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Effect of heat treatment on lactoperoxidase activity in camel milk: A comparison with bovine lactoperoxidase
Small Ruminant Research 99 (2011) 187–190
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Effect of heat treatment on lactoperoxidase activity in camel milk: A comparison with bovine lactoperoxidase Hossein Tayefi-Nasrabadi a,∗ , Mohammad Ali Hoseinpour-fayzi b , Maryam Mohasseli b a b
Department of Biochemistry, Faculty of Veterinary Medicine, University of Tabriz, P.O. Box, 51666-16471, Tabriz, Iran Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran
a r t i c l e
i n f o
Article history: Received 27 January 2011 Received in revised form 19 April 2011 Accepted 19 April 2011 Available online 26 May 2011 Keywords: Camel milk Lactoperoxidase Bovine milk Thermal stability
a b s t r a c t The thermal inactivation of lactoperoxidase (LP) in camel and bovine milk was studied and compared in a temperature range of 67–73 ◦ C. The analysis of inactivation rate constant (k) data for the process of thermal denaturation of LP in camel and bovine milk showed monophasic inactivation pattern. Based on the thermal death time model, decimal reduction time (D) and inactivation rate constant (k) values of LP in camel milk were more decreased and increased, respectively with increasing temperature in respect of the bovine LP. The corresponding thermal sensitivity values (z) calculated for camel and bovine LP were 6.42 ◦ C and 4.7 ◦ C, respectively. Thermodynamic analysis of LP showed lower values for activation energy and change in enthalpy of denaturation in camel than bovine milk. Overall the results obtained in this study suggest a lower heat stability of camel LP than in its bovine counterpart. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Lactoperoxidase (LP) (EC 1.11.1.7) is a glycoprotein that occurs naturally in colostrum, milk, and many other human and animal secretions (Kussendrager and van Hooijdonk, 2000; Conner et al., 2002). It contributes to the nonimmune host defense system, exerting bacteriostatic and bactericidal activity mainly on gram negative bacteria (Touch et al., 2004). For antimicrobial function, LP needs the presence of hydrogen peroxide and thiocyanate, which have been called together “LP system”. Today this system is considered to be an important part of the natural host defense system in mammals (Boots and Floris, 2006). The antibacterial action of the LP system is due to the effect of reaction products of thiocyanate oxidation, OSCN− and
∗ Corresponding author at: Department of Biochemistry, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Postal Code: 5166614779, Iran. Tel.: +98 411 3290625; fax: +98 411 3357834. E-mail addresses: hossein tayefi[email protected], tayefi@tabrizu.ac.ir (H. Tayefi-Nasrabadi). 0921-4488/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2011.04.007
HOSCN, which are able to oxidize free SH – groups of various proteins that are important for the viability of pathogens, thereby, inactivating crucial enzyme and protein systems (Sermon et al., 2005). The LP system could be used as an alternative method for the preservation of raw milk, which is produced under high ambient temperature and low hygienic conditions when a cooling process is not found (Haddadin et al., 1996). Furthermore, LP has been used for preservation of cosmetics, foodstuffs and protection of growing flowers, fruits, tubers, etc. (Le Nguyen et al., 2005; Touch et al., 2004). LP-activity may be used as an indicator of a correct pasteurization process for bovine and camel milk. This enzyme can be activated in some cases after heat treatment, thus contributing to extend the shelf-life of pasteurized milk in locations with inefficient cold storage conditions (Barrett et al., 1999; Fox and Kelly, 2006). The effect of heat treatment on milk LP activity has been examined in buffalo (Tayefi-Nasrabadi and Asadpour, 2008), bovine (Hernandez et al., 1990; Ludikhuyze et al., 2001; Marin et al., 2003) and caprine (Trujillo et al., 2007), but detailed quantitative kinetic thermal
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inactivation studies in camel milk are lacking. The aim of this study was to determine the differences in lactoperoxidase heat-resistance in camel and bovine milk based on kinetic and thermodynamic analysis in the range 67–73 ◦ C. 2. Materials and methods 2.1. Chemicals Pyrogallol, hydrogen peroxide (30% solution) and all the other chemicals used in this research were obtained from Merck (Darmstadt, Germany) and were of reagent grade.
2.5. Kinetic data analysis Inactivation kinetics of milk LP toward thermal processes was subjected to reaction kinetic analysis. According to Eq. (1), loss of enzyme activity rate (−dA/dt) is proportional to the inactivation rate constant (k) and enzyme activity at each treatment time (A). −
dA = kAn dt
The experimental raw data are plotted according to the equation lnA/A0 = kt derived from Eq. (1), where A is the response value after heating treatment, A0 is the initial enzyme activity at time t0 , and t is the exposure time (min). We also calculated D-values (decimal reduction time) and zvalue (temperature necessary to reduce D-value by 1 logarithmic cycle) according to Eq. (2).
