THERMAL INACTIVATION KINETICS OF ALKALINE PHOSPHATASE IN BUFFER AND MILK

A detailed kinetic study on the thermal inactivation of alkaline phosphatase (ALP) added into buffer and pasteurized milk and for ALP naturally present in raw cow's milk has been performed. Kinetic parameters (rate constant, k; decimal reduction time, D; activation energy, E_a; and z value) were evaluated based on the first‐order rate model at 50–80C. The temperature sensitivity of the kinetic parameters was evaluated considering the Arrhenius‐type E_a model. All kinetic behaviors were well described by the first‐order model (r² > 0.91). The D values increased with increasing temperature. Higher temperatures resulted in higher rates of enzyme inactivation as indicated by lower D values and higher k values. There are significant differences (P < 0.01) among the D values for ALP in buffer and milk at treated temperatures. The rate of enzyme inactivation was much more rapid in buffer than in pasteurized milk. The evaluated E_a values for ALP added into the buffer and pasteurized milk, and for ALP naturally present in raw milk were 97.2, 149.9 and 207.8 kJ/mol, respectively. The inactivation kinetics of ALP during heat treatment was found to be dependent on the composition of the medium, and the time and temperature of the heat treatment.     

Kinetics of thermal inactivation of alkaline phosphatase in bovine and caprine milk and buffer

The thermal inactivation of alkaline phosphatase (ALP) in raw bovine and caprine milk was investigated in the temperature range 54 to 69 °C. To assess the stabilizing effect of milk compounds on ALP, inactivation experiments were also carried out in 0.1 m potassium phosphate buffer, pH 6.6. Each set of inactivation experiments was fitted simultaneously using kinetic models that were based on either one-step or two-step mechanisms. The parameters of the Arrhenius equation showed that the stabilization effect of milk compounds on ALP had an entropic character. They also indicated a different structure of bovine and caprine milk ALPs, which was reflected by a higher stability of the bovine milk enzyme.     

Comparison of the thermal inactivation of Bacillus subtilis spores in foods using the modified Weibull and Bigelow equations

The association of a modified Weibull model and Bigelow model was applied to the thermal inactivation of Bacillus subtilis spores heated in phosphate buffer, milk, kayu (a Japanese style rice porridge) and soy sauce as well. The inactivation kinetics presented a light downward concave profile, the acidic pH increased the efficiency of the heat treatment but on the opposite, lesser the water activity, weaker was the efficiency. The heat treatment kinetics observed in milk, soy sauce and kayu were greatly different from each other, while no large difference between sterilized whole milk, UHT whole milk, sterilized skim milk and UHT skim milk, were observed. The model established in buffer system allowed heat treatment in milk products to be simulated although it could not be employed to describe the inactivation of B. subtilis spores in soy sauce and kayu. For these two latter products, the food itself had to be introduced in the model as a parameter. Finally, this approach combining primary model (to simulate inactivation kinetics) and secondary model (to introduce temperature, pH, a_w and food matrix effect) seemed available for food application, nevertheless validations of results such as challenge-tests, must be performed before it is put to routine use.     

Kinetics of alkaline phosphatase and lactoperoxidase inactivation,and of beta-lactoglobulin denaturation in milk with different fat content

In the context of identifying intrinsic time temperature integrators (TTIs) for evaluating heat processing of milk, the extent to which milk fat content has an effect on alkaline phosphatase (ALP) and lactoperoxidase (Lpo) inactivation and on beta-lactoglobulin (beta-Ig) denaturation kinetics was studied. Inactivation and denaturation kinetics were analysed in whole, semi-skimmed and skimmed milk. In previous experiments (isothermal and non-isothermal heating conditions), heat inactivation of ALP and Lpo and heat denaturation of beta-Ig were found to follow first order kinetics. This allowed experimental design to be simplified. Data analysis was performed by non-linear regression and results were evaluated by construction of joint confidence regions. The possible effect of milk fat was illustrated by temperature time tolerance (TTT-) diagrams. Although initial ALP activity was lower in skimmed milk compared with semi-skimmed or whole milk, kinetics were comparable and fat content did not seem to substantially affect the ALP test result for pasteurized milk. Unlike ALP, Lpo inactivation and beta-Ig denaturation kinetics differed significantly in milk with different fat content. Differences between Lpo inactivation kinetics were relatively small and acceptable in the context of quantifying the process impact. Denaturation of beta-Ig, on the other hand, seemed to be enhanced at higher milk fat content (> 72 degrees C).     

