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LWT - Food Science and Technology 51 (2013) 325e330

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LWT - Food Science and Technology

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The effect of heat treatment on g-glutamyl transferase activity in non-bovine andbovine milk e A comparative kinetic and thermodynamic investigation

Loredana Dumitrascu 1, Nicoleta St�anciuc 1, Silvius Stanciu*,1

Dunarea de Jos University of Galati, Faculty of Food Science and Engineering, 111 Domneasca Street, 800201, Galati, Romania

a r t i c l e i n f o

Article history:Received 18 November 2011Received in revised form25 September 2012Accepted 27 September 2012

Keywords:g-Glutamyl transferaseHeat treatmentKineticSafetyNon-bovine milk

* Corresponding author. "Dunarea de Jos" UniversScience and Engineering, 111 Domneasca Street, BuGalati, Romania. Tel.: þ40 336 130 177; fax: þ40 4 02

E-mail address: Silvius.Stanciu@ugal.ro (S. Stanciu1 www.sia.ugal.ro, www.trasilact.ugal.ro

0023-6438/$ e see front matter � 2012 Elsevier Ltd.http://dx.doi.org/10.1016/j.lwt.2012.09.028

a b s t r a c t

The activities and degree of inactivation of g-glutamyl transferase (GGT) in raw milk from three species(goat, sheep and cow) were measured in relation to the heating process in order to determine thesustainability of this enzyme as a marker for the evaluation of pasteurization. GGT showed the highestactivity in the whole milk, in the following order: cow > sheep > goat milk. Kinetic and thermodynamicstudies were carried out in the temperature range of 60e77 �C and showed that the thermal inactivationfollowed the first-order kinetics. Based on the thermal death time model, decimal reduction time D andinactivation rate constant k values decreased and increased respectively, with increasing temperature,indicating a more rapid inactivation at higher temperatures in whole milk than in skimmed milk. Theinfluence of temperature on the inactivation rate constant was quantified using the Arrhenius andthermal death time models. The corresponding z-values for skimmed and whole milk were8.02 � 0.23 �C and 7.09 � 0.09 �C for goat milk, 5.97 � 0.08 �C, 5.88 � 0.027 �C for sheep milk and5.80 � 0.05 �C and 5.83 � 0.01 �C for cow milk respectively. The calculated values for activation energyand change in enthalpy of denaturation suggested that the enzyme is more stable toward thermaldenaturation in goat milk.

� 2012 Elsevier Ltd. All rights reserved.

1. Introduction

Milk provides the necessary nutrients and environmentalconditions for the growth of many microorganisms. Traditionally,milk is processed by thermal treatment to ensure the microbialsafety of the products and to extend the shelf-life compared withraw milk (Hammershøj, Hougaard, Vestergaard, Poulsen, & Ipsen,2010). When milk is heated the functional and nutritional proper-ties are affected and leads to numerous competitive and alsointerdependent reactions that depend on the intensity and dura-tion of the heat treatment as well as on milk composition,concentration or pH.

Heat-induced inactivation of indigenous enzymes in bovinemilk has been well studied. The importance of some indigenousenzymes to the dairy industry is correlated to their thermal inac-tivation characteristics. Due to their slightly more resistance toheating than the non-spore-forming pathogens found in milk on

ity of Galati, Faculty of Foodilding E, Room 304, 800201,36 460 165.).

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which thermal processes are based, they are considered goodindicators for the evaluation of the severity or effectiveness of heattreatment of milk.

The membrane spanning enzyme g-glutamyl transpeptidase org-glutamyl transferase (GGT) (EC 2.3.2.2) catalyzes the breakdownof the tripeptide glutathione using its g-glutamyl moiety asa shuttle to transport free amino acids across the membrane intothe cell (Martini, Salari, Pesi, & Tozzi, 2010). GGT is involved in thetransportation of the amino acids from blood to mammary glands,playing an important role for biosynthesis of milk proteins.

The study of GGT activity is of great interest due to its heatstability characteristics which recommends this enzyme formonitoring thermal processes treatments in the range of 70e80 �Cfor 15 s (Andrews, Anderson, & Goodneough, 1987). Zehetner,Bareuther, Henle, and Klostermeyer (1995) evaluated the thermalresistance of GGT activity as indicator of the severity of thermalprocessing and concluded that GGT represents a useful indicator forpasteurization at temperatures higher than 77 �C.

