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1.
The kinetics of beta-lactoglobulin (beta-LG) denaturation in reconstituted skim milk samples of various concentrations (9.6-38.4% total solids) over a wide temperature range (75-100 degrees C) was studied. The thermal denaturation of beta-LG had a reaction order of 1.5 at all milk solids concentrations and at all temperatures. The rate of denaturation of beta-LG was markedly dependent on the milk solids concentration and the heating temperature. At 75 degrees C, the thermal denaturation of beta-LG was retarded at higher milk solids concentrations. However, this retardation was less pronounced at higher temperatures so that a similar rate of denaturation was observed at all milk solids concentrations at 100 degrees C. From an examination of the level of disulfide-aggregated beta-LG, it was evident that most, but not all, of the denatured beta-LG was involved in disulfide-aggregated complexes, either with other denatured whey proteins or with the casein micelles. As with beta-LG denaturation, the rate of disulfide aggregation of beta-LG was markedly dependent on the milk solids concentration.  相似文献   

2.
The effect of milk concentration (10-40% TS) on the kinetics of the pressure-induced denaturation of β-lactoglobulin (β-LG) was studied. The denaturation was found to be a second-order process at all milk concentrations and pressures. There was a change in pressure dependence of the rate constants for denaturation at about 300 MPa, and this effect became more pronounced as the milk concentration increased. At pressures ≥300 MPa, a small effect of milk concentration was observed, with small decreases in the rate of denaturation as the milk concentration was increased above 20% TS. This was attributed to the lower pH as the milk concentration was increased. In contrast, at 200 MPa, β-LG denaturation was markedly retarded as the milk solids concentration was increased. This was attributed to the increased lactose concentration at higher milk concentrations. This would promote β-LG dimerization at this pressure and this would stabilize the β-LG to denaturation.  相似文献   

3.
Heat treatment during manufacturing of milk powder is one of the most important tools for manipulation of its functional properties, and it is the basis of the classification of these proteins into low-, medium-, and high-heat types. Slight differences in the sequences of the major proteins in milk (genetic variants) seem to have also a significant effect in milk powder processing (U.S. patent). Therefore, the effects of high-temperature storage and heat treatment on skim milk of defined genetic variants of beta-lactoglobulin (beta-LG) were measured. The samples had 45% total solids, the temperature of aging was 50 degrees C, and the heat treatment was 90 degrees C for 10 min prior to evaporation. Measurements on shear rate and on apparent viscosity were determined for each sample. During storage of the concentrated milk, the apparent viscosity and yield values increased markedly, and the age-dependent increase in viscosity in heat-treated concentrated skim milks was much more pronounced than in those prepared from unheated skim milks. The increase in apparent viscosity and yield value with storage time was notably different for milks containing different genetic variants. Unheated concentrated milks containing the B variant of beta-LG showed the most rapid increase in apparent viscosity with storage time, whereas the viscosity increase was slowest in the concentrate containing the A variant. In contrast, heat-treated concentrated milks containing the A variant of beta-LG showed the most rapid increase in viscosity with storage time, whereas the viscosity increase was slowest in the concentrate containing the AB variant. The changes in apparent viscosity of concentrated milk were largely reversible under high shear during the early stages of storage, but samples stored for a long time showed irreversible changes in apparent viscosity. Particle size analysis confirmed irreversible aggregation and fusion of casein particles during storage.  相似文献   

4.
The effect of protein, nonprotein-soluble components, and lactose concentrations on the irreversible denaturation of beta-lactoglobulin (beta-LG) and alpha-lactalbumin (alpha-LA) in reconstituted skim milk samples was studied over a wide temperature range (75-100 degrees C). The irreversible thermal denaturation of beta-LG had a reaction order of 1.5 and that of alpha-LA had a reaction order of 1.0 in all systems and under all conditions. The rates of irreversible denaturation of beta-LG and alpha-LA were markedly dependent upon the composition of the milk. At all temperatures, the irreversible denaturations of beta-LG and alpha-LA were enhanced at a higher protein concentration and were retarded when the nonprotein-soluble components and lactose concentrations were increased. The effects of increasing the concentrations of lactose and nonprotein-soluble components were interpreted using the preferential hydration theory and allowed for the interpretation of the changes in the denaturations of beta-LG and alpha-LA when the milk total solids concentration was increased.  相似文献   

