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1.
Apparent binding constants of aroma compounds limonene, alpha- and beta-ionone, and terpenyl acetate, with beta-lactoglobulin (BLG), were determined, using dynamic coupled column liquid chromatography, for pH values varying from 3 to 11. K(a) values varied from 2.61 to 3.21 x 10(3) M(-1) for limonene, indicating a strong interaction with BLG. Similarly, significant and close apparent binding constants were obtained for alpha- and beta-ionone, 1.7 x 10(2) and 4.5 to 5.4 x 10(2) M(-1), respectively. These data indicated that a similar mechanism is involved for the binding of these two molecules. The weaker values obtained at low pH, for alpha-ionone relative to beta-ionone, can be explained by the existence of steric hindrance. An increase of the apparent binding constant was observed, for all the compounds studied, when the pH was increased from 3 to 9. At this pH, an apparent binding constant was obtained for terpenyl acetate (1.04 x 10(2) M(-1)), whereas this determination was not possible at pH 3 and 6. The apparent binding constant increase was in agreement with the decrease of aroma compound relative activity coefficient in the presence of BLG, previously observed at this pH. It indicated a best accessibility to the same binding site. The binding constants of all the aroma compounds studied decreased at pH 11 as a result of the important release of the BLG structure previously reported.  相似文献   

2.
Interactions between volatile compounds and BLG in aqueous solution were studied using static and dynamic headspace techniques (exponential dilution). The intensity of interactions between methyl ketones (C7-C9), ethyl esters (C6-C9), limonene, myrcene, and beta-lactoglobulin (BLG) were estimated by determination of the relative infinite dilution activity coefficients (gamma(r)). For a constant pH value, the methyl ketones retention by BLG increased significantly with the hydrophobicity of the volatiles, whereas the retention reached a maximum for ethyl octanoate in the ester series, indicating a possible steric hindrance. For limonene and myrcene an unexpected increase in headspace concentration or "salting out" effect was noticed for acid pH. The variations of the retention according to the pH increase of the medium from pH 3 to pH 11 could be related to structural modifications of the BLG. The retention increase observed between pH 3 and pH 9 resulted from the flexibility modification of the protein, allowing better accessibility to the primary or the secondary hydrophobic sites, whereas the dramatic decrease observed at pH 11 was the consequence of the alkaline denaturation of BLG. Electrostatic interactions occurring at pH 7.5 could also explain the observed retention increase.  相似文献   

3.
Interactions between a well-characterized protein, beta-lactoglobulin, and two flavor compounds, beta-ionone and gamma-decalactone, were studied by 2D NMR spectroscopy. NMR spectra were recorded in aqueous solution (pH 2.0, 12 mM NaCl, 10% D(2)O) under conditions such that beta-lactoglobulin is present in a monomeric state. TOCSY and NOESY spectra were recorded on the protein and the complexes between protein and ligands. The spectra of the NH-CH(alpha) region showed the cross-signals due to the coupling between N- and C-bonded protons in the polypeptide backbone. The observed chemical shift variations in the presence of ligands can be assigned to changes in the protein conformation. It appears that the side chains of several amino acids are affected by binding of gamma-decalactone point into the central cavity (Leu46, Ile56, Met107, and Gln120), whereas binding of beta-ionone affects amino acids located in a groove near the outer surface of the protein (Leu104, Tyr120, and Asp129), as illustrated by molecular visualization. This NMR study provides precise information of the location of binding and confirms the existence of two different binding sites for aroma compounds on beta-lactoglobulin, which was suggested in previous competition studies by fluorometry or affinity chromatography and by structural information obtained from infrared spectroscopy.  相似文献   