GGT is more abundant in milk than alkaline phosphatase,rendering an enzymatic assay based on residual GGT activity moresensitive. GGT is also more heat resistant than alkaline phospha-tase, but less than lactoperoxidase (Claeys, Van Loey, & Hendrickx,2002). Alkaline phosphatase is universally recognized and used as

L. Dumitrascu et al. / LWT - Food Science and Technology 51 (2013) 325e330326

an index for HTST pasteurization, but may not be appropriate sincereactivation after heat treatment under certain conditions com-plicates interpretation of the test. Also, the relationship betweenlog10% initially activity and pasteurization equivalent is less linearthan the relationship of GGT and lactoperoxidase (Fox, 2003).

Most studies on GGT detection have been conducted on cowmilk and only few have focused on non-bovine milk. There are noreports available to provide foundational data for the application ofGGT assays in milk products of non-bovine origin (Moatsou, 2010).Growing markets for dairy products containing goat, sheep orbuffalo milk may increase the research interests to this area(Rankin, Christiansen, Lee, Banavara, & Lopez-Hernandez, 2010).Also, the importance that fat globules play in determining thequality of dairy products is known, and several components of milkfat globule membranes in milk have been recently identified asbeing beneficial for human health (Spitsberg, 2005).

To our knowledge there are no studies regarding detailed kineticinactivation and thermodynamic studies of GGT related to cow,sheep and goat milk under similar conditions. Therefore, the aim ofthis study was to provide information on the potential role of GGTactivity in sheep, goat and cow milk containing different levels offat as indicator of adequate pasteurization of milk based on kineticand thermodynamic analysis in the temperature range of 60 �C and77 �C.

2. Materials and methods

2.1. Materials

Bulk milk samples of indigenous goat (31 individuals, WhiteBanat Goats), sheep (25 individuals, Merino Sheep) and cow (20individuals, Romanian Simmental Cows) breeds were purchasedfrom different local farms (Galati, Romania). The samples werecollected from August to October. Each sample of raw milk wasobtained from a batch of 10 L. Milk composition was determinedusing PortableMilk Analyzer (Milk-Lab Ltd, Odham, Lancashire, UK)and pH measurements were carried out by means of Inolab pHmeter 730 (WTW, Weilheim, Germany). Each kind of milk wasskimmed by centrifugation (6000 rpm) with an FT 15 Disc BowelCentrifuge (Armfield Inc, England). By mixing skimmed milk andcream, milk was standardized to a 3.5% fat content. Then, both skimand whole milk were divided in small portions (2 mL) and storedfrozen at �20 �C until use. Table 1 shows the average compositionof bulk milk samples of the animal species used.

2.2. Isothermal inactivation of enzyme

Glass capillaries (length 100 mm, inner diameter 1 mm, wallthickness 0.15 mm) filled with milk samples were sealed and put ina water bath (Digibath-2 BAD 4, Raypa Trade, Spain) witha temperature ranging from 60 to 77 �C for different holding times(0e30 min). After thermal treatment, the capillaries were cooled inice water. The heating experiments were replicated for three times.

Table 1The composition of goat, sheep and cow raw milk.

Goat milk Sheep milk Cow milk

Total protein, g/l 34.5 � 2.5a 45.8 � 3.8 34.8 � 4.6Fat matter, g/l 46.2 � 8.7 79.0 � 5.7 37.0 � 3.5Lactose, g/l 45.9 � 5.5 54.2 � 4.5 43.3 � 3.4Ash, g/l 6.7 � 0.9 8.0 � 0.7 07.1 � 0.8Dry matter, g/l 130.6 � 1.8 182.1 � 1.2 126.8 � 9.5

a Standard deviations.

The heat-treated samples were stored at maximum 6 �C and noreactivation occurred during one week of storage in this condition.

