Anhydrous goat's milk fat: thermal and structural behavior. 1. Crystalline forms obtained by slow cooling

The thermal and structural behaviors of anhydrous goat's milk fat (AGMF) have been determined as a function of temperature using a powerful technique allowing simultaneous time-resolved synchrotron X-ray diffraction as a function of temperature (XRDT) and high-sensivity differential scanning calorimetry (DSC) measurements from the same sample. This first paper, aiming at the characterization of the physical properties of AGMF, we examine crystalline organizations made by triacylglycerols (TG) upon slow cooling at /dT/dt/ = 0.1 degrees C/min from 45 to -20 degrees C in order to approach system equilibrium. Three overlapped exotherms were observed by DSC upon cooling, whereas four endotherms were found on the subsequent heating at 1 degrees C/min. XRDT evidenced that AGMF crystallizes under four different lamellar structures, two with double-chain length packings at 41.5 and 38.2 angstroms and two with triple-chain lengths of 72 and 64.7 angstroms stacking. Simultaneous wide-angle XRDT has shown that initial nucleation mainly occurs in a packing of beta' type from approximately 26 degrees C, although some transient presence of alpha was detected. The absence of polymorphic transition, on heating, until final melting (approximately 40 degrees C) demonstrated the relative stability of the structures formed.     

Crystallization properties and polymorphism of triacylglycerols in goat's milk fat globules

The sensorial, functional, and nutritional properties of goat dairy products result from the specific fatty acid composition of goat's milk fat. However, information on the physical and thermal properties of goat's milk fat is scarce. In this study, crystallization of triacylglycerols (TG) in goat's milk fat globules was investigated using polarized light microscopy and the coupling of time-resolved synchrotron radiation X-ray diffraction (XRD) and high-sensitivity differential scanning calorimetry (DSC). The molecular organization of the solid fat phase was characterized for cooling rates between 3 and 0.1 degrees C/min. Quenching of goat's milk fat globules from 50 to -8 degrees C and 4 degrees C was also examined to identify the most unstable polymorphic forms of TG. Then, the melting behavior of fat crystals was studied on subsequent heating at 1 degrees C/min. Triple chain length (3L: 68.6-70 A) and double chain length (2L: 37-45.4 A) structures were characterized and 5 polymorphic forms, alpha, sub-alpha, beta' 1, beta' 2, and beta were identified. Polymorphic transitions were observed within goat's milk fat globules as a function of time after quenching and as a function of temperature on heating. From a technological point of view, this work will contribute to a better understanding of the rheological properties as well as on the flavor evolutions of goat's milk-based products.     

Syneresis rate of cow's, ewe's, and goat's curd. Effect of thermal treatment and ultrafiltration.

The influences of ultrafiltration (UF), the intensity of heat treatment (70 degrees C for 5 or 30 min) applied to milk prior to UF processing, and species on the syneresis rate of curd from cow's, ewe's, and goat's milk were analyzed. The influence of the species was significant (P < 0.05) in the syneresis rate. The syneresis rate depended significantly on the intensity of the heat treatment applied before UF and the degree of concentration; these two parameters showed significant (P < 0.05) differences in the syneresis rate depending on the species studied. These results could indicate a possible difference in the structure of the curd formed. The differences in the curd structure could be due to the different relative composition of the retentates of the different species as well as the possible effect of the UF process on the physicochemical composition of milk.     

Rheological and proteolytic properties of Monterey Jack goat's milk cheese during aging

To enhance the understanding of the quality traits of goat's milk cheeses, rheological and proteolytic properties of Monterey Jack goat's milk cheese were evaluated during 26 weeks of 4 degrees C storage. As expected with aging, beta-casein levels decreased with concomitant increases in peptide levels and were correlated with changes in rheological properties of the cheese. Hydrolysis of the protein matrix resulted in more flexible (increased viscoelastic properties) and softer (decreased hardness, shear stress, and shear rigidity) cheeses. During the first 4-8 weeks of storage, cheese texture changed significantly (P < 0.05) and then stabilized. Characterization of rheological and proteolytic properties of the goat's milk semihard cheese during aging provided insight into the changes occurring in the protein matrix, the relationship to structure, and a shift in cheese quality.     