4.
Structural and functional properties of two amaranth protein isolates as a function of pH were studied. Isolates, A9 and A11, were obtained by alkaline extraction at pH 9 and 11, respectively. Gel filtration chromatograms of A9 and A11 showed similar profiles. The A11 isolate contained mainly albumins and globulins, and a small proportion of globulin‐P aggregates, suggesting the presence of species with a higher degree of denaturation compared to A9. Differential scanning calorimetry (DSC) showed that A9 was characterized by two thermal transitions (65.8 and 98°C); A11 exhibited only a small endotherm (66.6°C) and a second, less defined one. DSC analysis of A9 at pH 2–4 did not show endotherms, but at pH 5, some protein structures were observed. A11 showed a greater degree of denaturation. FPLC results showed that the proteins in A9 are more folded and their conformation is closer to the native state than those in A11, which are more unfolded due to pH‐mediated denaturation, mainly in acid media. The surface hydrophobicity of the isolates in acid media was lower than in alkaline media. The fluorescence emission spectra of the isolates showed differences in acidic pH conditions. As expected, the highest solubility was at alkaline pH. The water‐holding capacity was similar for both isolates. The water‐imbibing capacity and speed of foaming was higher for A11 than for A9. In summary, intense pH treatment of amaranth isolates generated partial or total protein denaturation and differences in the functional properties.  相似文献   

5.
This study was conducted to assess the effect of high hydrostatic pressure on monomer beta-lactoglobulin (BLg) at acid pH by fluorescence spectroscopy under pressure and by circular dichroism (CD) and (1)H NMR spectroscopies after release of pressure. The intrinsic (tryptophan) fluorescence measurement and the study of 8-anilinonaphthalene-1-sulfonate (ANS) binding to BLg indicated that at pH 2.0 the recovery of center of spectral mass or ANS fluorescence was almost complete upon pressure release. No difference in (1)H NMR spectra was observed between pressurized and unpressurized BLg. In addition, NMR detection of the H/D exchange of aromatic protein indicated that the conformation of the vicinity of tryptophan residues could be refolded almost completely after release of pressure. These results seemingly confirm that the pressure-induced denaturation of BLg at pH 2.0 is reversible. However, cis-parinaric acid binding ability of pressurized BLg was largely lost, although its retinol binding ability was the same as its unpressurized one. Furthermore, CD spectra of the far-UV region and 2D NMR spectra evidently revealed the difference in the conformation of the molecule between unpressurized and pressurized BLg. These results are interpreted as an existence of partially fragile structure in the BLg molecule by high pressure.  相似文献   

6.
Two diffusion-based NMR techniques are presented and used to investigate the binding of selected flavor compounds to macromolecules. A pulsed field gradient NMR (PFG-NMR) method was applied to measure the apparent diffusion coefficients of four alkanone compounds as they associated with bovine serum albumin (BSA). The change in the apparent diffusion coefficient as a function of the BSA/alkanone ratio was fitted to yield binding constants (K(a)()) and binding stoichiometry (n) for each alkanone. The results showed that the apparent diffusion coefficients of alkanones increased with a decrease in the BSA/alkanone ratios, and the measured values of K(a)() and n were comparable with those obtained with other methods and depended on the alkanone structure. A diffusion-based nuclear Overhauser effect (called diffusion NOE pumping) method was also applied to screen mixtures of flavor compounds and identify those that have a binding affinity to complex macromolecules. Using this technique benzaldehyde and vanillin were observed to bind with bovine serum albumin, whereas 2-phenylethanol was identified as a nonbinding or weakly binding ligand with BSA. The diffusion NOE pumping method was also applied to a hydro alcoholic solution of cacao bean tannin extracts to which a mixture of ethylbenzoate, benzaldehyde, and 2-phenylethanol was added. The diffusion NOE pumping technique clearly indicated that ethylbenzoate had a stronger binding affinity to the polymeric (-)-epicatechin units of the cacao bean tannin extracts than the other two flavor compounds. The results successfully demonstrate the potential applications of diffusion-based NMR techniques for studying flavors and nonvolatile food matrix interactions.  相似文献   

