International Dairy Journal 14 (2004) 989–1001

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*Correspondin

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0958-6946/$ - see

doi:10.1016/j.ida

Effect of milk pasteurization temperature and in situ whey proteindenaturation on the composition, texture and heat-induced

functionality of half-fat Cheddar cheese

Nuala M. Rynnea, Thomas P. Beresforda, Alan. L. Kellyb, Timothy P. Guineea,*aDairy Products Research Centre, Teagasc Moorepark, Fermoy, Co. Cork, IrelandbDepartment of Food and Nutritional Sciences, University College, Cork, Ireland

Received 27 August 2003; accepted 12 March 2004

Abstract

In recent years, many dairy product manufacturers pasteurize milk at temperatures higher than 72�C, so as to reduce further the

risk of survival of Mycobacterium avium ssp. paratuberculosis. However, little information is available on the effect of these

temperatures on cheese quality. Half-fat Cheddar cheeses (B15%, w/w, fat) were manufactured from milk pasteurized at 72�C,

77�C, 82�C or 87�C for 26 s, and analyzed over a 360 d ripening period. The mean levels of whey protein denaturation in the

pasteurized milks were 2.8%, 8.4%, 20.2% and 34.1% of total whey protein, respectively. Increasing pasteurization temperature

significantly increased the levels of moisture and non-expressible serum and decreased the levels of protein, fat, calcium and free oil.

Pasteurization temperature did not significantly affect the levels of pH 4.6-soluble N but influenced the degradation profiles of as1-

and b-caseins. Increased pasteurization temperature significantly reduced fracture stress, fracture strain and firmness of cheeses.Raising the pasteurization temperature from 72�C to either 82�C or 87�C significantly reduced the flowability and stretchability of

the heated cheese and increased its apparent viscosity; in contrast, increasing pasteurization from 72�C to 77�C did not affect these

properties.

r 2004 Elsevier Ltd. All rights reserved.

Keywords: Pasteurization temperature; Whey protein denaturation; Half-fat Cheddar; Composition; Rheology; Functionality

1. Introduction

Milk for cheese manufacture is generally pasteurizedat 72�C, typically for 15–35 s. The work of Grant (2000)suggests that Mycobacterium avium ssp. paratuberculosis

(MAP), which is responsible for Johne’s disease incattle, may occasionally survive conventional pasteur-ization of milk at 72�C for 15 s, especially if the count inthe raw milk is >104CFUmL�1. In contrast, Pearce,Truong, Crawford, Yates, Cavaignac, & de Lisle, 2001found that MAP, at levels of 0.7� 103–16� 103CFUmL�1 did not survive heating at 72�Cfor 15 s in a pilot-scale pasteurizer operating underturbulent flow. Nevertheless, because of a possible linkbetween Johne’s disease and Crohn’s disease in humans,

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

s: tguinee@moorepark.teagasc.ie (T.P. Guinee).

front matter r 2004 Elsevier Ltd. All rights reserved.

iryj.2004.03.010

dairy product manufacturers have in recent yearsincreased pasteurization temperatures.Heretofore, pasteurization temperatures >72�C have

generally not been used in cheesemaking practice,because of their adverse effects on curd formation(Guinee, Gorry, O’Callaghan, O’Kennedy, O’Brien, &Fenelon, 1997; Singh & Waungana, 2001) and curdsyneresis (Pearse, Linklater, Hall, & MacKinlay, 1985;Pearse & MacKinlay, 1989). However, because of thepotential increase in cheese yield due to increasedmoisture content and more effective recovery of wheyproteins, the effects of increasing pasteurization tem-perature has been extensively studied at an experimentallevel (El-Koussy, Amer, & Ewais, 1977; Marshall, 1986;Green, 1990a, b; Guinee, Fenelon, Mulholland, O’Ken-nedy, O’Brien, & Reville, 1998; Lebeuf, Lacroix, &Paquin, 1998; Kelly, 1999). These studies have mainlyfocused on the effects of milk heat treatment on

ARTICLE IN PRESSN.M. Rynne et al. / International Dairy Journal 14 (2004) 989–1001990

composition, yield, texture and proteolysis. Guinee et al.(1998) concluded that high pasteurization temperature(HPT) of milk may play a role in reducing the firmnessof low-fat cheeses.Very little information is available on the effects of

HPT on the functional properties of heated cheese,particularly in relation to low-fat Cheddar cheese.However, the presence of whey proteins in cheese fromultrafiltered milks or the inclusion of whey proteins inprocessed cheese products can adversely influence theircooking properties (Guinee, 2003). In contrast, Schaferand Olson (1975) reported that increasing the pasteur-ization temperature of milk from 80�C to 130�C, for aresidence time of 2 s, resulted in increases in wheyprotein denaturation from B2% to 35% total wheyprotein, but did not influence the flow of Mozzarellacheese on melting.The objective of this study was to examine the effect

of pasteurization temperature on the composition,rheology, and heat-induced functionality of half-fatCheddar cheese.

2. Materials and methods

2.1. Cheese manufacture

Raw milk (2000L) was obtained from a local dairycompany, standardized to a protein to fat ratio of 2.35and stored overnight at 6�C. This milk was divided intofour portions, pasteurized at different temperatures(72�C, 77�C, 82�C and 87�C for 26 s), pumped to500L vats and cooled to 31�C. Defined strain startercultures Lactococcus lactis ssp. cremoris strain 303 andstrain 227 (Chr. Hansen Ireland Ltd., Rohan IndustrialEstate, Little Island, Cork) were grown separatelyovernight at 23�C in 10% (w/v) reconstituted, heat-treated (90�C for 30min), antibiotic-free skim milkpowder. Each strain was inoculated at a level of 0.7%(w/w) into the milk. The strain Lactobacillus helveticus

LB7 (Danisco Cultor Nieb .ull GmbH, Busch-Johannsen-Strabe 1, Germany) was added as an adjunct culture at arate of 10 g 100 kg�1 milk. After a 30min ripeningperiod at 31�C, rennet (Chymax Plus, Pfizer Inc.,Milwaukee, WI, USA), diluted 1:10 in de-ionized water,was added at a level of 0.18mLkg�1 to the milk. Thecurd was cut at a firmness of 54 Pa (see Section 2.2) andallowed to heal for 10min. The curd–whey mixture wascooked at a rate of 0.2�Cmin�1 to 38�C and pitched atpH 6.10. The curd was cheddared, milled at pH 5.75,salted at a level of 2.4% (w/w), mellowed for 20min,moulded and pressed overnight on a horizontal press at264.6 kPa. Cheeses were vacuum packed and stored at4�C for 30 d and at 8�C thereafter. Cheesemaking trialswere performed in triplicate.

