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Identification of Leguminosae gums and evaluation of carob-guar mixtures by capillary zone electrophoresis of protein extracts
Abstract
A procedure for the extraction and capillary zone electrophoresis (CZE) separation of proteins from carob, guar and tara gums in a background electrolyte (BGE) of pH 9 containing 0.1% polyvinyl alcohol is described. The CZE protein profiles exhibit characteristic peaks for each one of the Leguminosae gums, which can be used to construct models capable of identifying samples of carob, guar and tara gums, and predicting the guar content in binary carob-guar mixtures of different geographical origin and harvested in different years. The classification and prediction models are constructed by using linear discriminant analysis (LDA) and multiple linear regression (MLR), respectively. An excellent resolution between the three categories is obtained with LDA, the model being capable of classifying samples with recognition and prediction capabilities of 100%. For MLR models constructed with carob-guar mixtures with and without a common history, the average of the calibration residuals are +/- 0.50 and +/- 0.90%, respectively (average values for the 2-20% guar range). For the later model, the detection limit was 3.2% guar (from the standard deviation of 18 mixtures with 2-4% guar, and for alpha = beta = 0.05).
... Ruiz-Ángel et al. (49) separated the proteins of Leguminosae gums with capillary zone electrophoresis. The characteristic peaks of the resulting protein profiles were subjected to LDA, which was capable of correctly classifying all samples in both the calibration and prediction set in three classes, which were different types of Leguminosae gums, that is, carob, guar, and tara gum (Fig. 13.9). ...
... Projection of carob, tara, and guar gum samples on the plane of the two discriminant vectors showing the resolution between the three categories. Reproduced with permission from Ruiz-Ángel et al.(49). ...
Introduction Data pretreatment Exploratory data analysis Classification Conclusions Summary Acknowledgments References
... They differ in the ratio of mannose to galactose units, M/G. The more substituted of the commercial galactomannans is guar gum (M/Gw2:1); in tara gum, the M/G is w3:1 while in locust bean gum is w4:1 (Alais & Linden, 1991;Batlle & Tous, 1997;Mc Cleary & Neukom, 1982;Ruiz-Á ngel, Simó-Alfonso, Mongay-Fernández, & Ramis-Ramos, 2002) (Fig. 1). The great advantage of galactomannans is their ability, at relatively low concentrations, to form very viscous solutions that are only slightly affected by pH, added ions, and heat processing. ...
The rheological behaviour of aqueous galactomannan—tara gum (TG) and locust bean gum (LBG)—solutions was studied at 25°C, using steady-shear and dynamic oscillatory measurements performed with a controlled stress rheometer AR2000 (TA Instruments) fitted with cone-and-plate geometry. The intrinsic viscosity of LBG, [η]=11.03dl/g, was lower than those for crude ([η]=14.96dl/g) and purified ([η]=16.46dl/g) TG. The plot of the specific viscosity at zero shear rate vs the coil overlap parameter, C[η], revealed a similar behaviour for both galactomannans: a master curve could be obtained with a critical concentration C** (the upper limit of the semi-dilute regime) ∼7.76/[η] and a slope of 4.7 in the concentrated regime. Experimental data in steady shear (flow curves) have been correlated with the Cross and Carreau models. Both models described the apparent viscosity shear rate data well. In general, the Cross model provided a slightly better fit. In the concentrated regime, it was possible to obtain a master flow curve for both galactomannans after performing a concentration-dependent shift using the 1wt% LBG solution as the reference. Master curves were also obtained, in oscillatory shear, for G′(ω) and G″(ω) plots, by shifting along the two axes. The vertical and horizontal shift factors were the same for both G′(ω) and G″(ω) curves. Two mechanical models were used for fitting the experimental data (mechanical spectra): the generalized Maxwell model with four elements and the Friedrich–Braun model. Both models correlate the experimental data very well. The correlation between dynamic and steady-shear properties (Cox–Merz rule) was satisfactory for the two galactomannans.