7.
The binding properties of dry proteins are relatively poorly known. Many proteins are present in emulsions and suspensions and also in dry forms. This is particularly true of dairy proteins, which are often stored and sold in powdered form. In the present work, the binding of three terpenes (alpha-terpinene, gamma-terpinene, and terpinolene), which belong to the basic aroma components, and of decane by powdered beta-lactoglobulin (BLG) was studied at different hydration levels (0.05-0.40 g of H(2)O/g of protein) and temperatures (298 and 309.5 K), in the presence or absence of lipids and small concentrations of ethanol. Vapor sorption isotherms were determined for these systems by a static method of headspace gas chromatographic analysis. A cooperative effect of hydrophobic hydration was observed for the binding of aroma terpenes and decane by the solid BLG. The temperature increase from 298 to 309.5 K reduced the observed hydration threshold of BLG by 0.05-0.08 g of H(2)O/g of protein. Lipids (1.2% w/w) in hydrated BLG gave at least a 2-fold increase in its binding affinity for the hydrocarbons studied, and synergic effects of the hydration and lipid on this affinity were observed.  相似文献   

8.
The pepper alkaloid piperine is a nontoxic, natural dietary compound with a broad range of physiological activity. The present work is the first demonstration of its interaction with a mammalian protein. Circular dichroism (CD) spectroscopy was used to reveal and analyze the binding of piperine to a lipocalin protein. Induced CD spectra measured in pH 7.7 phosphate buffer at 37 degrees C demonstrated reversible, non-covalent association of piperine with bovine beta-lactoglobulin (BLG), the major whey protein in milk. The binding parameters (K(a) approximately 8 x 10(4) M(-1), n = 0.8) determined from the CD titration data showed no significant differences between the piperine binding properties of the two main genetic variants of BLG (A and B). The vanishing extrinsic CD signal obtained upon acidification of the piperine-BLG sample solution (Tanford transition) suggested that the ligand binds in the central hydrophobic cavity of the beta-barrel. The cavity binding concept was further supported by a CD displacement experiment using palmitic acid, the well-known hydrophobic ligand of BLG. Molecular docking calculations showed that piperine can be efficiently accommodated within the calyx of BLG. Additional molecular modeling calculations indicated that the beta-barrel of human tear lipocalin, human serum retinol binding protein, and human neutrophil gelatinase associated lipocalin might also accommodate a piperine molecule.  相似文献   

9.
The amino acid composition and the physicochemical and functional properties of quinoa protein isolates were evaluated. Protein isolates were prepared from quinoa seed by alkaline solubilization (at pH 9, called Q9, and at pH 11, called Q11) followed by isoelectric precipitation and spray drying. Q9 and Q11 had high levels of essential amino acids, with high levels of lysine. Both isolates showed similar patterns in native/SDS-PAGE and SEM. The pH effect on fluorescence measurements showed decreasing fluorescence intensity and a shift in the maximum of emission of both isolates. Q9 showed an endotherm with a denaturation temperature of 98.1 degrees C and a denaturation enthalpy of 12.7 J/g, while Q11 showed no endotherm. The protein solubility of Q11 was lower than that of Q9 at pH above 5.0 but similar at the pH range 3.0-4.0. The water holding capacity (WHC) was similar in both isolates and was not affected by pH. The water imbibing capacity (WIC) was double for Q11 (3.5 mL of water/g isolate). Analysis of DSC, fluorescence, and solubility data suggests that there is apparently denaturation due to pH. Some differences were found that could be attributed to the extreme pH treatments in protein isolates and the nature of quinoa proteins. Q9 and Q11 can be used as a valuable source of nutrition for infants and children. Q9 may be used as an ingredient in nutritive beverages, and Q11 may be used as an ingredient in sauces, sausages, and soups.  相似文献   

10.
This report has investigated the seasonal temperatures influences (winter and summer) of five vineyards at different altitudes on the concentrations of 2-methoxy-3-isobutylpyrazine (MIBP), alpha- and beta-ionone, and beta-damascenone in 2004 or 2005 vintages of Cabernet Sauvignon wines from Santa Catarina State, Brazil. Sensorial analyses were also carried out on the wine samples and compared to altitude and climate. Significant regression was observed between MIBP concentrations and the vineyard's altitude. No significant relation was observed between alpha- and beta-ionone and beta-damascenone with the vineyard's altitude. Principal component analysis positively correlated wines from higher altitudes with a "bell pepper" aroma. Conversely, the wines made with grapes from lower altitudes were correlated with "red fruits" and "jam" aromas. An important relation between the bell pepper aroma and the lower winter temperature was observed. A strong negative correlation was also observed between seasonal temperatures and vineyard altitude as well as between MIBP content and seasonal temperature of growing grapevines.  相似文献   