The cheeses made from the milks pasteurized at 72�C,77�C, 82�C and 87�C are denoted C72, C77, C82 andC87, respectively.

2.2. Rennet coagulation properties

At 2min after rennet addition and stirring, arepresentative sample of vat milk was placed in aninsulated glass container, and within 2min a sub-samplewas placed in the cell of a controlled-stress rheometer(CSL2500 Carri-Med; TA Instruments, Inc., New Castle,DE, USA). The elastic shear modulus, G0; which wasused as an index of curd firmness, was measureddynamically at 31�C as a function of time, using lowamplitude oscillation as previously described (Fenelon& Guinee, 1999). For each milk, cutting of the gel in thecheese vat was initiated when the firmness reached 54 Pa(Fenelon & Guinee, 1999).

2.3. Milk analysis

Raw, standardized and pasteurized milks were ana-lyzed using standard IDF methods as described byGuinee, Auty, and Fenelon (2000): fat using the R .ose-Gottlieb method, total protein using the macro-Kjeldahlmethod, casein number (CN) using sodium acetate–acetic acid precipitation, and non-protein N (NPN) bysolubility in 12% (w/v) tri-chloroacetic acid. The wheyprotein (WP), as a percentage of total protein, in rawand pasteurized milks were calculated using the formula:

WP ¼ ð100� CNÞ �NPNð% total NÞ

The whey protein denatured (WPD) on heating, as apercentage of total whey protein, was calculated usingthe following formula:

WPD ð%Þ ¼ 100ðWPrm �WPpmÞ=ðWPrmÞ;

where WPrm and WPpm are the levels of whey protein inthe raw and pasteurized milks, respectively. Thedenatured whey protein (DWP), expressed as % (w/w),was calculated using the following formula:

DWPð%;w=wÞ ¼ 100 PpmWPrm

100

� �WPDpm

100

� �� �;

where Ppm is the protein content of the pasteurized milk,and WPDpm is the WPD (%) in the pasteurized milk.

2.4. Cheese analysis

2.4.1. Composition

Cheese samples were grated to yield particles ofo1mm, as described by Guinee, Feeney, Auty, and Fox(2002). Samples were analyzed at 14 d for salt, protein,fat and moisture using standard IDF methods (Guineeet al., 2000); the pH was determined at 1 d on a slurry

ARTICLE IN PRESSN.M. Rynne et al. / International Dairy Journal 14 (2004) 989–1001 991

prepared from 20 g cheese and 12 g de-ionized water(Guinee et al., 2000).Grated cheese (1 g) was weighed into a porcelain

crucible, heated over a naked flame until it had stoppedsmoking, and placed in a muffle furnace for 12 h at550�C (Fenelon & Guinee, 2000). The resulting ash wasweighed and analyzed for calcium and phosphorus(Guinee et al., 2000).

2.4.2. Non-expressible serum

The level of expressible cheese serum (ES) wasobtained by centrifugation at 12,500 g for 75min at25�C, as described by Guinee et al. (2002). The serumand fat were weighed into a graduated cylinder andstored at 4�C until the fat layer had solidified. The fatlayer was then punctured to release the serum, whichwas collected and weighed.The level of non-expressible serum (NES) per 100 g

cheese was obtained by subtracting the level of ES fromthe percentage moisture, and expressed per unit weightof protein (NESP; g g�1 protein).

2.4.3. Proteolysis

The levels of pH 4.6-soluble cheese nitrogen (pH4.6-SN) and 5% (w/v) phosphotungstic acid-soluble cheesenitrogen (PTA-SN), expressed as percentages of totalnitrogen (TN), were measured as described by Fenelon,O’Connor, and Guinee (2000b). Urea–polyacrylamidegel electrophoresis (urea–PAGE) of cheese samples wasperformed on a PROTEANs II xi cell vertical slab gelunit (Bio-Rad Laboratories Ltd., Hemel Hempstead,Herts, UK), using a separating and stacking gel system,as described by Fenelon, Guinee, Delahunty, Murray,and Crowe (2000a). The sample buffer (pH 8.7) wasprepared by dissolving 0.75 g Tris (hydroxymethyl)-methylamine, 49 g urea, 0.7mL mercaptoethanol and0.15 g bromophenol blue in de-ionized water to a finalvolume of 100mL. Cheese (i.e., B15mg for C72 toB16.5mg for C87 cheese) was dissolved on a proteinbasis (4.9mg protein) in 1mL of sample buffer and wereincubated at 55�C for 15min. The gels (1mm thick)were pre-run at 280V for 35min prior to sampleloading.Cheese samples were loaded, via micro-syringe, both

on a total protein basis and on a total casein basis ontoseparate gels. The level of casein in the cheese wasestimated by calculating the whey protein in the cheesemilk, expressing as a fraction of total casein plusdenatured whey protein, and multiplying the totalprotein content in the cheese by this factor. Theoperating voltage during separation was at 280V inthe stacking gel and 300V in the separating gel. Whenthe tracking dye was approximately 1 cm from thebottom of the gel plates, the gels were removed andstained directly in Coomassie Blue G250 dye, as

described by Fenelon and Guinee (2000), and then de-stained in repeated changes of distilled water.

2.4.4. Plasmin activity

Plasmin activity in cheese was determined by themethod of Richardson and Pearce (1981).

2.4.5. Free oil

Free oil was measured using a modified Gerber Test,as described by Kindstedt and Fox (1991). This involvedextraction of the fat from a cheese at 60�C using a 1:1distilled water:methanol (v/v) solution, followed bycentrifugation at 362 g.

