ARTICLE IN PRESS

International Dairy Journal 14 (2004) 161–172

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 acidified, 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 gÀ1 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 gÀ1protein), and lower levels of protein. These changes were paralleled by higher mean values for stretchability and flowability, and lower mean values for firmness 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 flowability (as measured using the modified 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 reflected by a reduction in flowability 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, preacidification 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 preacidification of milk with organic acids to a pH of 5.8–5.6, in combination with a starter culture, resulted in a reduction in calcium level of low-fat (6%, w/w) LMM but did not significantly 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 flowability of the melted cheeses, to an extent depending on the preacidification pH, the type of acid used and final pH of the cheese. More recently, Guinee, Feeney, Auty, and

. approximately 14 loaves per vat. Villeurbanne Cedex-Lyon. Ca. 500 L vats with automated variable-speed cutting and stirring (APV Schweiz AG. CMT. & Mullins. Owing to the high tendency of the DA and DAS curds to mat following cutting of the gel. Almena-Aliste. Switzerland).4 in the control LMM made using starter culture (Guinee et al. B5. Model d. the curds– whey mixture was allowed to heal for 10 min and then stirred continuously.2 C minÀ1 and continually stirred until the pH of the curd decreased to 6. the curds (B36–39 kg) were cheddared in the finishing vat. pH of the curd at whey drainage. calibrated at 38 C with reference buffer solutions. which would be expected to increase the degree of casein hydration (Sood. Milk (454 kg) was inoculated with a starter culture consisting of Streptococcus thermophilus and Lactobacillus helveticus. Pfizer Inc. CL). & Ge. Sheehan. apart from the differences as outlined in Table 1. 2001b.1. Moreover. After whey drainage.8 to 5. DAS1 and DAS2). w/w) (Wardle Chemicals. 0. NaCl. The cultures were grown separately overnight at 37 C in 10%. Almena-Aliste. mellowed for 20 min. & Kindstedt. The resultant blocks were cooled in dilute brine (10%..6 with dilute lactic acid (5%. and pH of the curd at milling/plasticisation (Table 1). T.2%.4 increases the ratio of solubleto-colloidal calcium (Guinee.J. The four cheeses were further differentiated according to pH of milk at rennet addition (setting). jacketed. 2000c) with certain modifications. w/w. as measured at 38 C by inserting a pH probe (Radiometer Analytical S. or by a combination of added lactic acid and starter culture (directly acidified cheeses with starter culture. reducing the pH of cheese from 5. stainless steel. resulted in higher levels of moisture and MNFS.4:1. Gaind. B50 Chymosin units mLÀ1. with the speed of stirring being increased gradually from 11 to 25 rpm over 22 min. added at levels of 1. The cheesemaking procedure was otherwise similar to that for the CL cheese. i. After cutting (B30 min later). 2002). w/w. and transferred to the stretching unit (Automatic Stretching Machine. respectively. where the curd–whey mixture was allowed to settle for 2 min prior to drainage of the whey.1%. Chesire. Milwaukee. CW6 9JW). Mulholland. Mulholland.1) at BÀ4. addition of lactic acid (directly acidified cheese.A. The mixture was then pumped to rectangular jacketed stainless 500 L finishing vats (warmed to 38 C by circulating water). w/w. by preacidification of milk prior to renneting.18 mL kgÀ1 milk and the milk allowed to set at 36 C. Kindstedt. w/w. WI. The curds–whey mixture was cooked to 38 C.. Italy). In general. Nr. Calveley. Acidification was achieved either by: a starter culture as in conventional production (control cheese. the milk (29 C) was acidified directly to pH 5. as described below. All gels were cut at a firmness that was equivalent to an elastic shear modulus (G 0 ) value of 60 Pa. Pudja. Cheese manufacture In each of three trials. an occurrence.23. The curd was heated to 58 C by dosing with hot water at 78 C (used at a rate of B1. The milled curd was dry salted at a level of 4. While the DA milk was not inoculated with starter culture. 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. The CL cheese was manufactured as described previously (Guinee. 1994). Chymosin (Single strength Chy-max plus.P.4 kg kgÀ1 curd and added over a period of 8 min) and batch plasticised mechanically.1. France). the hydration of para-casein in dilute model systems increases with decreasing pH in the range 6–5.. and milled at pH 5. pH 5. DA). DAS1 and DAS2 cheeses. Lorenzo di Peveragno CN. at a rate of 0. & Dewan.3 kg portions (24 cm long) that were filled into rectangular moulds. & Mulholland. 