Phase transitions, solubility, and crystallization kinetics of phytosterols and phytosterol-oil blends

The thermal properties, solubility characteristics, and crystallization kinetics of four commercial phytosterol preparations (soy and wood sterols and stanols) and their blends with corn oil were examined. Differential scanning calorimetry (DSC) revealed narrow melting peaks between 138 and 145 degrees C for all phytosterol samples, reversible on rescan. Broader and less symmetrical melting transitions at lower temperatures with increasing oil content were observed for two samples of phytosterol-oil admixtures. The estimated, from the solubility law, deltaH values (34.7 and 70.7 mJ/mg for wood sterols and stanols, respectively), were similar to the DSC experimental data. Fatty acid esters of soy stanols differing in the chain length of the acyl groups (C2-C12) exhibited suppression of the melting point and increase of the fusion enthalpy with increasing chain length of the acyl group; the propionate ester exhibited the highest melting point (Tm: 151 degrees C) among all stanol-fatty acid esters. Solubility of phytosterols in corn oil was low (2-3% w/w at 25 degrees C) and increased slightly with a temperature rise. Plant sterols appeared more soluble than stanols with higher critical concentrations at saturation. The induction time for recrystallization of sterol-oil liquid blends, as determined by spectrophotometry, depended on the supersaturation ratio. The calculated interfacial free energies between crystalline sediments and oil were smaller for sterol samples (3.80 and 3.85 mJ/m2) than stanol mixtures (5.95 and 6.07 mJ/m2), in accord with the higher solubility of the sterol crystals in corn oil. The XRD patterns and light microscopy revealed some differences in the characteristics among the native and recrystallized in oil phytosterol preparations.     

Polymorphic transformation in mixtures of high- and low-melting fractions of milk fat

The kinetics of crystallization of high-melting fraction (HMF) and a mixture of 40% HMF and 60% low-melting fraction (LMF) of milk fat were studied at 5 degrees C by time-resolved in-situ synchrotron X-ray diffraction. HMF crystallized in the alpha polymorph, had a longer lifetime than the ones previously reported in pure milk fat, and was almost completely solid. The HMF/LMF mixture crystallized initially in the alpha form and transformed into the beta' polymorph, with a solid fat content much lower than that of HMF. The polymorphic change was therefore attributed to a delayed sudden formation of beta' mixed crystals from the uncrystallized melt. These findings are important for the food industry and as fundamental knowledge to improve our understanding of the origin of the macroscopic physical properties of solid milk fat fractions used in many manufacturing processes.     

Multiscale characterization of the organization of triglycerides and phospholipids in emmental cheese: from the microscopic to the molecular level

The chemical composition and properties of lipids, both triglycerides and phospholipids, play a major role in the functional and nutritional properties of food products. In this study, the suprastructure of fat, solid fat content, and crystallographic properties of triglycerides were investigated in hard-type cheeses from the microscopic scale to the molecular level using the combination of relevant techniques. Two industrial cheeses with different oiling off properties were compared with experimental cheeses manufactured in the laboratory. Microstructural analysis performed using confocal laser scanning microscopy showed that milk processing led to the disruption of fat globules with the formation of nonglobular fat. For a similar fatty acid composition, oiling off was mainly related to the fat in dry matter content and to the suprastructure of fat in cheese. An exogenous fluorescent phospholipid permitted the localization of milk phospholipids in the cheese matrix, which mainly remain around fat inclusions after disruption of the milk fat globule membrane, and to show heterogeneities. We also showed using differential scanning calorimetry that the suprastructure of fat did not affect the solid fat content in cheese at 4 degrees C: 71.6 +/- 4.9%. The organization of triglyceride molecules in fat crystals, elucidated at a molecular level using X-ray diffraction, corresponded to the coexistence of 2 lamellar structures (2L 40.5 angstroms and 3L 54.6 angstroms) with four polymorphic forms: alpha, two beta' and beta. A schematic representation of the multiscale organization of triglycerides and phospholipids in cheese is proposed.     

