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Journal of Food Engineering 113 (2012) 4752

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Journal of Food Engineering


journal homepage: www.elsevier.com/locate/jfoodeng

Thermal inactivation of lactoperoxidase in goat, sheep and bovine milk A


comparative kinetic and thermodynamic study
Loredana Dumitrascu, Nicoleta Stanciuc , Silvius Stanciu, Gabriela Rpeanu
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 19 August 2011
Received in revised form 18 March 2012
Accepted 18 May 2012
Available online 27 May 2012
Keywords:
Lactoperoxidase
Heat treatment
Kinetic
Safety

a b s t r a c t
Using isothermal heating, inactivation of lactoperoxidase (LPO) in goat, sheep and cow milk was studied
in the temperature range of 7077 C. Kinetic and thermodynamics studies were carried out at different
timetemperature combination in order to evaluate the suitability of LPO as marker for the heat-treatment of milk and dairy products from different species. The thermal inactivation of LPO followed the
rst-order kinetics. D- and k-values decreased and increased, respectively with increasing temperature,
indicating a more rapid LPO inactivation at higher temperatures. The inuence of temperature on the
inactivation rate constant was quantied using the Arrhenius and thermal death time models. The corresponding z-values were 3.38 0.013, 4.11 0.24 and 3.58 0.004 C in goat, sheep and cow milk,
respectively. Activation energy values varied between milk species with 678.96 21.43 kJ mol1 in goat
milk, 560.87 28.18 kJ mol1 in sheep milk and 641.56 13.12 kJ mol1 in cow milk, respectively.
2012 Elsevier Ltd. All rights reserved.

1. Introduction
Milk represents an ideal medium for the growth of microorganisms since it provides all the necessary nutrients and environmental conditions. Since milk is a very perishable foodstuff special
measures and considerations are necessary to ensure that it
reaches the market in an acceptable condition. Heat treatment of
milk is an important critical control point for ensuring the microbial safety and stability of the product (Claeys, 2003). Also, the collection of milk from the farmers and transportation to the dairy is
the most critical link in the total handling chain of milk (Seifu et al.,
2005).
Heating affects the functional and nutritional properties and
leads to numerous chemical and physical reactions that depend
on the temperature of the heat treatment applied as well as on
milk composition, concentration or pH. To control if heating was
adequate from a safety point of view, criteria need to be dened
(Claeys et al. 2002). Due to their slightly more resistance to heating
than the non-spore-forming pathogens found in milk on which
thermal processes are based, some enzymes are considered good
indicators for the evaluation of the severity or effectiveness of heat
treatment of milk (Wilinska et al., 2007). In addition, the enzymatic
indicators provide fast, simple and often less expensive screening
methods.
Lactoperoxidase (LPO) (EC 1.11.1.7) is a heme-containing glycoprotein (Boots and Floris, 2006) and is thought to be an important

Corresponding author. Tel.: +40 336 130 177; fax: +40 236 460 165.
E-mail address: Nicoleta.Sava@ugal.ro (N. Stanciuc).
0260-8774/$ - see front matter 2012 Elsevier Ltd. All rights reserved.
http://dx.doi.org/10.1016/j.jfoodeng.2012.05.028

component in the natural host-defence systems against bacterial


infections (Kussendrager and van Hooijdonk, 2000). The study of
LPO activity is of great interest because it has been proposed for
monitoring thermal treatments above 72 C for 15 s, but also as a
bactericidal agent, an index of mastitis and pro-oxidant (Fox,
2003). Additionally, LPO system can be used as an alternative
method to increase the storage stability of milk at high ambient
temperatures (FAO, 1999). The European Union currently species
a set of semi-quantitative standards for heat treated milk in order
to guarantee the correctness in terms of processing using as indicators the alkaline phosphatase activity, lactoperoxidase activity and
growth of microorganisms in UHT milk after certain conditions of
storage (Commission Regulations (EC) No. 2074/2005, 1664/2006).
Grifths (1986) evaluated the thermal resistance of several
indigenous enzymes as indices of the severity of thermal processing in milk and concluded that assay of LPO activity was the most
promising method for detecting high pasteurization treatments in
the order of 76 C for 15 s.
Most studies on LPO detection have been performed with cow
milk and only few have focused on non-bovine milk. Limited literature is available to provide data for the application of LPO assays
in milk products of non-bovine origin. Growing markets for dairy
products containing goat, sheep or buffalo milk may increase the
research interests to this area (Rankin et al., 2010). Certainly there
are several works performed with goat and cow milk, but due to
the different conditions used in the different works, it is important
to perform detailed quantitative kinetic studies in the three species
using the same experimental conditions.
Therefore, the aim of this work was to provide information on
the potential role of LPO activity in sheep and goat milk compared