1979. as measured using low-amplitude strain oscillation rheometry (Guinee.e. reconstituted skim milk powder that had been heat-treated at 90 C for 30 min. The cheeses. These changes resulted in marked increases in protein hydration in the unheated cheese and in the flowability and stretchability of the heated cheeses. & Corcoran. CH-3076 Worb 1. Mullins. held overnight at o10 C. pasteurised at 72 C Â 15 s. by the addition of skim milk. even though the pH of the LMM from pre-acidified milk was relatively high. USA) was added at a level of 0. Mid-lactation milk from the autumn-calving Friesian herd at the Dairy Production Centre. The raw milk (2200 L) was standardised to a protein-to-fat ratio of 3.5%. Corcoran. the healing period (3 min) was shorter than that of the . four types of reduced-fat LMM were manufactured by alteration of the make-procedure. For the manufacture of DA. 2002). 2000b). the DAS1 and DAS2 cheesemilks were inoculated with the same type and level of starter culture as the CL cheesemilk. 2. and a lower protein level in the resultant directly acidified LMM. Guinee / International Dairy Journal 14 (2004) 161–172 Fox (2002) found that reducing the calcium-to-casein ratio from B28 to B21 mg gÀ1. were then vacuum-wrapped and stored at 4 C.0% and 0. and pumped into cylindrical.0 C for 30 min to allow cooling to a surface temperature of 24 C and a core temperature of o45 C. Ge. 1985). Tarporley. 2001. Harrington.ARTICLE IN PRESS 162 J. Metzger et al. 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. S. Zielinski.8 compared to 5. directly into the curd. Materials and methods 2.3 (Creamer. w/w. Moorepark was collected on three separate occasions over a 4-week period.

ARTICLE IN PRESS J. the DA and DAS curd–whey mixtures were held for a period of 7–11 min to allow the curd particles to attain the desired resilience. Bodengraven. was used. Cylindrical samples (weight.6 (pH4.41 6.0 38 5.5 C minÀ1) for the DA and DAS curds was similar and higher than that of the CL curds. w/v). B13 mm diameter and 33.61 DAS1 5. or a combination of lactic acid and a starter culture (DAS1 and DAS2). & Crowe. Kista. Bos Kaasgereedschap.5 mm thick slices from which discs (45.64 5.23 29 29 0. for moisture.40 29 29 0. Netherlands) giving 6. The pH was measured on a cheese slurry prepared from 20 g cheese and 12 g distilled water (British Standards Institution. CL curd. 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. & Fenelon. Murray. d The values given are the means of the three trials.6 38 5. w/v) with distilled water. the DA curd was cut into slabs.13 5. The DAS1 and DAS2 curds were treated similarly and held until the pH reached pH 5.60 5. D5650 Solingen 19. 1999).60 5. prepared by diluting concentrated lactic acid (80%.60 5. 1570.5 mm) were cut using a stainless steel borer. Sampling of cheese Cheese was grated to yield particles of o1 mm. in a Krups Rotary 350 food processor fitted with a universal blade (Robert Krups GmbH & Co.23 DA 5. c NA.1. b Full details of cheese manufacture are given in text. salt.40 (DAS1) or pH 5. Germany). which were trenched. Shredded cheese was prepared by cutting the block. piled and held for B60 min (Table 1).7 mm height) were procured using custommade stainless steel borers. Following whey drainage. fat. into 25 mm cubes (Cheese Blocker.3.6SN) and in 5% phosphotungstic acid soluble nitrogen (PTAN) were measured.6 38 5. . calcium.20 (DAS2). Auty.40 DAS2 5.60 29 29 0. at 5 d. lactic acid (DA). Sweden) using the raw food grating disc (K) to yield shreds E25 mm long and E4 mm diameter. The cooking rate (0.54 5. Cheese was cut with a Unika cutter (model WG-300.05 g. BOS Kaasgereedschap. Analysis of cheese 2. Composition and proteolysis Grated cheese was analysed in triplicate. as described previously (Fenelon.3. After reaching the scald temperature.P. Guinee / International Dairy Journal 14 (2004) 161–172 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 Acidification 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. 2. T. The DA and DAS curds were treated as for CL from this stage of cheesemaking onwards. Netherlands) and immediately shredding in a Hallde RG-350 machine (AB Hallde Maskiner. Dilute lactic acid solution (5%. and phosphorous using standard IDF methods (Guinee.2. Postfach 190460.J.60 5.6 38 5. protein.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. The levels of total nitrogen soluble at pH 4. 1976). not applicable. Delahunty. Sheehan. 2000a). Postbus 2410 AC.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 Acidification during cheese manufacture was achieved by addition of a starter culture (CL). stored at 4 C. Guinee. Bodegraven. 2.

