. Age-related changes in NES, expressed as g g1 protein, in reduced-fat Mozzarella cheese made using different procedures: conventional starter culture acidication (CL, K), direct acidication by lactic acid (DA, J) or a combination of both (DAS1, m; DAS2, n). Details of make-procedures and cheese composition are given in the text. Values presented are the means from three replicate trials.

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levels for the DA and DAS cheeses were similar and signicantly higher than that for CL. Moreover, the level of NESP in the CL at 70 d was substantially lower than that of the DA or DAS cheeses. The relatively high NESP in the DA and DAS cheeses is consistent with their lower calcium levels (Sood et al., 1979) and with the observations of previous studies on LMM (Kindstedt & Guo, 1997a; Metzger et al., 2001b; Guinee et al., 2002).

3.4. Age-related changes in pH The pH of the CL cheese at 5 d was typical of the values (5.455.69) reported for low-moisture Mozzarella cheeses commercially available on the European market (Guinee et al., 2000b). The pH of all cheeses at 5 d was higher (by B0.150.3 units) than that of the corresponding curds at salting (Table 2, Fig. 2). The increase in pH between salting and 5 d has been observed in many studies on LMM (Guo, Gilmore, & Kindstedt, 1997; Walsh et al., 1998; Feeney et al., 2001; Guinee et al., 2000c, 2002). This increase may be due to losses of lactic acid, soluble calcium and phosphate in the stretch water, and to resolubilisation of micellar calcium phosphate on cooling the cheese after plasticisation (Guinee et al., 2002). Calcium phosphate is a major determinant of the buffering capacity of cheese and its solubilisation contributes increasingly to
6.0

buffering capacity as the pH is reduced from 6.0 to 5.1 (Lucey & Fox, 1993). Make-procedure had a signicant effect on pH (Table 3), with the mean values over the 70-d storage period for the different make-procedures being in the following order: CLBDAS2oDAS1oDA. These pH values generally reect the pH at curd milling. Storage time at 4 C resulted in a slight (B0.1 unit) but signicant (Po0:05) decrease in the mean pH of all cheeses (Table 3). This trend agrees with that of Barbano et al. (1994) for LLM and with Metzger et al. (2001b) for some low fat LMMs (from pre-acidied milk). However, the trend contrasts with the age-related increases in pH of LMM, as observed by Guo et al. (1997) and Guinee et al. (2002), and of low-fat LMM made using starter culture, as noted by Metzger et al. (2001b). The different interstudy pH/storage time trends may reect differences in the make-procedure, buffering capacity, contents of moisture and lactate, ratio of soluble-to-colloidal calcium phosphate, and thermal inactivation of the starter culture (Czulak et al., 1969; Huffman & Kristofferson, 1984; Lucey & Fox, 1993; Kindstedt, Guo, Vitto, Yun, & Barbano, 1995; Fox & Wallace, 1997). 3.5. Proteolysis 3.5.1. Ureapolyacrylamide gel electrophoresis The UreaPAGE gel electrophoretogram of the cheese from trial 2 is shown in Fig. 3 and is typical of the cheeses from trials 1 and 3 as well. Storage at 4 C resulted in a progressive degradation of as1 - and bcaseins with the extent of breakdown of as1 -casein being greater than that of the latter (Fig. 3). The breakdown products, as1 -casein (f 124-199), b-casein (f 1-192) and gcaseins, accumulated during storage to an extent dependent on the make-procedure and storage time. These degradation patterns are consistent with those of previous studies for LMM (Yun, Kiely, Barbano, & Kindstedt, 1993a; Feeney, Guinee, & Fox, 2002). At all storage times, the levels of as1 -casein breakdown for DAS1 and DAS2 were higher than that in CL and DA, as reected by the higher intensities of the as1 casein (f 124-199). At the end of the 70-d storage period, most of the as1 -casein was converted into as1 -casein (f 124-199) in the former cheeses. The higher degree of as1 casein breakdown in the DAS1 and DAS2 cheeses compared to DA may be due to their lower nal pH, which would be more favourable to proteolytic activity by residual chymosin (Tam & Whitaker, 1972; Vanderpoorten & Weckx, 1972; Mulvihill & Fox, 1977). Moreover, the low pH of DAS1 and DAS2, compared to DA, would be expected to reduce the ratio of colloidal to soluble calcium (Guinee et al., 2002) and the degree of aggregation of the casein, and thereby increase the susceptibility of the casein to hydrolysis by chymosin

5.8

pH

5.6

5.4

5.2 0 10 20 30 40 50 60 70 Storage time (d)

Fig. 2. Age-related changes in pH of reduced-fat Mozzarella cheeses made using different procedures: conventional starter culture acidication (CL, K), direct acidication by lactic acid (DA, J) or a combination of both (DAS1, m; DAS2, n). Details of makeprocedures and cheese composition are given in the text. Values presented are the means from three replicate trials.