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L. Dumitrascu et al. / Journal of Food Engineering 113 (2012) 4752

with cow milk with the object of being able to monitor heat treatments in a temperature range specic or even more severe than
typical pasteurization conditions. Also, has taken into account that
processing of non-bovine milk may involve thermal processing
parameters other than those for cows milk. In addition, it should
be kept in mind that timetemperature combinations may be specic for traditional and authentic milk products. Hereto, kinetic
and thermodynamic analyses were performed in the temperature
range between 70 and 77 C. Assay procedures were limited to
simple, colorimetric method that would be most likely to form
the basis of a quick, simple and inexpensive test.
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
in May. 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). The milk was divided in small portions (2 mL) and
stored frozen at 20 C until use.
0
2,2 -Azinobis (3-ethylbenzothiazoline)-6-sulfonic acid (ABTS)
was purchased from SigmaAldrich (Germany). All solvents and
chemical reagents were of analytical grade.
2.2. Isothermal inactivation of enzyme
Thermal inactivation kinetics of LPO experiments in raw milk
were performed using glass capillaries for quick temperature
transfer. Glass capillaries (length 100 mm, inner diameter 1 mm,
wall thickness 0.15 mm) lled with milk samples were sealed
and immersed in a water bath (Digibath-2 BAD 4, Raypa Trade,
Spain) at temperatures between 70 and 77 C for different holding
times (040 min). After thermal treatment, the capillaries were
immediately immersed in ice water to allow rapid cooling. A reactivation step was performed in order to check the (ir)reversibility
of the reactions. The (un)treated samples were stored at maximum
6 C and no reactivation occurred after one week of storage. For the
inactivation experiments, only the amount of the enzyme naturally
found in milk was considered.
2.3. Assay of enzyme activity
The LPO activity was measured spectrophotometrically at
412 nm, using a UVVIS GBC Cintra spectrophotometer (Australia),
as described by Kumar and Bathia (1999). In brief, the enzyme
sample (0.1 mL) was added to fresh substrate solution (1 mmol L1
ABTS in 0.1 mmol L1 phosphate buffer, pH 6.0). To initiate the
reaction, fresh prepared H2O2 solution (3.2 mmol L1) was added
and immediately the measurement of absorbance started up as a
function of time for 5 min. The blank solution used in measurements was prepared by heating 1 mL of raw milk for 1 min at
95 C. One unit of activity (U) is dened as the amount of enzyme
that catalyses the oxidation of l lmol of ABTS per min at 20 C. All
tests were performed at least in triplicate and the errors associated
with which experiment were lower than 2%.
2.4. Kinetic data analysis
Thermal LPO inactivation was described by the rst-order
kinetics reaction as we reported elsewhere (Stanciuc et al., 2011).