in each.3. UK). (2002).5 kg mÀ2) onto a pizza base.2. the shredded cheese was distributed uniformly at a fixed loading (2. Fracture stress was defined as the force per unit area to induce fracture. i. The pizza was clamped. DA. Surrey GU7 1YL.5 min in an electric fan oven.3. CH-7402. Samples were withdrawn from 8 C and immediately compressed to 30% of original height at a rate of 60 mm minÀ1 at room temperature. 3.. storage time and their interaction on the response variables measured at regular intervals during storage. Fox. the pizza was placed on the platform unit of a custombuilt stretch apparatus.1.ARTICLE IN PRESS 164 J. which was pre-cut in half. and 70 d. 6. Godalming. NESP.066 m sÀ1 (Guinee & O’Callaghan. Herts. PTAN. 2. Watford. forming gels that were sufficiently firm (60 Pa) to cut in B14 min. Duncan’s multiple-comparison test was used as a guide for pair comparisons of the make-procedure means. Prior to heating. The level of significance was determined at Po0:05: A split-plot design was used to determine the effects of make-procedure. Cheesemaking Both DA and DAS milk coagulated rapidly. respectively. which consisted of fixed and rolling elements. Six samples (25 Â 25 Â 25 mm cubes) were obtained from each cheese. 2. defined as the percentage increase in the diameter of a disc of cheese (45. . the other to the rolling element. four different cheeses (CL. Results and discussion 3.J. The gels were stained overnight in Coomassie brilliant blue G250 and de-stained in repeated changes of distilled water. fractured over the 70-d storage period. 2. apart from CL.e. T. where the effect of makeprocedure and replicates were estimated for all response variables. After baking. The rolling element was drawn along a rail system at a constant speed of 0. The data for both PAGE and fracture stress (for CL) are presented as supportive data but were not analysed statistically. Evaluation of cheese functionality on cooking Flowability was measured by (i) a modified Schreiber method. resulting in stretching of the molten cheese mass.5 mg cheese protein) were dissolved in 1 mL of sample buffer and incubated at 55 C for B10 min. PAGE was performed on a Protean II xi vertical slab gel unit (BIORAD Laboratories Ltd. The base with cheese was then baked at 280 C for 4 min in an electric fan oven.066 m sÀ2 by a motor-driven winch system.P. Analysis of variance (ANOVA) was carried out using a SAS procedure (SAS. was measured as described by Guinee et al. 2. enclosed in a graduated glass cylindrical tube fitted with a holed rubber bung. The levels of non-expressible serum (NES) were then calculated from data on moisture content and expressible serum. placed in an airtight plastic bag and held at 8 C overnight..6SN. Cheese rheology All cheeses were analysed using compression on a TAHDi Texture Profile analyser (Stable Micro Systems.3. Switzerland). 2000b) and (ii) a modified Olson & Price method (Olson & Price. Statistical analysis Three replicate cheesemaking trials were undertaken. The four cheeses from each trial were analysed at various times for fracture stress and for proteolysis. 35. None of the cheeses. and Guinee (2001). Non-expressible serum The levels of cheese serum expressed by centrifugation at 12 500g at 20 C.7 mm height). 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. Sheehan.3. one-half to the fixed element. and three replicate trials (blocks) was used for analysis of the response variables relating to cheese composition (Table 2). England). 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. pH. with the two halves aligned to form a flush interface. 1997).. on melting at 180 C for 7. and firmness as the force required to compress the cheese to 30% of its original height.4.5 mm diameter. DAS1 and DAS2) were produced using different make-procedures designed to vary the calcium level and pH in the cheese (Table 1). pH4. defined as the percentage increase in the length of a 15 g cylinder of cheese (13 mm diameter and 33. Statistically significant differences (Po0:05) between means were determined by Fisher’s least significant difference. Bonaduz. as described by Feeney. 1958). ANOVA for the split-plot design was carried out using a general linear model (GLM) procedure of SAS (1995). Guinee / International Dairy Journal 14 (2004) 161–172 All cheeses were analysed by urea–polyacrylamide gel electrophoresis (PAGE) at 1. using a separating and a stacking gel.5 mm thick) on melting at 280 C for 4 min in an electric fan oven (Guinee et al. 1995). The samples were run through the stacking gel and separating gel at 280 and 300 V. flowability and stretchability (Tables 3 and 4). Stretch was defined 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. by urea–PAGE. A randomised complete block design which incorporated the four make-procedures (treatments). Cheese samples (equivalent to 2.4. The stretchability of the molten cheese on a pizza base was measured by uniaxial extension at a velocity of 0.