Ultrasonic spectroscopy and differential scanning calorimetry of liposomal-encapsulated nisin

The thermal stability of phosphatidylcholine (PC) liposomes (colloidal dispersions of bilayer-forming polar lipids in aqueous solvents) in the presence and absence of the antimicrobial polypeptide nisin was evaluated using differential scanning calorimetry (DSC) and low-intensity ultrasonic spectroscopy (US). PC liposome mixtures with varying acyl chain lengths (C16:0 and C18:0) were formed in buffer with or without entrapped nisin. Gel-to-liquid crystalline phase transition temperatures (T(M)) of liposomes determined from DSC thermograms were in excellent agreement with those determined by ultrasonic velocity and attenuation coefficient measurements recorded at 5 MHz. The dipalmitoylphosphatidylcholine (DPPC) T(M) measured by DSC was approximately 41.3 and approximately 40.7 degrees C when measured by ultrasonic spectroscopy. The T(M) of distearoylphosphatidylcholine (DSPC) and DPPC/DSPC 1:1 liposomes was 54.3 and 54.9 degrees C and approximately 44.8 and approximately 47.3 degrees C when measured by DSC and US, respectively. The thermotropic stability generally increased upon addition of nisin. Analysis of the stepwise decrease in ultrasonic velocity with temperature indicated an increased compressibility corresponding to a loss of structure upon heating.     

Can the thermodynamic melting temperature of sucrose, glucose, and fructose be measured using rapid-scanning differential scanning calorimetry (DSC)?

The loss of crystalline structure in sucrose, glucose, and fructose has been shown to be due to the kinetic process of thermal decomposition (termed apparent melting), rather than thermodynamic melting. The purpose of this research was to investigate whether or not it is possible to scan quickly enough to suppress the kinetic process of thermal decomposition and reach the thermodynamic melting temperature of these sugars using a new rapid-scanning DSC. Indium, a thermodynamic melting material, and sucrose, glucose, and fructose were analyzed at three heating rates from 1 to 25 °C/min using standard DSC and at seven heating rates from 50 to 2000 °C/min using rapid-scanning DSC. Thermodynamic melting was achieved when the onset temperature (T(m onset)) of the endothermic peak leveled off to a constant value independent of heating rate. The T(m onset) for indium was constant (156.74 ± 0.42 °C) at all heating rates. In the case of fructose, the T(m onset) increased considerably until a heating rate of approximately 698 °C/min, after which the average T(m onset) for the remaining three heating rates was constant at 135.83 ± 1.14 °C. Thus, 135.83 °C is proposed to be the thermodynamic melting temperature of fructose. It is important to note that the heating rate at which this thermodynamic melting temperature is achieved is most likely influenced by the type and amount of trace components (e.g., water and salts) contained in the fructose, which are known to vary widely in sugars. In the case of sucrose and glucose, thermodynamic melting temperatures were not able to be obtained, because the upper limit heating rate used was not fast enough to suppress thermal decomposition and achieve thermodynamic melting, perhaps due to the higher apparent T(m onset) for sucrose and glucose compared to that for fructose.     

Effects of heat and high hydrostatic pressure treatments on disulfide bonding interchanges among the proteins in skim milk

Traditionally, milk has been heat treated to control microorganisms and to alter its functionality, for example, to increase its heat stability. Pressure treatment has been considered as a possible alternative for microorganism control, but some of the functionality-related milk protein interactions have not been explored. The present study used two novel two-dimensional polyacrylamide gel electrophoresis (2D PAGE) methods to explore the differences in the irreversible disulfide bond changes among the milk proteins after four common heat treatments and after 30-min pressure treatments of milk at 200, 400, 600, and 800 MPa at ambient temperature (22 degrees C). The pasteurizing heat treatment (72 degrees C for 15 s) denatured and aggregated only a few minor whey proteins, but the high heat treatments (100 degrees C for 120 s, 120 degrees C for 120 s, and 140 degrees C for 5 s) formed disulfide-bonded aggregates that included a high proportion of all of the whey proteins and kappa-casein (kappa-CN) and a proportion of the alpha(s2)-CN. Pressure treatment of milk at 200 MPa caused beta-lactoglobulin (beta-LG) to form disulfide-bonded dimers and incorporated beta-LG into aggregates, probably disulfide-bonded to kappa-CN. The other whey proteins appeared to be less affected at 200 MPa for 30 min. In contrast, pressure treatment at 800 MPa incorporated beta-LG and most of the minor whey proteins, as well as kappa-CN and much of the alpha(s2)-CN, into aggregates. The accessibility of alpha(s2)-CN and formation of complexes involving alpha(s2)-CN, kappa-CN, and whey proteins in the pressure treated milk is an important novel finding. However, only some of the alpha-lactalbumin was denatured or incorporated into the large aggregates. These and other results show that the differences between the stabilities of the proteins and the accessibilities of the disulfide bonds of the proteins at high temperature or pressure affect the formation pathways that give the differences among the resultant aggregates, the sizes of the aggregates, and the product functionalities.     

