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1.
The influence of type of fluorescent probe on the surface hydrophobicity values determined for three native and heated proteins was assessed using uncharged [6-propionyl-2-(N, N-dimethylamino)naphthalene or PRODAN] versus anionic aliphatic (cis-parinaric acid or CPA) and aromatic (1-anilinonaphthalene-8-sulfonic acid or ANS) probes. Surface hydrophobicities of whey protein isolate, beta-lactoglobulin, and bovine serum albumin under heated (80 degrees C for 30 min) and unheated conditions and at varying pH values (3.0, 5.0, 7.0, and 9. 0) were measured using ANS, CPA, and PRODAN. ANS and CPA yielded opposing results for the effects of pH and heating on protein hydrophobicity. Hydrophobicity was lower at pH 3.0 than at other pH values for all proteins measured by PRODAN, whereas the values measured by ANS and CPA at pH 3.0 were quite high compared to those at other pH values, suggesting the influence of electrostatic interactions on anionic probe-protein binding. These results suggest that the presence or absence of a permanent charge as well as the aromatic and aliphatic nature of fluorescent probes can affect protein hydrophobicity values measured under various pH conditions.  相似文献   

2.
Surface hydrophobicity of whey protein concentrate (WPC) under heated (85 degrees C for 5, 10, 20, 30, 40, and 60 min) and unheated conditions was measured using cis-parinaric acid (CPA), 1-anilino-8-naphthalenesulfonate (ANS), and a fluorescence quenching method using acrylamide as a quencher. This last method evaluates the degree of exposure of tryptophanyl residues in proteins to the solvent. The initial slope of Stern-Volmer plots, K(app), was used as an index of protein hydrophobicity. Surface hydrophobicity of WPC exhibited good relation with surface functional properties such as emulsifying and foaming. Analysis of the data obtained in this work showed that the fluorescence quenching method gave results similar to those obtained using CPA and ANS. Therefore, this simple technique is satisfactory in effectively obtaining information about the hydrophobicity of whey proteins.  相似文献   

3.
Prolamin extracted from rice flour using 55% n-propanol contained protein impurities. Reverse phase high-performance liquid chromatography (HPLC) on a perfusion column R2/H was used to separate rice prolamin from other proteins in less than 5 min. Prolamin eluted as the major peak. The isolated prolamin migrated as a single band on sodium dodecyl sulfate polyacrylamide gel electrophoresis using a 4-12% Bis-Tris gel. Matrix-assisted laser desorption ionization mass spectrometry identified the rice prolamin as a 15 013 Da protein. The surface hydrophobicity (S(o)) of the HPLC-separated protein fractions was measured using the hydrophobic fluorescent probe PRODAN. A comparison was made with the surface hydrophobicity (S(o)) of corn prolamin and bovine serum albumin. Surface hydrophobicity values and solubility in 90% ethanol assisted in rice prolamin identification from other chromatographic peaks. The advantage of perfusion chromatography in purifying rice prolamin from other rice proteins included the reduced separation time, the speed at which the separation was carried, and the ability to regenerate the column in a short period of time and allow for more samples to be purified and separated.  相似文献   

4.
The effects of gamma-irradiation on starch gels were characterized at the molecular level by Fourier transform (FT) Raman spectroscopy. Starches from five different sources were gelatinized and irradiated at 3, 5, and 10 kGy using a Co60 gamma-irradiator. Gamma-irradiation effects on starch gels were noted by the C-H stretch (2800-3000 cm(-1)) and O-H stretch (3000-3600 cm(-1)) and bend (1600-1800 cm(-1)) regions of the FT-Raman spectra. FT-Raman molecular fingerprints obtained through spectral analyses were used for discrimination of the gels based on the extent of irradiation by means of two different pattern-recognition techniques: canonical variate analysis (CVA) and soft modeling of class analogy (SIMCA). A complete discrimination of irradiated starches was attained using a hybrid partial least-squares (PLS) and CVA model, using the spectral variations in the C-H stretch and O-H stretch and bend regions of FT-Raman spectra. Using the same spectral regions, SIMCA predicted 84% of samples correctly.  相似文献   

