Estudio de Los Niveles de Calcio y pH en Queso Mozzarella

8/13/2019 Estudio de Los Niveles de Calcio y pH en Queso Mozzarella

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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 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 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 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 low-

moisture 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 heat-

induced functional properties of Mozzarella cheese.

These include homogenisation of cheesemilk to increase

the surface area of the fat phase (Jana & Upadhyay,

1991; Tunick et al., 1993b; Poudaval &Mistry, 1999);

alterations of make-procedure (e.g., varying pasteurisa-

tion 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 (Per-

ry, 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 5.85.6, in

combination with a starter culture, resulted in a

reduction in calcium level of low-fat (6%, w/w) LMM

but did not significantly affect the levels of moisture or

MNFS; the experimental cheeses generally had a lower

pH (p5.2) than the control (B5.45) at timesX15 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 pre-

acidification pH, the type of acid used and final pH of

the cheese. More recently, Guinee, Feeney, Auty, and

ARTICLE IN PRESS

*Corresponding author. Tel.: +353-25-42204; fax: +353-25-42340.

E-mail address: [email protected] (T.P. Guinee).

0958-6946/$- see front matter r 2003 Elsevier Ltd. All rights reserved.

doi:10.1016/S0958-6946(03)00167-5


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Fox (2002) found that reducing the calcium-to-casein

ratio from B28 to B21mgg1, by preacidification of

milk prior to renneting, resulted in higher levels of

moisture and MNFS, and a lower protein level in the

resultant directly acidified LMM. These changes re-

sulted in marked increases in protein hydration in the

unheated cheese and in the flowability and stretchabilityof the heated cheeses, even though the pH of the LMM

from pre-acidified 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 soluble-

to-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). More-

over, 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 LMMwere manufactured by alteration of the make-procedure.

Acidification was achieved either by: a starter culture as

in conventional production (control cheese, CL), addi-

tion of lactic acid (directly acidified cheese, DA), or by a

combination of added lactic acid and starter culture

(directly acidified 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 o10C, pasteurised at

72C 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 modifications, as de-

scribed below. Milk (454 kg) was inoculated with a

starter culture consisting of Streptococcus thermophilus

andLactobacillus helveticus, added at levels of 1.0% and

0.5%, w/w, respectively. The cultures were grown

separately overnight at 37C in 10%, w/w, reconstituted

skim milk powder that had been heat-treated at 90C

for 30 min. Chymosin (Single strength Chy-max plus,

B50 Chymosin units mL1; Pfizer Inc, Milwaukee, WI,

USA) was added at a level of 0.18 mLkg1

milk andthe milk allowed to set at 36C. All gels were cut at a

firmness that was equivalent to an elastic shear modulus

(G0) value of 60 Pa, as measured using low-amplitude

strain oscillation rheometry (Guinee, Pudja,& Mulhol-

land, 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 38C, at a rate of

0.2Cmin1 and continually stirred until the pH of the

curd decreased to 6.1. The mixture was then pumped to

rectangular jacketed stainless 500 L finishing vats

(warmed to 38C 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 finishing vat, and

milled at pH 5.23, as measured at 38C by inserting a

pH probe (Radiometer Analytical S.A., Villeurbanne

Cedex-Lyon, France), calibrated at 38C 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 (Auto-

matic Stretching Machine, Model d; CMT, S. Lorenzo

di Peveragno CN, Italy). The curd was heated to 58C

by dosing with hot water at 78

C (used at a rate ofB1.4kgkg1 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 filled into

rectangular moulds. The resultant blocks were cooled

in dilute brine (10%, w/w, NaCl; 0.2%, w/w, Ca; pH

5.1) at B4.0C for 30 min to allow cooling to a surface

temperature of 24C and a core temperature ofo45C.

The cheeses, approximately 14 loaves per vat, were then

vacuum-wrapped and stored at 4C.

For the manufacture of DA, DAS1 and DAS2

cheeses, the milk (29C) was acidified 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

inTable 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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CL curd. The cooking rate (0.5Cmin1) 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

B60min (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 o1mm, in a

Krups Rotary 350 food processor fitted with a universal

blade (Robert Krups GmbH & Co, Postfach 190460, D-

5650 Solingen 19, Germany). Shredded cheese was

prepared by cutting the block, stored at 4C, 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 dia-

meter and 33.7 mm height) were procured using custom-

made 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 20g cheese and 12g 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 pre-

viously (Fenelon, Guinee, Delahunty, Murray, &

Crowe, 1999).

