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1.
Raman spectroscopic study of oat globulin conformation   总被引：2，自引：0，他引：2  
Analysis of Raman spectra of oat globulin showed that extreme pH values caused an increase in the amide and C-H stretching band intensity, indicating changes in the secondary structures of the protein due to denaturation. Similar changes were observed when oat globulin was treated with chaotropic salts and several protein perturbants. Sodium dodecyl sulfate, beta-mercaptoethanol, and ethylene glycol also caused a shift in the amide III' band, suggesting a transition from beta-sheet to a random coil conformation. Heating at temperatures near the denaturation temperature of oat globulin led to increases in the amide and C-H band intensity, indicating unfolding of the protein. The data indicate that FT-Raman spectroscopy is suitable for studying the secondary structure of plant proteins such as oat globulin.  相似文献   

2.
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   

3.
Changes in protein secondary structure and conformation of ovalbumin and beta-lactoglobulin (15% protein w/w) were investigated by Fourier transform Raman spectroscopy and self-deconvolution. The amounts of alpha-helix, beta-sheets, random coil, and beta-turns in native beta-lactoglobulin were 15, 54, 6, and 25%, respectively, and those for ovalbumin (41, 34, 13, and 12%) compared well with published values obtained by X-ray crystallography. The proteins were heated at 90 degrees C for 30 min and high-pressure-treated at 600 MPa for 20 min. Heating increased beta-sheet structures in both proteins at the expense of alpha-helix; for beta-lactoglobulin beta-sheet structures increased from 54 to 70% and for ovalbumin, from 34 to 54%. Random coil increased from 6% in the native protein to 30% in high-pressure-treated beta-lactoglobulin. However, for ovalbumin, the contribution from beta-turns doubled in high-pressure-treated samples, with little change in random coil. Further examination of the deconvoluted amide I band in heated samples revealed several component bands. Bands at 1626 and 1682 cm(-1) for ovalbumin and at 1625 and 1680 cm(-1) for beta-lactoglobulin were observed and are associated with aggregated, intermolecular beta-sheet (beta-aggregation), indicative of heat denaturation. The band seen at 1632-1640 cm(-1) corresponded to intramolecular beta-sheet structures, whereas the band at 1625 cm(-1) is associated with exposed beta-sheets (for example, beta-strands with strong hydrogen bonding that are not part of the core of beta-sheets). In high-pressure-treated samples bands were also observed at 1628 and 1680 cm(-1) for ovalbumin and at 1626 and 1684 cm(-1) for beta-lactoglobulin, suggesting involvement of beta-sheet structures in protein aggregation. Raman bands were observed at 1665-1670 cm(-1) for ovalbumin and at 1663-1675 cm(-1) for beta-lactoglobulin due to random coil structures. The bands at 1650-1660 cm(-1) due to alpha-helices were observed in both heated and high-pressure-treated samples. In addition, in heated samples of both ovalbumin and beta-lactoglobulin, peak intensity increased for beta-sheet in the amide III region, 980-990 cm(-1), and decreased for helix structures (900-960 cm(-1)). In contrast, there was no peak at 1240 cm(-1) (amide III beta-sheet structures) in either high-pressure-treated ovalbumin or beta-lactoglobulin, suggesting that high-pressure denaturation at 600 MPa for 20 min is less extensive than heat denaturation at 90 degrees C for 30 min.  相似文献   

4.
Oat globulin was modified by a calcium-independent microbial transglutaminase (TG). The TG-polymerized protein had higher solubility than the control at acidic pH and had improved water- and fat-binding properties. Incubation of 10% (w/v) oat globulin dispersions in the presence of TG at 37 degrees C led to the formation of a well-developed viscoelastic gel network with a microstructure characterized by thick strands and large clusters. The TG-induced gels had higher modulus values, lower loss tangent values, and lower frequency dependency than the heat-induced gels. The TG-induced gel system has the characteristics of classical polymer gel with permanent "chemical" cross-links, whereas the heat-denatured system has the characteristics of a temporary "physical" gel with breakable cross-links. Fourier transform infrared spectroscopy showed marked shift and intensity changes in several major bands, suggesting pronounced changes in protein conformation during TG-induced gelation. Aggregation of protein molecules was also indicated by the progressive increases in two infrared bands (1679-1682 and 1622-1625 cm(-)(1)) associated with the formation of intermolecular beta-sheets and strands. Results suggest that new food polymers with unique functionality can be produced from oat globulin treated with TG and that elastic gels can be formed near neutral pH, instead of the alkaline pH required for thermally induced oat globulin gels.  相似文献   