5.
Near-infrared Fourier transform Raman (FT-Raman) spectroscopy was employed to study the molecular structure of edible zein films/coatings, which were fabricated directly from zein protein. The secondary structure of zein protein was mainly in alpha-helix and remained unaltered during film formation as evidenced by the vibrational modes of amide I at 1656 cm(-1) and amide III at 1274 cm(-1). Raman results indicated that hydrophobic interaction played an important role in the formation of zein film and disulfide bonding might be responsible for the structural stability of zein protein during film formation. To enhance its antimicrobial property, an antimicrobial zein film was manufactured by incorporating zein protein with benzoic acid whose structure was then characterized by FT-Raman. It showed that physical entrapment or hydrophobic interaction was crucial to the incorporation of benzoic acid with zein protein, and the secondary structure of the antimicrobial film was still maintained in alpha-helical form. In addition, FT-Raman exhibits its preference in directly determining the thickness of zein films/coatings. By correlating the Raman intensity ratio of nu(1003) to nu(84) (I(1003/84)) versus the thickness of zein film, a linear relationship with high coefficient (R(2) = 0.9927) was obtained, which was then used pragmatically to determine the thickness of zein coatings on apple. It showed that the FT-Raman result (thickness = 0.27 +/- 0.01 mm) was consistent with that of classical micrometric measurement (thickness = 0.28 +/- 0.02 mm). Consequently, FT-Raman provides a direct, simple, and reagent-free method to characterize the structure and the thickness of zein films/coatings.  相似文献   

6.
Fourier transform infrared (FTIR) and Fourier transform Raman (FT-Raman) methods were used for rapid characterization and classification of selected irradiated starch samples. Biochemical changes due to irradiation were detected using the two vibrational spectroscopic techniques, and canonical variate analysis (CVA) was applied to the spectral data for discriminating starch samples based on the extent of irradiation. The O-H (3000-3600 cm(-1)) stretch, C-H (2800-3000 cm(-1)) stretch, the skeletal mode vibration of the glycosidic linkage (900-950 cm(-1)) in both Raman and infrared spectra, and the infrared band of water adsorbed in the amorphous parts of starches (1550-1750 cm(-1)) were employed in classification analysis of irradiated starches. Spectral data related to water adsorbed in the noncrystalline regions of starches provided a better classification of irradiated starches with 5 partial least-squares (PLS) factors in the multivariate model.  相似文献   

7.
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   

8.
任姮烨  司炳成  李敏  胡优 《土壤学报》2021,58(2):391-400
热脉冲双探针技术被广泛应用于土壤热参数和含水率的测定.然而长度为2.8 cm的常规热脉冲双探针空间测试范围太小,加长探针可增加测定结果的代表性,但容易出现探针倾斜(探针间距改变),进而影响测量精度.设计制作了长度为10 cm的热脉冲双探针,通过室内土柱试验测定了四种倾斜方式(共面外倾、非共面外倾、共面内倾以及非共面内倾...  相似文献   

9.
为探究脉冲强光(IPL)对果蔬内源酶活性的抑制效果及相关机理,采用分光光度法研究了IPL处理对多酚氧化酶(PPO)活性的影响,并通过ANS荧光探针法、内源荧光光谱法、凝胶电泳及圆二色(CD)光谱等方法探讨了不同能量IPL处理对PPO结构特性的影响。结果表明,PPO活性随着IPL单次脉冲能量、脉冲次数的增加及脉冲板间距的减小而降低。IPL处理后,PPO表面疏水性及游离巯基含量上升,色氨酸荧光强度下降,最佳发射波长红移,α-螺旋含量下降,β型结构含量上升。同时,凝胶电泳结果显示,PPO蛋白氧化降解。综上,IPL处理可改变PPO二级和三级结构,促使蛋白氧化变性,酶活性下降。本研究结果为IPL技术在抑制食品中内源酶活性及其相关机理的研究提供了一定的理论依据。  相似文献   