2.4.6. Rheological properties

Cheeses were analyzed using compression on a TA-HDi Texture Profile analyzer (Stable Micro Systems,Vienna Court, Lammas Road, Godalming, Surrey, UK)located at room temperature. Six samples (25mm cubes)were prepared from each cheese, placed in an airtightplastic bag, held at 4�C overnight, and compressed at arate of 60mmmin�1 in one compression bite. Fracturestress was defined as the force per unit area to inducefracture of the cheese cube (i.e., to form cracks and/orbreak the cube into pieces) and was calculated from theforce at the minimum slope of the force–displacementcurve. The fracture strain was defined as the fractionaldisplacement at fracture, and firmness as the forcerequired to compress the cheese to 30% of its originalheight.

2.4.7. Physical characteristics of the heated cheese

Flowability was measured by determining the percen-tage increase in the diameter of a disc of cheese (45mmdiameter, 6.5mm thick) on melting at 280�C for 4min(Guinee et al., 2002). Flowability was also measured bya modification of the Olson/Price method (Olson &Price, 1958). This involved placing a 15 g cylindricalcheese sample (diameter, 22mm; height, 35mm) in thecenter of a 100-mL graduated glass tube, one end ofwhich was closed and the other fitted with a holedrubber bung. The tube was placed in a horizontalposition, on a stainless steel tray, in an electric fan ovenat 180�C for 7.5min. The tray was then removed andallowed to cool to room temperature (B20min), and thepercentage flow was defined as the percentage increasein the length of the cylinder of cheese.The stretchability of the melted cheese on a baked

pizza base was defined as the length of strings at failureon uniaxial extension of molten cheese, melted on apizza base, at a velocity of 0.066m s�1, as described byGuinee et al. (2002).The maximum apparent viscosity (AV) of the molten

cheese (at 80�C) was measured using a helipathviscometer (Brookfield, Mode DV-II; Brookfield En-gineering Laboratories Inc., Stoughton, MA), using a

ARTICLE IN PRESSN.M. Rynne et al. / International Dairy Journal 14 (2004) 989–1001992

spindle speed of 5 revmin�1 and a helipath speed of22mmmin –1 (Guinee et al., 2000).

2.5. Statistical analysis

Three replicate cheesemaking trials were undertaken;in each, cheeses were produced from milk pasteurized ateach of 4 temperatures (72�C, 77�C, 82�C or 87�C).

Table 2

The effect of pasteurization temperaturea on the compositionb of half-

fat Cheddar cheese

Pasteurization temperature (�C)c

Composition72 77 82 87 SEDd

Moisture (%, w/w) 45.2e 47.3ef 48.8fg 49.9g 1.03

Fat (%, w/w) 16.6e 15.3e 15.1e 13.5f 0.63

Protein (%, w/w) 32.5e 31.4ef 30.8ef 30.1f 0.75

Casein (%, w/w) 32.3e 30.8ef 29.4fg 28.0g 0.73

DWP (%, w/w)d 0.21e 0.59f 1.35g 2.17h 0.11

S/M (%, w/w)d 3.11e 3.41e 2.81e 2.96e 0.26

FDM (%, w/w)d 30.35e 29.06e 29.46e 28.85e 1.00

MNFS (%, w/w)d 54.2e 55.9ef 57.5ef 61.0f 2.37

Ash (%, w/w) 4.03ef 4.22e 3.84f 3.92f 0.08

Ca (mg 100 g�1) 927ef 990e 850fg 813g 44.55

P (mg 100 g�1) 657e 633ef 607f 586f 18.88

pH at 1 d 5.06e 5.11e 4.95f 4.99f 0.03

aThe residence time at the pasteurization temperature was 26 s; data

presented are the means of three replicate trials.bComposition was determined at 14 d and pH at 1 d.cValues within a row not sharing a common superscript differ

significantly, Po0:05:dAbbreviations: SED: standard error of difference across the 4

treatment means; DWP, denatured whey protein; S/M, salt in

moisture; FDM, fat in dry matter; MNFS, moisture in non-fat

substances.

Table 1

Composition of raw milk and cheesemilks pasteurized at different

temperaturesa

Composition Raw milk [Pasteurized milksb]

72�C 77�C 82�C 87�C

Protein (%, w/w) 3.27 3.35d 3.36d 3.36d 3.35d

Fat (%, w/w) 3.89 1.47d 1.47d 1.46d 1.42d

Lactose (%, w/w) 4.64 4.77e 4.78de 4.79de 4.79d

Casein no. 76.52 77.75d 78.74e 80.67f 82.84g

Casein (%, w/w) 2.50 2.56d 2.57d 2.57d 2.56d

NPN (%, w/w)c 6.66 5.83d 5.94d 5.98d 6.13d

Native whey protein (% total) 16.75 16.43d 15.32e 13.35f 11.03g

WPD (% total whey protein)c NAc 2.79d 8.39e 20.16f 34.10g

DWP (%, w/w)c NAc 0.02d 0.05e 0.12f 0.20g

aThe residence time at the pasteurization temperature was 26 s; data

presented are the means of three replicate trials.bValues within a row not sharing a common superscript differ

significantly, Po0:05:cAbbreviations: NPN, non-protein nitrogen; WPD, whey protein

denaturation on heating; DWP, denatured whey protein; NA, not

applicable.

A randomized complete block design which incorpo-rated the four treatments (pasteurization temperatures),and 3 blocks (replicate trials) was used for analysis ofthe response variables relating to compositions of thecheesemilk and cheese (Tables 1 and 2). Analysis ofvariance (ANOVA) was carried out using a SASprocedure (SAS, 1995) where the effect of treatmentand replicates were estimated for all response variables.Duncan’s multiple-comparison test was used as a guidefor pair comparisons of the treatment means. The levelof significance was determined at Po0:05:A split plot design was used to monitor the effects of

treatment, storage time and their interaction on theresponse variables measured at regular intervals duringstorage, i.e., free oil, non-expressible serum, pH 4.6-SN,PTA-SN, firmness, stress, strain, flowability, apparentviscosity and stretchability. Analysis of variance for thesplit plot design was carried out using a general linearmodel (GLM) procedure of SAS (SAS, 1995). Statisti-cally significant differences (Po0:05) between differenttreatment levels were determined by Fisher’s leastsignificant difference.