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Table 1

Treatments and details of the make-procedures used for manufacture of experimental reduced-fat Mozzarella cheeses

Cheese code

CL DA DAS1 DAS2

pH at different stages of manufacture

At set 6.41 5.60 5.60 5.64

At whey drainage 6.13 5.60 5.60 5.54

At curd milling 5.23 5.61 5.40 5.20

Acidification procedurea

pH adjustment before set Starter culture Lactic acid Lactic acid Lactic acid

pH decrease between setting and whey drainage Starter culture None Starter culture Starter culture

pH decrease between whey drainage and salting Starter culture None Starter culture Starter culture

Details of cheesemaking stepsb

Temperature of milk on adding acid (C) NAc 29 29 29

Set temperature (C) 36 29 29 29

Stirring speed, rate of increase (rpm/min) 1.0 0.6 0.6 0.6

Temperature at scald (C) 38 38 38 38

pH at milling 5.23 5.60 5.40 5.20

Times for cheesemaking stages (min)d

Ripening period (starter addition to set) 30 NA 10 10

Set (rennet addition) to cut 31 14 13 13

End of cut to stirring (healing) 10 3 3 3

Start of stirring to scald 14 22 22 22

Scald to start of whey drainage 41 7 9 11

End of drain to milling 85 56 77 165

aAcidification 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.bFull details of cheese manufacture are given in text.cNA, not applicable.dThe values given are the means of the three trials.

J.J. Sheehan, T.P. Guinee / International Dairy Journal 14 (2004) 161172 163


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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 (BIO-

RAD 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 samplebuffer and incubated at 55C for B10 min. The gels

(1 mm thick) were pre-run at 280V for B40 min prior to

sample application and 9 mL samples were loaded using a

micro-syringe (Hamilton, CH-7402, Bonaduz, Switzer-

land). 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 TA-

HDi Texture Profile analyser (Stable Micro Systems,

Godalming, Surrey GU7 1YL, England). Six samples

(25 25 25mm cubes) were obtained from each

cheese, placed in an airtight plastic bag and held at

8C overnight. Samples were withdrawn from 8C and

immediately compressed to 30% of original height at a

rate of 60mm min1 at room temperature. Fracture

stress was defined as the force per unit area to induce

fracture, and firmness 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 12500g at 20

C, was measured as described byGuinee 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 modified Schreiber

method, defined as the percentage increase in the

diameter of a disc of cheese (45.5 mm diameter,

6.5 mm thick) on melting at 280C for 4min in an

electric fan oven (Guinee et al., 2000b) and (ii) a

modified Olson & Price method (Olson&Price, 1958),

defined 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 fitted with

a holed rubber bung, on melting at 180C 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

0.066ms1 (Guinee & OCallaghan, 1997). Prior to

heating, the shredded cheese was distributed uniformly

at a fixed loading (2.5 kg m2) onto a pizza base, which

was pre-cut in half, with the two halves aligned to form

a flush interface. The base with cheese was then baked at

280C for 4 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 wasdrawn along a rail system at a constant speed of

0.066ms2 by a motor-driven winch system, resulting in

stretching of the molten cheese mass. 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.

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 incorpo-

rated 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 make-

procedure 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 significance was determined atPo0:05:

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,

flowability 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). Statisti-

cally significant differences (Po0:05) between means

were determined by Fishers least significant 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 sufficiently firm (60 Pa) to cut in B14 min,

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compared with B31min for the CL gel. The rapid

coagulation of rennet-treated pre-acidified 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 netnegative charge of the casein (van Hooydonk, Hage-

doorn, & 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

p29C(Guinee et al., 1994).

The DA and DAS curds had a relatively high

tendency to mat following cutting. This tendency reflects

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 fluidity of curd particles and their tendency to

flow 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, flowable and fluid 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

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

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 final pH, reflect 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 significant 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 non-functional Mozzarella cheese to one which has the

desired melt, flow and stretch properties (Kindstedt,

1995; Kindstedt & Guo, 1997b; McMahon, Fife, &

Oberg, 1999). An adequate WHC prevents excessive

dehydration at the temperature (X98C) 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 significantly affected by storage time, make-

procedure and their interaction (Table 3). The mean

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1.3

1.5

1.8

2.0

0 10 20 30 40 50 60 70

Storage time (d)

Nonexpres

sibleserum,

NESP(gg-

1p

rotein)

Fig. 1. Age-related changes in NES, expressed as g g1 protein, in

reduced-fat Mozzarella cheese made using different procedures:

conventional starter culture acidification (CL, K), direct acidification

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

significantly 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 (Kind-stedt&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 (byB0.150.3

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

buffering capacity as the pH is reduced from 6.0 to 5.1

(Lucey&Fox, 1993).