11.
Interactions between beta-lactoglobulin (BLG) in its monomeric form and a wide range of aroma compounds were investigated by Fourier transform infrared (FT-IR) and 2D nuclear magnetic resonance (NMR) spectroscopies. A screening of the ligands was carried out by FT-IR through the amide I region changes of BLG upon binding. The location of two binding sites was determined by 2D NMR from the study of 10 selected ligands with different structures. All of the data suggest at least two binding behaviors as a function of the chemical class, the hydrophobicity, or the structure of the ligands. The binding of the elongated aroma compounds, such as 2-nonanone or ethyl pentanoate, within the central cavity involves residues located at the entrance of the calyx and Trp19. The binding onto the protein surface of aroma compounds that have or adopt a compact structure occurs in a site located between strand beta-G, alpha helix, and strand beta-I.  相似文献   

12.
Pulsed field gradient NMR spectroscopy was used to determine the poly(ethylene glycol) (PEG) self-diffusion coefficient (D(PEG)) as a function of NaCl concentration (C(NaCl)) and denaturation time (t(D)) in whey protein solutions and gels. D(PEG) in the gel decreased with increasing C(NaCl) concentrations and increased with increasing t(D); the increase ceased for all PEGs when the gel was fixed. This increase was more pronounced for the 82250 g/mol PEG than the 1080 g/mol PEG. Moreover, the diffusion coefficient of nonaggregated whey protein was measured and an increase for longer t(D) was also observed. Scanning electron microscopy images and (1)H spectra demonstrated that D(PEG) were related to the structure changes and to the percentage of beta-lactoglobulin denaturation.  相似文献   

13.
Functional properties of whey protein concentrates (WPC) are primarily dependent on the degree of denaturation of beta-lactoglobulin (beta-LG), the major globular whey protein. Irreversible modifications in the tertiary structure and association state of beta-LG after heat treatment were studied by partition in aqueous two-phase systems and fluorescence quenching. Partitioning of preheated beta-LG in two-phase systems containing 5% (w/w) poly(ethylene glycol) and 7% (w/w) dextran, between pH 6.0 and7.0, are appropriately related with the intensity of heat treatment. An increase in the partition coefficient of beta-LG was observed with increasing temperature of heat treatment. On the other hand, fluorescence quenching of beta-LG by acrylamide was used to study the conformational flexibility of the protein at pH values between 4. 0 and 9.0. The values of bimolecular quenching rate constant (k(q)) obtained showed that beta-LG appears to be more flexible at high pH values, while at low pH the protein assumes a more compact form. The efficiency of acrylamide quenching on preheated beta-LG was substantially more pronounced than for the untreated protein. This difference can be ascribed to the presence of unfolded monomers and aggregates of denatured molecules formed after heat treatment, whose tryptophanyl residues are more exposed to the solvent. In conclusion, the results suggest that partition studies in aqueous two-phase systems and fluorescence quenching are very useful tools to detect changes in conformation and aggregation of beta-LG induced by heat treatment.  相似文献   

