Inactivation of Salmonella enteritidis and Salmonella senftenberg in liquid whole egg using generally recognized as safe additives, ionizing radiation, and heat

The effect of combining irradiation and heat (i.e., irradiation followed by heat [IR-H]) on Salmonella Enteritidis and Salmonella Senftenberg inoculated into liquid whole egg (LWE) with added nisin, EDTA, sorbic acid, carvacrol, or combinations of these GRAS (generally recognized as safe) additives was investigated. Synergistic reductions of Salmonella populations were observed when LWE samples containing GRAS additives were treated by gamma radiation (0.3 and 1.0 kGy), heat (57 and 60 degrees C), or IR-H. The presence of additives reduced the initial radiation Dgamma -values (radiation doses required to eliminate 90% of the viable cells) by 1.2- to 1.5-fold, the thermal decimal reduction times (D,-values) by up to 3.5- and 1.8-fold at 57 and 60 degrees C, respectively, and the thermal D,-values after irradiation treatments by up to 3.4- and 1.5-fold at 57 and 60 degrees C, respectively, for both Salmonella serovars. Of all the additives investigated, nisin at a concentration of 100 IU/ml was the most effective at reducing the heat treatment times needed to obtain a 5-log reduction of Salmonella. Thus, while treatments of 21.6 min at 57 degrees C or of 5 min at 60 degrees C should be applied to achieve a 5-log reduction for Salmonella in LWE, only 5.5 min at 57 degrees C or 2.3 min at 60 degrees C after a 0.3-kGy radiation pretreatment was required when nisin at a concentration of 100 IU/ml was used. The synergistic reduction of Salmonella viability by IR-H treatments in the presence of GRAS additives could enable LWE producers to reduce the temperature or processing time of thermal treatments (current standards are 60'C for 3.5 min in the United States) or to increase the level of Salmonella inactivation.     

Site-directed mutagenesis improves the thermostability of a recombinant Picrophilus torridus trehalose synthase and efficiency for the production of trehalose from sweet potato starch

A new recombinant Picrophilus torridus TSase (PTTS) has the catalytic ability for the conversion of maltose to trehalose by intramolecular transglucosylation. For industrial applications, the high thermostability of the enzyme would be the most important property for reducing the microbial contamination and lower the production cost. Therefore, in this study, we substituted ten selected proline residues of PTTS which differ from two well-known thermostable TSases. Interestingly, we found that the N503 mutant type, namely N503P-PTTS, showed about 39% higher relative activity than that of the wild type at 65 °C for 120 min. The trehalose yield of mutant N503P-PTTS was 1.3-fold higher than that of the wild type with sweet potato starch as substrate at 50 °C for 4 h. This suggests that the proline site substitution technology used in this study is useful for altering enzyme properties and catalytic efficiency for possible industrial applications.     

A thermostable histamine oxidase from Arthrobacter crystallopoietes KAIT-B-007

A thermostable histamine oxidase (EC 1.4.3.-) was found in cells of Arthrobacter crystallopoietes KAIT-B-007 isolated from soil. The enzyme was purified about 715-fold over the cell free extracts with a yield of 55% by ammonium sulfate fractionation and various column chromatographies. The purified enzyme was homogeneous on polyacrylamide gel-electrophoresis (native-PAGE). When the enzyme was kept at 65 degrees C and 70 degrees C for 10 min, the activity was fully stable at 65 degrees C and decreased to 9% of the initial level at 70 degrees C. The enzyme was very thermostable. The optimum pH for histamine oxidase activity was found to be at 9.0, and the enzyme was stable over the pH range of 6 to 9. The purified enzyme showed a single protein band on SDS-PAGE and its molecular mass was estimated to be about 81 kDa. The enzyme showed potent activity toward histamine, whereas it was inactive toward putrescine, cadaverine, spermine, and spermidine. Histamine oxidase was inhibited by N,N-diethyldithiocarbamate (DDTC). The inactive enzyme was restored with Cu2+ to 65% of the initial activity, but Cu+ did not enhance the enzyme activity. It is suggested that Cu2+ is essential for expression of histamine oxidase activity. The enzyme was a copper-containing protein having one atom of copper per mol of the enzyme protein as a result of atomic absorption analysis. The N-terminal amino acid sequence of the purified enzyme was different from that of histamine oxidase from Arthrobacter globiformis IFO12137.     