Model for the differentiation of temperature and electric field effects during thermal assisted PEF processing

A model was developed that enables the quantification of thermal and electric field effects during the pulsed electric field (PEF) inactivation of alkaline phosphatase (ALP) and lactoperoxidase (LPO) in milk as well as Escherichia coli in apple juice.     

Effect of high pressure carbon dioxide on alkaline phosphatase activity and quality characteristics of raw bovine milk

Novel food processing techniques would always be pursuits of researchers and food industry to avoid unfavorable thermal effects, especially in dairy and milk processing. In this study, effects of high pressure carbon dioxide (HPCD) on the activity of alkaline phosphatase (ALP) and main quality indices of raw bovine milk at 20 MPa using a batch system were investigated. A complete inactivation of ALP activity as exposure to HPCD treatment at 50 °C and 20 MPa for 50 min was observed. The protein and lactose content of HPCD-treated bovine milk hold steady, while pH value and total solids content decreased, turbidity and average particle size increased significantly (p < 0.05). Although a significant decrease of viscosity (p < 0.05) was observed, the Newtonian flow behavior of raw bovine milk did not alter. More obvious change of quality characteristics of raw bovine milk were observed as subjected to HPCD treatment at higher temperature or treated for longer period. Therefore, a compromise between controlling endogenous enzymes (and/or spoilage and pathogenic microorganisms) and retention of original/fresh like quality of foods should be introduced due to the nature of HPCD processing. It's suggested to keep raw bovine milk with low ALP activity and great quality treated with a batch HPCD apparatus at 20 MPa and 50 °C for 20 min.     

Effects of high pressure homogenisation of raw bovine milk on alkaline phosphatase and microbial inactivation. A comparison with continuous short-time thermal treatments

Raw whole milk of high microbial quality (58 degrees C), but markedly decreased above 200 MPa when Tin=24 degrees C (T2>60 degrees C). In contrast to inactivation induced by continuous short-time thermal treatments, ALP inactivation induced by HP homogenisation was clearly due to mechanical forces (shear, cavitation and/or impact) in the HP valve and not to the short (<1 s) residence time at temperature T2 in the same valve. Inactivation of the three exogenous microorganisms led to similar conclusions. Homogenisation at 250 MPa or 300 MPa (Tin=24 degrees C) induced a 2-3 log cycle reduction of the total endogenous milk flora and a 1.5-1.8 log cycle reduction of inoculated List. innocua. Higher reduction ratios (2-4 log cycles) were obtained for the two other microorganisms. The highest levels of ALP inactivation corresponded to the highest extents of microbial reduction. Running the milk twice or three times through the homogeniser (recycling), keeping temperature T1 approximately 29 degrees C and pressure=200 MPa, increased homogenisation efficiency.     

Protease stability in bovine milk under combined thermal-high hydrostatic pressure treatment