2.3. Assay of enzyme activity

GGT activity was analyzed spectrophotometrically by quanti-fying p-nitroaniline according to a modification of the methoddescribed by Zehetner et al. (1995). In brief, after appropriatedilution with double distilled water, aliquots of 50 mL of enzymesolutionwere added to 2.5 mL fresh prepared substrate consisted of0.1 mol/L Tris, 89 mmol/L NaCl, 48 mmol/L glycyleglycine and4.8 mmol/L g-glutamyl-p-nitroanilide. Both samples and substratewere preheated separately before measurements at 40 �C in awaterbath. The enzyme activity was spectrophotometrically quantifiedusing a UVeVIS GBC Cintra spectrophotometer (Australia), bymeasuring the increase in absorption at 410 nm for 15 min. Asample containing 2.5 mL of buffer and 50 mL double distilled waterwas used as the blank. An extinction molar coefficient equal to8.8 L/cm/mmol was used for p-nitroanilide at 410 nm.

The activity was expressed in terms of enzymatic units. One unitof activity (U) is equivalent to 1 mmol/L p-nitroanilide released perminute and per mL of milk.

2.4. Kinetic and statistical analysis of data

The kinetic and thermodynamic parameters were calculated aspreviously reported by St�anciuc, Dumitrascu, Râpeanu, and Stanciu(2011). All standard deviations and linear regression errors werecalculated using Microsoft Excel.

3. Results and discussions

3.1. GGT activity

GGT showed different activities in all the milk samples tested.Compared with other enzymes like lactoperoxidase, GGT activity iscorrelated with fat globule membrane, therefore the enzymeactivity is higher in whole milk compared with skimmed milk. Theaverage enzymatic values in whole sheep, goat and cowmilk were:4025 � 294 U/L, 951 � 193 U/L and 7081 � 505 U/L respectively. Itcan be seen that non-bovine milk has 56.83 � 0.14% (sheep) and13.30 � 2.51% (goat) of initial GGT activity in cow milk. The corre-sponding average enzymatic values obtained in skimmed milkwere: 3727� 255 U/L, 437� 78 U/L and 5503� 654 U/L. Our resultsshow an increase in enzyme activity with 7.37 � 0.60% (sheep),53.80 � 1.66% (goat) and 22.50 � 5.25% (cow) in whole milk whencompared with skimmed milk. The difference in GGT activity ispossibly related to a difference in the location of the enzyme. It isimportant to note that during one week of storage at refrigerationtemperature, no reactivation of GGT activity occurred, which is ingood agreement with the results of St�anciuc et al. (2011). No datawere available regarding the reactivation of GGT in non-bovinemilk.

The values obtained for GGT activity in the present study aredifferent compared with those reported in literature. Duringlactation stage (fromMay to November), Lorenzen, Martin, Clawin-Radecker, Barth, and Knappstein (2010) determined values of4143 U/L, 1878 U/L, 603 U/L for whole cow, sheep and goat milkrespectively. Also, these authors revealed that during lactationcycle over a period of 300 days, there is a small seasonal variationon both whole and skimmed milk. Piga, Urgeghe, Piredda, Scintu,and Sanna (2009) using chromatographic procedure for thedetermination of GGT activity in Sardinian sheep milk obtainedvalues ranging from 2720 to 3460 U/L with a mean value of 3090 U/L. Elsewhere, we reported mean values for GGT activities of 4010

L. Dumitrascu et al. / LWT - Food Science and Technology 51 (2013) 325e330 327

and 3200 U/L in whole and skimmed cow milk (St�anciuc et al.,2011). The results of this study are in fair agreement with thoseobtained by Zehetner et al. (1995) who measured a GGT activity of5920 U/L for raw cowmilk. The differences in GGT activity in wholeand skimmed milk are dependent on breed or health state of theanimal (Fox, 2003).