Enhanced inactivation of bacterial lipases and proteinases in whole milk by a modified ultra high temperature treatment

Cultures of Pseudomonas spp. strains P10, P12 and P15 grown in whole milk which contained approximately 1 x 10(8) viable bacteria ml-1 demonstrated near linear increases in the concentration of short-chain free fatty acids and trichloroacetic acid soluble free amino groups at 20 degrees C, following either ultra high temperature (UHT) treatment (140 degrees C for 5 s) or dual heat treatments (140 degrees C followed by either 57, 60 or 65 degrees C). The dual heat treatments reduced the rates of lipolysis and proteolysis compared to the UHT treatment by up to 25-fold. The dual heat treatment utilizing 60 degrees C for 5 min also effectively limited both lipase and proteinase activities in raw milk culture samples which had contained either 6 x 10(6), 5 x 10(7) or 1 x 10(8) viable bacteria ml-1. In this system enzyme activities were reduced by up to 10-fold following dual heat treatment compared to UHT treatment alone.     

Asp238→Asn Creates a Novel Consensus N‐Glycosylation Site in Aspergillus awamori Glucoamylase

A single mutation, Asp238→Asn (D238N), of Aspergillus awamori glucoamylase (GA) was identified that increases extracellular production of the enzyme in Saccharomyces cerevisiae at 37 °C. The mutant was isolated as a suppressor of Gly396→Ser (G396S), a previously isolated temperature‐sensitive mutation that decreases the thermostability and extracellular production of GA expressed in S. cerevisiae. Culture supernatants of the double mutant G396S/D238N contained much more GA than supernatants of G396S at 33.5 and 37 °C but not at 30 °C. Additionally, culture supernatants of the D238N contained 1.5 to 2‐fold more GA than supernatants of wild‐type when grown at 37 °C but not at 30 or 33.5 °C. The D238N mutation creates a consensus N‐glycosylation site in GA. Mass spectrometry showed that the molecular weight of D238N was 2319 Da greater than that of the wild‐type GA and that of D238N/G396S was 3094 Da greater than that of G396S, suggesting the presence of an additional N‐linked glycan at residue 238. No difference in thermostability or activity was observed between the G396S and G396S/D238N mutants or between wild‐type and D238N GAs, and D238N did not affect intracellular GA levels at 30 or 37 °C.     

Thermal resistance of Listeria monocytogenes in inoculated and naturally contaminated raw milk

Raw whole milk inoculated with 10(5) CFU/ml of Listeria monocytogenes was thermally processed at 60-72 degrees C for a minimum holding time of 16.2 s with survival being observed at temperatures up to 67.5 degrees C. In addition, milk naturally contaminated with L. monocytogenes serotype 1 (around 10(4) CFU/ml) was pooled for 2 to 2.5 days and then run through an HTST pasteurizer at temperatures ranging from 60-78 degrees C. Viable L. monocytogenes were detected in the temperature range of 60-66 degrees C. No viable Listeria were detected after treatment at temperatures of 69 degrees C and above in any of five trials. Efficacy of pasteurization and widespread use of processing conditions well above the minimum HTST guidelines ensure the absence of Listeria in pasteurized milk products. However, survival of Listeria at sub-pasteurization temperatures (60-67.5 degrees C) is of concern with regard to heat-treated or raw-milk cheeses.     

Purification and characterization of the extracellular proteinase of Pseudomonas fluorescens NCDO 2085

Pseudomonas fluorescens NCDO 2085 produced a single heat-stable extracellular proteinase in Na caseinate medium at 20 degrees C and pH 7.0. The proteinase was purified to electrophoretic homogeneity using chromatofocusing, gel filtration and ion-exchange chromatography. The purification procedure resulted in a 158-fold increase in the specific activity and a yield of 3.5% of the original activity. The enzyme is a metalloproteinase containing Zn and Ca, with an isoelectric point at 5.40 +/- 0.05 and a mol. wt of 40 200 +/- 2100. It is heat-stable having D-values at 74 and 140 degrees C of 1.6 and 1.0 min respectively; 40 and 70% of the original activity remained after HTST (74 degrees C/17 s) and ultra high temperature (140 degrees C/4 s) treatments respectively. The amino acid composition of the proteinase was determined and compared with those from other Pseudomonas spp.     