At atmospheric pressure, inactivation of protease from B. subtilis in raw milk and pasteurized milk (with and without homogenization) was studied in a temperature range of 50–80 °C. Thermal inactivation followed a first order kinetic model in the temperature range tested. Temperature dependence of the first order inactivation rate constants could be accurately described by the Arrhenius equation, allowing Ea values to be calculated. Different milk systems did not show differences in enzyme thermo stability.The combined thermal (40, 50 and 60 °C)-high hydrostatic pressure (300–450 and 600 MPa) effect on protease activity was studied. Protease was very resistant to high pressures. Pressure stability was higher in raw milk than in pasteurized milk; homogenization appeared to have a protective effect on the enzyme. The separate effects of pressure and temperature on enzyme inactivation were related to changes in L?-values and milk appearance.A very pronounced antagonistic effect between high temperature and pressure was observed, i.e. at temperatures where thermal inactivation at atmospheric pressure occurs rapidly, application of pressure up to 600 MPa exerted a protective effect.Industrial relevanceHigh hydrostatic pressure (HHP) is an emerging technology that has been successfully applied as a minimal process for a variety of foods. Although the potential for the use of HHP treatment as an alternative method to heat treatment of milk was proposed almost a century ago, the suitability of this innovative technology to extend the shelf-life of milk hinges not only on its ability to inactivate pathogenic vegetative microorganisms but also on its effectiveness to inactivate indigenous and endogenous enzymes. This work examines the combined effects of temperature, pressure and homogenization on the protease (exogenous enzyme from B. subtilis) activity in milk. Inactivation of protease could extend the shelf life of milk.     

Development of a monoclonal antibody-based immunoassay for specific quantification of bovine milk alkaline phosphatase

The detection of alkaline phosphatase (ALP) activity is used as a legal test to determine whether milk has been adequately pasteurized or recontaminated with raw milk. However, a wide variety of microorganisms produce both heat labile and heat stable ALPs which cannot be differentiated from the milk ALP by current enzymatic methods. Monoclonal antibodies specific of the bovine milk ALP were obtained in mice from a raw bovine milk ALP preparation. Coated in microtitre plates, these antibodies specifically capture the bovine milk ALP from dairy products. After washing, the enzymatic activity of the captured ALP is revealed by adding p-nitrophenyl-phosphate as a substrate. This simple immunoassay does not react with ALPs of intestinal or bacterial origin and, once optimized, was found to be the first immunoassay suitable to detect raw milk in boiled milk down to a 0.02% dilution. Moreover, in contrast with competitive indirect ELISA formats, the capture immunoassay does not require purified ALP.     

Inactivation of Cronobacter sakazakii by manothermosonication in buffer and milk

The inactivation of Cronobacter sakazakii by heat and ultrasound treatments under pressure at different temperatures [manosonication (MS) and manothermosonication (MTS)] was studied in citrate-phosphate pH 7.0 buffer and rehydrated powdered milk. The inactivation rate was an exponential function of the treatment time for MS/MTS treatments (35−68 °C; 200 kPa of pressure; 117 μm of amplitude of ultrasonic waves) in both media, and for thermal treatments alone when buffer was used as heating media. Survival curves of C. sakazakii during heating in milk had a concave downward profile. Up to 50 °C, the lethality of ultrasound under pressure treatments was independent of the treatment temperature in both media. At temperatures greater than 64 °C in buffer and 68 °C in milk, the inactivating effect of MTS was equivalent to that of the thermal treatments alone at the same temperature. Between 50 and 64 ºC for buffer and 50 and 68 °C for milk, the lethality of MTS was the result of a synergistic effect, where the total lethal effect was higher than the lethal effect of heat added to that of ultrasound under pressure at room temperature. The maximum synergism was found at 60 °C in buffer and at 56 °C in milk. A heat treatment of 12 min (60 °C) or 4 min of an ultrasound under pressure at room temperature treatment (35 °C; 200 kPa; 117 μm) would be necessary to guarantee the death of 99.99% of C. sakazakii cells suspended in milk. The same level of C. sakazakii inactivation can be achieved with 1.8 min of a MTS treatment (60 °C; 200 kPa; 117 μm). Damaged cells were detected after heat treatments and after ultrasound under pressure treatments at lethal but not at non-lethal temperatures.     