2.2. Milk sampling Log Fresh raw camel (Camelus bactrianus) and bovine (Holstein) milk were supplied from Khorkhor (Tabriz, East-Azerbaijan province, Iran) and was analysed for total protein (IDF, 1993), fat matter (ISO, 1976), ash content (AOAC, 1995) and dry matter (IDF, 1970). The milk was divided into small portions (50 mL) and stored at −20 ◦ C until analysis.
(1)
A A0
=−
1 ×t D
(2)
For estimation of z-value, the linear regression of log D-values versus corresponding temperatures was performed using the SigmaPlot for windows version 10.0 (Systat software, Germany). In a denaturation process, the rate constant (k) and the temperature of treatment are related according to the Arrhenius equation: Ea RT
2.3. Enzymatic activity assay
ln k = ln A −
Milk LP activity was measured by following the H2 O2 -dependent oxidation of pyrogallol at 430 nm, using an extinction coefficient of 2470 M−1 cm−1 (Pruitt et al., 1990). 3 mL of TS buffer (0.1 M citrate–phosphate–borate buffer, pH 6.5), 0.15 mL pyrogallol (200 mM) and milk sample (0.05 mL) were added together in cuvette. The reaction was initiated by the addition of 0.03 mL hydrogen peroxide solution (61 mM) and immediately the measurement of absorbance started at 430 nm as a function of time for 2 min at 15 s intervals using an UNICO UV-2100 PC spectrophotometer (UNICO, China). Measurements were carried out against the reagent blank containing pyrogallol and enzyme solution only. Reaction velocity was computed from linear slopes of absorbance–time curve. One unit of activity is defined as the amount of enzyme that catalyzes the oxidation of 1 mol of pyrogallol per min at room temperature (∼22–25 ◦ C).
where A is the Arrhenius constant, Ea the apparent activation energy, R the universal gas constant, and T the absolute temperature. Activation energy can be calculated from the slope of the line. From activation energy (Ea ), different thermodynamic parameters such as variations in enthalpy (H◦ ), Gibbs free energy (G◦ ) and entropy (S◦ ) can be estimated according to the following expressions:
2.4. Heat incubation study Thermal stability of milk LP was studied by incubating aliquots of milk at various temperatures (67, 69, 71 and 73 ◦ C) up to 60 min in a thermostatic water bath and measuring their activity at room temperature after brief cooling in ice. The incubation was carried out in sealed vials to prevent change of volume of the sample and, hence, the enzyme concentration due to evaporation. Assays at the different temperatures were done at least in 3 separate experiments and the mean values of data were used to obtain the different kinetic and thermodynamic parameters.
(3)
H ◦ = Ea − RT ◦
G = −RT ln S ◦ =
kh kB T
H ◦ − G◦ T
(4) (5) (6)
where h and kB are the Planck’s and the Boltzmann’s constants, respectively.
3. Results and discussion Composition of camel and bovine milk used in this study was: total protein (3.4%, 3.1%), fat matter (4.2%, 3.5%), ash content (1.33%, 0.71%) and dry matter (12.14%, 11.82%), respectively. Effects of heat treatment on the enzymatic activity of camel and bovine milk LP at different temperatures are
Fig. 1. Effect of heat treatment on camel (A) and bovine (B) milk lactoperoxidase activity as a function of treatment time at different temperatures: 67 ◦ C (䊉), 69 ◦ C (), 71 ◦ C (), 73 ◦ C (). The activity expressed as the percentage of initial activity.
H. Tayefi-Nasrabadi et al. / Small Ruminant Research 99 (2011) 187–190
189
Table 1 Inactivation kinetic parameters of camel and bovine milk lactoperoxidases toward thermal processes, assuming a 1st-order reaction. Camel D-Values (min) Temperature (◦ C) 17.24 67 7.30 69 71 3.16 73 2.09 z (◦ C) 6.426 Ea (kJ/mol) 349.04
Bovine 2
R
a
0.91 0.98 0.91 0.99 0.98 0.98
8.1 4.45 1.68 1.08
t1/2 (min)
k-Values (×10−2 min−1 )
D-Values (min)
R2
13.35 31.51 72.81 100.1
116.27 31.05 9.78 6.77 4.7 634.56
0.93 0.99 0.91 0.90 0.95 0.99
t1/2 (min)
37 13.75 4.4 2
k-Values (×10−2 min−1 ) 1.96 7.3 23.78 100
a: time required for decay of original activity by 50%; R2 : coefficients of correlation; D: decimal reduction time; k: rate constant; z: temperature necessary to reduce D-value by 1 logarithmic cycle; Ea : activation energy.