Thermal inactivation of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in breaded pork patties

ABSTRACT:  Decimal reduction times ( D -values) and thermal resistance constants ( z -values) for 3 foodborne pathogenic bacteria in formulated ready-to-eat breaded pork patties were determined with thermal inactivation studies. Meat samples, inoculated with Escherichia coli O157:H7, Salmonella , and Listeria monocytogenes cultures or uninoculated controls, were packaged in sterile bags, immersed in circulated water bath, and held at 55, 57.5, 60, 62.5, 65, 67.5, and 70 °C for different durations of time. The D - and z -values were determined by using a linear regression model. Average calculated D -values for E. coli O157:H7, Salmonella , and L . monocytogenes at a temperature range of 55 to 70 °C were 32.11 to 0.08 min, 69.48 to 0.29 min, and 150.46 to 0.43 min, respectively. Calculated z -values for E. coli O157:H7, Salmonella , and L. monocytogenes were 5.4, 6.2, and 5.9 °C, respectively. The results of this study will be useful to food processors to validate thermal lethality of the studied foodborne pathogens in ready-to-eat breaded pork patties.     

Thermal inactivation studies of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef patties

Thermal inactivation studies were used to determine the D- and z-values of Escherichia coli O157:H7, Salmonella, and Listeria monocytogenes in ready-to-eat chicken-fried beef patties. Inoculated meat was packaged in sterile bags, which were immersed in a circulated water bath and held at 55, 57.5, 60, 62.5, 65, 67.5, and 70 degrees C for different lengths of time. D- and z-values were determined with a linear regression model. Average D-values at temperatures 55 to 70 degrees C were 27.62 to 0.04 min for E. coli 0157:H7, 67.68 to 0.22 min for Salmonella, and 81.37 to 0.31 min for L. monocytogenes. The z-values were 5.2 degrees C for E. coli O157:H7, 6.0 degrees C for Salmonella, and 6.1 degrees C for L. monocytogenes. The results of this study can be used by food processors to validate their processes and help eliminate pathogenic bacteria associated with chicken-fried beef products.     

Inactivation of Salmonella serovars in liquid whole egg by heat following irradiation treatments

Salmonella is a frequent contaminant on eggs and is responsible for foodborne illnesses in humans. Ionizing radiation and thermal processing can be used to inactivate Salmonella in liquid whole egg, but when restricted to doses that do not affect egg quality, these technologies are only partially effective in reducing Salmonella populations. In this study, the effect of ionizing radiation in combination with thermal treatment on the survival of Salmonella serovars was investigated. Of the six Salmonella serovars tested, Salmonella Senftenberg was the most resistant to radiation (Dgamma = 0.65 kGy) and heat (D(55 degrees C) = 11.31 min, z = 4.9 degrees C). Irradiation followed by thermal treatment at 55 or 57 degrees C improved the pasteurization process. Radiation doses as low as 0.1 kGy prior to thermal treatments synergistically reduced the D(55 degrees C) and D(57 degrees C) of Salmonella Senftenberg 3.6- and 2.5-fold, respectively. The D(55 degrees C) and D(57 degrees C) of Salmonella Typhimurium were reduced 2- and 1.4-fold and those of Salmonella Enteritidis were reduced 2- and 1.6-fold, respectively. Irradiation prior to thermal treatment would enable the reduction of heat treatment times by 86 and 30% at 55 and 57 degrees C, respectively, and would inactivate 9 log units of Salmonella serovars.     

Purification, characterization, and steady-state kinetics of a meta-cleavage compound hydrolase from Pseudomonas fluorescens IPO1

2-Hydroxy-6-oxo-7-methylocta-2,4-dienoate (6-isopropyl-HODA) hydrolase (CumD), an enzyme of the cumene biodegradation pathway encoded by the cumD gene of Pseudomonas fluorescens IP01, was purified to homogeneity from an overexpressing Escherichia coli strain. SDS-polyacrylamide gel electrophoresis and gel filtration demonstrated that it is a dimeric enzyme with a subunit molecular mass of 32 kDa. The pH optima for activity and stability were 8.0 and 7.0-9.0, respectively. The enzyme exhibited a biphasic Arrhenius plot of catalysis with two characteristic energies of activation with a break point at 20 degrees C. The enzyme has a K(m) of 7.3 microM and a k(cat) of 21 s(-1) for 6-isopropyl-HODA (150 mM phosphate, pH 7.5, 25 degrees C), and its substrate specificity covers larger C6 substituents compared with another monoalkylbenzene hydrolase, TodR Unlike TodF, CumD could slightly hydrolyze 2-hydroxy-6-oxo-6-phenylhexa-2,4-dienoate (6-phenyl-HODA). A mutant enzyme as to a putative active site residue, S103A, had 10(5)-fold lower activity than that of the wild-type enzyme.     