011 0. as measured by both the Schreiber or modified Olson & Price methods. proteolysis as measured by pH 4.03 165 The values within row not followed by common alphabet differ (Po0:05).84b 30.27 0.5801 a The make-procedures differed with respect to type of milk and curd acidification.021 0.449 P 0.003 0.19b 63.48b 5. a The values presented are the means of three replicates. as described in Table 1.002 o0.253 27.6SN MS 15. and stretch of heated reduced-fat Mozzarella cheese made using different make-procedures and with different pH and calcium levels.89a DAS1 57.0001 pH4.03 0. w/w) Fat (%.21b 10. Sheehan.b Factor Flow: Schreiber MS Main plot Make-procedure Error Subplot Storage time Interaction (make-procedure  time) Error 697 51 P 0.02 Stretch MS 2907 274 P 0.001 o0. b Acification during cheese manufacture was achieved by addition of a starter culture (CL).076 0.P. whey drainage and curd milling. respectively.62a 9.15a 57.12b 21.646 0.47a 8.82a 4.581 0. w/w)d FDM (%.38b 15.167 0. w/w) MNFS (%. as described in Table 1.0015 0.49c SEDc 1. b For both the response variables pH and pH4.23 0.6-soluble N (pH4. lactic acid (DA).13 0. FDM=fat-in-dry matter.0082 452 22 36 o0.0001 o0.02b 5. respectively. and stored for 70 d at 4 Ca.67 1. and stored for 70 d at 4 Ca.066 P 0. and for both Olson & Price flow and stretch the corresponding values were 6 and 31.09b 18.68b 5.0001 a The make-procedures differed with respect to type of milk and curd acidification. for both PTAN and firmness were 6 and 31.0002 10269 2342 1808 0.011 P 0. w/w)d S/M (%. Guinee / International Dairy Journal 14 (2004) 161–172 Table 2 Composition of reduced-fat Mozzarella cheeses made using different make-procedures and with different pH and calcium levelsa. . firmness 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.006 0.90 0.25b 19.71a 3.040 0.0036 Firmness MS 257493 7372 P 0. and pH at set. respectively.39b 2.42c DA 57.54b 15.610 0.004 Flow: Olson & Price MS 32164 4414 P 0. Table 4 Mean squares (MS) and probabilities (P) for aggregated changes in flow.31 0. the df for the main plot and the subplot were 6 and 27.64b DAS2 56.62b 62. w/w) Protein (%. and for non-expressible serum were 6 and 32. b For the response variable Schreiber flow the df for the main plot and the subplot were 6 and 27.38a 5.72ab 2.6088 530 114 131 0. respectively.0093 0.0001 0.b Factor pH MS Main plot Make-procedure Error Subplot Storage time Interaction (make-procedure  time) Error 0.01a 20.72b 63.18a 2.015 0.80b 29.19 0.J.0003 NESP MS 0. S/M=salt in moisture.6SN) and 5% phosphotungstic acid-soluble N (PTAN).35 0. Table 3 Mean squares (MS) and probabilities (P) for aggregated changes in pH.31b 16.0004 PTAN MS 0. respectively.08a 28.0001 0.68 0.200 0.32a 21.0001 0.33a 33.01b 29.003 o0.ARTICLE IN PRESS J. Details are described in Section 2 and in Table 1.12a 20. or a combination of lactic acid and a starter culture (DAS1 and DAS2).63a 8.007 P o0.0001 0.25a 2. d MNFS=Moisture-in-non-fat-substances. c SED=standard error of difference.766 19071 683 811 o0.58a 19.175 0.63a 3. T.0001 0.b Composition Cheese code CL Moisture (%. whey drainage and curd milling.263 0. w/w)d Ash (% w/w) Ca (mg gÀ1 protein) P (mg gÀ1 protein) pH at 1 d 51.07b 18.6SN.60a 3.443 o0. and pH at set.