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Fig. 3. UreaPAGE analysis of sodium caseinate (C) (lanes 1 and 14) and reduced-fat Mozzarella cheeses made using different procedures: conventional starter culture acidication (CL, lanes 1, 5 and 9), or direct acidication by lactic acid (DA, lanes 2, 6 and 10), or a combination of both (DAS1, lanes 3, 7 and 11; DAS2, lanes 4, 8 and 12). Details of make-procedures and cheese composition are given in the text.

(Fox, 1970; Feeney et al., 2002). The lower level of as1 casein breakdown in CL compared to DAS1 and DAS2, despite its pH being lower than that of DAS1, may be due to a lower residual chymosin activity, which decreases with increasing pH at rennet addition and whey drainage (Creamer et al., 1985), and to its lower MNFS (Creamer, 1971; Pearce & Gilles, 1979; Lawrence & Gilles, 1980) (Table 2). The breakdown product, b-casein (f1-192), accumulated to a slightly higher level in the DA and DAS cheeses than in the CL cheese. The higher level in DA and DAS1 may be associated with their higher pH value which would be closer to the optimal value for plasmin activity (pH 7.5; McSweeney & Sousa, 2000), to which b-casein degradation in cheese is usually attributed (Fox, OConnor, McSweeney, Guinee, & OBrien, 1996). 3.5.2. Changes in pH 4.6-soluble N and 5% phosphotungstic acid-soluble N The levels of pH4.6SN and PTAN increased in all cheeses during maturation (Fig. 4). There was a signicant effect of storage time and make-procedure on the concentration of pH4.6SN (Table 3), with the values for DAS1 and DAS2 generally being similar and signicantly higher than those of DA and CL at storage times X21 d. The high levels in DAS1 and DAS2 are consistent with their relatively high levels of as1 -casein breakdown (Fig. 3), as discussed above. The mean level of PTAN, which reects the concentrations of small peptides and free amino acids (Jarrett, Aston, & Dulley, 1982), in CL was signicantly higher than that of the DA and DAS cheeses which had similar levels. This trend agrees with that previously noted for LMM made using starter culture or direct acidication (Feeney et al., 2002), and may be due to the proteolytic contribution of starter culture proteinases and pepti-

dases in the CL cheese. Starter cultures and their enzymes were absent in the DA cheese and expected to be present at lower levels in DAS1 and DAS2 than in CL (T.M. Cogan, Dairy Products Research Centre, Teagasc Moorepark, Ireland, pers. comm.). In this context, it is noteworthy that the pH decrease in the CL, DAS1 and DAS2 curds between cutting and milling/ salting, which was attributed to starter culture, was B0.9, 0.2 and 0.35 units, respectively (Table 1). 3.6. Rheology The mean rmness of all cheeses decreased signicantly between 1 and 70 d (Fig. 5a, Table 3), with the magnitude of the decrease (B77 N) being similar to that reported previously for LMM and reduced-fat LMM stored under similar conditions (Yun et al., 1993a; Yun, Kiely, Kindstedt, & Barbano, 1993b; Tunick, Malin, Smith, & Holsinger, 1995; Guinee et al., 2002). The net decrease in rmness is consistent with the increase in primary proteolysis, the level of which has generally been found to be inversely correlated with rmness (de Jong, 1977; Guinee et al., 2000a). The mean rmness over the storage period was signicantly affected by make-procedure (Table 3), with the rmness of DA and DAS cheeses being generally similar and signicantly lower than that of CL at all storage times. The relatively high rmness of the CL is consistent with its lower levels of moisture and primary proteolysis (Luyten, 1988; Visser, 1991; Guinee et al., 1998) and higher levels of calcium and protein (de Jong, 1978; Guinee et al., 2000a, 2002). None of the cheeses, apart from CL, fractured (Fig. 5b). This trend suggests elastic deformation in the CL, compared to plastic deformation in the DA and DAS cheeses (van Vliet, 1991), a trend that is compatible with their higher levels of MNFS and NESP (Table 2).

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7 6

169

pH4.6SN (% of Total N)

5 4 3 2 1 0

(A)
0.80

10

20

30

40

50

60

70

PTAN (% of Total N)

0.60

0.40

0.20

Fig. 5. Age-related changes in the rmness (A) and fracture stress (B) of reduced-fat Mozzarella cheeses made using different procedures: conventional starter culture acidication (CL, ), direct acidication by lactic acid (DA, &) or a combination of both (DAS1, ; DAS2, ). Details of make-procedures and compositions are given in the text. The asterisk () denotes that the samples (DA, DAS1 and DAS2) did not fracture during compression. Values presented are the means from three replicate trials.

0.00 0 10 20 30 40 50 60 70

(B)

Storage time (d)

Fig. 4. Age-related changes in the concentration of pH4.6SN (A) and 5% PTAN (B) in reduced-fat Mozzarella cheeses made using different procedures: conventional starter culture acidication (CL, K), direct acidication by lactic acid (DA, J) or a combination of both (DAS1, m; DAS2, n). Details of make-procedures and compositions are given in the text. Values presented are the means from three replicate trials.