Inactivation-denaturation kinetics of bovine milk alkaline phosphatase during mild heating as determined by using a monoclonal antibody-based immunoassay

A monoclonal antibody based capture immunoassay has been recently developed for the specific quantitation of bovine milk alkaline phosphatase (ALP) without interference by contaminating microbial or fungal ALPs (Geneix et al. 2007). This immunoassay was used to study the kinetics of ALP heat denaturation in bovine milk over a range 50-60 degrees C for 5 to 60 min using a colorimetric quantification of the enzyme activity as a reference test. A denaturation midpoint was obtained at 56 degrees C for a 30 min heating. Thermal inactivation was found to follow first order kinetics and is characterized by z value of 6.7 deg C (D60 degrees C=24.6 min) and 6.8 (D60 degrees C=23.0 min) for respectively immunoassay and colorimetric assay. The high values of enthalpy of activation and the positive values of the entropy of activation and free energy of activation indicate that during denaturation ALP underwent a large change in conformation. The results of the immunoassay were highly correlated (r=0.994) with those obtained by the colorimetric assay. A similar high correlation (r=0.998) was obtained when industrially thermized milks (62-67 degrees C for 20-90 s) were analysed by both techniques. These results indicated that 1) thermally induced epitopic structural changes recognized by the capture monoclonal antibody are concomitant with or occur after the loss of enzymatic activity and 2) quantification of ALP by the specific immunoassay is appropriate for determining mild time/temperature treatment of milk and for the control of milk pasteurization.     

Residual chemicals in silicone rubber products for food contact use]

The residues of additives and other chemicals in 23 kinds of silicone rubber products for food contact use, including nipples, packing and spatulas, were investigated by GC/MS. The packing and spatulas contained 80-480 micrograms/g of BHT, 60-5,830 micrograms/g of di(2-ethylhexyl) phthalate (DEHP), and 60-80 micrograms/g of dibutyl phthalate, while the nipples contained no additives. All of the samples contained 15 to 20 peaks of polydimethylcyclosiloxanes. Dodecamethylcyclohexasiloxane (D6) to tetratriacomethylcycloheptadecasiloxane (D17) were confirmed, and other larger siloxanes up to pentacontamethylcyclopentacosasiloxane (D25) were estimated. A rough estimate of the total cyclosiloxane content was 3,310-14,690 micrograms/g. They might be mainly unreacted materials or by-products, and some of them might be additives. Based on the migration test, no chemicals were released into 20% ethanol at 60 degrees C for 30 min, though DEHP and the polydimethylcyclosiloxanes were released into n-heptane at 25 degrees C for 60 min.     

Thermal inactivation of Bacillus cereus and Clostridium perfringens vegetative cells and spores in pork luncheon roll

The aim of this study was to design a thermal treatment(s) for pork luncheon roll, which would destroy Bacillus cereus and Clostridium perfringens vegetative cells and spores. B. cereus and C. perfringens vegetative and spore cocktails were used to inoculate luncheon meat. Samples were subjected to different temperatures and removal times. The decimal-reduction times (D-values) were calculated by linear regression analysis (D = -1/slope of a plot of log surviving cells versus time). The log(10) of the resulting D-values were plotted against their corresponding temperatures to calculate (-1/slope of the curve) the thermal resistance (z-values) of each cocktail. The D-values for vegetative cells ranged from 1 min (60 degrees C) to 33.2 min (50 degrees C) for B. cereus and from 0.9 min (65 degrees C) to 16.3 min (55 degrees C) for C. perfringens. The D-values for B. cereus spores ranged from 2.0 min (95 degrees C) to 32.1 min (85 degrees C) and from 2.2 min (100 degrees C) to 34.2 min (90 degrees C) for C. perfringens. The z-values were calculated to be 6.6 and 8.5 degrees C for B. cereus vegetative and spores, respectively, and 7.8 and 8.4 degrees C for C. perfringens vegetative cells and spores, respectively. The D-values of B. cereus and C. perfringens suggest that a mild cook of 70 degrees C for 12s and 1.3 min would achieve a 6 log reduction of B. cereus and C. perfringens vegetative cells, respectively. The equivalent reduction of B. cereus and C. perfringens spores would require the pork luncheon meat to be heated for 36 s at 105 and 110 degrees C, respectively. The results of this study provide the thermal inactivation data necessary to design a cooking protocol for pork luncheon roll that would inactivate B. cereus and C. perfringens vegetative cells and spores. The data may also be used in future risk assessment studies.     