THERMAL INACTIVATION of PECTINESTERASE IN PAPAYA PULP (pH 3.8)

In order to establish the thermal process required by acified papaya pulp (pH 3.8) var "formosa", a study was carried out on the kinetics of thermal inactivation of the heat resistant enzymes present in the pulp. Since no peroxidase activity was detected, the study was focused on pectinesterase.
The heat inactivation curves at 75, 77 and 80C showed a change in slope indicating the presence of two different portions of the enzyme, one heat labile and the other heat resistant. the decimal reduction times (D value) of pectinesterase were 0.8, 0.3 and 0.2 min for the heat labile portion and for the heat resistant portion 16.7, 7.2 and 3.7 min, respectively.
The temperature-dependency factor for the heat labile portion was 9.2C and 7.8C for the thermostable portion, while the activation energies were 258.3 and 304.4 Kj/mol. These values were within the range of 167.5–418.7 Kj/mol reported in the literature for the thermal inactivation of enzymes. Thermal destruction studies with Clostridium pasteurianum, conducted at the same temperatures used for the inactivation of the enzyme, showed that the heat resistant portion of pectinesterase presented greater thermal resistance and should be used as target for the establishment of the required process.     

Alkaline phosphatase and microbial inactivation by pulsed electric field in bovine milk

The effects of pulsed electric field (PEF) treatments at field intensities of 25–37 kV cm^− 1 and final PEF treatment temperatures of 15 °C and 60 °C on the inactivation of alkaline phosphatase (ALP), Total Plate Count (TPC), Pseudomonas and Enterobacteriaceae counts were determined in raw skim milk. At 15 °C, PEF treatments of 28 to 37 kV cm^− 1 resulted in 24–42% inactivation in ALP activity and < 1 log reduction in TPC and Pseudomonas count, while the Enterobacteriaceae count was reduced by at least 2.1 log units to below the detection limit of 1 CFU mL^− 1. PEF treatments of 25 to 35 kV cm^− 1 at 60 °C resulted in 29–67% inactivation in ALP activity and up to 2.4 log reduction in TPC, while the Pseudomonas and Enterobacteriaceae counts were reduced by at least 5.9 and 2.1 logs, respectively, to below the detection limit of 1 CFU mL^− 1. Kinetic studies suggested that the effect of field intensity on ALP inactivation at the final PEF treatment temperature of 60 °C was more than twice that at 15 °C. A combined effect was observed between the field intensity and temperature in the inactivation of both ALP enzyme and the natural microbial flora in raw skim milk.Industrial relevanceMilk has been pasteurised to ensure its safety and extend its shelf life. However, the need for retaining heat-sensitive nutrient and sensory properties of milk has resulted in interest in the application of alternative technologies. The results of the current study suggest that PEF as a non-thermal process can be employed for the treatment of raw milk in mild temperature to achieve adequate safety and shelf life while preserving the heat-sensitive enzymes, nutrients and bioactive compounds.     

Kinetic and thermodynamic study of thermal inactivation of the antimicrobial peptide P34 in milk

The knowledge on thermal inactivation of biopreservatives in a food matrix is essential to allow their proper utilisation in food industry, enabling the reduction of heating times and optimisation of heating temperatures. In this work, thermal inactivation of the antimicrobial peptide P34 in skimmed and fat milk was kinetically investigated within the temperature range of 90–120 °C. The inactivation kinetic follows a first-order reaction with k-values between 0.071 and 0.007 min⁻¹ in skimmed milk, and 0.1346 and 0.0119 min⁻¹ in fat milk. At high temperatures, peptide P34 was less resistant in fat milk, with a significant decrease in residual activity as compared with skimmed milk. At temperatures below 110 °C, the fat globules seem to have protective effect to the peptide P34. Results suggest that peptide P34 is heat stable in milk with activation energy of 90 kJ mol⁻¹ in skimmed milk and 136 kJ mol⁻¹ in fat milk.     