3.2. Thermal inactivation

In the literature are very well described the microbial indicescorresponding to the currently applied heat treatments and theirinactivation. Collecting data for accurate computation of tempera-ture history represents the main disadvantage of this method(Dinella, Monteleone, Farenga, & Hourigan, 2004). For an indige-nous enzyme to be considered as a process marker in heat-treatedmilk, the major requirement is the equivalence of the inactivationbehavior between the enzyme and the microorganisms of concern.The application of enzymes as heat markers depends not only on

Fig. 1. First-order thermal inactivation of GGT in whole milk at different temperatures(60 �C (A), 65 �C (-), 70 �C (:), 72 �C (>), 73 �C (6), 75 �C (C), 77 �C (B)) a) sheepb) goat and c) cow milk. Three independent tests were carried out in each case andevaluations were based on a significant level of p < 0.05.

the inactivation kinetics, but also on the initial activity or concen-tration presented in milk. These informations are also valuable forvalidation and implementation of a timeetemperature indicatorstudied (Claeys, Indrawati, Van Loey, & Hendrickx, 2003). Thermalstability of GGT was examined in the temperature range of 60 �Ce77 �C by measuring the residual activity after heat treatment forvarious times in order to compare the kinetics of inactivation andto establish this enzyme as indices to control the efficacy of heattreatment. Inactivation experiments were carried out in wholeand skimmed milk in order to asses the influence of fat contentsince it’s considered to be the most obvious variable in milkcomposition.

Fig. 2. First-order thermal inactivation of GGT in skimmed milk at different temper-atures (60 �C (A), 65 �C (-), 70 �C (:), 72 �C (>), 73 �C (6), 75 �C (C), 77 �C (B)) a)sheep b) goat and c) cow milk. Three independent tests were carried out in each caseand evaluations were based on a significant level of p < 0.05.

L. Dumitrascu et al. / LWT - Food Science and Technology 51 (2013) 325e330328

The effect of thermal treatment on the inactivation behavior ofGGT expressed as residual activity, in the different species of milkplotted as a function of inactivation time, is given in Fig.1 and Fig. 2.

There are significant differences in the GGT activities dependingon fat content and applied temperatureetime combinations. Thedegree of GGT inactivation increased with increasing temperatureand holding time. After 5 min of heating at 65 �C, GGT activity inwhole sheep (Fig. 1-a), goat (Fig. 1-b) and cow milk (Fig. 1-c)decreased to 96.07 � 1.70%, 85.90 � 1.20% and 50.50 � 1.25%respectively. In skimmed milk (Fig. 2), the enzyme activity rangedfrom 93.20 � 0.70% to 70.78 � 2.80% (sheep milk), from91.70 � 1.20% to 44.02 � 1.20% (goat milk) and from 51.6 � 0.71% to14.91 � 0.10% in cow milk respectively, after 5 and 20 min ofholding.

Significant differences in inactivation patterns were observedwhen compared non-bovine milk with cow milk. The GGT isalmost inactivated in cow milk after 20 s at 77 �C, with residualactivities values of 1.80 � 0.02% in whole milk and 1.03 � 0.01% inskimmed milk respectively. In the same heating conditions,residual GGT activities were 32.10 � 0.20% for whole and33.20 � 0.40% for skimmed sheep milk, respectively. A similarenzyme activity was observed in whole goat’s milk (34.75 � 0.57%)while in skimmed milk, GGT presented a higher residual activity(45.52 � 0.37%).

The timeetemperature combination needed for completeinactivation of the enzyme in cow milk is similar with those re-ported in the literature. For example, McKellar, Emmons, andFarber (1991) reported that GGT is completely inactivated at77 �C for 16 s, whereas Dos Anjos, Machdo, Ferro, and Bogin (1998)suggested that complete inactivation of GGT in cow milk requires70 �C for 10 min.

From our results, it seems that enzyme is more heat stable inmilk in the following order: goat > sheep > cow milk. Lorenzen,Wernery, Johnson, Jose, and Wernery (2011) investigated theeffects of isochrone heating with different temperatureetimecombinations on the residual activities of different indigenousenzymes in cow, sheep and goat’s milk. These authors concludedalso that GGT has a higher thermal stability in goat’s and sheepmilkthan cow’s milk.

Table 2Decimal reduction time (D) values and temperature resistance value (z) for GGT inactiva

Temperature, �C Goat milk Shee

D (min) R2 D (m

a) Whole milk60 59.52 � 5.2a 0.87 16365 43.70 � 1.88 0.97 7870 7.09 � 1.08 0.95 672 2.27 � 0.86 0.97 473 1.44 � 0.006 0.9 175 0.73 � 0.18 0.98 077 0.33 � 0.009 0.97 0z (�C) 7.09 ± 0.09 5

b) Skimmed milk60 69.52 � 8.81 0.94 265 24.30 � 7.84 0.97 6070 8.40 � 0.09 0.96 572 2.35 � 0.74 0.94 273 1.87 � 0.43 0.97 175 0.82 � 0.04 0.98 177 0.52 � 0.03 0.97 0z (�C) 8.02 ± 0.23 5

D-value: time required for 1 log reduction in activity at a specific temperature; z-value:a Standard deviations.