14.
Helianthinin, the main storage protein of sunflowers, has low water solubility and does not form a gel when heated; this behavior is different from other 11S globulins and limits its food applications. To understand this particular behavior, changes on helianthinin association-dissociation state induced by modifications in pH and ionic strength were analyzed. The influence of these different medium conditions on its thermal stability and tendency to form aggregates was also studied. Helianthinin behavior at different pH values and ionic strengths is similar to other 11S globulins except that it remains in a trimeric form at pH 11. Helianthinin thermal stability is higher than other 11S globulins but is lower than oat 11S globulin. Alkaline pH produces a 10 degrees C decrease of its denaturation temperature and also of the cooperativity of denaturation process, but it does not affect the denaturation activation energy. The decrease in thermal stability with the pH increase is also manifested by its tendency to form aggregates by SH/SS interchange reactions. When thermal treatments at alkaline pH are performed, all helianthinin subunits form aggregates, characterized by a higher proportion of beta-polypeptides than alpha-polypeptides, which is an indication that aggregation is accompanied by dissociation. Treatments at 80 degrees C are sufficient to induce aggregation but not to produce denaturation, and in these conditions hexameric forms remain after the treatment.  相似文献   

15.
The properties of new and aged glycerol-plasticized vital wheat gluten films containing < or =4.5 wt % natural or quaternary ammonium salt modified montmorillonite clay were investigated. The films were cast from pH 4 or pH 11 ethanol/water solutions. The films, aged for < or =120 days, were characterized by tensile testing, X-ray diffraction, and transmission electron microscopy. In addition, water vapor permeability (11% relative humidity) and the content of volatile components were measured. The large reduction in the water vapor permeability with respect to the pristine polymer suggests that the clay platelets were evenly distributed within the films and oriented preferably with the platelet long axis parallel to the film surface. The film prepared from pH 11 solution containing natural clay was, as revealed by transmission electron microscopy and X-ray diffraction, almost completely exfoliated. This film was consequently also the strongest, the stiffest, and the most brittle and, together with the pH 11 film containing modified clay, it also showed the greatest decrease in water vapor permeability. The large blocking effect of the clay had no effect on the aging kinetics of the films. During aging, the pH 4 and pH 11 film strength and the pH 4 film stiffness increased and the pH 4 film ductility decreased at the same rate with or without clay. This suggests that the aging was not diffusion rate limited, that is, that the loss of volatile components or the migration of glycerol or glycerol/wheat gluten phase separation was not limited by diffusion kinetics. The aging rate seemed to be determined by slow structural changes, possibly involving protein denaturation and aggregation processes.  相似文献   

16.
The self-diffusion coefficient of water in soybean protein dispersion and tofu was measured by pulsed field gradient (PFG) NMR. A soy protein isolate (SPI) dispersion (6 and 12%, w/w) in water, calcium cross-linked precipitate, and tofu were used for comparison. The self-diffusion coefficient of water (D) in the SPI dispersion, 2.23 x 10(-9) m2/s, was estimated lower than that of free water, 2.6 x 10(-9) m2/s at 25 degrees C, and decreased as the SPI concentration increased. It further decreased by the addition of calcium chloride, reflecting the obstruction effect induced by the precipitates in addition to the hydration and hydrodynamic interaction in the protein dispersion. The two water regions in tofu were interpreted by the two-site K?rger model: D1 and D2 of soft tofu were 2.26 (+/-0.11) x 10(-9) and 6.84 (+/-0.34) x 10(-11) m2/s, respectively. The relative amount of proton (water) was p1 = 0.98 and p2 = 0.02 at 100 ms of diffusion time. The self-diffusion coefficients of water decreased in pressed tofu, and their relative amounts of water changed to p1 = 0.93 and p2 = 0.07. It was suggested that D1 corresponded to obstructed water in the network structure and D2 corresponded to hydrated water on the surface layer of pores formed in the protein network of tofu. The pore sizes estimated from the diffusion length of obstructed water were 21.3 microm in soft tofu and 20.8 microm in pressed tofu. The removal of fat from pressed tofu led to a decrease in D2 from 6.26 (+/-0.31) x 10(-11) to 3.53 (+/-0.18) x 10(-11) m2/s, and the relative amount of hydrated water increased from 0.07 to 0.14, which indicated hydrophobic hydration.  相似文献   