3.7. Functionality In agreement with previous studies (Guinee, Mulholland, Mullins, & Corcoran, 2000c), there was a signicant increase in the mean owability and stretchability of all cheeses over the 70-d storage period (Table 4). These changes coincide with the increase in casein hydration (Kindstedt & Guo, 1997b; McMahon et al., 1999; Guinee et al., 2002), as reected by the increase in the level of NESP (Fig. 1), and the decrease in the levels of intact casein (Figs. 3 and 4). Make-procedure signicantly affected the owability and stretchability (Fig. 6, Table 4). The mean owability

of CL, as measured using both methods, was signicantly lower than that of DA or DAS cheeses. The higher owability of the latter cheeses may be attributed to their lower calcium-to-casein ratios (Metzger et al., 2001b; Guinee et al., 2002) and protein levels (Guinee et al., 2000a), and higher levels of MNFS (Ruegg, . Eberhard, Popplewell, & Peleg, 1991; Kindstedt, 1995) and primary proteolysis (Yun et al., 1993b; Madsen & Qvist, 1998; Feeney et al., 2001). The latter compositional changes are conducive to a greater degree of casein hydration and would, therefore, be expected to enhance heat-induced displacement of adjacent layers of the para-casein matrix on heating. It is noteworthy, that the mean level of NESP in the DA and DAS cheeses was signicantly higher than that of CL. The mean owability of DA over the 70-d storage period, as measured using the modied Schreiber method, was signicantly lower than that of DAS1 or DAS2 (Fig. 6b). This trend may be due to the lower pH values of the latter cheeses, which would give a higher ratio of soluble-to-colloidal calcium for a given total calcium level (Guinee et al., 2000b; Kindstedt et al., 2001) and thereby allow a greater displacement of

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170
Flowability :modified Olson - Price method (%) 350 300 250 200 150 100 50 0

J.J. Sheehan, T.P. Guinee / International Dairy Journal 14 (2004) 161172 Table 5 Statistical summary for effects of make-procedure, storage time and their interaction on characteristics of reduced-fat Mozzarella cheesea,b Parameter Makeprocedure Storage time Makeprocedure storage time interaction NS NS NS NS NS

(A)

10

20

30

40

50

60

70

pH pH4.6SNc PTANc Firmness NESPc Flow (Schreiber method) Flow (Olson & Price method) Stretch
a

NS Signicance levels: , Po0:05; , Po0:01; , Po0:001; NS,

Flowability : modified Schreiber method (%)

40

30

non-signicant (P > 0:05). b Details of make-procedure and storage conditions given in Table 1 and text. c pH4.6SN, pH 4.6-soluble N (% total N); PTAN, 5% phosphotungstic acid-soluble N (% total N); NESP, non-expressible serum per gram protein.

20

10

0 80 70 60

(B)

10

20

30

40

50

60

70

50 40 30 20 10 0 0 10 20 30 40 50 Storage time (d) 60 70

contiguous layers of the casein matrix (e.g., ow) for a given heat-induced stress (Guinee et al., 2002). The stretchability of the heated cheese showed a trend similar to that noted for owability, with both the makeprocedure and storage time having signicant effects (Table 4) and the CL cheese having a signicantly lower mean stretchability over the 70-d storage period (Fig. 6c). The similarity of trends between the owability and stretchability were expected as both involve displacement of the para-casein matrix. A higher degree of paracasein aggregation in CL, owing to its higher levels of protein and calcium and lower levels of primary proteolysis, NESP, and MNFS, would be expected to reduce the degree of displacement for a given stress applied during extension.

Stretch (cm)

4. Conclusions Reduced-fat low-moisture Mozzarella cheese was made using starter culture (control, CL), lactic acid (directly acidied, DA) or a combination of starter culture and lactic acid (DAS1 and DAS2) to reduce the pH during manufacture. The resultant cheeses differed in pH and calcium content. In general, the lower calcium level in the DA and DAS cheeses, compared to the control cheese, resulted in higher levels of moisture, MNFS and NESP, and signicant improvements in the owability and stretchability of the cooked cheese (Table 5). By comparison, reducing the pH of the DAS cheeses had only a relatively minor effect.

(C)

Fig. 6. Age-related changes in owability, as measured by the modied Schreiber method (A) or Olson & Price method (B), and stretchability (C), of reduced-fat Mozzarella cheeses made using different procedures: conventional starter culture acidication (CL, K), direct acidication by lactic acid (DA, J) or a combination of both (DAS1,m; DAS2, n). Details of make-procedures and compositions are given in the text. Values presented are the means from three replicate trials.

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Acknowledgements This work was funded by the Department of Agriculture and Food, under the Food Institutional Research Measure (National Development Plan). The authors kindly acknowledge the technical assistance of E.O. Mulholland and C. Mullins and the advice of K. OSullivan, Department of Statistics, University College, Cork, Ireland on the statistical analysis of the data.

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