shown in Fig. 1A and B, respectively. The degree of LP denaturation increased with temperature and treatment time. Linear regression analysis showed high coefficients of correlation (R2 ) between residual LP activity and time for each temperature (Table 1). These results indicate that thermal inactivation of LP in camel and bovine milk follow a monophasic inactivation pattern. These results agree in general with those obtained by Marin et al. (2003), Trujillo et al. (2007) and Tayefi-Nasrabadi and Asadpour (2008) for bovine, caprine and buffalo milk LP, respectively. As shown in Table 1, LP activity in bovine has declined very slowly below 69 ◦ C, and at 71 ◦ C there was a remarkable increase in the rate of deactivation. For instance, at 67 ◦ C, 37 min were necessary to reduce lactoperoxidase activity to 50% (t1/2 = 37), while at 71 ◦ C only 4.4 min of treatment caused the same reduction. In camel milk, times necessary to reduce LP activity by 50% at 67 ◦ C and 71 ◦ C were found 8.1 min and 1.68 min, which was 4.6 and 2.6 fold lower than the bovine counterpart, respectively. These results suggest that camel milk LP is more sensitive to increases of temperature and duration of treatment in comparison with bovine LP. Results were further confirmed by the calculation of decimal reduction time (D) and k-values of LP from semilogarithmic plot of LP activity and activity retention (A/A0 ) against time, respectively. As shown in Table 1, D and kvalues of LP in camel milk more decreased and increased, respectively with increasing temperature in respect of the bovine counterpart. D-values obtained for camel milk LP at 71 ◦ C and 67 ◦ C were about 3.1 and 6.74 times lower than bovine counterpart. These remarkable decreases of Dvalues indicate a potential thermal denaturation in camel LP than bovine counterpart. For the range of temperatures studied, thermal sensitivity values (z) were found 6.42 ◦ C and 4.7 ◦ C for camel and bovine LP, respectively. In this study, the z-value for bovine LP is very close to that reported by Barrett et al. (1999) and Griffiths (1986), which was 5.1 ◦ C and 5.4 ◦ C, respectively. In general, high z-values mean more sensitivity to the duration of heat treatment (Barrett et al., 1999). Therefore, higher z-value for camel milk LP indicates that this enzyme is more sensitive to the extension of treatment time than bovine counterpart. In the present study, occurrence of a smaller time windows for thermo inactivation of camel milk LP as a function of treatment time (Fig. 1) proved this finding.
Fig. 2. Arrhenius plots for camel () and bovine (䊉) milk lactoperoxidase activity.
Inactivation rate constants were used to drawn the Arrhenius plot, from which slope activation energy were calculated and found 349.04 and 634.56 kJ/mol for camel and bovine milk LP, respectively (Fig. 2 and Table 1). Recently, Zelent et al. (2010) with differential scanning calorimetric method showed that activation energy of bovine milk LP was 664 kJ/mol, which is very close to the value obtained for bovine milk LP in this study (634.56 kJ/mol). In camel milk, activation energy of LP was found 349.04 kJ/mol, which was 1.82 fold lower than the bovine counterpart (Table 1). The lower value for the activation energy of camel milk LP than bovine counterpart means that a lower amount of energy is needed to initiate denaturation (Bjorck, 1992). Table 2 shows the comparison of the thermodynamic values of variation in enthalpy (H◦ ), entropy (S◦ ) and Gibbs free energy (G◦ ) between camel and bovine LP calculated for the different temperatures. The values of the change in enthalpy of denaturation obtained for camel milk LP (∼346 kJ/mol) are lower than the values obtained for bovine milk LP (∼631 kJ/mol). It can be assumed that camel milk LP is probably less stable than bovine milk LP toward thermal processes, as suggested by lower value of activation energy (Hendrix et al., 2000). In camel milk, change in entropy (S◦ ) of LP was found about +722 J/mol K, which
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Table 2 Thermodynamic parameters for inactivation of camel and bovine milk lactoperoxidases toward thermal processes, assuming a 1st-order reaction. Temperature (◦ C)
Camel a
67 69 71 73 a
◦
G (kJ/mol)
100.90 99.07 97.27 96.69
Bovine ◦
◦
H (kJ/mol)
S (J/mol K)
G◦ (kJ/mol)
H◦ (kJ/mol)
S◦ (J/mol K)
346.22 346.20 346.18 346.17
721.52 722.60 723.58 721.02
106.33 103.23 100.48 96.95
631.73 631.72 631.70 631.68
1545.30 1545.27 1544.25 1545.48
G◦ : variations in Gibbs free energy; H◦ : variations in enthalpy; S◦ : variations in entropy.