Effects of gas flow rate, inlet concentration and temperature on biofiltration of volatile organic compounds in a peat-packed biofilter

The effects of incoming gas concentration, empty bed residence time (EBRT), and column temperature on the removal efficiency of volatile organic compounds (isoprene, dimethyl sulfide, chloroform, benzene, trichloroethylene, toluene, m-xylene, o-xylene and styrene) were studied for 101 d in a biofilter comprising two glass columns (I.D. 5.0 cm x height 62 cm) packed with peat. At an EBRT of 3 min the removal efficiency increased up to 90% 34 d after start up at both 25 degrees C and 45 degrees C when the incoming gas concentration was raised stepwise to 65 g.m(-3). When the incoming gas concentration increased to 83 g.m(-3), the removal efficiency was 93% at 25 degrees C, but dropped to 74% at 45 degrees C. At an incoming gas concentration of 92 g.m(-3) and an EBRT of 1.5 min, the removal efficiencies were 91% and 94% at 25 degrees C and 32 degrees C, respectively. However, at 1 min of EBRT, the removal efficiencies decreased to 68% and 81% at 25 degrees C and 32 degrees C, respectively. The removal rate per unit time and per unit volume of the biofilter was proportional to the incoming gas rate up to 3483 g VOC.m(-3).h(-1). Further increase of the incoming gas rate lowered the removal rate as compared to that predicted by the proportionality. The maximum removal rate was 3977 g.m(-3).h(-1) at 32 degrees C. At an EBRT of 1.5 min, the removal efficiency was highest for isoprene (93%), and lowest for chloroform (84%). Aromatic compounds (benzene, toluene, and xylene) were removed by 93-94%. The cell concentration increased 100-fold from the initial value, and reached 1.12 x 10(8) cells.(g of dry peat)(-1). At 32 degrees C, 67% of the incoming VOC was removed in the first quarter of the column.     

Heat inactivation of Listeria monocytogenes and Salmonella enterica serovar Typhi in a typical bologna matrix during an industrial cooking-cooling cycle

The heat resistance of Salmonella enterica serovar Typhi PF-724 and Listeria monocytogenes 2812 was determined in a commercial bologna batter. The heat inactivation of the two bacterial species was also studied in a semiautomatic pilot smokehouse under cooking conditions that reproduced an industrial bologna process. S. enterica serovar Typhi PF-724 was less heat resistant than L. monocytogenes 2812. The D-values (times required to reduce the population by 1 logarithmic cycle) for S. enterica serovar Typhi PF-724 ranged from 10.11 to 0.04 min for temperatures of 50 to 70 degrees C, while for L. monocytogenes 2812, the D-values were 2.5-, 4.9-, 3.8-, 3.3-, and 2-fold higher at 50, 55, 60, 65, and 70 degrees C, respectively, than for S. enterica serovar Typhi PF-724. However, the z-value (temperature required to reduce log D by 1 logarithmic cycle) for S. enterica serovar Typhi PF-724 (5.72 degrees C) was not significantly different from the z-value for L. monocytogenes 2812 (7.04 degrees C), indicating that a given increase in temperature would have a similar effect on the decimal reduction time for both bacterial species in that meat emulsion. Our data on experimentally inoculated batter also showed that processing bologna at a cooking-cooling cycle commonly used in the industry resulted in a minimum 5-log reduction for both S. enterica serovar Typhi PF-724 and L. monocytogenes 2812.     

Thermal inactivation of Escherichia coli O157:H7 in cow manure compost

Rates of inactivation of a five-strain mixture of green fluorescent protein-labeled Escherichia coli O157:H7 in autoclaved and unautoclaved commercial cow manure compost with a moisture content of ca. 38% were determined at temperatures of 50, 55, 60, 65, and 70 degrees C. Trypticase soy agar with ampicillin was determined to be the best medium for the enumeration of heat-injured and uninjured cells of green fluorescent protein-labeled E. coli O157:H7. The results obtained in this study revealed that in autoclaved compost, E. coli O157:H7 reductions of ca. 4 log CFU/g occurred within 8 h, 3 h, 15 min, 2 min, and < 1 min at 50, 55, 60, 65, and 70 degrees C, respectively. At 65 and 70 degrees C, considerably less time was required to kill the pathogen in unautoclaved compost than in autoclaved compost. Decimal reduction times (D-values) for autoclaved compost at 50, 55, 60, 65, and 70 degrees C were 137, 50.3, 4.1, 1.8, and 0.93 min, respectively, and D-values for unautoclaved compost at 50, 55, and 60 degrees C were 135, 35.4, and 3.9 min, respectively. Considerable tailing was observed for inactivation curves, especially at 60, 65, and 70 degrees C. These results are useful for identifying composting conditions that will reduce the risk of the transmission of E. coli O157:H7 to foods produced in the presence of animal fecal waste.     