3. Results and discussion

3.1. Milk composition

Increasing the pasteurization temperature resulted inincreased levels of WPD, fromB2.8%, at 72�C for 26 s,to 34% of total at 87�C for 26 s (Table 1). Themagnitude of the increase was of the same order as thatreported in previous studies (Guinee et al., 1997; Guineeet al., 1998).

3.2. Rennet coagulation characteristics

Increasing the level of WPD (by elevating thepasteurization temperature) impaired the rennet coagu-lation properties of milk resulting in a slower develop-ment of the elastic shear modulus, G0; with time fromrennet addition (Fig. 1). The poor rennet coagulabilitycoincided with an increase in set-to-cut times duringcheese manufacture e.g. from a mean of 48min at 72�Cto 78min at 87�C. The adverse effect of HPT has beenobserved by numerous investigators (McMahon, You-sif, & Kalab, 1993; Guinee, O’Callaghan, Pudja, &O’Brien, 1996; Guinee et al., 1997; Pomprasirt, Singh, &Lucey, 1998; Singh & Waungana, 2001). The effect ofHPT on rennet coagulability has been attributed, inter

alia, to the following factors (van Hooydonk, de Koster,& Boerrigter, 1987; McMahon et al., 1993; Hyslop,2003): (i) complex formation between denatured wheyproteins and k-casein at the micelle surfaces; (ii) theconcomitant reduction in accessibility of the rennet tothe k-casein; (iii) heat-induced deposition of calcium

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05

1015202530354045505560

0 1000 2000 3000 4000

Time (s)

Ela

stic

she

ar m

odul

us, G

', (P

a)

Fig. 1. The effect of milk pasteurization temperature (72�C, �; 77�C,J; 82�C, m and 87�C, n), with holding for 26 s, on the elastic shear

modulus of half-fat cheese milks.

N.M. Rynne et al. / International Dairy Journal 14 (2004) 989–1001 993

phosphate and the consequent reduction in nativecalcium phosphate, which is important for cross-linkingpara-casein micelles, on cooling; (iv) steric hindranceprovided by the casein–whey protein aggregates to closeapproach and fusion of the para-casein micelles.

3.3. Cheese composition

Increasing pasteurization temperature from 72�C to87�C resulted in significant increases in the levels ofmoisture, moisture in non-fat substance (MNFS) andreduction in the contents of fat, protein, calcium andphosphorus in the cheese (Table 2). Similar observationshave been reported previously (Schafer & Olson, 1975;Banks, Law, Leaver, & Horne, 1994; Guinee et al.,1998). Likewise, the addition of partially denaturedwhey protein preparations to cheese milk increase yieldsof Cheddar and Gouda cheeses (Guinee, 2003). Theincreases in moisture and MNFS are of a similarmagnitude to those reported previously for a similarrange of temperature and holding times for milks usedto make full-fat Cheddar cheese (Guinee et al., 1998).The positive relationship between pasteurization tem-perature and cheese moisture concurs with the results ofPearse et al. (1985), which showed an adverse effect ofheat treatment on syneresis of rennet-induced milk gelsin model systems. The adverse effects of HPT onsyneresis are probably due, in part, to the presence ofdenatured whey protein-k-casein complexes on thesurface of the rennet-treated micelles that retardaggregation and fusion of the rennet-treated micelles.Thus, it is noteworthy that syneresis of rennet-treatedsynthetic milk not containing whey proteins is scarcelyaffected by HPT (Walstra, van Dijk, & Geurts, 1985)and that the decrease in syneresis of rennet-treated milkwas linearly correlated with degree of b-lactoglobulindenaturation (Pearse et al., 1985).

The decrease in levels of protein and fat withpasteurization temperature are due to concomitantincrease in moisture, and hence, the reduction in levelof cheese dry matter. The estimated level of denaturedwhey protein (DWP) in the cheese increased significantlyin all cheeses with increasing pasteurization temperature(Table 2).

3.4. Age-related changes in pH

In agreement with an earlier study (Guinee et al.,1998), increasing pasteurization temperature of the milksignificantly reduced the pH of the cheese at 1 d (Table2); this trend continued throughout ripening (data notshown) with a significant effect of pasteurizationtemperature on the mean pH over the 360-day ripeningperiod. The lower pH in the HPT cheeses is due to theirhigher level of moisture and, thus, lactic acid (Wallace &Fox, 1997; Walstra, Noomen, & Geurts, 1993) and thelower levels of protein, casein as a percentage of thetotal protein, and thus buffering capacity (Lucey & Fox,1993).

3.5. Non-expressible serum and free oil

The level of non-expressible serum, in g g�1 protein(NESP), has been used as an index of the water holdingcapacity (WHC) of cheese, higher levels indicatinghigher WHC (Guinee et al., 2002). The NESP of cheeseincreased significantly between 1 and 30 d and thereafterremained essentially constant (data not shown). Increas-ing pasteurization temperature significantly increasedthe level of NESP, with the relationship being almostlinear (Table 3); the mean values at 365 d were 1.4, 1.5,1.6 and 1.7 g g�1protein for C72, C77, C82 and C87,respectively. The increased NESP correlates well withthe positive relationship between moisture content andpasteurization and the adverse effect of WPD onsyneresis (Pearse et al., 1985; Green, 1990a). Thecomplexing of DWP with k-casein in high heat-treatedmilk probably leads to a finer-structured gel with asmaller pore size than the gel from milk pasteurized atnormal temperature (Green, 1990b; Guinee et al., 1995).Such a gel would have a greater ability to retainmoisture by an increased capillary effect.The mean free oil content in cheese over the 360-d

ripening period decreased with increasing milk pasteur-ization temperature, with the mean value for C87 beingsignificantly lower than that of C72 and C77 (Fig 2;Table 3). The quantity of free oil for C72 was lower(B50%) than that reported by Rudan, Barbano, Yun,and Kindstedt (1999) for reduced-fat Mozzarella cheesewith a similar fat content. Differences in free oil cannotbe attributed to the decrease in fat content aspasteurization temperature increased, as free oil, ex-pressed as a percentage of total cheese fat, gave a similar

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0

1

2

3

4

5

6

120600 180 240 300 360Ripening time (day)

Fre

e oi

l (%

tota

l che

ese

fat)

Fig. 2. The effect of milk pasteurization temperature (72�C, �; 77�C,J; 82�C, m and 87�C, n), with holding for 26 s, on the levels of free

oil in half-fat Cheddar cheese.