was 2 fold lower than the bovine counterpart (Table 2). The positive values for the changes in entropy of LP in both camel and bovine milk mean that no process of aggregation has occurred during thermal denaturation (Anema and McKenna, 1996). 4. Conclusion The investigation of thermal denaturation of LP in camel and bovine milk in a temperature range of 67–73 ◦ C showed a first-order kinetics model. The lower values of activation energy and change in enthalpy of denaturation for camel milk LP than bovine counterpart suggest that camel milk LP is less stable than bovine milk LP toward thermal denaturation. The higher z-value (6.42 ◦ C) for camel milk LP than bovine counterpart (4.7 ◦ C) indicates that camel milk LP is more sensitive to the extension of thermal treatment time than bovine counterpart. Acknowledgment The financial support of the research affair of the University of Tabriz is highly appreciated. References Anema, S.G., McKenna, A.B., 1996. Reaction kinetics of thermal denaturation of whey proteins in heated reconstituted whole milk. J. Agric. Food Chem. 44, 422–428. AOAC, 1995. Dairy products. Determination of ash content. Standard 945.46. In: Official Methods of Analysis ,. Association of Official Analytical Chemists, Washington, DC, USA. Barrett, N.E., Grandison, A.S., Lewis, M.J., 1999. Contribution of the lactoperoxidase system to the keeping quality of pasteurised milk. J. Dairy Res. 66, 73–80. Bjorck, L., 1992. In: Fox, P.F. (Ed.), Indigenous Enzyme in Milk lactoperoxidase. Advanced Dairy Chemistry. , 1st ed. Springer, London, pp. 323–338, ISBN: 978-1851667611. Boots, J.W., Floris, R., 2006. Lactoperoxidase: from catalytic mechanism to practical applications. Int. Dairy J. 16, 1272–1276. Conner, G.E., Salathe, M., Forteza, R., 2002. Lactoperoxidase and hydrogen peroxide metabolism in the airway. Am. J. Respir. Crit. Care. Med. 66, S57–S61. Fox, P.F., Kelly, A.L., 2006. Indigenous enzymes in milk: overview and historical aspects. Part 1. Int. Dairy J. 16, 500–516. Griffiths, M., 1986. Use of milk enzymes as indices of heat treatment. J. Food Prot. 49, 696–705.
Haddadin, M.S., Ibrahim, S.A., Robinson, R.K., 1996. Preservation of raw milk by activation of the natural lactoperoxidase systems. Food Cont. 7, 149–152. Hendrix, T., Griko, Y.V., Privalov, P.L., 2000. A calorimetric study of the influence of calcium on the stability of bovine ␣-lactalbumin. Biophys. Chem. 84, 27–34. Hernandez, M., Van Markwijk, B., Vreeman, H., 1990. Isolation and properties of lactoperoxidase from bovine milk. Neth. Milk Dairy J. 44, 213–231. IDF, 1970. Milk. Determination of total solids. In: International Dairy Federation Standard 70 ,. International Dairy Federation, Brussels, Belgium. IDF, 1993. Milk. Determination of the nitrogen (Kjeldahl method) and calculation of the crude protein content. In: International Dairy Federation Standard 20B ,. International Dairy Federation, Brussels, Belgium. ISO, 1976. Milk. Determination of Fat Content-Gerber Method. Standard 2446. International Organization for Standardization, Leusden, The Netherlands. Kussendrager, K.D., van Hooijdonk, A.C.M., 2000. Lactoperoxidase: physico-chemical properties, occurrence, mechanism of action and applications. Br. J. Nutr. 84, S19–S25. Le Nguyen, D.D., Ducamp, M.N., Dornier, M., Montet, D., Loiseau, G., 2005. Effect of the lactoperoxidase system against three major causal agents of disease