5.
Studying the secondary structure of proteins leads to an understanding of the components that make up a whole protein. An understanding of the structure of the whole protein is often vital to understanding its digestive behavior in animals and nutritive quality. Usually protein secondary structures include alpha-helix and beta-sheet. The percentages of these two structures in protein secondary structures may influence feed protein quality and digestive behavior. Feathers are widely available as a potential protein supplement. They are very high in protein (84%), but the digestibility of the protein is very low (5%). The objective of this study was to use synchrotron-based Fourier transform infrared (FTIR) microspectroscopy to reveal chemical features of feather protein secondary structure within amide I at ultraspatial resolution (pixel size = 10 x 10 microm), in comparison with other protein sources from easily digested feeds such as barley, oat, and wheat tissue at endosperm regions (without destruction of their inherent structure). This experiment was performed at beamline U2B of the Albert Einstein Center for Synchrotron Biosciences at the National Synchrotron Light Source (NSLS) in Brookhaven National Laboratory (BNL), U.S. Dept of Energy (NSLS-BNL, Upton, NY). The results showed that ultraspatially resolved chemical imaging of feed protein secondary structure in terms of beta-sheet to alpha-helix peak height ratio by stepping in pixel-sized increments was obtained. Using synchrotron FTIR microspectroscopy can distinguish structures of protein amide I among the different feed protein sources. The results show that the secondary structure of feather protein differed from those of other feed protein sources in terms of the line-shape and position of amide I. The feather protein amide I peaked at approximately 1630 cm(-1). However, other feed protein sources showed a peak at approximately 1650 cm(-1). By using multicomponent peak modeling, the relatively quantitative amounts of alpha-helix and beta-sheet in protein secondary structure were obtained, which showed that feather contains 88% beta-sheet and 4% alpha-helix, barley contains 17% beta-sheet and 71% alpha-helix, oat contains 2% beta-sheet and 92% alpha-helix, and wheat contains 42% beta-sheet and 50% alpha-helix. The difference in percentage of protein secondary structure may be part of the reason for different feed protein digestive behaviors. These results demonstrate the potential of highly spatially resolved infrared microspectroscopy to reveal feed protein secondary structure. Information from this study by the infrared probing of feed protein secondary structure may be valuable as a guide for feed breeders to improve and maintain protein quality for animal use.  相似文献   

6.
Oat globulin was polymerized by a microbial transglutaminase (TG), and some physicochemical and functional properties of polymers were studied. Reversed-phase HPLC revealed that the number of epsilon-(gamma-glutamyl) lysine isopeptide bonds formed after 4 h of enzyme incubation was 2.21 micromol/g of protein. SDS-PAGE showed that the oat globulin acidic polypeptides (AP) were more susceptible to polymerization than the basic polypeptides (BP), and the reactivities of both AP and BP were enhanced by the addition of other substrate proteins. Differential scanning calorimetry showed that both the denaturation temperature and denaturation enthalpy were decreased after TG treatment. Fourier transform infrared spectroscopy revealed marked increases in the intensity of two intermolecular beta-sheet bands associated with aggregate formation but little conformational changes in the polymerized protein. TG incubation led to progressive changes in flow properties of oat globulin dispersions, indicating enhanced pseudoplasticity and increased viscosity and yield stress.  相似文献   