Table 3

Probabilities (P) and significance of effects of milk pasteurization temperature and ripening time on free-oil (% total cheese fat), non-expressible

serum (g g�1 protein), pH4.6-SN (% total N) and PTA-SN (% total N) in half-fat Cheddar cheesesa

Free oil (% (w/w) total cheese fat) NESPb (g g�1 protein) pH4.6-SNb, % total N PTA-SNb, %total N

Factors P Significancec P Significancec P Significancec P Significancec

Main plot

Pasteurization temperature 0.022 � 0.023 � 0.009 �� 0.0501 NS

Sub-plot

Ripening time o0.0001 ��� o0.0001 ��� o0.0001 ��� o0.0001 ���Interaction 0.04 � 0.227 NS 0.531 NS 0.002 ��(Past. temp� ripening time)

aMilks were pasteurized at 72�C, 77�C, 82�C or 87�C and held for 26 s.bAbbreviations: NESP, non-expressible serum (expressed as g g�1 protein); pH4.6-SN, pH 4.6-soluble N; PTA-SN, 5% phosphotungstic acid-

soluble N.cStatistical significance: �Po0:05; ��Po0:01; ���Po0:001; NS, not significant.

N.M. Rynne et al. / International Dairy Journal 14 (2004) 989–1001994

trend as to when free oil was expressed as g 100 g�1

cheese (results not shown). The reduction in free fat levelas milk pasteurization temperature increased may beassociated with some chemical change in the milk fatglobule membrane which renders it more stable in thecheese, e.g., disulphide interchange reactions betweendenatured whey proteins and cysteine residues in themembrane proteins (van Boekel & Walstra, 1995).

3.6. Proteolysis

3.6.1. Changes in pH 4.6-soluble N (pH4.6-SN) and 5%

phosphotungstic acid-soluble N (PTA-SN)

The degree of primary proteolysis was measured bymonitoring the level of pH 4.6-SN, which includesmainly peptides with molecular mass p10 kDA (Fene-lon et al., 2000b). Ripening was accompanied by asignificant increase in the level of pH4.6-SN (Table 3),the mean value of which increased from 2.8% TN at 1 dto 27.9% TN at 360 d (data not shown). The increasewas of the same order of magnitude as that previously

reported for half-fat Cheddar cheese (Guinee et al.,2000; Fenelon et al., 2000b; Fenelon, Beresford, &Guinee, 2002). Pasteurization temperature significantlyaffected the formation of pH4.6-SN, with the mean levelin C72 over the 360-d ripening period being slightly, butsignificantly, lower than that in the C82 and C87cheeses. While there was a numerical increase in theconcentration of the pH4.6-SN in the C77, C82 and C87cheeses with pasteurization temperature, the mean levelsdid not significantly differ. The higher level of pH4.6-SNin the C82 and C87 cheeses than in C72 may be due tothe higher levels of MNFS (Creamer, 1971; Pearce &Gilles, 1979), which would be conducive to higher levelsof available moisture and, hence, increases in microbialand enzymatic activities (Lawrence & Gilles, 1980).Similar trends for the effect of milk heat treatment onpH4.6-SN or water-soluble N have been reported inGouda-type cheeses from ultrafiltered milk (Guineeet al., 1995) and Cheddar cheese (Kelly, 1999). Theseresults are also consistent with those of Lo and Bastian(1998), who reported that increasing the pasteurizationtemperature of ultrafiltered milk retentate from 72�C to85�C resulted in reduced hydrolysis of as1-casein in UFHavarti cheese.The degree of secondary proteolysis was quantified by

measuring the levels of PTA-SN, which includes lowmolecular mass peptides (o15 kDa) and free aminoacids (Jarrett, Aston, & Dulley, 1982). The levelsof PTA-SN increased significantly during ripening(Table 3), from a mean value of 0.7% TN in all cheesesat 1 d, to values of 13.4, 11.3 and 8.8 in the C72, C77 andC87, respectively, at 360 d (data not shown). The meanlevels over the 360-d ripening period were significantlylower in C87 than in the other cheeses. While the levelsin the other cheeses did not differ statistically, the meanlevels in C72 were numerically higher at most ripeningtimes. A similar trend was noted by Guinee et al. (1995)who reported that increasing the heat treatment ofultrafiltered milk retentate from 72�C to 85�C resulted

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D1 D60 D120

D180 D270 D360

γ2-CNγ1-CNγ3-CN

β-CN(f1-192)αS2-CN

αS1-CN

αS1-CN(f99-199)

αS1-CN(f24-199)αS1-CN(f121-199)

αS1-CN f80-

C 72 77 82 87 72 77 82 87 72 82 8777

C 72 77 82 87 72 77 82 87 72 82 8777

β-CN

γ2-CNγ1-CNγ3-CN

β-CN(f1-192)αS2-CN

αS1-CN

αS1-CN(f99-199)

αS1-CN(f24-199)αS1-CN(f121-199)

αS1-CN f80-

β-CN

(a)

(b)

Fig. 3. (a,b)Urea–polyacrylamide gel electrophoresis analysis of

sodium caseinate (C) and samples of half-fat cheddar cheese made

from milk pasteurized at different temperatures and ripened at 1, 60,

120, 180, 270 or 360 d.