in mangoes. J. Food Prot. 68, 1497–1500. Ludikhuyze, L.R., Claeys, W.L., Hendrickx, M.E., 2001. Inactivation kinetics of alkaline phosphatase and lactoperoxidase, and denaturation of lactoglobulin in raw milk under isothermal and dynamic temperature conditions. J. Dairy Res. 68, 625–637. Marin, E., Sanches, L., Perez, M.D., Puyol, P., Calvo, M., 2003. Effect of heat treatment on bovine lactoperoxidase activity in skim milk: kinetic and thermodynamic analysis. J. Food Sci. 68, 89–93. Pruitt, K.M., Kamau, D.N., Miller, K., Mansson-Rahemtulla, B., Rahemtulla, F., 1990. Quantitative, standardized assays for determining the concentrations of bovine lactoperoxidase, human salivary peroxidase, and human myeloperoxidase. Anal. Biochem. 191, 278– 286. Sermon, J., Vanoirbeek, K., De Spiegeleer, P., Van Houdt, R., Aertsen, A., Michiels, C.W., 2005. Unique stress response to the lactoperoxidasethiocyanate enzyme system in Escherichia coli. Res. Microbiol. 156, 225–232. Tayefi-Nasrabadi, H., Asadpour, R., 2008. Effect of heat treatment on buffalo (Bubalus bubalis) lactoperoxidase activity in raw milk. J. Boil. Sci. 8, 1310–1315. Touch, V., Hayakawa, S., Yamada, S., Kaneko, S., 2004. Effects of a lactoperoxidase–thiocyanate–hydrogen peroxide system on Salmonella enteritidis in animal or vegetable foods. Int. J. Food Microbiol. 93, 175–183. Trujillo, A.J., Pozo, P.I., Guamis, B., 2007. Effect of heat treatment on lactoperoxidase activity in caprine milk. Small Ruminant Res. 67, 243–246. Zelent, B., Sharp, K.A., Vanderkooi, J.M., 2010. Differential scanning calorimetry and fluorescence study of lactoperoxidase as a function of guanidinium–HCl, urea, and pH. Biochim. Biophys. Acta. 1804, 1508–1515.
Contents lists available at ScienceDirect
Small Ruminant Research journal homepage: www.elsevier.com/locate/smallrumres
Short communication
Effect of heat treatment on lactoperoxidase activity in camel milk: A comparison with bovine lactoperoxidase Hossein Tayefi-Nasrabadi a,∗ , Mohammad Ali Hoseinpour-fayzi b , Maryam Mohasseli b a b
Department of Biochemistry, Faculty of Veterinary Medicine, University of Tabriz, P.O. Box, 51666-16471, Tabriz, Iran Faculty of Natural Sciences, University of Tabriz, Tabriz, Iran
a r t i c l e
i n f o
Article history: Received 27 January 2011 Received in revised form 19 April 2011 Accepted 19 April 2011 Available online 26 May 2011 Keywords: Camel milk Lactoperoxidase Bovine milk Thermal stability
a b s t r a c t The thermal inactivation of lactoperoxidase (LP) in camel and bovine milk was studied and compared in a temperature range of 67–73 ◦ C. The analysis of inactivation rate constant (k) data for the process of thermal denaturation of LP in camel and bovine milk showed monophasic inactivation pattern. Based on the thermal death time model, decimal reduction time (D) and inactivation rate constant (k) values of LP in camel milk were more decreased and increased, respectively with increasing temperature in respect of the bovine LP. The corresponding thermal sensitivity values (z) calculated for camel and bovine LP were 6.42 ◦ C and 4.7 ◦ C, respectively. Thermodynamic analysis of LP showed lower values for activation energy and change in enthalpy of denaturation in camel than bovine milk. Overall the results obtained in this study suggest a lower heat stability of camel LP than in its bovine counterpart. © 2011 Elsevier B.V. All rights reserved.