Investigate the efficacy of UV pretreatment on thermal inactivation of Bacillus subtilis spores in different types of milk

Shortwave ultraviolet (UVC) radiation is commonly used for sterilization of drinking water. However, its low transmittance within opaque liquids limits its use for milk and other liquid food products. The objective of this study was to assess the efficacy of UV pretreatment on thermal inactivation of B. subtilis spores in skim cow milk, whole cow milk and whole sheep milk. In this work, UV treatment was applied by using a coiled tube UV unit with a perfluoroalkoxy tube around a quartz sleeve containing a 254-nm UV lamp. It was observed that UV pretreatment (D _Act 2.37 ± 0.126 J/ml) combined with thermal treatment at 110 °C for 30 s resulted in a reduction of approximately 6 log CFU/mL in bovine skim milk, 2.90 log CFU/ml in whole bovine milk and 1.1 log CFU/ml in ovine milk. The results showed that UV in combination with heat can possibly be an alternative to sterilization of skim milk at lower temperatures compared to ultra high temperature (UHT) treatment (135 °C, 3 s).     

Thermal inactivation of lactoperoxidase in goat,sheep and bovine milk – A comparative kinetic and thermodynamic study

Using isothermal heating, inactivation of lactoperoxidase (LPO) in goat, sheep and cow milk was studied in the temperature range of 70–77 °C. Kinetic and thermodynamics studies were carried out at different time–temperature 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 first-order kinetics. D- and k-values decreased and increased, respectively with increasing temperature, indicating a more rapid LPO inactivation at higher temperatures. The influence of temperature on the inactivation rate constant was quantified 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 mol⁻¹ in goat milk, 560.87 ± 28.18 kJ mol⁻¹ in sheep milk and 641.56 ± 13.12 kJ mol⁻¹ in cow milk, respectively.     

Influence of the suspension media on the thermal treatment of mesophilic lactococcal bacteriophages

The thermal resistances of the Lactococcus lactis phages P008 (a heat-sensitive wide-spread phage) and P680 (a heat-resistant phage) suspended in milk and in solutions supplemented with milk components were studied to elucidate the protective role of milk on phage inactivation. For both phages a decrease of inactivation was observed in casein solution. Furthermore, the inactivation kinetics of the phages in whey, in whey cream (3.5%, 20%, 30% fat) and in whey protein concentrate (0.7%, 5%, 10% protein) were tested. The inactivation experiments in whey cream and in whey protein concentrate having different concentrations of fat and protein revealed that fat had no influence on the inactivation, while, in contrast, the presence of protein had a protective effect.     

Seasonal and regional occurrence of heat-resistant spore-forming bacteria in the course of ultra-high temperature milk production in Tunisia

Spore-forming bacteria, principally Bacillus species, are important contaminants of milk. Because of their high heat resistance, Bacillus species spores are capable of surviving the heat treatment process of milk and lead to spoilage of the final product. To determine the factors influencing the contamination of milk, spore-forming bacteria occurrence throughout the UHT milk production line during winter, spring, and summer was studied. The obtained results confirm that the total viable rate decreases rapidly throughout the production line of UHT milk showing the efficiency of thermal treatments used. However, the persistent high rate of spore-forming bacteria indicates their high heat resistance, especially in spring and summer. In addition, a significant variation of the quality of raw milk according to the location of the collecting centers was revealed. The molecular identification showed a high degree of diversity of heat-resistant Bacillus species, which are isolated from different milk samples. The distribution of Bacillus species in raw milk, stored milk, bactofuged milk, pasteurized milk, and UHT milk were 28, 10, 16, 13, and 33%, respectively. Six Bacillus spp. including Bacillus licheniformis (52.38%), Bacillus pumilus (9.52%), Bacillus sp. (4.76%), Bacillus sporothermodurans (4.76%), Terribacillus aidingensis (4.76%), and Paenibacillus sp. (4.76%) were identified in different milk samples.     