N.M. Rynne et al. / International Dairy Journal 14 (2004) 989–1001 995

in lower concentrations of PTA-SN, low molecular mass(o 1.5 kDa) peptides and free amino acids in semi-hardGouda-type cheese prepared from the retentate. Thelower pH in the C87 cheese may partly explain the lowerPTA-SN and higher pH4.6-SN in the C87 cheeses. ThepH optimum for many peptidases, which are mainlyresponsible for the production of low molecular masspeptides that account for the bulk of PTA-SN fractionof cheese (Jarrett et al., 1982), is in the pH range 6.7–7.5(Hickey, van Leeuwen, Hillier, & Jago, 1983; Yan,Azuma, Kaminogawa, & Yamauchi, 1987; Gobbetti,Lanciotti, De Angelis, Corbo, Massini, & Fox, 1999).Conversely, the proteolytic activity of chymosin, whichis generally considered to be the principal agentcontributing to the formation of primary proteolysisand formation of pH4.6-SN in Cheddar cheese (Sousa,Ard .o, & McSweeney, 2001), increases as the pHdecreases (Fox, 1969; Tam & Whitaker, 1972; Mulvihill& Fox, 1980).

3.6.2. Polyacrylamide gel electrophoresis (PAGE)

Urea–PAGE gel electrophoretograms of the differentcheeses, from trial 3, applied to the gel on a total proteinbasis are shown in Fig. 3; similar trends were obtainedfor the other trials (data not shown). In agreement withthe trends reported in previous studies for reduced-fatand full-fat Cheddar cheeses (Phelan, Guiney, & Fox,1973; Creamer & Olson, 1982; Kelly, Fox, & McSwee-ney, 1996; Fenelon & Guinee, 2000; Fenelon et al.,2000a), both as1- and b-casein (CN) were progressivelydegraded during maturation, with the rate of extent ofbreakdown of former being much higher than that of thelatter. A band, which appeared between the b-casein andas1�casein, and which probably corresponds toas2�casein, (McSweeney, Olson, Fox, & Healy, 1994),was present in all cheeses at 1 d but had virtuallydisappeared by 60 d.For a fixed weight of protein loaded onto the gel,

increasing pasteurization temperature of milk resulted ina more rapid degradation of as1-CN to as1-CN(f24-199)during the early stages of ripening (e.g., p120 d). AttimesX120 d, hydrolysis of as1-CN(f24-199) to peptidesof higher electrophoretic mobility increased as thepasteurization temperature increased, resulting in nota-bly lower levels of the former in C82 and C87 than inC72 and C77 at 270 and 360 d. The two bands betweenthe as2-CN and as1-CN probably correspond to peptidesas1-CN(f80-) and as1-CN(f99-199), as described byMooney, Fox, Healy, and Leaver (1998). The intensityof the former increased with pasteurization temperatureand with ripening time, while the latter was present onlyin the C87 at 360 d.The overall degradation pattern of b-CN in C72 was

very similar to that noted by Fenelon and Guinee (2000)for reduced-fat Cheddar cheese. Increasing pasteuriza-tion temperature resulted in a slight decrease in the

extent of b-CN degradation; this trend agrees with theobservations of Benfeldt and Sørensen (2001) andBenfeldt, Sørensen, Elleg(ard, and Petersen (1997) onDanbo cheese and with those of Somers and Kelly(2002) for full-fat Cheddar cheese. In accordance withthe observations of Somers and Kelly (2002), theintensity of the b-CN fractions, f(106-128), f(106-209),f(29-209), f(108-209) and f(30-), as identified by Mooneyet al. (1998), was highest in C72 and C77 and lowest inC87. The levels of the latter peptides in the cheeses wereconsistent with the levels of plasmin activity, which wassignificantly higher in the C72 and C77 cheeses than inthe C87 cheese (Table 4). Hence, the reduction indegradation of b-CN as the milk pasteurizationtemperature increased may reflect the concomitantinactivation of plasmin and plasminogen which resultsin a decrease in plasmin activity (Benfeldt et al., 1997;Somers & Kelly, 2002). Interestingly, the plasminactivity increased in all cheeses during ripening (Table 4),

ARTICLE IN PRESSN.M. Rynne et al. / International Dairy Journal 14 (2004) 989–1001996

suggesting activation of plasminogen which is perhapsnot surprising as plasminogen activators are known tobe very heat-stable (Lu & Nielsen, 1993). The reductionin cheese pH as the pasteurization temperature of themilk was increased would also be unfavourable to theactivity of plasmin, an alkaline proteinase (Dulley, 1972;Benfeldt et al., 1997).Similar trends were found when cheese samples were

loaded onto gels on a casein basis (data not shown).

3.7. Rheology

Similar to previous results for reduced-fat Cheddarcheese (Fenelon & Guinee, 2000), the firmness (smax),fracture stress (sf ), and fracture strain (ef ) decreasedsignificantly with ripening time (Table 5; Fig. 4). Thistrend is consistent with the increases in proteolysis(Fig. 3) and the concomitant decrease in intact caseincontent that have been found to be inversely correlatedwith smax and sf (de Jong, 1976, 1977; Creamer &Olson, 1982; Guinee et al., 2000).Increasing milk pasteurization temperature signifi-

cantly decreased the mean values for smax; sf ; and efover the 360-d ripening period. This trend, which

Table 4

The effect of milk pasteurization temperature on the plasmin activity

(AMCa units per g cheese) of half-fat Cheddar cheeseb

Cheese age (d) Pasteurization temperature (�C)

72 77 82 87

1 0.87 0.87 0.55 0.31

(70.13) (70.16) (70.15) (70.04)180 1.15 1.19 0.86 0.40

(70.34) (70.35) (70.15) (70.00)360 1.29 1.48 1.07 0.53

(70.39) (70.36) (70.32) (70.07)

aAMC, 7-amino-4-methyl-courmarin (AMC).bPresented data are the mean of three replicate cheese trials;

standard deviations are given in parenthesis.