Quantitative NMR has become a well-recognized and widely applied analytical tool for the quantification of very diverse classes of compounds in a large variety of samples. The advantages of quantitative NMR upon chromatography are (i) it requires almost no preparation time allowing high-throughput quantification; (ii) it is independent of chemical nature of compound of interest or the matrix in which it should be determined, therefore minimal or no sample preparation or derivatization is needed; and (iii) it is a primary ratio quantification method and therefore does not need reference compounds for quantification. Over decades, NMR has been used for the structural characterization of polysaccharides from different origin. Only the past 15 years, publications are available in which NMR is applied for the quantification of polysaccharides. This chapter shows an overview of liquid 1D and 2D methods used to determine the amount of polysaccharides in food-related samples or to quantify characteristics such as degree of polymerization and substitution degrees. These examples confirmed the versatility, robustness, and straightforwardness of qNMR as analytical tool for polysaccharide quantification. © Springer International Publishing AG, part of Springer Nature 2018.
Quantitative NMR has become a well-recognized and widely applied analytical tool for the quantification of very diverse classes of compounds in a large variety of samples. The advantages of quantitative NMR upon chromatography are (i) it requires almost no preparation time allowing high-throughput quantification; (ii) it is independent of chemical nature of compound of interest or the matrix in which it should be determined, therefore minimal or no sample preparation or derivatization is needed; and (iii) it is a primary ratio quantification method and therefore does not need reference compounds for quantification. Over decades, NMR has been used for the structural characterization of polysaccharides from different origin. Only the past 15 years, publications are available in which NMR is applied for the quantification of polysaccharides. This chapter shows an overview of liquid 1D and 2D methods used to determine the amount of polysaccharides in food-related samples or to quantify characteristics such as degree of polymerization and substitution degrees. These examples confirmed the versatility, robustness, and straightforwardness of qNMR as analytical tool for polysaccharide quantification.
This study aimed at characterizing the rheological properties and molecular weight of tara gum fractionated with ammonium sulfate. Tara gum was separated into six fractions (F1-F6) at different concentrations of ammonium sulfate, ranging from 12.21 to 28.67% (w/w). The yield of the tara gum fractions ranged between 4.98 and 17.47%, and their intrinsic viscosity ranged from 9.38 to 12.44 dL/g. The highest values of Huggins coefficient (k') and viscositymolecular mass were observed in fraction F3. The shear viscosity of the tara gum fractions was measured by a cone-plate viscometer, clearly showing shear thinning behavior. Size-exclusion chromatography results showed that the molecular weight ranged between 635.42 and 776.71 kg/mol, and the F3 fraction exhibited higher values of molecular weight.
Intact protein profiles established by CZE have been used to predict the cultivar of olive leaves and pulps. For this purpose, proteins were extracted using a mild enzyme-assisted extraction method, which provided higher protein recoveries and a lower environmental impact than other previously described methods. These extracts were subjected to CZE determination under basic conditions using a BGE composed by 50 mM phosphate, 50 mM tetraborate and 0.1% PVA at pH 9. Nine and fourteen common peaks, for leaf and pulp samples, respectively, were identified in the nine cultivars studied in this work. In addition, and using linear discriminant analysis of the CZE data, olive leaf and pulp samples belonging to nine cultivars from different Spanish regions were correctly classified with an excellent resolution among all categories, which demonstrated that intact protein profiles are characteristic of each cultivar. This article is protected by copyright. All rights reserved
A strategy for the unambiguous identification and selective quantification of xanthan gum and locust bean gum (LBG) in gelled food concentrates is presented. DNA detection by polymerase chain reaction (PCR) showed to be a fast, sensitive, and selective method that can be used as a first screening tool in intact gelled food concentrates. An efficient isolation procedure is described removing components that may interfere with subsequent analyses. NMR spectroscopy enabled the direct identification of xanthan gum and the discrimination between different galactomannans in the isolated polysaccharide fraction. An enzymatic fingerprinting method using endo-β-mannanase, in addition to being used to differentiate between galactomannans, was developed into a selective, quantitative method for LBG, whereas monosaccharide analysis was used to quantify xanthan gum. Recoveries for xanthan gum and LBG were 87% and 70%, respectively, with in-between day relative standard deviations below 20% for xanthan gum and below 10% for LBG.