Influence of seasonal variation on kinetics of time temperature integrators for thermally processed milk

In the context of identifying intrinsic time temperature integrators (TTIs) for evaluating thermal processing of milk, the possible influence of seasonal variation in milk composition on the applicability of alkaline phosphatase (ALP), hydroxymethylfurfural (HMF), lactulose and furosine for process impact assessment was studied. Hereto inactivation and formation kinetics of these indicators were analysed in milk samples collected over a one year period. Based on previous research (isothermal and non-isothermal heating conditions) it was assumed that thermal inactivation of ALP followed first order kinetics, and formation of HMF, lactulose and furosine could be described by pseudo-zero order kinetics. This allowed the use of a simplified experimental design to obtain the kinetic parameters. Based on 90% joint confidence regions, kinetic parameter values differed significantly between different batches of milk for all four indicators. Additionally, variable ALP activities and HMF concentrations were observed in the raw milk samples. Nevertheless, the observed differences did not seem to restrict the applicability of the selected TTIs as illustrated by the construction of temperature time tolerance (TTT-) diagrams. Through these diagrams, use of the TTIs implied an uncertainty of approximately 2 degrees C between equivalent processes.     

Effect of sweet solutes and potassium sorbate on the thermal inactivation of Z. bailii in model aqueous systems

The effect of solute type (glucose and polyols) and potassium sorbate (KS) on Zygosaccharomyces bailii thermal inactivation was evaluated in acidified aqueous model systems. Thermal inactivation curves obtained were fitted with Baranyi equation and parameters of this model were estimated and used to establish the effect of water activity (a_w), solute added and KS on Z. Bailii survival. Results obtained showed that addition of KS (0.025%, w/w) in general promoted an increase in the rate of heat inactivation. The use of polyols to depress a_w to 0.985 in the absence of KS produced no effect on rate of heat inactivation. On the contrary, the use of glucose (10%, w/w) enhanced it. This trend was also observed when a_w was depressed to 0.971 by glucose or xylitol or glucose-polyols. Moreover, a_w depression in systems containing KS produced different effects on thermal inactivation rate depending on system composition. A synergic effect on the rate of inactivation of Z. bailii was observed by the combined use of KS and sorbitol, xylitol or glucose to depress a_W to 0.985–0.988. This behavior might allow to decrease the severity of the thermal treatment with no detrimental effect on sterility.     

Evaluation of pulsed electric field and minimal heat treatments for inactivation of pseudomonads and enhancement of milk shelf-life

Pulsed Electric Field (PEF) treatment of milk provides the opportunity to increase the shelf-life of fresh milk for distribution to distant markets. PEF treatments were evaluated in sterile (UHT) milk to determine the inactivation of added spoilage Pseudomonas isolates and the subsequent gains in microbial shelf-life (time taken to reach 10⁷ CFU mL^− 1). Little inactivation of Pseudomonas was achieved at 15 or 40 °C compared with 50 or 55 °C. The greatest inactivation (> 5 logs) was achieved by processing at 55 °C with 31 kV cm^− 1 (139.4 kJ L^− 1). Heat treatment at the application temperature without PEF treatment caused minimal inactivation of Pseudomonas (only 0.2 logs), demonstrating that the inactivation of the Pseudomonas was due to the PEF treatment rather than the heat applied to the milk. At added Pseudomonas levels of 10³ and 10⁵ CFU mL^− 1, the microbial shelf-life of PEF-treated milk was extended by at least 8 days at 4 °C compared with untreated milk. The total microbial shelf-life of the PEF-treated milk was 13 and 11 days for inoculation levels of 10³ and 10⁵ CFU mL^− 1 respectively. The results indicate that PEF treatment is useful for the reduction of pseudomonads, the major spoilage bacteria of milk.Industrial relevancePseudomonads are the major psychrotrophic spoilage microflora of refrigerated, stored HTST pasteurised milk. Long-life (UHT) products are an important component of milk sales in South-East Asia, but in recent years there has been an increasing demand for less processed milk products with extended shelf-life. The recent practice of shipping fresh bulk milk from Australia to South-East Asian countries has necessitated additional heat treatment prior to export and on arrival, to achieve the required shelf-life. Pulsed electric field treatment of HTST milk, applied alone or in combination with mild heat under optimised conditions, offers the opportunity of shelf-life extension, while limiting the reduction in quality attributes of milk associated with more severe additional heat treatments.