All statistical procedures were carried out using the SAS software
package, version 9.2 (SAS Institute, Cary, USA).
The thermodynamic parameters enthalpy (DH, kJ mol1), entropy (DS, kJ mol1), and free energy of activation (DG, kJ mol1) were
calculated according to the following expressions:

DH Ea  RT

DS RlnA  lnKb=hp  lnT

DG DH  T DS

where lnA is the ordinate intersection of the straight line obtained


by linear regression for Ea calculation, Kb is the Boltzmann constant
(1.38066  1023 J K1), hp the Planck constant (6.62618  1034
J s1), R the gas constant and T the absolute temperature.
3. Results and discussion
3.1. LPO activity
The composition of goat, sheep and bovine raw milk used in this
study was: total protein (3.35 0.74%, 4.28 0.50%, 3.56 0.42%),
fat matter (4.62 0.65%, 7.90 0.90%, 3.70 0.96%), lactose content
(4.39 0.84%, 5.63 0.41%, 4.67 0.67%), ash content (0.67 0.06%,
0.8 0.09%, 0.71 0.04%) and dry matter (13.06 0.40%, 18.21
0.85%, 12.68 0.61%). LPO showed different activity in three types
of tested raw milk samples. The average of LPO activity in goat, sheep
and cows raw milk was 0.81 0.05, 1.72 0.05 and 0.97
0.012 U mL1, respectively.
Because of the various chromogens used for its assay and the
variability in the assay conditions, data for LPO activity in the literature vary widely. For example, Medina et al. (1989) determined in
raw sheep milk at mid-lactation values ranging from 0.14 to
2.38 U mL1 with a mean value of 0.77 U mL1. Althaus et al.
(2001) reported mean LPO activity of 3.46 U mL1 during the lactation cycle, whereas Trujillo et al. (2007) have measured LPO activity of 1.73 U mL1 in goat milk. The lactation point related to the
highest activity in the milk from various goat breeds is mid-lactation (Zapico et al., 1998; Fonteh et al., 2002) compared with milk
from Verata goats, in which the highest activity of LPO is correlated
with the end of lactation. In Romania there are two local goat
breeds: Carpathian goat and Banat White goat. Data regarding
LPO activity in these breeds were not found in literature.
Values obtained for LPO activity in cow milk are lower than
those reported in literature. According to Fonteh et al. (2002),
LPO activity ranges from 1.5 to 2.7 U mL1. Seifu et al. (2005) indicated that LPO activity in cow milk varies from 1.2 to 19.4 U mL1.
These variations in enzyme level depend on the sexual cycle of the
cow, season, feeding regime and breed (Kussendrager and van
Hooijdonk, 2000).
3.2. Thermal inactivation
As a consequence of the above reported compositional differences, the micellar systems of different milk types differ noticeably
from that of cow milk by several properties, more specically micelle composition, size, mineralization and hydration (Pellegrini
et al., 1994). Furthermore, due to a lower colloidal stability when
compared to cow milk, goat and sheep milk are characterized by
a short clotting time during renneting coagulation, lower heat
stability at high temperature (Muir et al., 1993), and even more
severe fouling process during milder treatments such as pasteurization (De Raphael and Calvo, 1996).
The thermal stability of LPO in goat, sheep and cow milk was
examined in the temperature range from 70 to 77 C by measuring

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L. Dumitrascu et al. / Journal of Food Engineering 113 (2012) 4752

100

heating time (min)

(a)

90

10

20

30

40

Residual activity of LPO (%)

80
-0.5

70

60
-1

ln (A/A0)

50
40
30

-1.5
-2

20

-2.5

10
0
70

71

72

73

74

75

76

-3

77

Temperature (0C)
Fig. 1. Thermal stability of LPO in cow raw milk (diamonds), goat milk (squares)
and in sheep milk (triangles). Residual activity was measured after 5 min treatment
at different temperatures. Experimental points were the average of three
repetitions.

heating time (min)

(b)

10

10

20

30

40

50

0
-0.5
-1

ln (A/A0)

(a)

-1.5

0.9
0.8

-2
-2.5

A/A0

0.7

-3

0.6

-3.5

0.5
-4

0.4

-4.5

0.3
0.2

heating time (min)

0.1

(c)

0
0

10

20

30

heating time (min)


1

-1

0.9

-1.5

0.8
0.7

A/A0

30

40

50

-0.5

ln(A/A0)

(b)

20

40

0.6

-2
-2.5

0.5

-3

0.4

-3.5

0.3
0.2

-4

0.1
0
0

10

20

30

40

heating time (min)

(c)

Fig. 3. First order thermal inactivation of LPO at different temperatures: 70 C (),


71 C (N), 72 C (j), 73 C (d), 75 C (e), 77 C (4) in (a) cow, (b) goat, (c) sheep
milk. (A is the enzyme activity at time t, A0 the initial enzyme activity. For details,
see Kinetic data analysis section).