Heterogenous samples of locust bean gum (galactomannan) were prepared into homogeneous substances. Locust bean gum was fractioned using ammonium sulfate (14.11-23.08%, w/w). The intrinsic viscosity was obtained by extrapolating reduced viscosity versus concentration by using an Ubbelohde viscometer. The ranges of intrinsic viscosity for fractions that not included protein (F3-F6) and fractions that included protein (F1-F2) were 9.89-8.10 and 8.44-4.59, respectively. Values for Huggins' coefficient (k'), which depends on physical interactions, were 0.46-0.78. Increasing ammonium sulfate concentration was associated with a weak trend towards lower molecular weight and intrinsic viscosity by size-exclusion chromatography (SEC): ranged from 674 to 617 kg/mol and [] from 9.80 to 8.10 dL/g between F3 and F6. The evaluations of those fractions by using SEC and the Ubbelohde viscometer produced very similar values, as predicted. We verified the application of a gradient of ammonium sulfate to precipitate locust bean gum into fractions of different molecular size and show structural variations.
A capillary zone electrophoresis procedure for the prediction of curing time of Spanish hams using peptide profiles has been developed. Excellent resolution between the seven peptide peaks was achieved within 30min analysis time with a BGE containing 60mM MgSO4 and 50mM phosphate at pH 2.5. Using hams with curing times of 6, 8 and 12months, both linear discriminant analysis (LDA) and multiple linear regression (MLR) models were constructed. In both cases, two different normalisation procedures of the peak areas were compared. Using LDA, all the ham samples corresponding to the three categories were correctly classified. Using MLR, the ham curing time could be predicted with average prediction errors below 2.5%.
A quantitative assay for guar gum in carob gum, based on the extraction of proteins in acetonitrile–water (7:3), separation by capillary electrophoresis and multiple linear regression (MLR) using the areas of nine selected peaks as predictors, was improved by performing the extraction in the presence of gamanase. In the absence of the enzyme, peak migration times and areas depended on the guar content, which complicated peak identification and evaluation. Manual correction of the migration times by comparison with standard electropherograms obtained with pure carob and carob–guar mixtures was required; however, when the proteins were extracted under sonication at 60 °C for 30 min in the presence of gamanase, the migration times and peak areas did not vary with the composition of the carob–guar mixtures, and its reproducibility improved largely. These effects were attributed to the reduction of the viscosity of the extracts and the removal of the galactomannose interactions with the proteins and the capillary walls. Peak identification and evaluation were easily and directly performed on these electropherograms without further processing. An MLR model constructed with 36 carob–guar mixtures containing up to 20% guar, and by measuring the areas of 12 selected peaks and using eight of them as predictors, yielded a detection limit of 2.8% guar (α=β=0.05 criterion). A model of similar quality was obtained by partial least-squares (PLS) regression.
A simple method was developed that enabled the enzymatic determination of the galactose distribution in galactomannans. endo-Mannanase of Aspergillus niger was used to degrade the galactomannan polymers and the degradation products were determined with high-performance anion-exchange chromatography. A whole range of commercial high-to-low substituted galactomannans was analyzed in this way. It was found that differences in the anion-exchange chromatograms reflected dissimilarities in the distribution of galactose and could be used directly to discern these dissimilarities. The differences among the various elution profiles were used to construct a similarity distance tree. In addition to this approach, the absolute amount of non-substituted mannose released by the enzyme was found to be a good discriminating factor. In this way, galactomannans with regular, blockwise, and randomly distributed galactose could be discerned. All guars and the highly substituted gum of Prosopis juliflora were found to have a blockwise distribution of galactose. For different batches of tara gum both random and blockwise distributions were found. Among batches of locust bean gum the greatest variation was observed: both random, blockwise, and ordered galactose distributions were present. Cassia gum was found to have a highly regular distribution of galactose.
Capillary electrophoresis (CE) and polarized light microscopy (PLM) were utilized in the detection of the adulteration of locust bean gum with guar gum. For CE analyses, standards of locust bean and guar gums were extracted with 30% CH3CN, removing the residual proteins from the gum matrix. A 8.75 mM NaH2PO4-20.6 mM Na2B4O7 buffer, pH 9, was used to separate these proteins and to identify marker proteins that were present in the guar gum. These markers did not co-migrate with components in the extracts of mechanically processed locust bean gum, and are used as indicators of adulteration. Using PLM with toluidine blue and iodine staining techniques, unadulterated locust bean gum samples were distinguished from mixed samples through the differential staining of components in locust bean versus guar and tara gums. These experiments in the use of CE and PLM provide orthogonal and complementary methods for the verification of 'true' positives and the elimination of 'false' positives.