Changes in the physical state of sucrose during dark chocolate processing

Dark chocolate tablets were manufactured using 100% crystalline sucrose, 50% crystalline and 50% amorphous sucrose, and 100% amorphous sucrose. The physical state of sucrose was determined by differential scanning calorimetry (DSC) and X-ray diffraction. DSC scans of dark chocolate samples containing amorphous sucrose were characterized by a glass transition at 63 degrees C, a sucrose crystallization peak at 105 degrees C, and a melting endotherm at 188 degrees C. Independent of the amount of amorphous or crystalline sucrose used for the preparation of dark chocolate, all final chocolate products provided a single melting endotherm at 188 degrees C and a crystalline X-ray diffraction pattern. These results indicated that sucrose crystallized during production of dark chocolate and that no amorphous sucrose was present in the final chocolate products.     

A DSC study on the gel-sol transition of a starch and hsian-tsao leaf gum mixed system

Differential scanning calorimetric analysis was performed on the admixture of 4% hsian-tsao leaf gum and 8% wheat starch as a function of salt types and concentrations. The salt concentrations (C(s)) studied were 5-100 mM for sodium and potassium chloride, and 3.4-34 mM for calcium and magnesium chloride. It was found that hsian-tsao leaf gum or starch alone did not present a readily recognizable exothermic peak or endothermic peak during cooling or heating in DSC. However, mixing these two polymers promoted the intermolecular binding and subsequent gelation of the mixtures as evidenced by the DSC exothermic and endothermic peaks during cooling and heating, respectively. The setting and melting temperatures of such a mixed system shifted progressively to higher temperatures with increasing concentrations of added salts. It was considered that the aggregated mixed polymers formed thermally stable junction zones with higher binding energies. The thermal behavior change was more remarkable by the addition of K(+) than by Na(+), and by Ca(2+) than by Mg(2+). For monovalent cations, the DSC heating and cooling curves showed a single endothermic and exothermic peak. For divalent cations at low concentration, the DSC curves showed a single peak. However, with sufficient divalent cations, the DSC curves eventually developed a bimodal character. A mixed system with sufficient Ca(2+) could form firm gel that was difficult to remelt completely upon heating to 130 degrees C, indicating the possibility of the formation of ionic bonds through cross-links with the carboxyl groups in hsian-tsao leaf gum.     

Elimination of trypsin inhibitor activity and beany flavor in soy milk by consecutive blanching and ultrahigh-temperature (UHT) processing

Soy foods contain significant health-promoting components but also may contain beany flavor and trypsin inhibitor activity (TIA), which can cause pancreatic disease if present at a high level. Thermal processing can inactivate TIA and lipoxygenase. Ultrahigh-temperature (UHT) processing is relatively new for manufacturing soy milk. Simultaneous elimination of TIA and soy odor by UHT processing for enhancing soy milk quality has not been reported. The objective was to determine TIA in soy milk processed by traditional, steam injection, blanching, and UHT methods and to compare the products with commercial soy milk products. Soybean was soaked and blanched at 70-85 degrees C for 30 s-7.5 min. The blanched beans were made into base soy milk. The hexanal content of the base soy milk was determined by gas chromatography to determine the best conditions for further thermal processing by indirect and direct UHT methods at 135-150 degrees C for 10-50 s using the Microthermics processor. Soy milk was also made from soaked soybeans by traditional batch cooking and steaming methods. Eighteen commercial products were selected from the supermarket. Residual TIA in soy milk processed by the traditional and steam injection to 100 degrees C for 20 min was approximately 13%. Blanching could inactivate 25-50% of TIAs of the raw soy milk. The blanch conditions of 80 degrees C and 2 min were selected for UHT processing because these conditions produced blanched soy milk without hexanal, indicating a complete heat inactivation of lipoxygenases. The TIA decreased with increased temperature and time of UHT heating. The maximal trypsin inhibitor inactivation was achieved by UHT direct and indirect methods with residual activities of approximately 10%. Some commercial soy milk products contained high TIAs. The results are important to the food industry and consumers. Kinetic analysis showed that heat inactivation (denaturation) of TIA, under the continuous processing conditions of the Microthermics processor, followed first-order reaction kinetics, and the activation energy of the inactivation was 34 kJ/mol.     