1
0.9
0.8

A/A0

0.7
0.6
0.5
0.4

0.3
0.2
0.1
0
0

10

20

30

40

heating time (min)


Fig. 2. Thermal inactivation of LPO activity (A/A0) at different temperatures: 70 C
(), 71 C (N), 72 C (j), 73 C (d), 75 C (e), 77 C (4) in a) cow, b) goat, c) sheep
milk.

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.

Fig. 1 shows the relative residual LPO activities in the different


species of milk plotted as a function of inactivation temperature
after 5 min of holding. For example, at 75 C the residual enzyme
activity was 16.2 2.0% (goat), 8.75 1.3% (sheep) and 38.9
1.8% (cow).
The decrease in LPO activity after heating, expressed as residual
activity, is given in Fig. 2. There are signicant differences in the
activities of LPO depending on the temperature applied and the
species of origin. At 77 C after 15 s and 1.5 min of holding time,
LPO residual activity decreased from 81.87 0.001% to 18.52
0.01% in goat milk (Fig. 2a), from 86.23 0.003% to 12.58 0.02%
in sheep milk (Fig. 2b), and from 79.20 0.04% to 14.25 0.03%
in cow milk (Fig. 2c), respectively. The timetemperature combination needed for complete inactivation of the enzyme is higher than
those reported in the literature. For example, Marks et al. (2001)
reported that the classical pasteurization process (72 C, 15 s) does
not inactivate the LPO in milk, whereas de Wit and van Hooydonk

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L. Dumitrascu et al. / Journal of Food Engineering 113 (2012) 4752

Table 1
Decimal reduction time (D) values and temperature resistance value (z) for LPO.
Temperature, C

Goat milk

Sheep milk
2

D (min)
70
71
72
73
75
77
z (C)

107.97 9.85
43.85 8.98
24.55 8.78
8.90 1.63
2.97 0.18
0.88 0.11
3.38 0.01

Cow milk
2

D (min)

0.87
0.97
0.95
0.97
0.9
0.98
4.11 0.24

38.44 7.38
21.41 2.86
8.67 1.18
5.25 0.97
1.86 0.45
0.68 0.04
3.58 0.004

0.92
0.95
0.9
0.97
0.98
0.95

D (min)

R2

89.52 10.69
42.88 3.38
22.97 0.70
13.59 1.02
3.68 0.14
0.77 0.14

0.94
0.95
0.97
0.94
0.97
0.97

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 error of regression.

(1996) suggested that complete inactivation of LPO in cow milk requires 78 C for 15 s.
From our results, it seems that the enzyme is more heat labile in
goat and sheep milk in the lower temperature range (7075 C),
whereas at higher temperature the enzymes is more stable in goat
milk when compared with cow and sheep milk. Lorenzen et al.
(2010) concluded that a heat treatment at 75 C for 28 s led to a
reduction of LPO activity between 50% and 60% without signicant
differences between species.
These differences may due to different composition of the milk
(matrix effects), but the increased heat stability may also be due to
molecular properties of the enzyme itself. The stability of LPO may
be attributed to its monomeric and highly ordered structure which
is also stabilized by eight disulde bonds and a calcium ion (Kussendrager and van Hooijdonk, 2000). Additionally studies are necessary to verify this hypothesis.

Table 2
Inactivation rate constant (k) and volume change of activation energy for LPO.
Temperature, C

70
71
72
73
75
77
Ea (kJ/mol)
a

Goat milk

Sheep milk

Cow milk

2.14 0.1a
5.26 1.2
10.01 3.5
26.30 4.8
77.53 3.5
262.17 33.6
678.96 21.43

5.2 0.11
10.75 0.2
29.36 0.4
96.0 0.6
123.55 2.2
335.78 21.2
560.87 28.18

2.59 0.03
5.68 0.3
10.02 0.3
16.98 1.2
64.19 1.4
302.36 5.7
641.56 13.12

Standard error of regression.