5.
Bovine beta-lactoglobulin B (beta-LG) is susceptible to pressure treatment, which unfolds it, allowing thiol-catalyzed disulfide bond interchange to occur, facilitating intermolecular bonding (both noncovalent and disulfide). In the present study, beta-LG was mixed with sodium dodecyl sulfate (SDS), all-trans-retinol (retinol), or 8-anilino-1-naphthalenesulfonate (ANS) on a 1:1.1 molar basis, and aliquots were held at pressures between 50 and 800 MPa for 30 min at pH 7.2 and 20 degrees C. Polyacrylamide gel electrophoresis (PAGE) showed that beta-LG alone (control) was converted into a non-native monomer and a series of dimers, trimers, etc., at pressures beyond 100 MPa; SDS inhibited the formation of non-native species up to 200 MPa, and neither retinol nor ANS inhibited the formation of the non-native species as effectively as SDS. At pressures beyond 350 MPa, SDS ceased to have any inhibitory effect, but both ANS and retinol showed significant inhibition. The near- and far-UV CD patterns and the ANS fluorescent data were consistent with the PAGE data, but the retinol fluorescent data did not show sufficient change to interpret. The results suggested that there were three discernible structural stages. In Stage I (0.1-150 MPa), the native structure is stable; in Stage II (200-450 MPa), the native monomer is reversibly interchanging with non-native monomers and disulfide-bonded dimers; and in Stage III (>500 MPa), the free CysH in non-native monomer and dimer interacts with -S-S- bonds to produce high molecular weight aggregates of beta-LG. SDS inhibited the Stage I to Stage II transition at 200 MPa, and ANS and retinol inhibited the Stage II to Stage III transition at 600 MPa.  相似文献   

6.
Heat treatment of bovine beta-lactoglobulin B (beta-LG) causes it to partially unfold and aggregate via hydrophobic association and intra- and interprotein disulfide bonds. The first stage, which involves a "loosening" of the native structure, is influenced by the environmental conditions, such as pressure, pH, and added solutes. In the present study, four potential beta-LG ligands [palmitate, sodium dodecyl sulfate (SDS), 8-anilino-1-naphthalenesulfonate (ANS), and all-trans-retinol (retinol)] were added to beta-LG solutions prior to heat treatment for 12 min at temperatures between 40 and 93 degrees C. The extent of the changes in secondary and tertiary structures, unfolding, and aggregation at 20 degrees C were determined by circular dichroism, fluorescence, and alkaline- and SDS-polyacrylamide gel electrophoresis (PAGE). Both palmitate and SDS stabilized the native structure of beta-LG against heat-induced structural flexibility, subsequent unfolding, and denaturation. Retinol was less effective, probably because of its lower affinity for the calyx-binding site, and ANS did not stabilize beta-LG, suggesting that ANS did not bind strongly in the calyx. It was also noted that holding a beta-LG solution with added SDS or ANS promoted the formation of a hydrophobically associated non-native dimer.  相似文献   

7.
The denaturation, aggregation, and rheological properties of chicken breast muscle myosin, beta-lactoglobulin (beta-LG), and mixed myosin/beta-LG solutions were studied in 0.6 M NaCl, 0.05 mM sodium phosphate buffer, pH 7.0, during heating. The endotherm of a mixture of myosin and beta-LG was identical to that expected if the endotherm of each protein was overlaid on the same axis. The maximum aggregation rate (AR(max)) increased, and the temperature at the AR(max) (T(max)) and initial aggregation temperature (T(o)) decreased as the concentration of both proteins was increased. The aggregation profile of <0.5% myosin was altered by the presence of 0.25% beta-LG. Addition of 0.5-3.0% beta-LG decreased storage moduli of 1% myosin between 55 and 75 degrees C, but increased storage moduli (G') when heated to 90 degrees C and after cooling. beta-LG had no effect on the gel point of > or =1.0% myosin, but enhanced gel strength when heated to 90 degrees C and after cooling. After cooling, the G' of 1% myosin/2%beta-LG gels was about 1.7 times greater than that of gels prepared from 2% myosin/1% beta-LG.  相似文献   

