The effect of heat treatment on g-glutamyl transferase activity in non-bovine and

bovine milk e A comparative kinetic and thermodynamic investigation

Loredana Dumitras cu
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 2011
Received in revised form
25 September 2012
Accepted 27 September 2012
Keywords:
g-Glutamyl transferase
Heat treatment
Kinetic
Safety
Non-bovine milk
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 the
sustainability of this enzyme as a marker for the evaluation of pasteurization. GGT showed the highest
activity in the whole milk, in the following order: cow > sheep > goat milk. Kinetic and thermodynamic
studies were carried out in the temperature range of 60e77

C and showed that the thermal inactivation
followed the rst-order kinetics. Based on the thermal death time model, decimal reduction time D and
inactivation 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. The
inuence of temperature on the inactivation rate constant was quantied using the Arrhenius and
thermal death time models. The corresponding z-values for skimmed and whole milk were
8.02 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 and
5.80 0.05

C and 5.83 0.01

C for cow milk respectively. The calculated values for activation energy
and change in enthalpy of denaturation suggested that the enzyme is more stable toward thermal
denaturation in goat milk.
2012 Elsevier Ltd. All rights reserved.
1. Introduction
Milk provides the necessary nutrients and environmental
conditions for the growth of many microorganisms. Traditionally,
milk is processed by thermal treatment to ensure the microbial
safety of the products and to extend the shelf-life compared with
raw milk (Hammershj, Hougaard, Vestergaard, Poulsen, & Ipsen,
2010). When milk is heated the functional and nutritional proper-
ties are affected and leads to numerous competitive and also
interdependent 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 bovine
milk has been well studied. The importance of some indigenous
enzymes to the dairy industry is correlated to their thermal inac-
tivation characteristics. Due to their slightly more resistance to
heating than the non-spore-forming pathogens found in milk on
which thermal processes are based, they are considered good
indicators for the evaluation of the severity or effectiveness of heat
treatment of milk.
The membrane spanning enzyme g-glutamyl transpeptidase or
g-glutamyl transferase (GGT) (EC 2.3.2.2) catalyzes the breakdown
of the tripeptide glutathione using its g-glutamyl moiety as
a shuttle to transport free amino acids across the membrane into
the cell (Martini, Salari, Pesi, & Tozzi, 2010). GGT is involved in the
transportation 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 heat
stability characteristics which recommends this enzyme for
monitoring thermal processes treatments in the range of 70e80

C
for 15 s (Andrews, Anderson, & Goodneough, 1987). Zehetner,
Bareuther, Henle, and Klostermeyer (1995) evaluated the thermal
resistance of GGT activity as indicator of the severity of thermal
processing and concluded that GGT represents a useful indicator for
pasteurization at temperatures higher than 77

C.
GGT is more abundant in milk than alkaline phosphatase,
rendering an enzymatic assay based on residual GGT activity more
sensitive. 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
* Corresponding author. "Dunarea de Jos" University of Galati, Faculty of Food
Science and Engineering, 111 Domneasca Street, Building E, Room 304, 800201,
Galati, Romania. Tel.: 40 336 130 177; fax: 40 4 0236 460 165.
E-mail address: Silvius.Stanciu@ugal.ro (S. Stanciu).
1
www.sia.ugal.ro, www.trasilact.ugal.ro
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http://dx.doi.org/10.1016/j.lwt.2012.09.028
LWT - Food Science and Technology 51 (2013) 325e330
an index for HTST pasteurization, but may not be appropriate since
reactivation after heat treatment under certain conditions com-
plicates interpretation of the test. Also, the relationship between
log10% initially activity and pasteurization equivalent is less linear
than the relationship of GGT and lactoperoxidase (Fox, 2003).
Most studies on GGT detection have been conducted on cow
milk and only few have focused on non-bovine milk. There are no
reports available to provide foundational data for the application of
GGT assays in milk products of non-bovine origin (Moatsou, 2010).
Growing markets for dairy products containing goat, sheep or
buffalo 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 the
quality of dairy products is known, and several components of milk
fat globule membranes in milk have been recently identied as
being benecial for human health (Spitsberg, 2005).
To our knowledge there are no studies regarding detailed kinetic
inactivation and thermodynamic studies of GGT related to cow,
sheep and goat milk under similar conditions. Therefore, the aim of
this study was to provide information on the potential role of GGT
activity in sheep, goat and cow milk containing different levels of
fat as indicator of adequate pasteurization of milk based on kinetic
and thermodynamic analysis in the temperature range of 60

