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ABSTRACT: A new colorimetric method based on the bleaching of the iodoplatinate ion has been developed for fast and easy determination of γ-glutamyl-S-ethenyl-cysteine (GEC) in narbon vetch (Vicia narbonensis L.) seeds. The calibration curve showed a good correlation (r(2)=0.9959) between absorbance and GEC amounts from 5.5 to 33 μg (10-59.78 μmol/L). The limits of detection and quantification were 1.16 and 3.55 μmol/L, respectively, and no significant interferences from other sulfur-containing compounds were observed. The method showed excellent repeatability (relative standard deviation [RSD]=0.28%), reproducibility (RSD=4.4%), and accuracy (94%). Determination of GEC in 20 narbon vetch accessions yielded values that were in agreement with those reported previously using capillary electrophoresis and high-performance liquid chromatography methods. The method could be especially valuable for determination of GEC during the process of production of new low-GEC narbon vetch varieties.
Analytical Biochemistry 07/2011; 418(2):180-3. · 3.00 Impact Factor
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F Raposo,
M A de la Rubia,
R Borja, M Alaiz,
J Beltrán,
C Cavinato,
M Clinckspoor,
G Demirer,
E Diamadopoulos,
B Helmreich,
P Jenicek,
N Martí,
R Méndez,
J Noguerol,
F Pereira,
S Picard,
M Torrijos
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ABSTRACT: In 2008, the first Proficiency Testing Scheme of Chemical Oxygen Demand (1(st)COD-PT(ADG)) was conducted to assess the results obtained for different research groups whose field work is mainly anaerobic digestion. This study was performed using four samples, two solid samples as raw materials and two solid samples to prepare high concentration suspended solid solutions. Invitations were sent to a large number of laboratories, mainly to anaerobic digestion research groups. Finally, thirty labs from sixteen countries agreed to participate, but for different reasons four participants could not send any data. In total, twenty-six results were reported to the COD-PT coordinator. This study showed the importance of continuous participation in proficiency testing (PT) schemes in order to compare the results obtained. Taking into account the lack of a general standard method and high quality certified reference materials (CRMs), the traceability of COD determination is not currently easy to check. In addition, the spread of participants' results obtained was high and pointed to the advisability of using consensus values due to their unreliability. Therefore, the theoretical oxygen demand (ThOD) values were considered as assigned values for all the samples analysed. On the other hand, in this PT the established standard deviation (ESD) has been determined by the Horwitz modified function. Participants of this 1(st)COD-PT(ADG) were asked to give a short report on the analytical method used. Although all the participants used potassium dichromate as their oxidant reagent, their experimental procedures were very different. With the purpose of comparing the results obtained, the different experimental conditions used were classified into five methods, corresponding to two main categories, open and closed reflux. The performance of laboratories was expressed by the z-score, whose value is considered satisfactory when z-score <or=+/-2. The overall analytical data evaluation showed that 64% of z-scores obtained were outside the accepted limits.
Talanta 11/2009; 80(1):329-37. · 3.79 Impact Factor
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ABSTRACT: A modified approach to determine the chemical oxygen demand (COD) of solid substrates based on the DIN 38414-S9 standard method is proposed. The adapted procedure is assessed and compared with standard methods widely used for water and wastewater such as the American Public Health Association-American Water Works Association-Water Pollution Control Federation (APHA-AWWA-WPCF) standard methods 5220 B-open reflux (SM-OR) and 5220 D-closed reflux colorimetric (SM-CR). Solutions with high suspended concentration of solids, as well as digestates from an anaerobic reactor, were used during the comparative test. For solid substrates, the COD recovery was about 100% when the proposed method was used. For solutions with solid content higher than 20 g TS L(-1), the recovery was only completed when the proposed method was used, showing that the methods traditionally employed are not very appropriate for samples with the described characteristics. For instance, percentages of COD recovery in the ranges of 77.3-87.1% and 89.4-94.1% were achieved when the SM-OR and SM-CR methods were used, respectively.