and hence. Guinee et al. owing to the low pH of these curds at whey drainage. 1997a. An adequate WHC prevents excessive dehydration at the temperature (X98 C) normally reached during pizza baking. 3. Hagedoorn.. n). Guinee et al. Olson. T. Non-expressible serum The level of NES. Fife. This tendency reflects the reduced level of calcium in the curd. by reducing the milk temperature at set to p29 C (Guinee et al. 2002). m. & Oberg. 1979).. The mean level of NESP over the 70-d storage period was significantly affected by storage time. has been used as an index of the water holding capacity (WHC) of cheese. However. The increase in the levels of moisture and MNFS in the DA and DAS cheeses. 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. 1. is in agreement with earlier studies (Shehata. 3. and lower levels of protein. makeprocedure and their interaction (Table 3). 1999). 1999. 2000. Creamer. Cheese composition in which the soluble Ca is removed from the curds at whey drainage (Czulak. Sutherland. 1997a. This trend. & Boerrigter. Kindstedt & Guo. There was a significant increase in the mean level of NESP during storage (Fig.. Age-related changes in NES.ARTICLE IN PRESS 166 J. 1995. 1986) within the curd particles while still in contact with the whey. expressed as g gÀ1 protein. 1979. Factors contributing to the low calcium level in the latter cheeses included the low pH at setting and at whey drainage. Heap. 1967. 1). Rudan et al. Guinee et al. Compared to CL.. McMahon. . 2002). DAS2. 1974. Barbano. The rapid coagulation of rennet-treated pre-acidified milk (at pH values o6.0) is in agreement with the results of previous studies (Keller. The similar concentrations of calcium in the DA and DAS cheeses.3 0 10 20 30 40 50 60 70 Storage time (d) Fig. Metzger et al. & Gilles. direct acidification by lactic acid (DA.5 1. ash and calcium. K). which was also noted previously (Kindstedt & Guo. reflect the similar pH values at setting and at whey drainage... Keller et al.. 1974. which resulted in a high degree of solubilisation of micellar calcium phosphate (van Hooydonk et al.. & Gilles. & Richardson. Details of make-procedures and cheese composition are given in the text. The composition of CL was similar to that reported for reduced-fat LMM in previous studies (Tunick et al. and thereby minimises the risk of crusting and associated defects. smooth. Guinee et al. 2002). 1985). Metzger. 1994). with the magnitude of the increase being most pronounced for CL. The mean 2.2. Olson. the coagulation times of the DA and DAS milks could be lengthened. given as g gÀ1 protein (NESP). 1969). 1993b. The lower calcium content of the DA and DAS cheeses concurs with the results of previous studies (Keller et al. 2002) and is consistent with the concomitant reduction in calcium content (Sood et al.. Conochie.P. Poudaval & Mistry. Lawrence. again suggests a greater degree of casein hydration in the DA and DAS curds. in reduced-fat Mozzarella cheese made using different procedures: conventional starter culture acidification (CL. during healing and the initial stages of stirring. Both of these factors are expected to increase casein hydration (Sood et al. The heated DA and DAS curds appeared more pliable. if required. especially when allowed to settle or collide at low speed. Sheehan. Kindstedt & Guo. The whey acts as a vehicle Nonexpressible serum..0 The gross composition of the cheeses is summarised in Table 2.. as the set pH was lowered..8 1. Values presented are the means from three replicate trials. 1997b. factors which are the major determinants of the total Ca content of cheese (Lawrence. J) or a combination of both (DAS1. 1984).. 2000. 2000b).. NESP (g g protein) -1 1. 1974. 1999). the fluidity of curd particles and their tendency to flow and coalesce (knit) together. & Kindstedt. e. and the higher proportion of total curd calcium that was soluble (see Guinee et al.J.. & van Leeuwen. 1986). Rudan. the DA and DAS cheeses had higher levels of moisture and MNFS. flow and stretch properties (Kindstedt. despite the differences in plasticisation pH and final pH. higher levels indicating higher WHC (Guinee et al.3.g. Guinee / International Dairy Journal 14 (2004) 161–172 compared with B31 min for the CL gel. flowable and fluid than the CL curd during the cooking and stretching (plasticisation) of the salted curd in hot water.. Iyer. 2002) and may be attributed to the increased level of [Ca2+ ] and the reduction in the net negative charge of the casein (van Hooydonk. The DA and DAS curds had a relatively high tendency to mat following cutting. & Richardson.