Stability of sterols in phytosterol-enriched milk under different heating conditions

Commercially available phytosterol-enriched milk was subjected to usual and drastic heating conditions to evaluate the stability of the sterols at different treatments. Products showed 422.2 mg of phytosterols/100 g of milk and 132 microg of sterol oxidation products (SOPs)/g of fat (277 microg of SOPs/100 g of milk). Schaal oven conditions (24 h/65 degrees C, equivalent to 1 month of storage at room temperature) reduced the phytosterol content by only 4%. Drastic heating treatments (2 min of microwave heating at 900 W or 15 min of electrical heating at 90 degrees C) led to a 60% decrease of total phytosterol content, with a significant increase of TBARs. The oxysterol amount under those conditions (which was higher in microwave-treated samples) was lower than expected, probably because of the degradation of the oxidation products. Usual heating conditions (1.5 min of microwaves) maintained phytosterol content on physiologically active values (301 mg/100 g of milk) with oxidation percentages around 0.12-0.40% for phytosterols and 1.13% for cholesterol.     

Acrylamide formation from asparagine under low-moisture Maillard reaction conditions. 1. Physical and chemical aspects in crystalline model systems

The formation of acrylamide in crystalline model systems based on asparagine and reducing sugars was investigated under low-moisture reaction conditions. The acrylamide amounts were correlated with physical changes occurring during the reaction. Molecular mobility of the precursors turned out to be a critical parameter in solid systems, which is linked to the melting behavior and the release of crystallization water of the reaction sample. Heating binary mixtures of asparagine monohydrate and anhydrous reducing sugars led to higher acrylamide amounts in the presence of fructose compared to glucose. Differential scanning calorimetry measurements performed in open systems indicated melting of fructose at 126 degrees C, whereas glucose and galactose fused at 157 and 172 degrees C, respectively. However, glucose was the most reactive and fructose the least efficient sugar in anhydrous liquid systems, indicating that at given molecular mobility the chemical reactivity of the sugar was the major driver in acrylamide formation. Furthermore, reaction time and temperature were found to be covariant parameters: acrylamide was preferably formed by reacting glucose and asparagine at 120 degrees C for 60 min, whereas 160 degrees C was required at shorter reaction time (5 min). These results suggest that, in addition to the chemical reactivity of ingredients, their physical state as well as reaction temperature and time would influence the formation of acrylamide during food processing.     

Effects of glycerol and moisture gradient on thermomechanical properties of white bread

The thermomechanical properties of breadcrumb were investigated using dynamic mechanical analysis (DMA) and differential scanning calorimetry (DSC). The main transition (T(1), near 0 degrees C) shifted to lower temperature with added glycerol due to freezing point depression. The low-temperature transition (T(3), approximately -50 degrees C), found only in high-glycerol (8.8%) bread, suggested that of excess or phase-separated glycerol. The high-temperature transition (T(2), 60-85 degrees C) appeared only in aged breadcrumbs; its temperature range was correlated well with the amylopectin melting transition (DSC) but its tan delta amplitude did not correlate well with the amylopectin melting enthalpy (r(2) = 0.72). On the other hand, the change of E' ' (viscous behavior) suggested that T(2) might be related to the change in the amorphous region. Domain-to-domain (amorphous) and crumb-to-crust moisture migrations are two critical phenomenological changes associated with aging and could lead to significant local dehydration of some amorphous regions contributing to mechanical firming during storage.     

Inactivation of soybean trypsin inhibitors and lipoxygenase by high-pressure processing

Trypsin inhibitors (TIA), one of the antinutritional factors of soy milk, are usually inactivated by heat treatment. In the current study, high-pressure processing (HPP) was evaluated as an alternative for the inactivation of TIA in soy milk. Moreover, the effect of HPP on lipoxygenase (LOX) in whole soybeans and soy milk was studied. For complete LOX inactivation either very high pressures (800 MPa) or a combined temperature/pressure treatment (60 degrees C/600 MPa) was needed. Pressure inactivation of TIA was possible only in combination with elevated temperatures. For TIA inactivation, three process parameters, temperature, time, and pressure, were optimized using experimental design and response surface methodology. A 90% TIA inactivation with treatment times of <2 min can be reached at temperatures between 77 and 90 degrees C and pressures between 750 and 525 MPa.     