Talanta 08/2008; 76(2):448-53. · 3.79 Impact Factor
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ABSTRACT: The protein profile and the amino acid composition of eleven amaranth species have been studied. The following species were taken into account: A. viridis, A. powellii, A.muricatus, A. deflexus, A. graecizans, A. blitoides, A. retroflexus, A. blitum, A. albus, A. cruentus and A. hypochondriacus . Seed samples were obtained from wild populations located in the southwest of Spain. The protein profile was studied by gel filtration chromatography and denaturing electrophoresis. Profiles were similar in all taxa, with small variations in the molecular weights and amounts of the main seed proteins. Thus, after gel filtration chromatography six main fractions of around 300 kDa, 180 kDa, 120 kDa, between 40 and 50 kDa, 20 and 30 kDa and below 10 kDa were observed. On the other hand, the electrophoretic analysis showed peptides grouped into three main fractions, between 50 and 64 kDa, 33 and 37 kDa and 18 and 25 kda. The most balanced amino acid compositions were observed in the wild taxa A. muricatus, A. blitum and A. powellii showed the most equilibrated amino acid composition. A. hypochondriacus and A. graecizans showed the most deficient amino acid composition with limitations in five essential amino acids. These results show the potential of wild amaranthus taxa for their introduction as crops or their use in the improvement by hybridization mechanisms of other crops such as A. hypochondriacus.
El objetivo de este estudio fue determinar la composición aminoacídica y el perfil proteico de las semillas de once especies de amaranto. Las especies estudiadas fueron A. viridis, A. powellii, A. muricatus, A. deflexus, A. graecizans, A. blitoides, A. retroflexus, A. blitum, A. albus, A. cruentus y A. hypochondriacus . Se estudiaron poblaciones silvestres de estos taxones localizadas en el suroeste de España. El perfil proteico se estudió mediante cromatografía de filtración en gel y electroforesis desnaturalizante. Este perfil fue similar en todas las especies, con ligeras variaciones en los pesos moleculares y abundancia de las principales proteínas. Así, mediante cromatografía de filtración en gel se apreciaron seis fracciones mayoritarias de alrededor de 300 kDa, 180 kDa, 120 kDa, entre 40 y 50 kDa, entre 20 y 30 kDa y menores de 10 kDa. Por otro lado, el estudio electroforético mostró tres grupos de péptidos mayoritarios con pesos comprendidos entre 50 y 64 kDa, entre 33 y 37 kDa y entre 18 y 25 kDa. Las especies con la composición aminoacídica más equilibrada correspondieron a taxones no cultivadas. A. muricatus, A. blitum y A. powellii mostraron la composición aminoácidica más equilibrada. A. hypochondriacus y A. graecizans mostraron la composición aminoacídica más deficitaria, con carencias en cinco aminoácidos esenciales. Estos resultados muestran el potencial de los taxones silvestres de amaranto para su introducción como cultivos o su uso para la mejora mediante hibridación de otros cultivados, como A. hypochondriacus .
Grasas y Aceites. 01/2007;
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ABSTRACT: Incubation experiments in order to study the influence of some parameters in the fluorescence products formation have been carried out. It has been observed a greater efficiency with the relation linoleic acid hydroperoxide:glutathione, 2:1, and a temperature of 37 °C. The complex formed working with labelled linoleic acid has been separated in two main fractions. The first one (without radioactivity) is fluorescent with excitation and emission maxima at 350 nm and 440 nm, respectively. It has been confirmed that this fraction consists of a complex of glutathione and short chain aldehydes. The fluorescence of the complex did not decrease by treatment with NaBH4 or with pH. The NH2 and SH groups take part in the lipid-peptide complex formation. The second, highly radioactive fraction includes free hydroperoxides and short chain hydroxy fatty acids.
Food / Nahrung 10/2006; 33(3):283 - 288.
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ABSTRACT: Plant species are considered as a good source of dietary proteins, although the nutritional quality of proteins depends on their amino acid composition. In this work the protein content and amino acid composition of nutlets of 21 Teucrium taxa (Lamiaceae) from Spain were analysed and their nutritional quality was compared with the minimum values established by the Food and Agriculture Organization of the United Nations (FAO). In addition, the amino acid composition was evaluated as a chemical character to clarify the taxonomic complexity in this genus.
Amino acid content of nutlets was determined after derivatization with diethyl ethoxymethylenemalonate by high-performance liquid chromatography. Previously, nutlets samples were hydrolysed and incubated in an oven at 110 degrees C for 24 h.
The protein content was variable, ranging from 6.4 % in T. dunense to 43.8 % in T. algarbiense. According to the FAO values all taxa contain satisfactory amounts of leucine, threonine and valine and are deficient in lysine. The similarity analysis of Teucrium taxa using amino acid composition data did not clearly reflect the infrageneric classification of this genus.
Annual species, such as T. spinosum, T. aristatum and T. resupinatum showed a better balanced amino acid composition. The dendrogram partly matched with the karyological complexity of Teucrium. No correlation between amino acid composition and habitat has been observed, showing that Teucrium nutlet amino acid composition may not be strongly influenced by the environment.