8.
Thermal denaturation, rheological, and microstructural properties of gels prepared from native beta-lactoglobulin (beta-LG) and preheated or heat-denatured beta-LG (HDLG) aggregates were compared. The HDLG was prepared by heating solutions of 4% beta-LG in deionized water, pH 7.0, at 80 degrees C for 30 min and then diluted to the desired concentration in 0.6 M NaCl and 0.05 M phosphate buffer at pH 6.0, 6.5, and 7.0. When reheated to 71 degrees C, HDLG formed a gel at a concentration of 2% protein. At pH 7.0, 3% HDLG gelled at 52.5 degrees C and had a storage modulus (G') of 2200 Pa after cooling. beta-LG (3%) in 0.6 M NaCl and 0.05 M phosphate buffer, pH 7.0, did not gel when heated to 71 degrees C. The gel point of 3% HDLG decreased by 10.5 degrees C and the G' did not change when the pH was decreased to 6.0. The HDLG gel microstructure was composed of strands and clumps of small globular aggregates in contrast to beta-LG gels, which contained a particulate network of compacted globules. The HDLG formed a gel at a lower concentration and lower temperature than beta-LG in the high-salt buffer, suggesting an application in meat systems or other food products prepared with salt and processed at temperatures of < or =71 degrees C.  相似文献   

9.
The combined high pressure/thermal (HP/T) inactivation of tomato pectin methyl esterase (PME) and polygalacturonase (PG) was investigated as a possible alternative to thermal processing classically used for enzyme inactivation. The temperature and pressure ranges tested were from 60 degrees C to 105 degrees C, and from 0.1 to 800 MPa, respectively. PME, a heat-labile enzyme at ambient pressure, is dramatically stabilized against thermal denaturation at pressures above atmospheric and up to 500-600 MPa. PG, however, is very resistant to thermal denaturation at 0.1 MPa, but quickly and easily inactivated by combinations of moderate temperatures and pressures. Selective inactivation of either PME or PG was achieved by choosing proper combinations of P and T. The inactivation kinetics of these enzymes was measured and described mathematically over the investigated portion of the P/T plane. Whereas medium composition and salinity had little influence on the inactivation rates, PME was found less sensitive to both heat and pressure when pH was raised above its physiological value. PG, on the other hand, became more labile at higher pH values. The results are discussed in terms of isoenzymes and other physicochemical features of PME and PG.  相似文献   

10.
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.  相似文献   

11.
High-pressure processing (HPP) was utilized to induce unfolding of beta-lactoglobulin (beta-LG). beta-Lactoglobulin solutions at concentrations of 0.5 mg/mL, in pH 7.5 phosphate buffer, were pressure treated at 510 MPa for 10 min at either 8 or 24 degrees C. The secondary structure, as determined by circular dichroism (CD), of beta-LG processed at 8 degrees C appeared to be unchanged, whereas beta-LG processed at 24 degrees C lost alpha-helix structure. Tertiary structures for beta-LG, as determined by near-UV CD, intrinsic protein fluorescence spectroscopy, hydrophobic fluorescent probe binding, and thiol group reactivity, were changed following processing at either temperature. The largest changes to tertiary structure were observed for the samples processed at 24 degrees C. Model solutions containing the pressure-treated beta-LG showed significant decreases in surface tension at liquid-air interfaces with values of 54.00 and 51.69 mN/m for the samples treated at 24 and 8 degrees C, respectively. In comparison, the surface tension for model solutions containing the untreated control was 60.60 mN/m. Changes in protein structure during frozen and freeze-dried storage were also monitored, and some renaturation was observed for both storage conditions. Significantly, the sample pressure-treated at 8 degrees C continued to display the lowest surface tension.  相似文献   