10.
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   

11.
Peptide nucleic acid (PNA) microarrays for the detection of Roundup Ready soybeans in food have been prepared. PNA probes are known to be more efficient and selective in binding DNA sequences than the analogous oligonucleotides and are very suitable to be used for diagnostics in food. PNAs of different lengths were carefully designed and synthesized by solid-phase synthesis on an automatic synthesizer adopting the BOC strategy. PNAs were purified by HPLC and characterized by HPLC/MS. The probes were spotted on a functionalized surface to produce a microarray to be hybridized with PCR products. DNA extracted from reference material was amplified using Cy3- and Cy5-labeled primers, and the fluorescent PCR products obtained were hybridized on the microarray. Two protocols were adopted: the hybridization with dsDNA or with ssDNA obtained by digestion with the enzyme lambda exonuclease. The best results were obtained using a 15-mer PNA probe in combination with the ssPCR product derived from enzymatic digestion. The method was applied to the analysis of a sample of certified transgenic soybean flour.  相似文献   

12.
A simple and rapid method for the quantitative determination of four major components found in oregano and thyme essential oils is presented. The method correlates the Raman peak intensity in the spectral region from 1800 to 600 cm(-1) and the concentration percentage of each particular constituent in the sample. To achieve accurate quantification results and avoid the risk of overlapping peaks of unknown Raman-active substances in natural essential oils, the peaks must be analyzed. For this purpose, PEAKSOLVE software (Ver. 1.0.5) was used. Unknown samples were measured with the FT-Raman method, and the results were compared to those of the gas chromatographic (GC) analysis. The comparison was made at a confidence level of 99%, and the two methods scored equally in terms of repeatability and accuracy even at the edge of the method specifications. The new method can provide accurate results in very short times once the setup is complete and could be utilized in areas where vast amounts of samples must be analyzed.  相似文献   

13.
The determination of conjugated linoleic acids (CLA) in cow milk fat was studied by using UV (210-250 nm) and Fourier transform (FT)-Raman (900-3400 cm (-1)) spectroscopy in order to determine the best spectrophotometric technique for routine analysis of milk fat. A collection of 57 milk fat samples was randomly divided into two sets, a calibration set and a validation set, representing two-thirds and one-third of the samples, respectively. All calculations were performed on the calibration set and then applied to the validation set. The CLA content ranged from 0.56 to 4.70%. A comparison of various spectral pretreatments and different multivariate calibration techniques, such as partial least-squares (PLS) and multiple linear regression (MLR), was done. This paper shows that UV spectroscopy is as reliable as FT-Raman spectroscopy to monitor CLA in cow milk fat. The best calibration for FT-Raman was given by a PLS model of seven factors with a standard error of prediction (SEP) of 0.246. For UV spectroscopy, PLS models were also better than MLR models. The most robust PLS model was constructed with only one factor and with SEP=0.288.  相似文献   

14.
Structural modifications of ovalbumin, ovotransferrin, and lysozyme at the air-water interface have been investigated using SDS-PAGE, both intrinsic and ANS fluorometry, and circular dichroism experiments. Ovalbumin contact with an interface induced an exposure of aromatic residues, a slight decrease in alpha-helix structures (-1.7%), and an increase in both beta-sheet (+3.4%) and beta-turn (+7.9%) structures. Moreover, these conformational changes led to the formation of insoluble polymers of ovalbumin through intermolecular disulfide bonds. Ovotransferrin contact with an interface led to an increase in its surface hydrophobicity (+30%) and modifications of its secondary structure (-33% of alpha-helices, +96.4% of beta-sheets, +13.2% of beta-turns, and +21.2% of random coils), characteristic of major conformational changes. On the other hand, lysozyme did not undergo any structural modification. These results clearly underscore that at the air-water interface proteins are susceptible to denaturation.  相似文献   