C and
77

C.
2. Materials and methods
2.1. Materials
Bulk milk samples of indigenous goat (31 individuals, White
Banat Goats), sheep (25 individuals, Merino Sheep) and cow (20
individuals, Romanian Simmental Cows) breeds were purchased
from different local farms (Galati, Romania). The samples were
collected from August to October. Each sample of raw milk was
obtained from a batch of 10 L. Milk composition was determined
using Portable Milk Analyzer (Milk-Lab Ltd, Odham, Lancashire, UK)
and pH measurements were carried out by means of Inolab pH
meter 730 (WTW, Weilheim, Germany). Each kind of milk was
skimmed by centrifugation (6000 rpm) with an FT 15 Disc Bowel
Centrifuge (Armeld Inc, England). By mixing skimmed milk and
cream, milk was standardized to a 3.5% fat content. Then, both skim
and whole milk were divided in small portions (2 mL) and stored
frozen at 20

C until use. Table 1 shows the average composition
of bulk milk samples of the animal species used.
2.2. Isothermal inactivation of enzyme
Glass capillaries (length 100 mm, inner diameter 1 mm, wall
thickness 0.15 mm) lled with milk samples were sealed and put in
a water bath (Digibath-2 BAD 4, Raypa Trade, Spain) with
a temperature ranging from 60 to 77

C for different holding times
(0e30 min). After thermal treatment, the capillaries were cooled in
ice water. The heating experiments were replicated for three times.
The heat-treated samples were stored at maximum 6

C and no
reactivation 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 modication of the method
described by Zehetner et al. (1995). In brief, after appropriate
dilution with double distilled water, aliquots of 50 mL of enzyme
solutionwere added to 2.5 mL fresh prepared substrate consisted of
0.1 mol/L Tris, 89 mmol/L NaCl, 48 mmol/L glycyleglycine and
4.8 mmol/L g-glutamyl-p-nitroanilide. Both samples and substrate
were preheated separately before measurements at 40