Table 5

Probabilities (P) and significance of effects of milk pasteurization tempera

Cheddar cheesea

Factors Firmness (N)

P Significancec

Main plot

Pasteurization temperature 0.0037 ��Sub-plot

Ripening time o0.0001 ���Interaction 0.1884 NS

(Past. temp� ripening time)

aMilks were pasteurized at 72�C, 77�C, 82�C or 87�C and held for 26 s.bFracture strain is a dimensionless quantity.cStatistical significance: �Po0:05; ��Po0:01; ���Po0:001; NS, not signi

concurs with previous studies (El-Koussy et al., 1977;Marshall, 1986; Guinee et al., 1998), was expectedbecause of the increase in moisture content andreduction in protein level as the pasteurization tempera-ture was increased (de Jong, 1978; Guinee et al., 2000).Another factor contributing to reduction in smax and sfwith pasteurization temperature is the increase in thelevel of DWP, as a percentage of total protein in thecheese. DWP that are complexed with the caseinmicelles during heating reduce the degree of caseinaggregation during subsequent rennet coagulation andcurd formation (Pearse et al., 1985; Green, 1990a, b) andlead to a finer gel matrix structure; hence, the strands ofthe matrices in cheeses from HPT milks may be lesscapable of bearing stress than those in cheese made frommilk pasteurized at 72�C.Increasing the milk pasteurization temperature

also significantly decreased the mean value of ef overthe 360-d ripening period (Table 5, Fig. 4c). Thisdecrease coincides with a significant increase in DWP asa fraction of the total cheese protein (Table 2). TheDWP, which are embedded within the casein matrix ofthe cheese, may reduce the degree of casein–caseininteraction and thereby reduce the continuity of thecasein matrix per se and contribute to a shorterconsistency. This trend in the current study contrastswith the observations of Watkinson, Crawford, andDodds (2002) and Visser (1991) for model Cheddar-typeand Gouda cheeses, respectively, in which increases inmoisture content were paralleled by increases in ef :However, in contrast to the current study, the moisturecontents in cheeses in the latter studies were adjusted bymeans other than HPT of milk.

3.8. Physical characteristics of the heated cheese

The changes in the heat-induced functional character-istics are shown in Figs. 5 and 6. As reported in previousstudies on reduced-fat Cheddar cheese (Guinee et al.,2000), maturation significantly increased the mean

ture and ripening time on the rheological characteristics of half-fat

Fracture stress (kPa) Fracture strain (�)b

P Significancec P Significancec

0.0007 ��� o0.0001 ���

o0.0001 ��� o0.0001 ���0.0987 NS 0.1655 NS

ficant.

ARTICLE IN PRESS

200

250

300

350

400

450

500

0 60 120 180 240 300 360

Firm

ness

(N

)

150

250

350

450

550

650

0 60 120 180 240 300 360

Fra

ctur

e st

ress

(kP

a)

0.2

0.3

0.4

0.5

0.6

0.7

0 60 120 180 240 300 360Ripening time (day)

Fra

ctur

e st

rain

(-)

(a)

(b)

(c)

Fig. 4. The effect of milk pasteurization temperature (72�C, �; 77�C,J; 82�C, m and 87�C, n), with holding for 26 s, on age-related

changes in the firmness (a), fracture stress (b) and fracture strain (c) of

half-fat Cheddar cheese.

0

10

20

30

40

50

60

70

0 60 120 180 240 300 360

Sch

reib

er fl

ow (

%)

0

50

100

150

200

250

300

350

400

0 60 120 180 240 300 360Ripening time (day)

Ols

on/P

rice

flow

(%

)(b)

(a)

Fig. 5. The effect of milk pasteurization temperature (72�C, �; 77�C,J; 82�C, m and 87�C, n), with holding for 26 s, on age-related

changes in the flowability of half-fat Cheddar cheese, as measured by

the (a) Schreiber method or (b) Olson/Price method.

0

500

1000

1500

2000

2500

3000

0 60 120 180 240 300 360

App

aren

t vi

scos

ity (

Pa

s)

0

10

20

30

40

50

60

0 60 120 180 240 300 360Ripening time (day)

Str

etch

abili

ty (

cm)

(a)

(b)

Fig. 6. The effect of milk pasteurization temperature (72�C, �; 77�C,J; 82�C, m and 87�C, n), with holding for 26 s, on age-related

changes in the apparent viscosity (a) and stretchability (b) of half-fat

Cheddar cheese.

N.M. Rynne et al. / International Dairy Journal 14 (2004) 989–1001 997

flowability and decreased the mean apparent viscosity(AV) of all cheeses (Figs. 5 and 6(a), Table 6). Theincrease in flowability during the first 30 d, as measuredusing both methods, was much larger than that in theremainder of the ripening period. These changes infunctionality coincide with increases in proteolysis andnon-expressible serum, which was most pronounced inthe first 30 d. It is noteworthy that the level of intactcasein in Cheddar cheeses of different fat contents hasbeen found to be negatively correlated with flowabilityand positively correlated with AV (Guinee et al., 2000).Other studies have reported a positive correlationbetween flowability and proteolyis for Cheddar cheese(Arnott, Morris, & Combs, 1957), Mozzarella (Yun,Barbano, & Kindstedt, 1993; Yun, Kiely, Kindstedt, &Barbano, 1993; Guinee, Feeney, & Fox, 2001) and UFMozzarella (Madsen & Qvist, 1998). The stretchabilityof all cheeses increased during the first 30 d and thendecreased to an extent dependent on the pasteurizationtemperature of the milk. While a similar trend was

ARTICLE IN PRESS

Table 6

Probabilities (P) and significance of effects of milk pasteurization temperature and ripening time on the heat-induced functionality of half-fat

Cheddar cheesea

Schreiber flow (%) Olson/Price flow (%) Stretchability (cm) Apparent viscosity (Pa.s)

Factors P Significanceb P Significanceb P Significanceb P Significanceb

Main plot

Pasteurization temperature o0.0001 ��� o0.0001 ��� 0.0001 ��� 0.0502 NS

Sub-plot

Ripening time o0.0001 ��� o0.0001 ��� o0.0001 ��� o0.0001 ���Interaction 0.0013 �� 0.0002 ��� 0.2015 NS 0.011 �(Past. temp� ripening time)

aMilks were pasteurized at 72�C, 77�C, 82�C or 87�C and held for 26 s.bStatistical significance: �Po0:05; ��Po0:01; ���Po0:001; NS, not significant.