7.
Fourier transform horizontal attenuated total reflectance (FT-HATR) was used to examine changes in the secondary structure of gluten proteins in a flour-water dough system during mixing. Midinfrared spectra of mixed dough revealed changes in four bands in the amide III region associated with secondary structure in proteins: 1317 (alpha-helix), 1285 (beta-turn), 1265 (random coil), and 1242 cm (-1) (beta-sheet). The largest band, which also showed the greatest change in second derivative band area (SDBA) during mixing, was located at 1242 cm (-1). The bands at 1317 and 1285 cm (-1) also showed an increase in SDBA over time. Conversely, the band at 1265 cm (-1) showed a corresponding decrease over time as the doughs were mixed. All bands reached an optimum corresponding to the minimum mobility of the dough as determined by the mixograph. Increases in alpha-helix, beta-turn, and beta-sheet secondary structures during mixing suggest that the dough proteins assume a more ordered conformation. These results demonstrate that it is possible, using infrared spectroscopic techniques, to relate the rheological behavior of developing dough in a mixograph directly to changes in the structure of the gluten protein system.  相似文献   

8.
The potential of near-infrared (NIR) spectroscopy in detailed food analysis was tested in a model system consisting of gluten powder treated with moisture and heat. Second-derivative transformation and extended multiplicative signal correction were applied for improving the band resolution and removing physical and quantitative spectral variations. Subsequent chemometric analyses gave loading spectra, which were interpreted as spectral effects of altered protein structures, induced by the treatments. Moistening of the gluten powder resulted in shifts and intensity changes in the protein bands, which could be explained by a combination of minor secondary structure changes, water binding, and changed microenvironments of the amino acid side chains. Heat denaturation induced increases at 2209 nm and decreases at 2167-2182 nm, indicating an alpha-helix to beta-sheet transformation, in agreement with the expectations.  相似文献   

9.
Pressure-assisted thermal processing (PATP) is being widely investigated for processing low acid foods. However, its microbial safety has not been well established and the mechanism of inactivation of pathogens and spores is not well understood. Fourier transform infrared (FT-IR) spectroscopy was used to study some of the biochemical changes in bacterial spores occurring during PATP and thermal processing (TP). Spore suspensions (approximately 10(9) CFU/mL of water) of Clostridium tyrobutyricum, Bacillus sphaericus, and three strains of Bacillus amyloliquefaciens were treated by PATP (121 degrees C and 700 MPa) for 0, 10, 20, and 30 s and TP (121 degrees C) for 0, 10, 20, and 30 s. Treated and untreated spore suspensions were analyzed using FT-IR in the mid-infrared region (4000-800 cm(-1)). Multivariate classification models based on soft independent modeling of class analogy (SIMCA) were developed using second derivative-transformed spectra. The spores could be differentiated up to the strain level due to differences in their biochemical composition, especially dipicolinic acid (DPA) and secondary structure of proteins. During PATP changes in alpha-helix and beta-sheets of secondary protein were evident in the spectral regions 1655 and 1626 cm(-1), respectively. Infrared absorption bands from DPA (1281, 1378, 1440, and 1568 cm(-1)) decreased significantly during the initial stages of PATP, indicating release of DPA. During TP changes were evident in the bands associated with secondary proteins. DPA bands showed little or no change during TP. A correlation was found between the spore's Ca-DPA content and its resistance to PATP. FT-IR spectroscopy could classify different strains of bacterial spores and determine some of the changes occurring during spore inactivation by PATP and TP. Furthermore, this technique shows great promise for rapid screening PATP-resistant bacterial spores.  相似文献   