C in a water
bath. The enzyme activity was spectrophotometrically quantied
using a UVeVIS GBC Cintra spectrophotometer (Australia), by
measuring the increase in absorption at 410 nm for 15 min. A
sample containing 2.5 mL of buffer and 50 mL double distilled water
was used as the blank. An extinction molar coefcient equal to
8.8 L/cm/mmol was used for p-nitroanilide at 410 nm.
The activity was expressed in terms of enzymatic units. One unit
of activity (U) is equivalent to 1 mmol/L p-nitroanilide released per
minute and per mL of milk.
2.4. Kinetic and statistical analysis of data
The kinetic and thermodynamic parameters were calculated as
previously reported by St anciuc, Dumitras cu, Rpeanu, and Stanciu
(2011). All standard deviations and linear regression errors were
calculated 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 is
correlated with fat globule membrane, therefore the enzyme
activity is higher in whole milk compared with skimmed milk. The
average enzymatic values in whole sheep, goat and cow milk were:
4025 294 U/L, 951 193 U/L and 7081 505 U/L respectively. It
can be seen that non-bovine milk has 56.83 0.14% (sheep) and
13.30 2.51% (goat) of initial GGT activity in cow milk. The corre-
sponding average enzymatic values obtained in skimmed milk
were: 3727 255 U/L, 437 78 U/L and 5503 654 U/L. Our results
show 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 when
compared with skimmed milk. The difference in GGT activity is
possibly related to a difference in the location of the enzyme. It is
important to note that during one week of storage at refrigeration
temperature, no reactivation of GGT activity occurred, which is in
good agreement with the results of St anciuc et al. (2011). No data
were available regarding the reactivation of GGT in non-bovine
milk.
The values obtained for GGT activity in the present study are
different compared with those reported in literature. During
lactation stage (from May to November), Lorenzen, Martin, Clawin-
Radecker, Barth, and Knappstein (2010) determined values of
4143 U/L, 1878 U/L, 603 U/L for whole cow, sheep and goat milk
respectively. Also, these authors revealed that during lactation
cycle over a period of 300 days, there is a small seasonal variation
on both whole and skimmed milk. Piga, Urgeghe, Piredda, Scintu,
and Sanna (2009) using chromatographic procedure for the
determination of GGT activity in Sardinian sheep milk obtained
values ranging from2720 to 3460 U/L with a mean value of 3090 U/
L. Elsewhere, we reported mean values for GGT activities of 4010
Table 1
The composition of goat, sheep and cow raw milk.
Goat milk Sheep milk Cow milk
Total protein, g/l 34.5 2.5
a
45.8 3.8 34.8 4.6
Fat matter, g/l 46.2 8.7 79.0 5.7 37.0 3.5
Lactose, g/l 45.9 5.5 54.2 4.5 43.3 3.4
Ash, g/l 6.7 0.9 8.0 0.7 07.1 0.8
Dry matter, g/l 130.6 1.8 182.1 1.2 126.8 9.5
a
Standard deviations.
L. Dumitras cu et al. / LWT - Food Science and Technology 51 (2013) 325e330 326
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 those
obtained by Zehetner et al. (1995) who measured a GGT activity of
5920 U/L for rawcowmilk. The differences in GGT activity in whole
and skimmed milk are dependent on breed or health state of the
animal (Fox, 2003).
3.2. Thermal inactivation
In the literature are very well described the microbial indices
corresponding to the currently applied heat treatments and their
inactivation. 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-treated
milk, the major requirement is the equivalence of the inactivation
behavior between the enzyme and the microorganisms of concern.
The application of enzymes as heat markers depends not only on
the inactivation kinetics, but also on the initial activity or concen-
tration presented in milk. These informations are also valuable for
validation and implementation of a timeetemperature indicator
studied (Claeys, Indrawati, Van Loey, & Hendrickx, 2003). Thermal
stability of GGT was examined in the temperature range of 60

Ce
77

C by measuring the residual activity after heat treatment for
various times in order to compare the kinetics of inactivation and
to establish this enzyme as indices to control the efcacy of heat
treatment. Inactivation experiments were carried out in whole
and skimmed milk in order to asses the inuence of fat content
since its considered to be the most obvious variable in milk
composition.
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) sheep
b) goat and c) cow milk. Three independent tests were carried out in each case and
evaluations were based on a signicant level of p < 0.05.
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 case
and evaluations were based on a signicant level of p < 0.05.
L. Dumitras cu et al. / LWT - Food Science and Technology 51 (2013) 325e330 327
The effect of thermal treatment on the inactivation behavior of
GGT expressed as residual activity, in the different species of milk
plotted as a function of inactivation time, is given in Fig. 1 and Fig. 2.
There are signicant differences in the GGT activities depending
on fat content and applied temperatureetime combinations. The
degree of GGT inactivation increased with increasing temperature
and holding time. After 5 min of heating at 65

C, GGT activity in
whole 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 ranged
from 93.20 0.70% to 70.78 2.80% (sheep milk), from
91.70 1.20% to 44.02 1.20% (goat milk) and from 51.6 0.71% to
14.91 0.10% in cow milk respectively, after 5 and 20 min of
holding.
Signicant differences in inactivation patterns were observed
when compared non-bovine milk with cow milk. The GGT is
almost inactivated in cow milk after 20 s at 77

C, with residual
activities values of 1.80 0.02% in whole milk and 1.03 0.01% in
skimmed milk respectively. In the same heating conditions,
residual GGT activities were 32.10 0.20% for whole and
33.20 0.40% for skimmed sheep milk, respectively. A similar
enzyme activity was observed in whole goats milk (34.75 0.57%)
while in skimmed milk, GGT presented a higher residual activity
(45.52 0.37%).
The timeetemperature combination needed for complete
inactivation of the enzyme in cow milk is similar with those re-
ported in the literature. For example, McKellar, Emmons, and
Farber (1991) reported that GGT is completely inactivated at
77