3.3. Kinetic and thermodynamic parameters

In terms of reaction kinetic, GGT followed a first-order inacti-vation model for all temperatures tested. Linear regression analysisshowed good correlation coefficients between residual GGT activityand time for each temperature. The relationship between heatsensitivity of the target microorganism and of the thermal intrinsicindicator was expressed in terms of D and z-values.

The corresponding D-values for GGT inactivation in whole andskimmed milk samples together with the standard errors and r2

values are given in Table 2. The D-values decreased with increasingtemperature from 60 �C to 77 �C, indicating a faster inactivation ofGGT at higher temperatures.

For the experiments performed in sheepmilk, it can be seen thatthe D-values are higher for GGT inactivation in whole milk whencompared with skimmed milk in the temperature range of 65 �Ce73 �C. For goat milk, it seems that the fat content did notsignificantly affect the D-values at temperatures between 70 �Cand 75 �C. In whole milk, the highest value for D was calculatedin sheep milk (163.93 � 22.98 min at 60 �C), followed by cow andgoat milk where the decimal reduction time was about 2.5 timeslower. At lower temperature, GGT seems to be more heat stablein skimmed sheep milk, followed by goat and cow milk, whereasat higher temperatures (77 �C) the GGT seems to be more heatstable in goat milk when compared with sheep and cow milk.Our results for cow milk are in good agreement with thosereported elsewhere (St�anciuc et al., 2011), but different thanthose obtained by Blel, Guingamp, Gaillard, and Humbert (2002).These authors obtained D-values of 7.2 min,1.7 min and 0.34 min at69 �C, 73 �C and 75 �C respectively. The differences may beexplained by the different heating conditions and method used forthe determination of GGT activity. The increase in stability may alsobe due to molecular properties of the enzymes itself.

Thermal sensitivity values (z) calculated in the temperaturerange studied for skimmed and whole milk were: 8.02 � 0.23 �Cand 7.09 � 0.09 �C (goat milk), 5.97 � 0.08 �C and 5.88 � 0.027 �C(sheep milk) and 5.83 � 0.01 �C and 5.80 � 0.05 �C (cow milk).Andrews et al. (1987) reported z-value of 5.4 �C for GGT inactivationin cow milk in the temperature range of 71e75 �C. In general, high

tion in different types of milk.

p milk Cow milk

in) R2 D (min) R2

.93 � 22.98 0.92 68.75 � 5.99 0.94

.74 � 9,94 0.95 7.18 � 1.44 0.95

.60 � 0,09 0.9 1.53 � 0.04 0.97

.33 � 0,58 0.97 0.42 � 0.07 0.94

.84 � 0,18 0.98 0.26 � 0.001 0.97

.59 � 0.25 0.95 0.18 � 0.002 0.97

.28 � 0.07 0.97 0.07 � 0.003 0.98

.88 ± 0.027 5.80 ± 0.05

00 � 9.02 0.95 59.35 � 0.74 0.92.60 � 18.59 0.98 10.11 � 3.74 0.95.08 � 0.55 0.94 2.03 � 0.20 0.96.10 � 0.10 0.99 0.50 � 0.06 0.94.69 � 0.07 0.97 0.34 � 0.08 0.98.03 � 0.04 0.97 0.18 � 0.02 0.98.31 � 0.03 0.96 0.07 � 0.01 0.97.97 ± 0.08 5.83 ± 0.01

increase in temperature required for 1 log change in D-value.

Table 3Inactivation rate constant (k) and volume change of activation energy for GGT in goat, sheep and cow milk.