10.
Lipase inactivation is an essential treatment for oat processing, because of the negative effects of lipase on nutrient preservation and storage extension. The effects of different lipase inactivation treatments including hot air roasting, infrared roasting, normal‐pressure steaming, and high‐pressure steaming on the physicochemical properties of oat globulins were investigated. Results showed that normal‐pressure steaming had little effect on solubility of oat globulins; hot air roasting increased foaming capacity of oat globulins but did not change their foaming stability; and all the inactivation treatments increased the surface hydrophobicity and content of total sulfhydryls of oat globulin but decreased exposed sulfhydryl groups. In addition, oat globulin granules from the hot air roasting treatment were distributed more evenly in oat globulin powder compared with the control group. All treatments except normal‐pressure steaming changed the molecular weight of oat globulin subunits, which made the bands of 66,000 and 45,000 disappear from SDS‐PAGE. These results indicated that normal‐pressure steaming was ideal to maintain good solubility of oat globulins, and hot air roasting was ideal to maintain relatively good foaming properties. The treatments changed physicochemical properties of oat globulins by influencing protein aggregation and subunit composition that resulted in different content of sulfhydryl groups and surface hydrophobicity.  相似文献   

11.
Raman spectroscopy was used to elucidate structural changes of beta-lactoglobulin (BLG), whey protein isolate (WPI), and bovine serum albumin (BSA), at 15% concentration, as a function of pH (5.0, 7.0, and 9.0), heating (80 degrees C, 30 min), and presence of 0.24% kappa-carrageenan. Three data-processing techniques were used to assist in identifying significant changes in Raman spectral data. Analysis of variance showed that of 12 characteristics examined in the Raman spectra, only a few were significantly affected by pH, heating, kappa-carrageenan, and their interactions. These included amide I (1658 cm(-1)) for WPI and BLG, alpha-helix for BLG and BSA, beta-sheet for BSA, CH stretching (2880 cm(-1)) for BLG and BSA, and CH stretching (2930 cm(-1)) for BSA. Principal component analysis reduced dimensionality of the characteristics. Heating and its interaction with kappa-carrageenan were identified as the most influential in overall structure of the whey proteins, using principal component similarity analysis.  相似文献   

12.
Low-field NMR T(2) and Fourier transform infrared (FT-IR) measurements were performed on meat samples of two qualities (normal and high ultimate pH) during cooking from 28 degrees C to 81 degrees C. Pronounced changes in both T(2) relaxation data and FT-IR spectroscopic data were observed during cooking, revealing severe changes in the water properties and structural organization of proteins. The FT-IR data revealed major changes in bands in the amide I region (1700-1600 cm(-)(1)), and a tentative assignment of these is discussed. Distributed NMR T(2) relaxation data and FT-IR spectra were compared by partial least-squares regression. This revealed a correlation between the FT-IR peaks reflecting beta-sheet and alpha-helix structures and the NMR relaxation populations reflecting hydration water (T(2B) approximately 0-10 ms), myofibrillar water (T(21) approximately 35-50 ms), and also expelled "bulk" water (T(2) relaxation times >1000 ms). Accordingly, the present study demonstrates that definite structural changes in proteins during cooking of meat are associated with simultaneous alterations in the chemical-physical properties of the water within the meat.  相似文献   

13.
Lysozyme (25% in D2O, corn oil, and their emulsions (10% w/w oil/D2O solution) were examined by Fourier transform Raman spectroscopy. Emulsions showed three layers, namely, top oil, middle cream, and bottom aqueous layers. Raman spectral analysis revealed hydrophobic interactions involving both protein and lipid components. Compared to lysozyme in D2O, the difference spectrum obtained after subtraction of oil from the cream layer spectrum showed reduced intensity of tryptophan bands at 760, 1013, 1340, and 1360 cm(-)(1), reduced intensity ratio of the tyrosine doublet at 850 and 830 cm(-)(1), and increased intensity of the C-H bending band at 1455 cm(-)(1). Compared to corn oil, the difference spectrum after subtraction of lysozyme from the cream layer spectrum indicated decreased intensity at 2855 cm(-)(1) (lipid CH(2) symmetric stretch) and 3011 cm(-)(1) (unsaturated fatty acid hydrocarbon chain =C-H stretch) and a higher intensity ratio of the C-H stretching band at 2900 cm(-)(1) to bands at 2885 and 2933 cm(-)(1). Spectra of the top and bottom layers resembled corn oil and lysozyme, respectively, except for changes in the D2O band. Raman spectroscopy can be used to detect structural changes in proteins, lipids, and D2O due to protein-lipid interactions.  相似文献   