N.M. Rynne et al. / International Dairy Journal 14 (2004) 989–1001998

previously reported for half-fat Cheddar cheese (Guineeet al., 2000), the time at which stretchability decreasedwas B90 d in that study. However, in both studiesstretchability began to decrease when the level of pH4.6-SN was B7–8% of total N.Increasing the milk pasteurization temperature re-

sulted in a significant decrease in the mean flowabilityof cheese over the 360-d maturation period (Fig. 5;Table 6); the mean flowability of C87 was significantlylower than that of C82, which in turn was significantlylower than that of C77 and C72 cheeses, which hadsimilar flow. There was a significant effect of theinteraction between heat treatment and ripening time,with the magnitude of the difference between the variouscheeses (apart from C72 and C77) at times X30 d beinggreater than that at 1 d. A similar trend was noted forstretchability of the heated cheese (Fig. 6b), except thatthere was no interaction between pasteurization tem-perature and ripening time (Table 6). In contrast,increasing the pasteurization temperature significantlyincreased the mean AV of the melted cheese throughoutripening (Fig. 6a, Table 6). The mean AVs of C87 andC82 were similar and significantly higher than that ofC77 and C72 cheeses. There was a significant effect ofthe interaction between pasteurization temperature andripening time on AV, with the differences between thecheeses diminishing with maturation. The adverse effectof high milk pasteurization temperatures on cheese flowand viscosity is perhaps surprising on consideration ofthe concomitant increase in moisture and decrease inprotein level of the cheese, both of which would beexpected to enhance flow and reduce AV (Guinee, 2002).To our knowledge, little or no information is

available on the effects of HPT on the AV andstretchability of heated cheese. However, the currentresults are consistent with those showing that theflowability of Mozzarella cheese from ultrafiltered milkretentate (e.g., 42–48% dry matter) (Madsen & Qvist,1998) was markedly lower than that of the correspond-ing cheeses made from control milks. Similarly, the

addition of whey proteins (undenatured or denatured)to cheese milk markedly reduced the heat-inducedflowability of Mozzarella cheese (Punidadas, Feirtag,& Tung, 1999; Meade & Roupas, 2001). The inclusion ofwhey proteins also impairs the flow of pasteurizedprocessed cheese products and analogue pizza cheeses(Guinee 2003).Hence, the negative impact of HPT on the flowability

of the current cheeses is probably related to the increasein the WPD and the inclusion of para-k-CN/b-lactoglo-bulin complexes and/or aggregates of DWP within thecheese. The inclusion of DWP in these forms probablymodifies the matrix structure and thereby impedes flow,which may be considered as a heat-induced rheologicaldisplacement (Guinee, 2002, 2003). Modifications couldresult from para-k-CN/b-lactoglobulin complexes actingas inclusions in the matrix which impart more continuityto the matrix per se, and hence, impair displacement onheating the cheese. Moreover, aggregation of DWP orfree sections of b-lactoglobulin molecules attached topara-k-CN, e.g., through increased hydrophobic inter-actions (Harwalkar & Ma, 1990), may further enhancethe continuity and the integrity of the matrix on heatingthe cheese. The tendency towards aggregation and/orpseudo-gelation of whey proteins on heating (grilling orbaking) cheeses from high heat-treated milk is probablyaccentuated by the high contents of protein and solublecalcium in the cheese (Jelen & Rattray, 1995). Theaggregates or pseudo-gels would impede the flow of theheated cheese as the fat phase melts and coalesces.Evidence for some form of pseudo-gelation or aggrega-tion of whey proteins on heating cheeses from HPTmilks is corroborated by the dynamic viscoelasticanalysis of experimental Cheddar cheeses with differentlevels of DWP (Guinee, 2003). Increasing the level ofDWP from 0.3% to 3.4%, w/w, by altering the heattreatment of the milk used for cheese manufacture,resulted in a large decrease in phase angle (d), and thusfluidity, on heating the cheese at temperatures >65�C(Guinee, 2002).

ARTICLE IN PRESSN.M. Rynne et al. / International Dairy Journal 14 (2004) 989–1001 999

4. Conclusions

The current study corroborates the results of previousstudies showing that increasing the pasteurizationtemperature of milk leads to higher levels of DWP andmoisture in the cheese and lower levels of protein andfat. These compositional changes may prove attractiveas a means of enhancing cheese yield, especially inrapidly ripened, mild-flavored hard, or semi-hard,cheese types. Moreover, the reductions in cheesefirmness and fracture stress/strain that parallel theelevation of pasteurization temperature would undoubt-edly be beneficial in moderating the undesirablerheological and textural characteristics of reduced-fatcheeses. However, the current study indicates thatincreasing pasteurization temperature from 72�C or77�C to 82�C significantly reduces the flowability andstretchability of the melted cheese. The deterioration inflow would be undesirable in most cooking applicationsfor which the melted cheese is expected to flow and forpizza where the ability of the melted cheese topping tostretch is a major quality criterion (Guinee, 2003).However, the flow resistance of cheeses from milkspasteurized at 87�C may be suited to particularapplications where maintenance of the visual identityof cheese shape following cooking is important, e.g.,deep fried breaded cheese sticks or burger inserts, or inethnic cuisine in which cheese is fried or used in curry.The changes in the rheological and cooking properties

associated with HPT of milk are, undoubtedly, due inpart to the concomitant changes in gross composition,the increases in protein hydration and the reduction infree oil. The effects of HPT may also be associated witha modification of the protein matrix which affects itsdeformation characteristics. Moreover, DWP and de-natured whey protein–casein complexes probablyinfluence the behavior of the protein matrix duringheating to an extent dependent on the degree ofdenaturation. However, the mechanism by whichHPT influences structure of the unheated cheese or thetype/extent of structural changes during heating thecheese is not clear and is an area that merits furtherinvestigation. Analyses of concentrated cheese-likemodel systems may help to clarify the contribution ofwhey proteins and whey–protein casein interaction tocheese deformation and cooking properties. This mayprovide a basis for optimizing the role of whey proteinsin cheese products.

Acknowledgements

This work was supported by United States/Ireland co-operation programme in Agricultural Science andTechnology.

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