C for 16 s, whereas Dos Anjos, Machdo, Ferro, and Bogin (1998)
suggested that complete inactivation of GGT in cow milk requires
70

C for 10 min.
From our results, it seems that enzyme is more heat stable in
milk in the following order: goat > sheep > cow milk. Lorenzen,
Wernery, Johnson, Jose, and Wernery (2011) investigated the
effects of isochrone heating with different temperatureetime
combinations on the residual activities of different indigenous
enzymes in cow, sheep and goats milk. These authors concluded
also that GGT has a higher thermal stability in goats and sheep milk
than cows milk.
3.3. Kinetic and thermodynamic parameters
In terms of reaction kinetic, GGT followed a rst-order inacti-
vation model for all temperatures tested. Linear regression analysis
showed good correlation coefcients between residual GGT activity
and time for each temperature. The relationship between heat
sensitivity of the target microorganism and of the thermal intrinsic
indicator was expressed in terms of D and z-values.
The corresponding D-values for GGT inactivation in whole and
skimmed milk samples together with the standard errors and r
2
values are given in Table 2. The D-values decreased with increasing
temperature from 60

C to 77

C, indicating a faster inactivation of
GGT at higher temperatures.
For the experiments performed in sheep milk, it can be seen that
the D-values are higher for GGT inactivation in whole milk when
compared with skimmed milk in the temperature range of 65

Ce
73

C. For goat milk, it seems that the fat content did not
signicantly affect the D-values at temperatures between 70

C
and 75

C. In whole milk, the highest value for D was calculated
in sheep milk (163.93 22.98 min at 60

C), followed by cow and
goat milk where the decimal reduction time was about 2.5 times
lower. At lower temperature, GGT seems to be more heat stable
in skimmed sheep milk, followed by goat and cow milk, whereas
at higher temperatures (77

C) the GGT seems to be more heat
stable in goat milk when compared with sheep and cow milk.
Our results for cow milk are in good agreement with those
reported elsewhere (St anciuc et al., 2011), but different than
those obtained by Blel, Guingamp, Gaillard, and Humbert (2002).
These authors obtained D-values of 7.2 min, 1.7 min and 0.34 min at
69

C, 73

C and 75

C respectively. The differences may be
explained by the different heating conditions and method used for
the determination of GGT activity. The increase in stability may also
be due to molecular properties of the enzymes itself.
Thermal sensitivity values (z) calculated in the temperature
range studied for skimmed and whole milk were: 8.02 0.23

C
and 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 inactivation

in cow milk in the temperature range of 71e75

C. In general, high
Table 2
Decimal reduction time (D) values and temperature resistance value (z) for GGT inactivation in different types of milk.
Temperature,

C Goat milk Sheep milk Cow milk
D (min) R
2
D (min) R
2
D (min) R
2
a) Whole milk
60 59.52 5.2
a
0.87 163.93 22.98 0.92 68.75 5.99 0.94
65 43.70 1.88 0.97 78.74 9,94 0.95 7.18 1.44 0.95
70 7.09 1.08 0.95 6.60 0,09 0.9 1.53 0.04 0.97
72 2.27 0.86 0.97 4.33 0,58 0.97 0.42 0.07 0.94
73 1.44 0.006 0.9 1.84 0,18 0.98 0.26 0.001 0.97
75 0.73 0.18 0.98 0.59 0.25 0.95 0.18 0.002 0.97
77 0.33 0.009 0.97 0.28 0.07 0.97 0.07 0.003 0.98
z (