Temperature, �C k � 10�2 (min�1)

Goat milk Sheep milk Cow milk

Whole Skimmed Whole Skimmed Whole Skimmed

60 3.97 � 0.14a 3.89 � 0.25 1.40 � 0.35 1.15 � 0.01 3.36 � 0.29 3.88 � 0.0465 5.04 � 0.09 9.73 � 0.30 2.92 � 0.58 3.32 � 0.66 37.97 � 0.84 24.43 � 0.9070 40.29 � 5.48 30.89 � 4.60 34.84 � 0.96 43.20 � 2.89 137.55 � 21.20 113.79 � 11.6972 90.17 � 14.57 136.53 � 1.48 53.10 � 5.21 105.07 � 6.3 582.65 � 48.85 457.60 � 54.7173 185.80 � 38.35 146.97 � 0.65 124.79 � 15.01 135.88 � 0.01 862.30 � 1.38 679.15 � 159.9175 326.49 � 78.65 286.59 � 1.09 384.57 � 102.1 225.17 � 5.04 1216.18 � 22.52 1222.37 � 147.2977 688.26 � 9.78 438.44 � 1.57 796.83 � 146.5 761.71 � 43.15 3207.38 � 115.3 3072.89 � 545.86Ea (kJ/mol) 313.7 ± 3.98b 277.59 ± 2.32 377.62 ± 1.39 373.16 ± 2.58 384.24 ± 1.45 381.44 ± 2.19

a Standard deviations.b Standard errors of regressions.

Fig. 3. Arrhenius plot for thermal inactivation of GGT in sheep (A), goat (-) and cowmilk (:). Filled squares corresponds to whole milk and the empty for skimmed milk.

L. Dumitrascu et al. / LWT - Food Science and Technology 51 (2013) 325e330 329

z-values mean more sensitivity to the duration of the heat treat-ment (Barret, Grandison, & Lewis, 1999).

The inactivation rate values (k), calculated from the slope of theregression line obtained by plotting the natural logarithm of rela-tive residual activity as a function of inactivation time (t) are givenin Table 3. In general, fat is considered to have a protective effect onmicroorganisms inactivation (van Asselt & Zwietering, 2006). At60 �C, in both bovine and non-bovine milk, the fat content had nomajor influence on k values. At higher temperature (77 �C), GGTinactivates faster in whole milk when compared with skimmedcounterpart. However, regarding the interspecies comparison, theresults obtained in our study indicate a faster inactivation of GGT incowmilk. In non-bovinemilk, onewould have expected an increasein D-values while increasing fat content. This statement is not

Table 4Changes in enthalpy of activation (DH), free energy of activation (DG) and entropy of act

Temperature Goat milk Sheep milk

�C K DH (kJ mol�1) DG (kJ mol�1) DS (kJ mol�1) DH (kJ mol�1) D

sa wb s w s w s w s

60 333 274.82 310.93 90.68 90.73 0.261 0.262 370.4 374.85 965 338 274.78 310.89 89.16 91.57 0.261 0.262 370.35 374.81 970 343 274.74 310.85 87.47 86.74 0.261 0.262 370.31 374.76 872 345 274.72 310.83 83.81 84.90 0.261 0.262 370.30 374.75 873 346 274.71 310.83 84.06 83.00 0.261 0.262 370.29 374.74 875 348 274.70 310.81 82.81 82.80 0.261 0.262 370.27 374.72 877 350 274.68 310.79 81.89 80.61 0.261 0.262 370.25 374.71 8

a Skimmed milk.b Whole milk.

supported, probably due to the different physico-chemical struc-ture and composition of milk fats (Park, 2006). It has been reportedthat heat treatment has only a minor effect on milk fat (Claeys,2003). The different thermal sensitivity of GGT observed in ourstudy, in non-bovine milk can be correlated with specific heatsensitive protein compounds in fat globule membrane (Spreer,1998). Only a limited number of studies on the effect of milk faton a milk compound evaluated as an index for heat treatment arereported and the results are contradictory (van Boekel & Walstra,1989). However, it is difficult to predict the complex mechanismsby which milk fat affects heat-induced modifications. Additionalstudies to verify this effect are needed.