17.
Fourier transform infrared (FTIR) spectroscopy and differential scanning calorimetry (DSC) were used to study changes in the conformation of globulin from common buckwheat (Fagopyrum esculentum Moench) (BWG) under various environmental conditions. The IR spectrum of the native BWG showed several major bands from 1691 to 1636 cm(-1) in the amide I' region, and the secondary structure composition was estimated as 34.5% beta-sheets, 20.0% beta-turns, 16.0% alpha-helices, and 14.4% random coils. Highly acidic and alkaline pH conditions induced decreases in beta-sheet and alpha-helical contents, as well as in denaturation temperature (Td) and enthalpy of denaturation (DeltaH), as shown in the DSC thermograms. Addition of chaotropic salts (1.0 M) caused progressive decreases in ordered structures and thermal stability following the lyotropic series of anions. The presence of several protein structure perturbants also led to changes in IR band intensities and DSC thermal stabilities, suggesting protein unfolding. Intermolecular antiparallel beta-sheet (1620 and 1681 cm(-1)) band intensities started to increase when BWG was heated to 90 degrees C, suggesting the initiation of protein aggregation. Increasing the time of the preheat treatment (at 100 degrees C) caused progressive increases in Td and pronounced decreases in DeltaH, suggesting partial denaturation and reassociation of protein molecules.  相似文献   

18.
The effect of protein oxovanadium(V) ion concentration and pH on the ratio of diffusion current (id/id0) was studied in vanadium(V) ovalbumin-S and denatured ovalbumin systems. In both the cases marked decrease in diffusion current was observed at the respective pH values, indicating that binding takes place with cationic groups of the proteins. The binding sites (n) were found to be pH dependent. The uniformity of logK and ΔG 0 value at all pH values indicated the involvement of same sites in interaction. Furthermore, the linear scatchard plots in both the systems supported the involvement of single class of independent sites in oxovanadium(V) anion interaction. The difference in binding sites (n) has been attributed to the folded structure of ovalbumin-S while unfolded one of denatured ovalbumin.  相似文献   

19.
The reactions of Ni, Zn and Cd with goethite were studied over a range of initial metal concentrations (10−6 to 10−4M), pH values (4 to 8), reaction times (2h to 42d) and temperatures (5 to 35°C). The adsorption of metals increased with pH, reaction time and temperature. Adsorption of Ni increased relative to Zn and Cd with increasing time and temperature. The initially rapid adsorption of metals within a few hours was followed by a much slower reaction linearly related to time1/2, interpreted as diffusion–controlled penetration of goethite. The pH–dependent relative diffusion rates (Ni > Zn > Cd) were influenced by both affinity for goethite surfaces and by ionic radius. Diffusion coefficients of the three metals ranged from about 10−19to 10−20cm2s−1. The corresponding activation energies of diffusion were also calculated (Ni 35, Cd 55, Zn 90 kJ mol−1). Our view about the reactions of heavy metals with goethite involves (i) adsorption of metals on external surfaces, (ii) solid–state diffusion of metals from external to internal binding sites, and (iii) metal binding and fixation at positions inside the goethite particles. The general parameters of these processes are related to the hydrolytic properties (p K values) and the ionic radii of the metals. The results show that goethite may be an efficient sink for trace metals.  相似文献   

20.
Previous studies have provided unequivocal evidence for the symmetry of beta-endosulfan and the corresponding asymmetry of alpha-endosulfan; the conversion of beta-endosulfan to alpha-endosulfan was identified. In this study, evidence from differential scanning calorimetry (DSC) and nuclear magnetic resonance (NMR) experiments combined with computational chemistry calculations was used to propose a molecular mechanism for the corresponding conformational changes that occur in this process. DSC and NMR data of mixtures indicated that both isomers can influence the conformer populations in the solid, solution, and vapor phase. Computational chemistry demonstrated that the relative S==O configuration between alpha- and beta-isomers can be the intermediate state through which the conformations of alpha- and beta-isomers affect each other. Furthermore, calculations for mixtures indicated that the asymmetrical conformation of the sulfite in alpha-endosulfan can induce asymmetry in beta-endosulfan, and conversion to alpha-endosulfan occurs from this transition state.  相似文献   

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