C) 7.09 0.09 5.88 0.027 5.80 0.05

b) Skimmed milk
60 69.52 8.81 0.94 200 9.02 0.95 59.35 0.74 0.92
65 24.30 7.84 0.97 60.60 18.59 0.98 10.11 3.74 0.95
70 8.40 0.09 0.96 5.08 0.55 0.94 2.03 0.20 0.96
72 2.35 0.74 0.94 2.10 0.10 0.99 0.50 0.06 0.94
73 1.87 0.43 0.97 1.69 0.07 0.97 0.34 0.08 0.98
75 0.82 0.04 0.98 1.03 0.04 0.97 0.18 0.02 0.98
77 0.52 0.03 0.97 0.31 0.03 0.96 0.07 0.01 0.97
z (

C) 8.02 0.23 5.97 0.08 5.83 0.01

D-value: time required for 1 log reduction in activity at a specic temperature; z-value: increase in temperature required for 1 log change in D-value.
a
Standard deviations.
L. Dumitras cu et al. / LWT - Food Science and Technology 51 (2013) 325e330 328
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 the
regression line obtained by plotting the natural logarithm of rela-
tive residual activity as a function of inactivation time (t) are given
in Table 3. In general, fat is considered to have a protective effect on
microorganisms inactivation (van Asselt & Zwietering, 2006). At
60

C, in both bovine and non-bovine milk, the fat content had no
major inuence on k values. At higher temperature (77

C), GGT
inactivates faster in whole milk when compared with skimmed
counterpart. However, regarding the interspecies comparison, the
results obtained in our study indicate a faster inactivation of GGT in
cowmilk. In non-bovine milk, one would have expected an increase
in D-values while increasing fat content. This statement is not
supported, probably due to the different physico-chemical struc-
ture and composition of milk fats (Park, 2006). It has been reported
that heat treatment has only a minor effect on milk fat (Claeys,
2003). The different thermal sensitivity of GGT observed in our
study, in non-bovine milk can be correlated with specic heat
sensitive protein compounds in fat globule membrane (Spreer,
1998). Only a limited number of studies on the effect of milk fat
on a milk compound evaluated as an index for heat treatment are
reported and the results are contradictory (van Boekel & Walstra,
1989). However, it is difcult to predict the complex mechanisms
by which milk fat affects heat-induced modications. Additional
studies to verify this effect are needed.
To calculate the activation energy (Ea), the natural logarithm of
the inactivation rate constant k was plotted against the reciprocal of
the absolute temperature in Kelvin (T). Ea can be dened as the
energy absorbed or released needed to the molecules to be able to
react (van Boekel, 2008). The temperature dependence of the rate
constants for thermal inactivation of GGT in goat, sheep and cow
milk is depicted in Fig. 3. In skimmed and whole milk, Ea values
were 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 and
384.24 1.45 kJ/mol and 381.44 2.19 kJ/mol for cow milk
respectively. In goat skimmed milk, activation energy is signicant
different when compared with whole milk, whereas the Ea values
calculated for sheep and cow milk with different fat content are
similar.
The value of activation energy for GGT inactivation in skimmed
goat milk indicates that a lower amount of energy is needed to
initiate the denaturation process, to re-arrange the initial folded
conformation into the unfolded, inactive state (Dinella et al., 2004).
The activation energy values for sheep and cows milk indicates
a less compact structure, probably with a slow denaturation
process at lowtemperature, but relatively fast at high temperatures
(van Boekel, 2008).
Table 3
Inactivation 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 0.14
a
3.89 0.25 1.40 0.35 1.15 0.01 3.36 0.29 3.88 0.04
65 5.04 0.09 9.73 0.30 2.92 0.58 3.32 0.66 37.97 0.84 24.43 0.90
70 40.29 5.48 30.89 4.60 34.84 0.96 43.20 2.89 137.55 21.20 113.79 11.69
72 90.17 14.57 136.53 1.48 53.10 5.21 105.07 6.3 582.65 48.85 457.60 54.71
73 185.80 38.35 146.97 0.65 124.79 15.01 135.88 0.01 862.30 1.38 679.15 159.91
75 326.49 78.65 286.59 1.09 384.57 102.1 225.17 5.04 1216.18 22.52 1222.37 147.29
77 688.26 9.78 438.44 1.57 796.83 146.5 761.71 43.15 3207.38 115.3 3072.89 545.86
Ea (kJ/mol) 313.7 3.98
b
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 cow
milk (:). Filled squares corresponds to whole milk and the empty for skimmed milk.
Table 4
Changes in enthalpy of activation (DH), free energy of activation (DG) and entropy of activation (DS) for GGT inactivation in goat, sheep and cow milk.
Temperature Goat milk Sheep milk Cow milk