To calculate the activation energy (Ea), the natural logarithm ofthe inactivation rate constant kwas plotted against the reciprocal ofthe absolute temperature in Kelvin (T). Ea can be defined as theenergy absorbed or released needed to the molecules to be able toreact (van Boekel, 2008). The temperature dependence of the rateconstants for thermal inactivation of GGT in goat, sheep and cowmilk is depicted in Fig. 3. In skimmed and whole milk, Ea valueswere 277.59 � 2.32 kJ/mol and 313.7 � 3.98 kJ/mol for goat milk,373.16 � 2.58 kJ/mol and 377.62 � 1.39 kJ/mol for sheep milk and384.24 � 1.45 kJ/mol and 381.44 � 2.19 kJ/mol for cow milkrespectively. In goat skimmed milk, activation energy is significantdifferent when compared with whole milk, whereas the Ea valuescalculated for sheep and cow milk with different fat content aresimilar.

The value of activation energy for GGT inactivation in skimmedgoat milk indicates that a lower amount of energy is needed toinitiate the denaturation process, to re-arrange the initial foldedconformation into the unfolded, inactive state (Dinella et al., 2004).The activation energy values for sheep and cow’s milk indicatesa less compact structure, probably with a slow denaturationprocess at low temperature, but relatively fast at high temperatures(van Boekel, 2008).

ivation (DS) for GGT inactivation in goat, sheep and cow milk.

Cow milk

G (kJ mol�1) DS (kJ mol�1) DH (kJ mol�1) DG (kJ mol�1) DS (kJ mol�1)

w s w s w s w s w

4.23 93.68 0.264 378.67 381.47 90.86 91.26 0.2642.33 93.06 0.264 378.63 381.43 87.10 86.28 0.2646.67 87.42 0.264 378.59 381.39 84.04 83.26 0.2644.66 86.73 0.264 378.57 381.37 80.56 80.03 0.2644.29 84.54 0.264 378.56 381.37 79.66 78.97 0.2643.38 81.78 0.264 378.54 381.35 78.44 78.45 0.2640.40 80.15 0.264 378.53 381.33 76.22 76.10 0.264

L. Dumitrascu et al. / LWT - Food Science and Technology 51 (2013) 325e330330

Estimation of thermodynamic parameters is essential tounderstand the probable mechanism of denaturation, which is veryimportant in thermal processes (Sant’Anna, Oliviera-Cardela, &Brandelli, 2012). The activation energy value enabled the deter-mination of enthalpy (DH), entropy (DG), and Gibbs free energy ofactivation (DS) for GGT inactivation in different milk calculated forthe different temperatures (Table 4). The thermodynamic param-eters decreased with increasing temperature.

For sheep and cow milk, no significant differences were ob-tained betweenwhole and skimmedmilk. The values of the changein enthalpy of denaturation obtained for skimmed milk at 70 �Cwere: 274.74 kJ/mol, 370.31 kJ/mol and 378.59 kJ/mol for goat,sheep and cow milk respectively. It can be assumed that GGT ingoat milk is more stable than in sheep and cowmilk during thermaltreatment and undergoes large heat-induced conformationalchanges. This conclusion is also supported by the lower value ob-tained for the activation energy. Data regarding thermodynamicparameters of GGT inactivation in sheep and goat milk were notfound in the literature.

4. Conclusions

The results presented have shown that there are significantvariations in the raw milk activities of GGT between species. GGTactivity varied as a function of species and fat content, as follow:cow milk > sheep milk > goat milk.

Investigation of GGT followed a first-order reaction in thetemperature range of 60 �Ce77 �C. The lower values obtained foractivation energy and change in enthalpy of denaturation suggestthat the enzyme is more stable toward thermal denaturation ingoat milk when compared with sheep or cow milk. Taking inconsideration the breed specific differences, further studies areneeded in order to establish limiting values for GGTactivities and toevaluate the possibilities of using this enzyme as indicator ofindustrial processing of non-bovine milk. Our results constitutetherefore one of the first reports on the detailed kinetic and ther-modynamic investigation of GGT inactivation in non-bovine milk.

Acknowledgments

The authors acknowledge financial support from the NationalUniversity Research Council (NURC, PN-II-ID-PCE-2008-2, Idea,ID 517) Romania (www.trasilact.ugal.ro). Bioaliment ResearchPlatform (www.bioaliment.ugal.ro) is also acknowledged forproviding technical support.

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