2
1

ln k

3.3. Kinetic and thermodynamic parameters


Thermal inactivation of LPO followed a rst order model (Fig. 3)
as indicated by the linearity obtained by plotting the retention values on a logarithmic scale as a function of heating time. High coefcient of correlation between residual LPO activity and time for
each temperature were obtained during linear regression analysis.
To verify the validity of the rst order kinetic model and to measure the linearity, coefcients of determination (R2) were calculated and residual plots checked for the absence of trends or
correlations. The model was appropriate for the data, since residuals represented only the experimental errors and were randomly
distributed when plotted. In all cases there was a good correlation
between experimental and predicted values (r2 = 0.99).
The corresponding D values for LPO in milk samples together
with the standard errors and r2 values are given in Table 1. The D-values decreased with increasing temperature from 70 to 77 C, indicating a faster inactivation of LPO at higher temperatures. For LPO
inactivation in goat milk, D-values ranged from 107.97 9.85 min
at 70 C to 0.88 0.11 min at 77 C. In sheep and cow milk, D-values
decreased from 38.44 7.38 min and 89.52 10.69 min at 70 C to
0.68 0.04 min and 0.77 0.14 min at 77 C. Trujillo et al. (2007)
reported D-values of 148.77 min at 69 C, 96.40 min at 71 C and
56.15 min at 73 C for LPO inactivation in goat milk. Thermal
sensitivity values (z) calculated in the temperature range studied
were: 3.38 0.01 C (goat milk), 4.11 0.24 C (sheep milk)
and 3.58 0.004 C (cow milk). Taye-Nasrabadi et al. (2011) reported z-value of 4.7 C for LPO inactivation in cow milk. Our results
for LPO inactivation in cow milk are in good correlation with those
obtained by Olszewski and Reuter (1992) and Claeys et al. (2002),
who reported z-value of 3.74 0.31 in the temperature range of
6973 C and 3.7 C in the temperature range of 7378 C, respectively. The obtained z-value for LPO inactivation in goat milk is lower

k  102 (min1)

-1

-2
-3

-4
-5
0.00285 0.00286 0.00287 0.00288 0.00289
1/T (K-1)

0.0029

0.00291 0.00292

Fig. 4. Arrhenius plot for thermal inactivation of LPO in cow (), goat (N) and sheep
(h) milk.

than that previously reported by Trujillo et al. (2007) who calculated


a z value of 9.45 C in the range of 6973 C. The differences in D and
z- values probably resulted from the different heating conditions
and also from acidic conditions under which LPO is less heat stable.
In general, low z-values indicate a more sensitivity to changes in
temperature (Barret et al., 1999). Therefore, higher z-values for
sheep milk indicate that this enzyme is less sensitive than in goat
or cow milk and this can be due to the matrix effects of the milk composition, as explained earlier.
The inactivation rate values (k), calculated from the slope of the
regression line obtained by plotting the natural logarithm of relative residual activity as a function of inactivation time (t) are given
in Table 2. The LPO inactivates faster in sheep milk, e.g. k-value at
73 C being 1.6 and 6 times higher when compared with goat and
cow milk, respectively.
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) according to the Arrhenius

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L. Dumitrascu et al. / Journal of Food Engineering 113 (2012) 4752


Table 3
Changes in enthalpy of activation (DH), free energy of activation (DG) and entropy of activation (DS) for LPO inactivation in goat, sheep and cow milk.
Temperature

Goat milk

Sheep milk
1

1

1

1

Cow milk

DH (kJ mol )

DG (kJ mol )

DS (kJ mol )

DH (kJ mol )

DG (kJ mol )

DS (kJ mol )

DH (kJ mol1)

DG (kJ mol1)

DS (kJ mol1)