1. Introduction Lactoperoxidase (LP) (EC 1.11.1.7) is a glycoprotein that occurs naturally in colostrum, milk, and many other human and animal secretions (Kussendrager and van Hooijdonk, 2000; Conner et al., 2002). It contributes to the nonimmune host defense system, exerting bacteriostatic and bactericidal activity mainly on gram negative bacteria (Touch et al., 2004). For antimicrobial function, LP needs the presence of hydrogen peroxide and thiocyanate, which have been called together “LP system”. Today this system is considered to be an important part of the natural host defense system in mammals (Boots and Floris, 2006). The antibacterial action of the LP system is due to the effect of reaction products of thiocyanate oxidation, OSCN− and
∗ Corresponding author at: Department of Biochemistry, Faculty of Veterinary Medicine, University of Tabriz, Tabriz, Postal Code: 5166614779, Iran. Tel.: +98 411 3290625; fax: +98 411 3357834. E-mail addresses: hossein tayefi[email protected], tayefi@tabrizu.ac.ir (H. Tayefi-Nasrabadi). 0921-4488/$ – see front matter © 2011 Elsevier B.V. All rights reserved. doi:10.1016/j.smallrumres.2011.04.007
HOSCN, which are able to oxidize free SH – groups of various proteins that are important for the viability of pathogens, thereby, inactivating crucial enzyme and protein systems (Sermon et al., 2005). The LP system could be used as an alternative method for the preservation of raw milk, which is produced under high ambient temperature and low hygienic conditions when a cooling process is not found (Haddadin et al., 1996). Furthermore, LP has been used for preservation of cosmetics, foodstuffs and protection of growing flowers, fruits, tubers, etc. (Le Nguyen et al., 2005; Touch et al., 2004). LP-activity may be used as an indicator of a correct pasteurization process for bovine and camel milk. This enzyme can be activated in some cases after heat treatment, thus contributing to extend the shelf-life of pasteurized milk in locations with inefficient cold storage conditions (Barrett et al., 1999; Fox and Kelly, 2006). The effect of heat treatment on milk LP activity has been examined in buffalo (Tayefi-Nasrabadi and Asadpour, 2008), bovine (Hernandez et al., 1990; Ludikhuyze et al., 2001; Marin et al., 2003) and caprine (Trujillo et al., 2007), but detailed quantitative kinetic thermal
188
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inactivation studies in camel milk are lacking. The aim of this study was to determine the differences in lactoperoxidase heat-resistance in camel and bovine milk based on kinetic and thermodynamic analysis in the range 67–73 ◦ C. 2. Materials and methods 2.1. Chemicals Pyrogallol, hydrogen peroxide (30% solution) and all the other chemicals used in this research were obtained from Merck (Darmstadt, Germany) and were of reagent grade.
2.5. Kinetic data analysis Inactivation kinetics of milk LP toward thermal processes was subjected to reaction kinetic analysis. According to Eq. (1), loss of enzyme activity rate (−dA/dt) is proportional to the inactivation rate constant (k) and enzyme activity at each treatment time (A). −
dA = kAn dt
The experimental raw data are plotted according to the equation lnA/A0 = kt derived from Eq. (1), where A is the response value after heating treatment, A0 is the initial enzyme activity at time t0 , and t is the exposure time (min). We also calculated D-values (decimal reduction time) and zvalue (temperature necessary to reduce D-value by 1 logarithmic cycle) according to Eq. (2).
2.2. Milk sampling Log Fresh raw camel (Camelus bactrianus) and bovine (Holstein) milk were supplied from Khorkhor (Tabriz, East-Azerbaijan province, Iran) and was analysed for total protein (IDF, 1993), fat matter (ISO, 1976), ash content (AOAC, 1995) and dry matter (IDF, 1970). The milk was divided into small portions (50 mL) and stored at −20 ◦ C until analysis.
(1)
A A0
=−
1 ×t D
(2)
For estimation of z-value, the linear regression of log D-values versus corresponding temperatures was performed using the SigmaPlot for windows version 10.0 (Systat software, Germany). In a denaturation process, the rate constant (k) and the temperature of treatment are related according to the Arrhenius equation: Ea RT
2.3. Enzymatic activity assay
ln k = ln A −
Milk LP activity was measured by following the H2 O2 -dependent oxidation of pyrogallol at 430 nm, using an extinction coefficient of 2470 M−1 cm−1 (Pruitt et al., 1990). 3 mL of TS buffer (0.1 M citrate–phosphate–borate buffer, pH 6.5), 0.15 mL pyrogallol (200 mM) and milk sample (0.05 mL) were added together in cuvette. The reaction was initiated by the addition of 0.03 mL hydrogen peroxide solution (61 mM) and immediately the measurement of absorbance started at 430 nm as a function of time for 2 min at 15 s intervals using an UNICO UV-2100 PC spectrophotometer (UNICO, China). Measurements were carried out against the reagent blank containing pyrogallol and enzyme solution only. Reaction velocity was computed from linear slopes of absorbance–time curve. One unit of activity is defined as the amount of enzyme that catalyzes the oxidation of 1 mol of pyrogallol per min at room temperature (∼22–25 ◦ C).
where A is the Arrhenius constant, Ea the apparent activation energy, R the universal gas constant, and T the absolute temperature. Activation energy can be calculated from the slope of the line. From activation energy (Ea ), different thermodynamic parameters such as variations in enthalpy (H◦ ), Gibbs free energy (G◦ ) and entropy (S◦ ) can be estimated according to the following expressions:
2.4. Heat incubation study Thermal stability of milk LP was studied by incubating aliquots of milk at various temperatures (67, 69, 71 and 73 ◦ C) up to 60 min in a thermostatic water bath and measuring their activity at room temperature after brief cooling in ice. The incubation was carried out in sealed vials to prevent change of volume of the sample and, hence, the enzyme concentration due to evaporation. Assays at the different temperatures were done at least in 3 separate experiments and the mean values of data were used to obtain the different kinetic and thermodynamic parameters.