14.
The conformational changes of whey proteins upon adsorption at the soy oil/water interface were investigated using Fourier transform infrared (FT-IR) spectroscopy. Significant changes were observed in the bands assigned to beta-sheets and alpha-helix structures following the adsorption of proteins at the oil/water interface. The remaining interfacial proteins after Tween 20 desorption revealed small changes in beta-sheet and alpha-helical structures, whereas in the desorbed whey proteins the unordered structures largely increased, and beta-sheet structures almost disappeared. These FT-IR results provide important knowledge about the conformational modifications in whey proteins occurring upon adsorption at the oil/water interface. Finally, specific conformational changes are necessary to stabilize emulsions: adsorption-induced unfolding, increase in alpha-helical structures to establish interactions with the oil phase, and aggregation between adsorbed whey proteins to form protein membranes. Moreover, the structural changes in whey protein adsorbed at the oil/water interface under high-pressure homogenization are irreversible.  相似文献   

15.
Raman spectra of amino acids showed complexity in the C-H stretching region (2800-3100 cm(-)(1)) attributed to diversity of CH, CH(2), and CH(3) groups in the side chains, ionization state, and microenvironment. The involvement of specific amino acids in the C-H stretching region of selected proteins, namely, lysozyme, alpha-lactalbumin, beta-lactoglobulin, and their binary mixtures, was investigated by deconvolution using maximum likelihood techniques. The main protein band near 2940 cm(-)(1) was attributed not only to aromatic and aliphatic amino acids but also to many other amino acids. A band near 3065 cm(-)(1) was assigned to aromatic residues, whereas bands near 2880 and 2900 cm(-)(1) corresponded primarily to aliphatic amino acids. Heating at 90 degrees C increased relative intensity near 2940 cm(-)(1) and decreased relative intensity at 2895-2902 cm(-)(1) for lysozyme and its mixtures with alpha-lactalbumin or beta-lactoglobulin. Additional bands at 2812 or 2838 and 3003 cm(-)(1) were observed after heating or in 8 M deuterated urea, reflecting changes upon denaturation.  相似文献   

16.
The structural, physicochemical, and biological properties of sulfated oat beta-glucan were characterized. The degree of substitution of the sulfated oat-beta glucan was obtained by elemental analysis, which was 0.68. Compared to native oat beta-glucan, the FT-IR spectra of the derivative showed two new absorption bands at 1250 and 810 cm(-)(1), which would be attributed to (S=O) and (C-O-S) groups, respectively. The molecular weight of the sulfated beta-glucan was determined to be 68 kDa and its viscosity decreased by almost 2 orders of magnitude while its solubility increased by more than 100% compared to that of the native beta-glucan. In addition, the sulfation caused the reduction of in vitro bile acid binding capacity of oat beta-glucan due to the new anionic character and decreased molecular weight. The sulfated derivative exhibited, however, anticoagulant activity which showed a concentration-dependent increase.  相似文献   

17.
Heating with microwave energy and tannin complexation of kafirin both increase the tensile strength of cast kafirin bioplastic films. The effects of these treatments on the molecular structure of kafirin and of kafirin in the film were investigated. SDS-PAGE of heated wet kafirin showed an increase in kafirin oligomers. Disulfide groups increased in heated kafirin and in films made from the heated kafirin. Fourier transform infrared (FTIR) spectroscopy of heated kafirin and films made from the heated kafirin indicated an increase in beta-sheet conformation. In contrast, kafirin complexation with tannic acid (TA) and sorghum condensed tannin (SCT) resulted in a slight decrease in beta-sheet conformation in the kafirin and a larger decrease in the kafirin in the films. Raman spectroscopy showed that, with TA, there was a shift in peak from 1710 to 1728 cm(-1) for kafirin-tannic acid complexes, indicating kafirin and tannic acid interaction. The protein conformational changes presumably facilitated cross-linking between kafirin molecules and/or between kafirin and the tannins. Thus, although both heating with microwave energy and tannin complexation cause cross-linking of kafirin to increase film tensile strength, their effects on kafirin structure appear to be different.  相似文献   