Make-procedure had a significant 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 reflect the pH at curd milling. Storage timeat 4C resulted in a slight (B0.1 unit) but significant

(Po0:05) decrease in the mean pH of all cheeses (Table

3). This trend agrees with that ofBarbano et al. (1994)

for LLM and with Metzger et al. (2001b) for some low

fat LMMs (from pre-acidified 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 byMetzger et al. (2001b). The different inter-

study pH/storage time trends may reflect 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 4C

resulted in a progressive degradation of as1- and b-caseins with the extent of breakdown ofas1-casein being

greater than that of the latter (Fig. 3). The breakdown

products,as1-casein (f 124-199),b-casein (f 1-192) andg-

caseins, 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 break-

down for DAS1 and DAS2 were higher than that in CL

and DA, as reflected 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 ofas1-

casein breakdown in the DAS1 and DAS2 cheeses

compared to DA may be due to their lower final pH,

which would be more favourable to proteolytic activity

by residual chymosin (Tam&Whitaker, 1972;Vander-

poorten & 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

ARTICLE IN PRESS

5.2

5.4

5.6

5.8

6.0

0 10 20 30 40 50 60 70

Storage time (d)

pH

Fig. 2. Age-related changes in pH of reduced-fat Mozzarella cheeses

made using different procedures: conventional starter culture acid-

ification (CL, K), direct acidification 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.



8/12


9/12

3.7. Functionality

In agreement with previous studies (Guinee, Mulhol-

land, Mullins, & Corcoran, 2000c), there was a

significant increase in the mean flowability and stretch-

ability 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 reflected 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 significantly affected the flowability

and stretchability (Fig. 6,Table 4). The mean flowability

of CL, as measured using both methods, was signifi-

cantly lower than that of DA or DAS cheeses. The

higher flowability 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 (R .uegg,

Eberhard, Popplewell, &Peleg, 1991; Kindstedt, 1995)

and primary proteolysis (Yun et al., 1993b; Madsen&

Qvist, 1998; Feeney et al., 2001). The latter composi-

tional changes are conducive to a greater degree of

casein hydration and would, therefore, be expected to

enhance heat-induced displacement of adjacent layers of

thepara-casein matrix on heating. It is noteworthy, that

the mean level of NESP in the DA and DAS cheeses was

significantly higher than that of CL.

The mean flowability of DA over the 70-d storage

period, as measured using the modified Schreiber

method, was significantly 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

ARTICLE IN PRESS

pH4.6

SN(%of

TotalN)

0.00

0.20

0.40

0.60

0.80

0 10 20 30 40 50 60 70

Storage time (d)

PTAN(%o

fTotalN)

0

1

2

3

4

5

6

7

0 10 20 30 40 50 60 70(A)

(B)

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 acidification (CL, K), direct

acidification 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.

Fig. 5. 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, ), direct acidification

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.



10/12

contiguous layers of the casein matrix (e.g., flow) for a

given heat-induced stress (Guinee et al., 2002).

The stretchability of the heated cheese showed a trend

similar to that noted for flowability, with both the make-

procedure 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.

6c). The similarity of trends between the flowability andstretchability were expected as both involve displace-

ment of thepara-casein matrix. A higher degree ofpara-

casein 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.

4. Conclusions

Reduced-fat low-moisture Mozzarella cheese was

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 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 significant improvements in the

flowability and stretchability of the cooked cheese

(Table 5). By comparison, reducing the pH of the

DAS cheeses had only a relatively minor effect.

ARTICLE IN PRESS

0

10

20

30

40

50

60

70

80

0 10 20 30 40 50 60 70

Storage time (d)

Stretch(cm)

0

10

20

30

40

0 10 20 30 40 50 60 70

Flowability:modifiedSchreiberm

ethod(%)

0

50

100

150

200

250

300

350

0 10 20 30 40 50 60 70F

lowability:modifiedOlson-Pricemethod(%)

(A)

(B)

(C)

Fig. 6. Age-related changes in flowability, as measured by the

modified Schreiber method (A) or Olson & Price method (B), and

stretchability (C), of reduced-fat Mozzarella cheeses made using

different procedures: conventional starter culture acidification (CL,

K), direct acidification by lactic acid (DA, J) or a combination of

both (DAS1,m; DAS2, n). Details of make-procedures and composi-

tions are given in the text. Values presented are the means from three

replicate trials.

Table 5

Statistical summary for effects of make-procedure, storage time and

their interaction on characteristics of reduced-fat Mozzarella cheesea,b

Parameter Make-

procedure

Storage

time

Make-

procedure storage

time interaction

pH

NSpH4.6SNc NS

PTANc

Firmness NS

NESPc

Flow (Schreiber

method)

NS

Flow (Olson &

Price method)

NS

Stretch NS

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

non-significant (P> 0:05).bDetails of make-procedure and storage conditions given inTable 1

and text.cpH4.6SN, pH 4.6-soluble N (% total N); PTAN, 5% phospho-

tungstic acid-soluble N (% total N); NESP, non-expressible serum per

gram protein.



11/12

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 Col-

lege, Cork, Ireland on the statistical analysis of the data.

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Estudio de Los Niveles de Calcio y pH en Queso Mozzarella