15.
Lysyl residues of rapeseed napin (2S) and cruciferin (12S) were acylated and sulfamidated by means of anhydrides and sulfonyl chlorides, respectively. The secondary and tertiary structures as well as the surface hydrophobicity of the modified proteins were studied using circular dichroism, intrinsic fluorescence, and binding of anilinonaphthalenesulfonic acid. The results showed clearly that grafting of hydrophobic chains induced different structural modifications and surface hydrophobicities on the monomeric (2S) and on the hexameric (12S) proteins. Thus, the original structure of the 2S modified protein seemed to be preserved. Therefore, the surface hydrophobicity increased proportionally with the number of groups grafted. Conversely, after modification, 12S was shown to be expanded. As a result, hydrophobic regions were exposed, leading to a much greater hydrophobization of the protein surface. Acylation and sulfamidation appeared, therefore, to be good methods to hydrophobize efficiently the surface of the two proteins and thus might probably induce new functional properties.  相似文献   

16.
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   

17.
The Fourier transform Raman (FT-Raman) spectra of pure terpenes and essential oils obtained by hydrodistillation of some Lamiaceae species, are presented. This study shows that principal components of an essential oil can be recognized by FT-Raman. Components predicted by FT-Raman spectrum of an essential oil correlate well with those found as major constituents by GC-MS. In this way the basic chemical character of an essential oil can be recognized. The results demonstrate that certain Raman intensities can be correlated to specific terpenes and therefore FT-Raman can discriminate between the essential oils of which main components belong to different classes of compounds.  相似文献   

18.
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   

19.
This paper presents a novel spectrofluorometric method using the novel fluorescent probe 2-(2-pyridyl)benzothiazoline for the determination of superoxide dismutase (SOD) activity. The fluorescent probe was synthesized in house and fully characterized by elemental analysis and by IR and (1)H NMR spectra. It could specially identify and trap superoxide anion radicals (O2(.-)), and then was oxidized by O2(.-) to form a strong fluorescence product. On the basis of this reaction, the spectrofluorometric method was proposed and successfully used to determine SOD activity. The proposed method has a better selectivity in the determination of reactive oxygen species, because the probe can be oxidized to afford a highly fluorescent product only by O2(.-) excluding hydrogen peroxide and hydroxyl radical. As a kind of simple, rapid, precise, and sensitive technique, it could avoid the errors caused by detection time and was applied to the measurement of SOD activity in scallion genus foods with satisfactory results.  相似文献   

20.
Calcium (Ca) is an essential plant nutrient and is important in determining quality of fruits in storage. The analytical method presently known to determine Ca concentration in vegetables measures total Ca, and there is a need to develop methods that can determine the distribution of Ca within vegetable tissues. The objective of this study was to determine the ability of fluorescent probes to visualize Ca location and distribution in vegetable tissues. We examined three protocols using fluorescent dyes developed to detect Ca. The first tested Fura 2 acetoximetil (AM) probe in fresh cuts from grapes at different combinations of temperatures and incubation times. The second protocol was developed with the aim of lowering the natural autofluorescense. In the third protocol, the Fura 2 probe was used in tissues previously fixed in formalin, acetic acid, and alcohol solution (FAA). Tissues were observed under an epifluorescence microscope and, to compare and complement the observations, under a scanning electron microscope (SEM). No sign of fluorescence was observed in fixed and fresh tissue using the Fura 2 AM. In fresh cuts, there was autofluorescent interference, provoked mainly by chlorophyll, but also from the vascular system and tannins. The Fura 2 probe was introduced to vegetable tissues by utilizing a pH of 4.5, and a more intense signal was observed in tissues with greater Ca concentration. This signal was also associated with Ca crystals, which might have had some degree of hydrolysis at low pH. Under a SEM microscope, Ca oxalate crystals were clearly observed in those tissues. Our results show that Fura 2 probe can be used to detect Ca and its distribution in vegetable tissues.  相似文献   

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