and bcaseins with the extent of breakdown of as1 -casein being greater than that of the latter (Fig. This increase may be due to losses of lactic acid. 2002). Guinee / International Dairy Journal 14 (2004) 161–172 167 levels for the DA and DAS cheeses were similar and significantly higher than that for CL. These degradation patterns are consistent with those of previous studies for LMM (Yun. Guo. and to resolubilisation of micellar calcium phosphate on cooling the cheese after plasticisation (Guinee et al. Moreover. The relatively high NESP in the DA and DAS cheeses is consistent with their lower calcium levels (Sood et al.3 units) than that of the corresponding curds at salting (Table 2. 1977).8 pH 5. the trend contrasts with the age-related increases in pH of LMM.0 to 5. Proteolysis 3. Vitto. (2002). 2000b). Mulvihill & Fox..15–0. . Fox & Wallace. as1 -casein (f 124-199). 1995. Yun. the level of NESP in the CL at 70 d was substantially lower than that of the DA or DAS cheeses. 2002)..1. accumulated during storage to an extent dependent on the make-procedure and storage time. Age-related changes in pH The pH of the CL cheese at 5 d was typical of the values (5. Guinee et al.4. At all storage times. Make-procedure had a significant effect on pH (Table 3).1 unit) but significant (Po0:05) decrease in the mean pH of all cheeses (Table 3). Walsh et al. 1998. and thereby increase the susceptibility of the casein to hydrolysis by chymosin 5. This trend agrees with that of Barbano et al. The increase in pH between salting and 5 d has been observed in many studies on LMM (Guo. 1997. Lucey & Fox. 1979) and with the observations of previous studies on LMM (Kindstedt & Guo. Feeney.. soluble calcium and phosphate in the stretch water. Kiely. 3 and is typical of the cheeses from trials 1 and 3 as well. Guinee et al. 2). b-casein (f 1-192) and gcaseins. The breakdown products. Sheehan. 1997a. as noted by Metzger et al. Kindstedt. 2002). Calcium phosphate is a major determinant of the buffering capacity of cheese and its solubilisation contributes increasingly to 6. & Barbano.J. Fig. Guinee. Moreover. 2002) and the degree of aggregation of the casein. 3. 1969. 1972. Age-related changes in pH of reduced-fat Mozzarella cheeses made using different procedures: conventional starter culture acidification (CL. and thermal inactivation of the starter culture (Czulak et al. Storage time at 4 C resulted in a slight (B0.. K).4 5. 2001.. 2000c. (1994) for LLM and with Metzger et al. 1972. At the end of the 70-d storage period. 1993.. m. However. most of the as1 -casein was converted into as1 -casein (f 124-199) in the former cheeses. Metzger et al.1 (Lucey & Fox. 1993). as observed by Guo et al. 3). Barbano. 3. Storage at 4 C resulted in a progressive degradation of as1 . buffering capacity.P. & Kindstedt. Urea–polyacrylamide gel electrophoresis The Urea–PAGE gel electrophoretogram of the cheese from trial 2 is shown in Fig.45–5. (1997) and Guinee et al. These pH values generally reflect the pH at curd milling. n). Gilmore. The different interstudy pH/storage time trends may reflect differences in the make-procedure.5. & Fox. 2001b.69) reported for low-moisture Mozzarella cheeses commercially available on the European market (Guinee et al. Values presented are the means from three replicate trials. 2.5.ARTICLE IN PRESS J.6 5.. 1997). 2002). the low pH of DAS1 and DAS2. would be expected to reduce the ratio of colloidal to soluble calcium (Guinee et al. compared to DA. direct acidification by lactic acid (DA. ratio of soluble-to-colloidal calcium phosphate. as reflected by the higher intensities of the as1 casein (f 124-199). DAS2. The pH of all cheeses at 5 d was higher (by B0. J) or a combination of both (DAS1.. Details of makeprocedures and cheese composition are given in the text. T.. (2001b). with the mean values over the 70-d storage period for the different make-procedures being in the following order: CLBDAS2oDAS1oDA.2 0 10 20 30 40 50 60 70 Storage time (d) Fig. & Kindstedt. Feeney et al. and of low-fat LMM made using starter culture. which would be more favourable to proteolytic activity by residual chymosin (Tam & Whitaker. 1993a. 1984. Vanderpoorten & Weckx.. (2001b) for some low fat LMMs (from pre-acidified milk). contents of moisture and lactate. the levels of as1 -casein breakdown for DAS1 and DAS2 were higher than that in CL and DA.0 buffering capacity as the pH is reduced from 6. The higher degree of as1 casein breakdown in the DAS1 and DAS2 cheeses compared to DA may be due to their lower final pH. Huffman & Kristofferson.