Gelation of edible blue-green algae protein isolate (Spirulina platensis Strain Pacifica): thermal transitions, rheological properties, and molecular forces involved

Proteins isolated from blue-green algae Spirulina platensis strain Pacifica were characterized by visible absorption, differential scanning calorimetry (DSC), viscometry, and dynamic oscillatory rheological measurements. Unique thermal unfolding, denaturation, aggregation, and gelation of the algal protein isolate are presented. DSC analysis showed that thermal transitions occur at about 67 and 109 degrees C at neutral pH. Calcium chloride stabilized the quaternary structure against denaturation and shifted the transitions at higher temperatures. Viscometric studies of Spirulina protein isolate as a function of temperature showed that the onset of the viscosity increase is closely related to the dissociation-denaturation process. Lower viscosities were observed for the protein solutions dissolved at pH 9 due to an increased protein solubility. Solutions of Spirulina protein isolate form elastic gels during heating to 90 degrees C. Subsequent cooling at ambient temperatures caused a further pronounced increase in the elastic moduli and network elasticity. Spirulina protein isolate has good gelling properties with fairly low minimum critical gelling concentrations of about 1.5 and 2.5 wt % in 0.1 M Tris buffer, pH 7, and with 0.02 M CaCl(2) in the same buffer, respectively. It is suggested that mainly the interactions of exposed hydrophobic regions generate the molecular association, initial aggregation, and gelation of the protein isolate during the thermal treatment. Hydrogen bonds reinforce the network rigidity of the protein on cooling and further stabilize the structure of Spirulina protein gels but alone are not sufficient to form a network structure. Intermolecular sulfhydryl and disulfide bonds were found to play a minor role for the network strength of Spirulina protein gels but affect the elasticity of the structures formed. Both time and temperature at isothermal heat-induced gelation within 40-80 degrees C affect substantially the network formation and the development of elastic modulus of Spirulina protein gels. This is also attributed to the strong temperature dependence of hydrophobic interactions. The aggregation, denaturation, and gelation properties of Spirulina algal protein isolate are likely to be controlled from protein-protein complexes rather than individual protein molecules.     

Characterization of anthocyanin-rich waste from purple corncobs (Zea mays L.) and its application to color milk

Pigment production from anthocyanin-rich purple corncobs generates a deeply colored waste precipitate. Our objectives were to characterize this anthocyanin-rich waste (ARW) and to find a suitable application in a food matrix. Composition and solubility characteristics of ARW were evaluated. Color (CIELAB) and pigment (monomeric anthocyanin and HPLC profiles) stability of ARW in milk (35 mg/100 mL) were evaluated using an accelerated test at 70 degrees C and phosphate buffer as a control. ARW provided milk an attractive purple hue (324-347 degrees ). Monomeric anthocyanin degradation followed zero-order kinetics in skim and whole milk and second-order kinetics in the control, with half-lives of 173, 223, and 44 min at 70 degrees C, respectively. ARW shows potential as a natural colorant for a pH range unusual for anthocyanin applications. A protective effect of matrix constituents on the stability of anthocyanins was evident. Anthocyanins may interact with different compounds in biological systems when the pH values are close to neutral.     

Evaluation of APHA and AOAC II methods for phosphatase in butter and differentiation of milk and microbial phosphatases by agarose-gel electrophoresis

Salted and unsalted butters with 3 levels of phosphatase were prepared with both raw and pasteurized cream containing 36% fat. Test samples were analyzed for phosphatase by the modified method of the American Public Health Association (APHA) and the official AOAC method, 16.256 (1984, 14th Ed., 1990 15th Ed., 946.02). In the APHA method, weighing of solid frozen butter for testing yielded repeatable results. Addition of 0.0-1.0 mg magnesium to the butter had little effect on phosphatase activity in the APHA modified rapid colorimetric method (MRCM), but caused the phosphatase activity to decrease in the AOAC method. Phosphatase in salted and unsalted butters was quite stable at -17 +/- 1 degrees C and at 3.0 +/- 0.5 degrees C; however, within 2 to 4 days, freshly prepared butters stored at 22 +/- 1 degrees C developed reactivated and/or microbial phosphatases that were both heat-labile and heat-stable. At 22 +/- 1 degrees C, frozen butters showed decreased milk phosphatase activity before producing microbial phosphatase. Heat-labile phosphatases in salted and unsalted butters were inactivated at 62.8 degrees C for 10 min, and the phosphatase lability was partially due to the heat-denaturing effect of NaCl in salted butter. Some heat-stable phosphatases in unsalted butter survived at 66 degrees C for 30 min. Differentiation of milk phosphatase from microbial phosphatases was difficult by both methods; however, they were successfully differentiated by the agarose-gel electrophoretic technique.