Preparation and characterization of immobilized beta-lactamase for destruction of penicillin in milk

beta-Lactamase I (Bacillus cereus) was covalently bound to cyanogen bromide-activated, crosslinked agarose. An initial 5.00 mg of soluble beta-lactamase were used in the immobilization reaction for each preparation, and average coupling yield was 80.5%. Of the enzyme immobilized on the matrix, an average 53.4% remained active. To minimize diffusional effects on immobilized enzyme activity, reaction mixtures were rotated at 250 rpm throughout the study. The shape of the pH activity curve of the immobilized enzyme was identical to that of the soluble enzyme; both exhibited optimum pH around 7.0. In general, only 2-fold differences in Michaelis constant and maximum volume were observed between native and immobilized enzyme when penicillin G was used as the substrate. However, the Michaelis constant of the immobilized enzyme increased up to 22-fold that of the native enzyme when cephaloridine was used as the substrate. The immobilized enzyme exhibited enhanced stability in the acidic pH region in contrast to the native enzyme, which had superior stability in the alkaline pH region. The heat stability of the immobilized enzyme was about twice that of native enzyme after heat treatment at 60 degrees C for 30 min. Approximately a 10% increase of storage stability on immobilization of beta-lactamase was observed when stored at room temperature (23 +/- 1 degree C) for up to 6 d in the absence of antimicrobial agents. Little loss of activity (less than 2%) was noted after repeated use of the immobilized enzyme up to seven times each in 10.0 ml of skim milk containing .5 U/ml penicillin G.(ABSTRACT TRUNCATED AT 250 WORDS)     

突变酶Y93S对嗜热地衣芽孢杆菌SR01葡聚糖酶热稳定性的影响

为提高嗜热地衣芽孢杆菌SR01葡聚糖酶的热稳定性,对其相关的氨基酸残基进行定点突变改造。通过对其结构的分析,构建了Y93S突变体,利用分子动力学模拟分析评估后,发现Y93S突变体可能具有较高的耐热能力,利用定点突变技术构建Y93S表达载体并分析温度对表达产物酶活的影响。试验结果表明,突变酶Y93S的最适温度由野生型酶的55 ℃提高至70 ℃;在90 ℃条件下,突变酶Y93S较野生型酶的半衰期由60 min提高到120 min以上;pH及pH稳定性较野生型变化不明显。突变酶Y93S极大的提高了野生型酶的热稳定性,具有潜在的工业应用价值,同时为葡聚糖酶的耐热机理提供有力依据。     

Inactivation kinetics of alkaline phosphatase and lactoperoxidase, and denaturation kinetics of beta-lactoglobulin in raw milk under isothermal and dynamic temperature conditions

A detailed kinetic study of alkaline phosphatase, lactoperoxidase and beta-lactoglobulin was carried out in the context of identifying intrinsic time-temperature indicators for controlling the heat processing of milk. The heat inactivation or denaturation of alkaline phosphatase, lactoperoxidase and beta-lactoglobulin under isothermal conditions was found to follow first order kinetics. Experimental results were analysed using both a two step linear regression and a one step non-linear regression method. Results obtained using the two statistical techniques were comparable, but the 95% confidence interval for the predicted values was smaller when the one step non-linear regression method was used, indicating its superiority for estimating kinetic parameters. Thermal inactivation of alkaline phosphatase and lactoperoxidase was characterized by z values of 5.3 deg C (D60 degrees C = 24.6 min) and 4.3 deg C (D71 degrees C = 38.6 min) respectively. For the denaturation of beta-lactoglobulin we found z values of 7.9 deg C (D7.5 degrees C = 49.9 min) in the temperature range 70-80 degrees C and 24.2 deg C (D85 degrees C = 3.53 min) in the range 83-95 degrees C. Dref and z were evaluated under dynamic temperature conditions. To estimate the statistical accuracy of the parameters, 90% joint confidence regions were constructed.