Guinee et al. Sheehan. Guinee / International Dairy Journal 14 (2004) 161–172 Fig. 6 and 10). was B0. which reflects the concentrations of small peptides and free amino acids (Jarrett. 1977. Feeney et al.. 2002). lanes 2. The net decrease in firmness is consistent with the increase in primary proteolysis. 2002).2. 5 and 9). with the firmness of DA and DAS cheeses being generally similar and significantly lower than that of CL at all storage times. T. as discussed above. apart from CL. accumulated to a slightly higher level in the DA and DAS cheeses than in the CL cheese. 1996). 3. and may be due to the proteolytic contribution of starter culture proteinases and pepti- dases in the CL cheese.6SN and PTAN increased in all cheeses during maturation (Fig.). b-casein (f1-192). compared to plastic deformation in the DA and DAS cheeses (van Vliet.9. & Barbano. The mean level of PTAN. The breakdown product.... Pearce & Gilles. pers. 1991). respectively (Table 1). In this context. 2000a.ARTICLE IN PRESS 168 J. Dairy Products Research Centre. it is noteworthy that the pH decrease in the CL. which was attributed to starter culture. 1982). 1979. in CL was significantly higher than that of the DA and DAS cheeses which had similar levels. & Dulley. (Fox. Lawrence & Gilles. Tunick. 8 and 12). and to its lower MNFS (Creamer. DAS1 and DAS2 curds between cutting and milling/ salting. DAS2. lanes 4. Changes in pH 4.P. 4). There was a significant effect of storage time and make-procedure on the concentration of pH4. the level of which has generally been found to be inversely correlated with firmness (de Jong. 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. 1998) and higher levels of calcium and protein (de Jong. McSweeney. 2000).. 5a..5. Guinee et al. None of the cheeses. 1991. 3.. Table 3). despite its pH being lower than that of DAS1. 5b). fractured (Fig. The high levels in DAS1 and DAS2 are consistent with their relatively high levels of as1 -casein breakdown (Fig. 7 and 11. The mean firmness over the storage period was significantly affected by make-procedure (Table 3). The lower level of as1 casein breakdown in CL compared to DAS1 and DAS2. Malin.6SN (Table 3). to which b-casein degradation in cheese is usually attributed (Fox. This trend suggests elastic deformation in the CL. Kindstedt. 1970. 1971. which decreases with increasing pH at rennet addition and whey drainage (Creamer et al. Guinee. 0.6-soluble N and 5% phosphotungstic acid-soluble N The levels of pH4. The relatively high firmness of the CL is consistent with its lower levels of moisture and primary proteolysis (Luyten. Rheology The mean firmness of all cheeses decreased significantly between 1 and 70 d (Fig. 2000a). lanes 3. O’Connor. Guinee et al.5. or direct acidification by lactic acid (DA. with the values for DAS1 and DAS2 generally being similar and significantly higher than those of DA and CL at storage times X21 d. 1993a. This trend agrees with that previously noted for LMM made using starter culture or direct acidification (Feeney et al. Yun. 1988. a trend that is compatible with their higher levels of MNFS and NESP (Table 2).J. Smith. Guinee et al. Kiely. & Holsinger. may be due to a lower residual chymosin activity. 3). McSweeney & Sousa. Visser.2 and 0. comm.6. or a combination of both (DAS1. Teagasc Moorepark. lanes 1. 1995. 1993b. 2002). 2002). Ireland. Cogan.. 1978. 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.M. 1980) (Table 2). . Details of make-procedures and cheese composition are given in the text. 1985). 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.35 units. & O’Brien. 3. Aston. Urea–PAGE analysis of sodium caseinate (C) (lanes 1 and 14) and reduced-fat Mozzarella cheeses made using different procedures: conventional starter culture acidification (CL.