(3)
H ◦ = Ea − RT ◦
G = −RT ln S ◦ =
kh kB T
H ◦ − G◦ T
(4) (5) (6)
where h and kB are the Planck’s and the Boltzmann’s constants, respectively.
3. Results and discussion Composition of camel and bovine milk used in this study was: total protein (3.4%, 3.1%), fat matter (4.2%, 3.5%), ash content (1.33%, 0.71%) and dry matter (12.14%, 11.82%), respectively. Effects of heat treatment on the enzymatic activity of camel and bovine milk LP at different temperatures are
Fig. 1. Effect of heat treatment on camel (A) and bovine (B) milk lactoperoxidase activity as a function of treatment time at different temperatures: 67 ◦ C (䊉), 69 ◦ C (), 71 ◦ C (), 73 ◦ C (). The activity expressed as the percentage of initial activity.
H. Tayefi-Nasrabadi et al. / Small Ruminant Research 99 (2011) 187–190
189
Table 1 Inactivation kinetic parameters of camel and bovine milk lactoperoxidases toward thermal processes, assuming a 1st-order reaction. Camel D-Values (min) Temperature (◦ C) 17.24 67 7.30 69 71 3.16 73 2.09 z (◦ C) 6.426 Ea (kJ/mol) 349.04
Bovine 2
R
a
0.91 0.98 0.91 0.99 0.98 0.98
8.1 4.45 1.68 1.08
t1/2 (min)
k-Values (×10−2 min−1 )
D-Values (min)
R2
13.35 31.51 72.81 100.1
116.27 31.05 9.78 6.77 4.7 634.56
0.93 0.99 0.91 0.90 0.95 0.99
t1/2 (min)
37 13.75 4.4 2
k-Values (×10−2 min−1 ) 1.96 7.3 23.78 100
a: time required for decay of original activity by 50%; R2 : coefficients of correlation; D: decimal reduction time; k: rate constant; z: temperature necessary to reduce D-value by 1 logarithmic cycle; Ea : activation energy.
shown in Fig. 1A and B, respectively. The degree of LP denaturation increased with temperature and treatment time. Linear regression analysis showed high coefficients of correlation (R2 ) between residual LP activity and time for each temperature (Table 1). These results indicate that thermal inactivation of LP in camel and bovine milk follow a monophasic inactivation pattern. These results agree in general with those obtained by Marin et al. (2003), Trujillo et al. (2007) and Tayefi-Nasrabadi and Asadpour (2008) for bovine, caprine and buffalo milk LP, respectively. As shown in Table 1, LP activity in bovine has declined very slowly below 69 ◦ C, and at 71 ◦ C there was a remarkable increase in the rate of deactivation. For instance, at 67 ◦ C, 37 min were necessary to reduce lactoperoxidase activity to 50% (t1/2 = 37), while at 71 ◦ C only 4.4 min of treatment caused the same reduction. In camel milk, times necessary to reduce LP activity by 50% at 67 ◦ C and 71 ◦ C were found 8.1 min and 1.68 min, which was 4.6 and 2.6 fold lower than the bovine counterpart, respectively. These results suggest that camel milk LP is more sensitive to increases of temperature and duration of treatment in comparison with bovine LP. Results were further confirmed by the calculation of decimal reduction time (D) and k-values of LP from semilogarithmic plot of LP activity and activity retention (A/A0 ) against time, respectively. As shown in Table 1, D and kvalues of LP in camel milk more decreased and increased, respectively with increasing temperature in respect of the bovine counterpart. D-values obtained for camel milk LP at 71 ◦ C and 67 ◦ C were about 3.1 and 6.74 times lower than bovine counterpart. These remarkable decreases of Dvalues indicate a potential thermal denaturation in camel LP than bovine counterpart. For the range of temperatures studied, thermal sensitivity values (z) were found 6.42 ◦ C and 4.7 ◦ C for camel and bovine LP, respectively. In this study, the z-value for bovine LP is very close to that reported by Barrett et al. (1999) and Griffiths (1986), which was 5.1 ◦ C and 5.4 ◦ C, respectively. In general, high z-values mean more sensitivity to the duration of heat treatment (Barrett et al., 1999). Therefore, higher z-value for camel milk LP indicates that this enzyme is more sensitive to the extension of treatment time than bovine counterpart. In the present study, occurrence of a smaller time windows for thermo inactivation of camel milk LP as a function of treatment time (Fig. 1) proved this finding.