The saccharide composition of a number of heteropolysaccharides has been successfully characterized by pyrolysis-gas chromatography. In addition, small residues of carbohydrate impurities in samples can be easily identified. The success of pyrolysis-gas chromatography to differentiate among carbohydrates was established by previous studies which found that rapid pyrolysis initiates glycosidic cleavage of saccharide units through transglycosidation thereby forming intact fragments which retain the stereoconfiguration of the saccharide, i.e., anhydro sugars. The high molecular weight product mixtures are resolved on a proper capillary column and the individual anhydro sugars identified by mass spectrometry. Bacterial and plant polysaccharides were selected having various compositions, consisting of hexoses, pentoses, deoxy sugars and uronic acids. The composition of a highly sulfated polysaccharide was also examined. It was found that uronic acid-containing polysaccharides require that the carboxylate functional group be protonated in order to obtain structurally-significant pyrolysis products.
A novel polymer network is described for efficient sieving of sodium dodecyl sulfate (SDS)-protein complexes: poly(vinyl alcohol) (PVA; average Mr 133 000). The entanglement threshold of these solutions was found to be at 3% (w/v) PVA. Solutions from 4 to 6% PVA offer excellent resolution in the 14 400-94 000 protein molecular mass interval. Ferguson plot analysis showed that the separation is indeed based on mass discrimination, as it should in SDS electrophoresis, with extrapolated (at 10% polymer) limit values of mobility for all particles in the range (2.34-2.87) · 10−8 m2 V−1s−1. The advantages of PVA are full transparency in the UV region down to 200 nm and extremely low viscosities (e.g., a 5% PVA solution has a viscosity 25 times greater than that of buffer at 30°C). A unique wall effect was found, by which, on decreasing the inner diameter of the capillary from 75 to 25 μm, the apparent entanglement threshold was shifted to extremely dilute PVA solutions, since in 25-μm capillaries efficient sieving was obtained below 1% PVA, i.e., at concentrations well below the entangled regime. It is hypothesized that residual, free silanols present (even in a coated capillary) act as nucleation sites for H-bond formation and aggregation of free PVA molecules.
Pyrolysis/capillary gas chromatography is used for the characterization of the monomer composition of various oligosaccharides and polysaccharides including glucose-containing disaccharides,glucans, a galactomannan and an arabinogalactan. The chromatograms showed many common pyrolysis products, but also unique anhydrosugar products (e.g., 1,6-anhydroglucopyranose, 1,4-anhydroarabinopyranose, 2,6-anhydrofructofuranose) derived from each type of saccharide unit present in the samples. Reasonable values of the monomer composition of the polysaccharide can also be obtained from th pyrograms. The method is rapid and direct, requiring no sample preparation.
J. Chromatogr. Vol.319, 90 - 98
Capillary electrophoresis (CE) was utilized in the characterization of various galactomannans. Standards of gums were extracted with 50% CH3CN to remove the residual proteins from the gum matrix. Separation buffers were optimized with respect to pH, buffer concentration and presence of sodium dodecyl sulphate, yielding protein profiles from which the desired information could be obtained. Examples are given of the profiles generated by various gums and gum blends to aid in the verification of component presence, and to demonstrate levels of adulteration detectable under the buffer conditions used.
A polymerase chain reaction (PCR) was developed to differentiate the thickening agents locust bean gum (LBG) and the cheaper guar gum in finished food products. Universal primers for amplification of the intergenic spacer region between trnL 3' (UAA) exon and trnF (GAA) gene in the chloroplast (cp) genome and subsequent restriction analysis were applied to differentiate guar gum and LBG. The presence of <5% (w/w) guar gum powder added to LBG powder was detectable. Based on data obtained from sequencing this intergenic spacer region, a second PCR method for the specific detection of guar gum DNA was also developed. This assay detected guar gum powder in LBG in amounts as low as 1% (w/w). Both methods successfully detected guar gum and/or LBG in ice cream stabilizers and in foodstuffs, such as dairy products, ice cream, dry seasoning mixes, a finished roasting sauce, and a fruit jelly product, but not in products with highly degraded DNA, such as tomato ketchup and sterilized chocolate cream. Both methods detected guar gum and LBG in ice cream and fresh cheese at levels <0.1%.