12.
Thermal, rheological, and microstructural properties of myosin (1 and 2% protein) were compared to mixtures of 1% myosin and 1% heat-denatured beta-lactoglobulin aggregates (myosin/HDLG) and 1% myosin and 1% native beta-lactoglobulin (myosin/beta-LG) in 0.6 M NaCl and 0.05 M sodium phosphate buffer, pH 6.0, 6.5, and 7.0 during heating to 71 degrees C. Thermal denaturation patterns of myosin and myosin/HDLG were similar except for the appearance of an endothermic peak at 54-56 degrees C in the mixed system. At pH 7.0, 2% myosin began to gel at 48 degrees C and had a storage modulus (G') of 500 Pa upon cooling. Myosin/HDLG (2% total protein) had a gel point of 48 degrees C and a G' of 650 Pa, whereas myosin/beta-LG had a gel point of 49 degrees C but the G' was lower (180 Pa). As the pH was decreased, the gel points of myosin and myosin/HDLG decreased and the G' after cooling increased. The HDLG was incorporated within the myosin gel network, whereas beta-LG remained soluble.  相似文献   

13.
The activity of tomato pectinesterase (PE) was studied as a function of pressure (0.1-900 MPa) and temperature (20-75 degrees C). Tomato PE was rather heat labile at atmospheric pressure (inactivation in the temperature domain 57-65 degrees C), but it was very pressure resistant. Even at 900 MPa and 60 degrees C the inactivation was slower as compared to the same treatment at atmospheric pressure. At atmospheric pressure, optimal catalytic activity of PE was found at neutral pH and a temperature of 55 degrees C. Increasing pressure up to 300 MPa increased the enzyme activity as compared to atmospheric pressure. A maximal enzyme activity was found at 100-200 MPa combined with a temperature of 60-65 degrees C. The presence of Ca(2+) ions (60 mM) decreased the enzyme activity at atmospheric pressure in the temperature range 45-60 degrees C but increased enzyme activity at elevated pressure (up to 300 MPa). Maximal enzyme activity in the presence of Ca(2+) ions was noted at 200-300 MPa in combination with a temperature of 65-70 degrees C.  相似文献   

14.
This study was conducted to determine whether the application of high hydrostatic pressure could modify the enzymatic activity and membrane integrity of lysosomes in muscle. Several combinations of pressure (0-600 MPa) and time (0-300 s) were applied to two types of samples: purified enzymes (cathepsin D and acid phosphatase) in buffer solution and intact muscle (biceps femoris). The enzymes studied showed varying degrees of susceptibility depending on the level of pressure, holding time, and environment. Acid phosphatase activity was minimally affected by pressure in buffer solution, whereas cathepsin D was modulated significantly by the pressure and time applied. The activities of the enzymes extracted from meat increased with pressure. The cytochemical observations showed the presence of primary and secondary lysosomes in muscles. After pressurization, the membrane integrity of the lysosomes was modified. A correlation could be established between lysosomal enzymatic activities and the lysosome membrane breakdown.  相似文献   

15.
Bovine beta-lactoglobulin-carboxymethyl cyclodextrin (beta-LG-CMCyD) conjugate was prepared using water-soluble carbodiimide, in an effort to improve the functional properties of the protein. The molar ratio of beta-LG to CMCyD in the conjugate was 1:2. The isoelectric point of the conjugate was 4.1-5.8. Spectroscopic studies indicated that the global conformation of the conjugate was similar to that of native beta-LG. Structural analyses with monoclonal antibodies indicated that there was a conformational change around (15)Val-(29)Ile (beta-sheet). The denaturation temperature of the conjugate was about 77 degrees C, which is about 4 degrees C higher than that of native beta-LG. The beta-LG-CMCyD conjugate maintained retinol-binding activity as strong as that of beta-LG. The emulsifying activity of beta-LG was much improved and the antioxidative activity of beta-LG was maintained after conjugation with CMCyD.  相似文献   