18.
Soybean glycinin groups I, IIa, and IIb were purified from soybeans composed of only glycinin groups I, IIa, and IIb, respectively. When these protein solutions were heated, the amount of the particulate protein formed in these solutions was greatest in the order of groups IIa, IIb, and I. The protein solubilities decreased upon the addition of magnesium chloride in the order of groups IIa, IIb, and I. It was determined by differential scanning calorimetry analysis that the denaturation temperatures of groups I, IIa, and IIb were 92.8, 96.0, and 97.9 degrees C, and that the enthalpies of their transitions were 24.2, 27.4, and 28.1 J g(-)(1), respectively. The alpha-helix rates of groups I, IIa, and IIb in aqueous solution were analyzed by circular dichroism and were 19, 16, and 15%, respectively. The beta-sheet rates of groups I, IIa, and IIb were 44, 38, and 39%, respectively. In all group proteins, the alpha-helix rates were decreased by heating and the beta-sheet rates were increased. The surface hydrophobicity of these group proteins increased as a result of heating, and those of groups IIa and IIb were larger than that of group I. The surface hydrophobicity of these protein groups increased by heating, and those of groups IIa and IIb were larger than that of group I and beta-conglycinin. Breaking stress of curds prepared from these group proteins containing more than 1 of beta-conglycinin ration showed similar values, but the order of those containing less than 1 in strength was groups I, IIb and IIa. These results suggest that the increase of particulate contents and the curd formation are related to the increase of surface hydrophobicity by heating.  相似文献   

19.
Phytocystatins are the plant thiol protease inhibitors involved in several reaction mechanisms of the plant system like regulation of proteolytic activity and storage of proteins. Biochemical and biophysical changes induced by fungicide SDD in phytocystatin purified from Phaseolus mungo have been investigated in terms of mass spectroscopy, Fourier transform infrared spectroscopy, and fluorescence spectroscopy, at pH 7.0, with varying fungicide concentrations (1-9 mM) and a time of incubation ranging from 2 to 8 h at 37 degrees C, with a fixed cystatin concentration (1.5 mM). Reactive oxygen species responsible for inhibitor damage were also investigated, and thiourea was found to scavenge the free radicals generated by SDD. FTIR analysis indicates a significant conformational transition from alpha-helix to beta-sheet structure; quenching of fluorescence is evident by fluorescence spectroscopy. The activity assay showed a decrease in inhibitory activity, as well as a fragmentation of the inhibitor was observed in electrophoresis. Results obtained implicate that exposure of phytocystatins to SDD involves physicochemical changes in cystatins leading to damage and a decrease in the activity of the inhibitor.  相似文献   

20.
The effects of water activity (A(w)) and lipid addition on the secondary structure of powdery zein were investigated using Fourier transform infrared spectroscopy. Two fatty acid esters, i.e., the linolenic and eicosapentaenoic acid ethyl esters (LAE and EPE), were mixed with the zein powder. The powders were stored in the "dry" state (with silica gel) and the "humid" state (A(w) = 0.9). The powdery zein without the lipids was shown to have a high content of the intermolecular hydrogen-bonded beta-sheet in the "dry" state, indicating the presence of protein aggregates. An increase in A(w) induced a decrease in this beta-sheet, concomitant with increases in the alpha-helix and beta-turn structures. The addition of LAE caused decreases in the alpha-helix and intermolecular hydrogen-bonded beta-sheet of zein when the powder was stored in the "humid" state, suggesting the strong interaction of LAE and zein molecules. However, LAE did not affect the secondary structure of zein in the "dry" state. The addition of EPE hardly influenced the secondary structure of zein, irrespective of A(w). These results are discussed in relation to the antioxidative activity of zein in the powder system, which had studied previously.  相似文献   

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