C K DH (kJ mol
1
) DG (kJ mol
1
) DS (kJ mol
1
) DH (kJ mol
1
) DG (kJ mol
1
) DS (kJ mol
1
) DH (kJ mol
1
) DG (kJ mol
1
) DS (kJ mol
1
)
s
a
w
b
s w s w s w s w s w s w s w s w
60 333 274.82 310.93 90.68 90.73 0.261 0.262 370.4 374.85 94.23 93.68 0.264 378.67 381.47 90.86 91.26 0.264
65 338 274.78 310.89 89.16 91.57 0.261 0.262 370.35 374.81 92.33 93.06 0.264 378.63 381.43 87.10 86.28 0.264
70 343 274.74 310.85 87.47 86.74 0.261 0.262 370.31 374.76 86.67 87.42 0.264 378.59 381.39 84.04 83.26 0.264
72 345 274.72 310.83 83.81 84.90 0.261 0.262 370.30 374.75 84.66 86.73 0.264 378.57 381.37 80.56 80.03 0.264
73 346 274.71 310.83 84.06 83.00 0.261 0.262 370.29 374.74 84.29 84.54 0.264 378.56 381.37 79.66 78.97 0.264
75 348 274.70 310.81 82.81 82.80 0.261 0.262 370.27 374.72 83.38 81.78 0.264 378.54 381.35 78.44 78.45 0.264
77 350 274.68 310.79 81.89 80.61 0.261 0.262 370.25 374.71 80.40 80.15 0.264 378.53 381.33 76.22 76.10 0.264
a
Skimmed milk.
b
Whole milk.
L. Dumitras cu et al. / LWT - Food Science and Technology 51 (2013) 325e330 329
Estimation of thermodynamic parameters is essential to
understand the probable mechanismof denaturation, which is very
important in thermal processes (SantAnna, Oliviera-Cardela, &
Brandelli, 2012). The activation energy value enabled the deter-
mination of enthalpy (DH), entropy (DG), and Gibbs free energy of
activation (DS) for GGT inactivation in different milk calculated for
the different temperatures (Table 4). The thermodynamic param-
eters decreased with increasing temperature.
For sheep and cow milk, no signicant differences were ob-
tained between whole and skimmed milk. The values of the change
in enthalpy of denaturation obtained for skimmed milk at 70

C
were: 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 in
goat milk is more stable than in sheep and cowmilk during thermal
treatment and undergoes large heat-induced conformational
changes. This conclusion is also supported by the lower value ob-
tained for the activation energy. Data regarding thermodynamic
parameters of GGT inactivation in sheep and goat milk were not
found in the literature.
4. Conclusions
The results presented have shown that there are signicant
variations in the raw milk activities of GGT between species. GGT
activity varied as a function of species and fat content, as follow:
cow milk > sheep milk > goat milk.
Investigation of GGT followed a rst-order reaction in the
temperature range of 60

Ce77

C. The lower values obtained for
activation energy and change in enthalpy of denaturation suggest
that the enzyme is more stable toward thermal denaturation in
goat milk when compared with sheep or cow milk. Taking in
consideration the breed specic differences, further studies are
needed in order to establish limiting values for GGTactivities and to
evaluate the possibilities of using this enzyme as indicator of
industrial processing of non-bovine milk. Our results constitute
therefore one of the rst reports on the detailed kinetic and ther-
modynamic investigation of GGT inactivation in non-bovine milk.
Acknowledgments
The authors acknowledge nancial support from the National
University Research Council (NURC, PN-II-ID-PCE-2008-2, Idea,
ID 517) Romania (www.trasilact.ugal.ro). Bioaliment Research
Platform (www.bioaliment.ugal.ro) is also acknowledged for
providing technical support.
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