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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
48
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
DG DH T DS
49
100
(a)
90
10
20
30
40
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.
(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
0.1
(c)
0
0
10
20
30
-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
(c)
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
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.
50
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
(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
2
1
ln k
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.
51
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
1
1
52
Lorenzen, P.Chr., Martin, D., Clawin-Radecker, I., Barth, K., Knappstein, K., 2010.
Activities of alkaline phosphatase, c-glutamyltransferase and lactoperoxidase
in cow, sheep and goats milk in relation to heat treatment. Small Ruminant Res.
89, 1823.
Ludikhuyze, L.R., Claeys, W.L., Hendrickx, M.E., 2001. Inactivation kinetics of
alkaline phosphatase and lactoperoxidase, and denaturation of b-lactoglobulin
in raw milk under isothermal and dynamic temperature conditions. J. Dairy Res.
68, 625637.
Marks, N.E., Grandison, A.S., Lewis, M.J., 2001. Challenge testing of the
lactoperoxidase system in pasteurized milk. J. Appl. Microbiol. 91, 735741.
Medina, M., Gaya, P., Nunez, M., 1989. The lactoperoxidase system in ewes; milk:
levels of lactoperoxidase and thiocyanate. Lett. Appl. Microbiol. 8, 147149.
Muir, D.D., Horne, D.S., Law, A.J.R., Sweetsur, A.W.M., 1993. Ovine milk. 2. Seasonal
changes in indices of stability. Milchwissenschaft 48, 442445.
Olszewski, E., Reuter, H., 1992. The inactivation and reactivation behaviour of
lactoperoxidase in milk at temperature between 50 C and 135 C. Zeitschrift
fr Lebensmittel-Untersuchung und Forschung 194, 235239.
Pellegrini, O., Remeuf, F., Rivemale, M., 1994. Evolution of physicochemical
characteristics and renneting properties of ewes milk collected in Roquefort
area. Lait 74, 425442.
Rankin, S.A., Christiansen, A., Lee, W., Banavara, D.S., Lopez-Hernandez, A., 2010.
Invited review: the application of alkaline phosphatase assays for the validation
of milk product pasteurization. J. Dairy Sci. 93, 55385551.
Seifu, E., Buys, E.M., Donkin, E.F., 2005. Signicance of lactoperoxidase system in the
dairy industry and its potential applications: a review. Trends Food Sci. Technol.
16, 137154.
Stanciuc, N., Dumitrascu, L., Rpeanu, G., Stanciu, S., 2011. C-Glutamyl transferase
inactivation in milk and cream: a comparative kinetic study. Innovative Food
Sci. and Emer. Technol. 12, 5661.
Taye-Nasrabadi, H., Hoseinpour- fayzi, M.A., Mohasseli, M. (, 2011. Effect of heat
treatment on lactoperoxidase activity in camel milk: a comparison with bovine
lactoperoxidase. Small Ruminant Research. http://dx.doi.org/10.1016/
j.smallrumres.2011.04.007.
Trujillo, A.J., Pozo, P.I., Guamis, B., 2007. Effect of heat treatment on lactoperoxidase
activity in goat milk. Small Ruminant Res. 67, 243246.
Wilinska, A., Bryjak, J., Illeov, V., Polakovic, M., 2007. Kinetics of thermal
inactivation of alkaline phosphatase in bovine and caprine milk and buffer.
Int. Dairy J. 17, 579586.
Zapico, P., Medina, M., Gaya, P., Nunez, M., 1998. Synergistic effect of nisin and
lactoperoxidase system on Listeria monocytogenes in skim milk. Int. J. Food
Microbiol. 40, 3542.
Zelent, B., Sharp, K.A., Vanderkooi, J.M., 2010. Differential scanning calorimetry and
uorescence study of lactoperoxidase as a function of guanidinium-HCl, urea,
and pH. Biochim. Biophys. Acta 1804, 15081515.