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Improvement of the electrophoretic protein profiles of Leguminosae gum extracts using gamanase and application to the evaluation of carob–guar mixtures
Abstract
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.
The suitability of protein profiles established by capillary gel electrophoresis (CGE) as a tool to discriminate between 11 cultivars of Citrus (orange and tangerine) peel and pulp was evaluated in this work. Before CGE analysis, different extraction buffers (which included enzyme-assisted treatments) were compared. The best results were achieved using 5% (v/v) Celluclast® 1.5 L and 5% (v/v) Palatase® 20,000 L buffers for Citrus peel and pulp protein extracts, respectively. The resulting protein profiles obtained were used to construct linear discriminant analysis (LDA) models able to distinguish Citrus peel and pulp samples according to their cultivar. In both cases, all samples were correctly classified with an excellent resolution among all categories, which demonstrated that protein patterns are a powerful tool to discriminate Citrus samples coming from different cultivars.
The improvement of protein extraction from olive leaves using an enzyme-assisted protocol has been investigated. Using a cellulase enzyme (Celluclast® 1.5L), different parameters that affect the extraction process, such as the influence and amount of organic solvent, enzyme amount, pH and extraction temperature and time, were optimised. The influence of these factors was examined using the standard Bradford assay and the extracted proteins were characterised by sodium dodecyl sulphate polyacrylamide gel electrophoresis (SDS–PAGE). The optimum extraction parameters were: 30% acetonitrile, 5% (v/v) Celluclast® 1.5L at pH 5.0 and 55 °C for 15 min. Under these conditions, several protein extracts from olive leaves of different genetic variety (with a total protein amount comprised between 1.87 and 6.64 mg g−1) were analysed and compared by SDS–PAGE, showing differences in their electrophoretic protein profiles. The developed enzyme-assisted extraction method has shown a faster extraction, higher recovery and reduced solvent usage with respect to the use of the non-enzymatic methods described in literature.
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
An efficient protein extraction protocol for proteins from olive pulp and stone by using enzymes was developed. For this purpose, different parameters that affect the extraction process, such as enzyme type and content, pH, and extraction temperature and time, were tested. The influence of these factors on protein recovery was examined using the standard Bradford assay, while the extracted proteins were characterized by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE). The best extraction conditions were achieved at pH 7.0 and 5% (v/v) Palatase® 20000 L (lipase) for pulp and Lecitase® Ultra (phospholipase) for stone proteins. The optimal extraction temperature and time were 30 and 40 °C for 15 min for pulp and stone tissues, respectively. Under these conditions, several protein extracts coming from olive fruits of different genetic variety were analyzed, their profiles being compared by SDS-PAGE. The developed enzyme-assisted extraction method showed faster extraction, higher recovery, and reduced solvent usage than the nonenzymatic methods previously described in the literature. In the case of stone proteins, different electrophoretic profiles and band intensities were obtained that could be helpful to distinguish samples according to their genetic variety.
Capillary electrophoresis, as an analytical tool for drugs, offers several advantages for pharmaceuticals and clinical applications; however, it suffers from poor detection limits. Concentration on the capillary (stacking) improves greatly this problem and is very easy to perform. One of the simple and practical methods to perform stacking is dissolving the sample in organic solvents and injecting a large volume of sample on the capillary. This leads to concentration of the sample 10-30 folds directly on the capillary, removes the excess of proteins found in biological fluids and overcomes the deleterious effects of salts. The stacking can be performed in both the hydrodynamic and electroinjection. This stacking brings the detection limits of the CE closer to that of the HPLC. The mechanism, practical applications, different factors, and optimum conditions for this type of stacking are reviewed and discussed.
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 sucess of the first edition of Thickening and Gelling Agents for Food underlined the keen interest in functional food ingredients. In this second edition, the text has been completely revised and updated to reflect the current market trends. New chapters have been included to broaden the scope of materials used by the food technologist. Agar and konjac gum (flour), probably the most traditional gelling and thickening agents, but most widely utilised in the Far East, have been given greater prominence. Microcrystalline cellulose, a relatively new food stabiliser used widely in the USA, has been included. The preparation of traditional products using formulations suited to bulk food processings is described while new areas focus on low fat and low calorie foods where there is an even greater demand for controlling the stability, viscosity, gelation and mouthfeel using a broad range of thickening and gelling agents. Recent legislative changes in the USA and EC impact the use of additives including gellan gum, konjac flour, carrageenan, tara gum and microcrystal line cellulose: some changes have increased the number of additives ap proved for foods, while others allow a broader range of materials to be used in foods. The detailed information on products, properties and applications given in this second edition will enable these highly functional thickening and gelling agents to be used to full advantage.
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.
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%.
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).
Smeyers-Verbeke, Handbook of Chemo-metrics and Qualimetrics: Part A
- D L Massart
- B G M Vandeginste
- L M C Buydens
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D.L. Massart, B.G.M. Vandeginste, L.M.C. Buydens, S. De Jong, P.J. Lewi, J. Smeyers-Verbeke, Handbook of Chemo-metrics and Qualimetrics: Part A, Elsevier, Amsterdam, 1997, p. 426.
Natural Plant Hydrocolloids
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H. Deuel, H. Neukmon, Natural Plant Hydrocolloids, American Chemical Society, Washington, DC, 1959.
Engineering Foundation Conferences: (8-AH) Enzyme Engineering XIV
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