16.
Beta-lactoglobulin (beta-LG) was treated with high hydrostatic pressure (HHP) at 600 MPa and 50 degrees C for selected times as long as 64 min. The intrinsic tryptophan fluorescence of beta-LG indicated that HHP treatment conditions induced a conformational change. HHP treatment conditions also promote a 3-fold increase in the extrinsic fluorescence of 1-anilinonaphthalene-8-sulfonate and a 2.6-fold decrease for cis-paraneric acid, suggesting an increase in accessible aromatic hydrophobicity and a decrease in aliphatic hydrophobicity. Far-ultraviolet circular dichroism (CD) spectra reveal that the secondary structure of beta-LG converts from native beta-sheets to non-native alpha-helices following HHP treatment, whereas near-ultraviolet CD spectra reveal that the native tertiary structure of beta-LG essentially disappears. Urea titrations reveal that native beta-LG unfolds cooperatively, but the pressure-treated molecule unfolds noncooperatively. The noncooperative state is stable for 3 months at 5 degrees C. The nonaccessible free thiol group of cysteine121 in native beta-LG became reactive to Ellman's reagent after adequate HHP treatment. Gel electrophoresis with and without beta-mercaptoethanol provided evidence that the exposed thiol group was lost concomitant with the formation of S-S-linked beta-LG dimers. Overall, these results suggest that HHP treatments induce beta-LG into hydrophobic molten globule structures that remain stable for at least 3 months.  相似文献   

17.
A combined high-pressure carbon dioxide (HP-CO 2) and thermal degradation reaction of betanin and isobetanin in aqueous solution was investigated and can be described by a first-order decay. At 45 degrees C, the degradation rate constant ( k) for each pigment component significantly increased (the half-life ( t 1/2) decreased, p < 0.05) with elevated pressure. Furthermore, HP-CO 2 treatment led to lower k values (higher t 1/2 values) than thermal treatment. However, k and t 1/2 values approached those of thermal treatment when the pressure was >30 MPa combined with temperatures exceeding 55 degrees C. Moreover, betanin was more stable than isobetanin under HP-CO 2. E a values ranged from 94.01 kJ/mol for betanin and 97.16 kJ/mol for isobetanin at atmospheric pressure to 170.83 and 142.69 kJ/mol at 50 MPa, respectively. A higher pressure and temperature as well as longer exposure time resulted in higher values of L*, b*, C*, and h degrees . HP-CO 2 induced more degradation products from betanin and isobetanin than thermal treatment with an identical temperature and exposure time.  相似文献   

18.
High hydrostatic pressure (HHP) at 500 MPa and 50 degrees C induces beta-LG into the molten globule state. Retinol, cis-parinaric acid (CPA), and 1-anilino-naphthalene-8-sulfonate (ANS) fluorescence from pH 2.5 to 10.5 in the presence of the native and molten globule states of beta-LG indicate that retinol binds to beta-LG in the calyx, CPA at the surface hydrophobic site, and ANS in multiple hydrophobic sites. HHP treatment results in a decrease of beta-LG affinity for retinol and CPA, suggesting conformational changes in the calyx and surface hydrophobic site of beta-LG during HHP treatment. beta-LG treated by HHP in the presence of N-ethylmaleimide (NEM) retains retinol affinity, suggesting that NEM protects the calyx conformation of beta-LG during HHP treatment. HHP treatment of beta-LG in the presence of KIO(3) exhibits a great decrease of CPA affinity compared to HHP-treated beta-LG in the absence of KIO(3), suggesting the formation of non-native disulfide bonding at the CPA binding site.  相似文献   

19.
Fish oil was incorporated into milk under different homogenization temperatures (50 and 72 degrees C) and pressures (5, 15, and 22.5 MPa). Subsequently, the oxidative stability of the milk and changes in the protein composition of the milk fat globule membrane (MFGM) were examined. Results showed that high pressure and high temperature (72 degrees C and 22.5 MPa) resulted in less lipid oxidation, whereas low pressure and low temperature (50 degrees C and 5 MPa) resulted in faster lipid oxidation. Analysis of protein oxidation indicated that especially casein was prone to oxidation. The level of free thiol groups was increased by high temperature (72 degrees C) and with increasing pressure. Furthermore, SDS-PAGE and confocal laser scanning microscopy (CLSM) indicated that high temperature resulted in an increase in beta-lactoglobulin adsorbed at the oil-water interface. This was even more pronounced with higher pressure. Less casein seemed to be present at the oil-water interface with increasing pressure. Overall, the results indicated that a combination of more beta-lactoglobulin and less casein at the oil-water interface gave the most stable emulsions with respect to lipid oxidation.  相似文献   

20.
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.  相似文献   

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