Fig. 2. Arrhenius plots for camel () and bovine (䊉) milk lactoperoxidase activity.
Inactivation rate constants were used to drawn the Arrhenius plot, from which slope activation energy were calculated and found 349.04 and 634.56 kJ/mol for camel and bovine milk LP, respectively (Fig. 2 and Table 1). Recently, Zelent et al. (2010) with differential scanning calorimetric method showed that activation energy of bovine milk LP was 664 kJ/mol, which is very close to the value obtained for bovine milk LP in this study (634.56 kJ/mol). In camel milk, activation energy of LP was found 349.04 kJ/mol, which was 1.82 fold lower than the bovine counterpart (Table 1). The lower value for the activation energy of camel milk LP than bovine counterpart means that a lower amount of energy is needed to initiate denaturation (Bjorck, 1992). Table 2 shows the comparison of the thermodynamic values of variation in enthalpy (H◦ ), entropy (S◦ ) and Gibbs free energy (G◦ ) between camel and bovine LP calculated for the different temperatures. The values of the change in enthalpy of denaturation obtained for camel milk LP (∼346 kJ/mol) are lower than the values obtained for bovine milk LP (∼631 kJ/mol). It can be assumed that camel milk LP is probably less stable than bovine milk LP toward thermal processes, as suggested by lower value of activation energy (Hendrix et al., 2000). In camel milk, change in entropy (S◦ ) of LP was found about +722 J/mol K, which
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Table 2 Thermodynamic parameters for inactivation of camel and bovine milk lactoperoxidases toward thermal processes, assuming a 1st-order reaction. Temperature (◦ C)
Camel a
67 69 71 73 a
◦
G (kJ/mol)
100.90 99.07 97.27 96.69
Bovine ◦
◦
H (kJ/mol)
S (J/mol K)
G◦ (kJ/mol)
H◦ (kJ/mol)
S◦ (J/mol K)
346.22 346.20 346.18 346.17
721.52 722.60 723.58 721.02
106.33 103.23 100.48 96.95
631.73 631.72 631.70 631.68
1545.30 1545.27 1544.25 1545.48
G◦ : variations in Gibbs free energy; H◦ : variations in enthalpy; S◦ : variations in entropy.
was 2 fold lower than the bovine counterpart (Table 2). The positive values for the changes in entropy of LP in both camel and bovine milk mean that no process of aggregation has occurred during thermal denaturation (Anema and McKenna, 1996). 4. Conclusion The investigation of thermal denaturation of LP in camel and bovine milk in a temperature range of 67–73 ◦ C showed a first-order kinetics model. The lower values of activation energy and change in enthalpy of denaturation for camel milk LP than bovine counterpart suggest that camel milk LP is less stable than bovine milk LP toward thermal denaturation. The higher z-value (6.42 ◦ C) for camel milk LP than bovine counterpart (4.7 ◦ C) indicates that camel milk LP is more sensitive to the extension of thermal treatment time than bovine counterpart. Acknowledgment The financial support of the research affair of the University of Tabriz is highly appreciated. References Anema, S.G., McKenna, A.B., 1996. Reaction kinetics of thermal denaturation of whey proteins in heated reconstituted whole milk. J. Agric. Food Chem. 44, 422–428. AOAC, 1995. Dairy products. Determination of ash content. Standard 945.46. In: Official Methods of Analysis ,. Association of Official Analytical Chemists, Washington, DC, USA. Barrett, N.E., Grandison, A.S., Lewis, M.J., 1999. Contribution of the lactoperoxidase system to the keeping quality of pasteurised milk. J. Dairy Res. 66, 73–80. Bjorck, L., 1992. In: Fox, P.F. (Ed.), Indigenous Enzyme in Milk lactoperoxidase. Advanced Dairy Chemistry. , 1st ed. Springer, London, pp. 323–338, ISBN: 978-1851667611. Boots, J.W., Floris, R., 2006. Lactoperoxidase: from catalytic mechanism to practical applications. Int. Dairy J. 16, 1272–1276. Conner, G.E., Salathe, M., Forteza, R., 2002. Lactoperoxidase and hydrogen peroxide metabolism in the airway. Am. J. Respir. Crit. Care. Med. 66, S57–S61. Fox, P.F., Kelly, A.L., 2006. Indigenous enzymes in milk: overview and historical aspects. Part 1. Int. Dairy J. 16, 500–516. Griffiths, M., 1986. Use of milk enzymes as indices of heat treatment. J. Food Prot. 49, 696–705.
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