70
71
72
73
75
77

343
344
345
346
348
350

676.1
676.09
676.09
676.08
676.06
676.04

95.38
93.09
91.71
89.06
86.42
83.41

0.258
0.258
0.258
0.258
0.258
0.258

558.01
558
558
557.99
557.97
557.95

92.44
91.04
88.72
86.66
85.07
82.67

0.266
0.266
0.266
0.266
0.266
0.266

638.7
638.69
638.69
638.68
638.66
638.64

95.19
93.75
90.99
90.22
85.93
83.84

0.268
0.268
0.268
0.268
0.268
0.268

equation. The temperature dependence of the rate constants for


thermal inactivation of LPO in goat, sheep and cow milk is depicted
in Fig. 4. Ea values were 678.96 21.43, 560.87 28.18 and
641.56 13.12 kJ mol1, respectively. Activation energy for LPO
inactivation in sheep milk is 12.57% and 17.39% lower, respectively
when compared with cow and goat milk meaning that a lower
amount of energy is needed to initiate the denaturation process.
Zelent et al. (2010) using differential scanning calorimetric method reported similar value of 664 kJ mol1 for activation energy in
cow milk. Taye-Nasrabadi et al. (2011) and Ludikhuyze et al.
(2001) reported activation energy values of 634.56 and
635.3 70.7 kJ mol1, respectively. Trujillo et al. (2007) reported a
signicant lower value for activation energy corresponding to LPO
inactivation in goat milk (225.98 kJ mol1). Information regarding
activation energy in sheep milk was not found in literature.
However, from Table 1 and 2 it can be concluded that LPO inactivates faster in sheep milk when compared to goat and cow milk
as indicated by the z- and Ea-value.
The activation energy value enabled the determination of
enthalpy (DH), entropy (DG), and Gibbs free energy of activation
(DS) for LPO inactivation (Table 3). The values of the change in
enthalpy of denaturation obtained for goat, sheep and cow milk
at 70 C were: 676.10, 558.01 and 638.70 kJ mol1, respectively.
It can be assumed that LPO in sheep milk is less stable than in goat
and cow milk during thermal treatment and undergoes large
heat-induced conformational changes. This conclusion is also supported by the lower value obtained for the activation energy, as
suggested by Hendrix et al. (2000). Positive values for change in
entropy were obtained in all the milk samples indicating that no
aggregation processes has occurred during thermal denaturation
(Anema and McKenna, 1996).
Data regarding thermodynamic parameters of LPO inactivation
in sheep were not found in the literature.
Comparing these thermodynamic parameters with data from
the literature (Dannenberg and Kessler, 1988, Levieux et al.,
2007; Stanciuc et al., 2011), it can be concluded that LPO is more
heat stable when compared with other intrinsic indicators of heat
treatments applied to milk, such as b-lactoglobulin, alkaline phosphatase and c-glutamyl transferase, respectively.
4. Conclusions
Lactoperoxidase is one of the most heat stable enzymes in milk.
According to the ndings of this study, the LPO activity of raw
sheep milk samples was the highest and that of goat samples
was the lowest among the three milk types tested.
The investigation of the thermal inactivation at a temperature
ranging from 70 to 77 C showed a rst-order kinetics model.
The lower values obtained for activation energy and change in enthalpy of denaturation suggest that the enzyme is less stable towards thermal denaturation in sheep milk when compared with
goat and cow milk.
Taking in consideration the breed specic differences, further
studies are needed in order to evaluate the posibilities to use this

1

1

enzyme as indicator of industrial processing of non-bovine milk.


Therefore, the kinetic data need to be compared with microbial
inactivation kinetic and with industrially applied and legally dened heat-treatment processess.
However, these data can be valuable in terms of implementing
food safety management systems, including traceability of the heat
treatment step as a critical control point for milk and dairy
products.
Acknowledgments
The authors acknowledge nancial support from the National
University Research Council (NURC, PN-II-ID-PCE-2008-2, Idea, ID
517) Romania (http://www.trasilact.ugal.ro). Bioaliment Research
Platform (http://www.bioaliment.ugal.ro) is also acknowledged
for providing technical support.
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