6SN (A) and 5% PTAN (B) in reduced-fat Mozzarella cheeses made using different procedures: conventional starter culture acidification (CL.00 0 10 20 30 40 50 60 70 (B) Storage time (d) Fig.60 0. Eberhard. Table 4).80 0 10 20 30 40 50 60 70 PTAN (% of Total N) 0. DAS1 and DAS2) did not fracture during compression. direct acidification by lactic acid (DA.P. 1). as measured using the modified Schreiber method. 2000c). 3 and 4). was significantly lower than that of DA or DAS cheeses. McMahon et al. 1995) and primary proteolysis (Yun et al. 1991. m.. 1997b. Madsen & Qvist. 2001). These changes coincide with the increase in casein hydration (Kindstedt & Guo. Values presented are the means from three replicate trials. 4. ).. Age-related changes in the firmness (A) and fracture stress (B) of reduced-fat Mozzarella cheeses made using different procedures: conventional starter culture acidification (CL. as measured using both methods. Mulholland.6SN (% of Total N) 5 4 3 2 1 0 (A) 0. & Corcoran. Details of make-procedures and compositions are given in the text. T. 1999. 2002) and protein levels (Guinee et al. 3. This trend may be due to the lower pH values of the latter cheeses. Kindstedt. 2002). be expected to enhance heat-induced displacement of adjacent layers of the para-casein matrix on heating. 6b). Sheehan. K). and higher levels of MNFS (Ruegg.40 0. that the mean level of NESP in the DA and DAS cheeses was significantly higher than that of CL. 0. direct acidification by lactic acid (DA. Age-related changes in the concentration of pH4. Guinee et al.ARTICLE IN PRESS J.. Guinee et al.. The latter compositional changes are conducive to a greater degree of casein hydration and would. 6. 1998. Popplewell. 2001) and thereby allow a greater displacement of . The asterisk (Ã) denotes that the samples (DA. The mean flowability of CL. DAS2. Values presented are the means from three replicate trials. and the decrease in the levels of intact casein (Figs. DAS2.. 5. It is noteworthy. Feeney et al. Kindstedt et al.... as reflected by the increase in the level of NESP (Fig. Make-procedure significantly affected the flowability and stretchability (Fig. . &) or a combination of both (DAS1. Details of make-procedures and compositions are given in the text. The higher flowability of the latter cheeses may be attributed to their lower calcium-to-casein ratios (Metzger et al. 1993b. )..J. 2000a). & Peleg. therefore. 2001b. there was a significant increase in the mean flowability and stretchability of all cheeses over the 70-d storage period (Table 4). J) or a combination of both (DAS1. Mullins. was significantly lower than that of DAS1 or DAS2 (Fig. The mean flowability of DA over the 70-d storage period.20 Fig. n). . 2000b. Functionality In agreement with previous studies (Guinee. Guinee / International Dairy Journal 14 (2004) 161–172 7 6 169 pH4.7. which would give a higher ratio of soluble-to-colloidal calcium for a given total calcium level (Guinee et al.

The stretchability of the heated cheese showed a trend similar to that noted for flowability. b Details of make-procedure and storage conditions given in Table 1 and text. NESP. owing to its higher levels of protein and calcium and lower levels of primary proteolysis. direct acidification by lactic acid (DA. pH 4. ÃÃ. and MNFS. Flowability : modified Schreiber method (%) 40 30 non-significant (P > 0:05). Po0:001.. lactic acid (directly acidified. J) or a combination of both (DAS1. would be expected to reduce the degree of displacement for a given stress applied during extension. Po0:05. storage time and their interaction on characteristics of reduced-fat Mozzarella cheesea.6-soluble N (% total N). compared to the control cheese. PTAN.J.Price method (%) 350 300 250 200 150 100 50 0 J. 6. 5% phosphotungstic acid-soluble N (% total N). Age-related changes in flowability.g. Values presented are the means from three replicate trials. Po0:01.6SN. NESP. . The similarity of trends between the flowability and stretchability were expected as both involve displacement of the para-casein matrix. of reduced-fat Mozzarella cheeses made using different procedures: conventional starter culture acidification (CL. flow) for a given heat-induced stress (Guinee et al. n). NS. and stretchability (C). 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. Stretch (cm) 4. Sheehan. resulted in higher levels of moisture. K). (C) Fig.. and significant improvements in the flowability and stretchability of the cooked cheese (Table 5). Conclusions Reduced-fat low-moisture Mozzarella cheese was made using starter culture (control. ÃÃÃ. Guinee / International Dairy Journal 14 (2004) 161–172 Table 5 Statistical summary for effects of make-procedure. In general. the lower calcium level in the DA and DAS cheeses.m. c pH4.ARTICLE IN PRESS 170 Flowability :modified Olson . 6c). MNFS and NESP.6SNc PTANc Firmness NESPc Flow (Schreiber method) Flow (Olson & Price method) Stretch a ÃÃà Ãà Ãà Ãà à à à à ÃÃà ÃÃà ÃÃà à ÃÃà ÃÃà ÃÃà Ãà ÃÃà Ãà NS Significance levels: Ã. By comparison. as measured by the modified Schreiber method (A) or Olson & Price method (B). 20 10 0 80 70 60 (B) 0 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. with both the makeprocedure and storage time having significant effects (Table 4) and the CL cheese having a significantly lower mean stretchability over the 70-d storage period (Fig. T. A higher degree of paracasein aggregation in CL. DAS2.P.b Parameter Makeprocedure Storage time Makeprocedure  storage time interaction NS NS NS NS NS (A) 0 10 20 30 40 50 60 70 pH pH4. non-expressible serum per gram protein. reducing the pH of the DAS cheeses had only a relatively minor effect. Details of make-procedures and compositions are given in the text. CL). 2002).

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