Annals of Botany 11/2004; 94(4):615-21. · 4.03 Impact Factor
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ABSTRACT: Extraction of proteins from defatted sunflower meal has been improved by addition of the protease alcalase during alkaline extraction. This method offers several additional advantages as compared to the traditional alkaline extraction without alcalase, which is usually carried out after a sedimentation/flotation step to remove the lignocellulosic fraction. As compared to extraction without alcalase, addition of 0.1% (v/v) alcalase improved the yield of protein extraction from 57.5% to 87.4%, providing an extract that is 22% hydrolyzed. In addition, an increment of up to 4.5 times in protein solubility at low pH values is achieved, which correlates with the degree of hydrolysis. The extracts that were obtained in the presence of alcalase had a higher proline and glycine content, suggesting that the protease improves extraction of proline-rich and glycine-rich cell wall proteins that are part of the lignocellulosic fraction. These protein extracts can be directly dried without generation of wastewater, and the resulting fiber-rich material could be used for animal feeding.
Se ha mejorado la extracción proteica de la harina desengrasa de girasol mediante la adición de la proteasa alcalasa durante la extracción alcalina. Este método ofrece varias ventajas adicionales en comparación con la extracción alcalina tradicional sin alcalasa, que se desarrolla normalmente mediante un proceso de flotación/sedimentación para retirar la fracción lignocelulósica. En comparación a la extracción sin alcalasa, la adicción de 0.1% (v/v) de alcalasa mejora los rendimientos de extracción proteica desde un 57.5% a un 87.4%, dando un extracto con un 22% de grado de hidrólisis. Además se obtiene un incremento de hasta 4.5 veces de la solubilidad proteica a bajos pHs, que se correlaciona con el grado de hidrólisis. Los extractos obtenidos con alcalasa tenían un mayor contenido de prolina y glicina, sugiriendo que la proteasa mejora la extracción de las proteínas ricas en prolina y glicina de la pared celular que forma parte de la fracción lignocelulósica. Este extracto proteico puede ser secado directamente sin generación de aguas residuales, y el material resultante rico en fibra podría ser usado para alimentación animal.
Grasas y Aceites. 01/2003;
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ABSTRACT: Proteins of olive fruit mesocarp are not very well-known at present. However, they have been shown to pass, at least partially, to the olive oil during its elaboration and therefore might be contributing to some of the special characteristics of this vegetable oil. In this study, protein content and composition were determined in olive fruits, cv. Arbequina and Picual, at three stages of ripening: green, spotted, and purple. Mesocarp proteins constituted 1.3-1.8% of the dry weight of the olive fruit, and cultivar and fruit ripening did not produce important changes in mesocarp protein content or composition. In addition, this composition was also similar to the amino acid composition of a 4.6-kDa polypeptide, which is the major protein component of olive oils and of oil bodies of olive fruit mesocarp, suggesting that this polypeptide is likely to be a major component of mesocarp proteins. There was, also, a relationship between the oil content of the olive fruit and the protein content determined, suggesting a stabilizing function of these proteins in the oil bodies of the olive fruit, analogously to the role suggested for oleosins. This stabilizing function does not seem to be extended to olive oils because when the polypeptides isolated were added at 20 ppm to soybean oil, the stability of the oil increased only slightly, suggesting that if these compounds play some role in the stability of the oils, this should be mostly a consequence of the possible interactions among these protein components and other olive oil antioxidant constituents.
Journal of Agricultural and Food Chemistry 10/2001; 49(9):4267-70. · 2.82 Impact Factor
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ABSTRACT: The consequences of oxidative stress on microsomal proteins were analyzed by studying their pyrrolization and the antioxidative activity of the modified proteins produced. The microsomal system consisted of freshly prepared trout muscle microsomes, which were oxidized in the presence of 5 microM Cu(2+), 1 mM Fe(3+)/5 mM ascorbate, or 1 mM Cu(2+)/10 mM H(2)O(2). Pyrroles on proteins were detected by forming Ehrlich adducts with p-(dimethylamino)benzaldehyde and by determination of epsilon-N-pyrrolylnorleucine (Pnl) by capillary electrophoresis. Their antioxidative activity was studied by testing two model pyrrolized proteins (dimeric and monomeric modified bovine serum albumin: DBSA and MBSA, respectively), which were produced in the reaction of BSA and 4,5(E)-epoxy-2(E)-heptenal. These proteins were assayed at a concentration of 10-40 microg/mL, which was selected because at this concentration both DBSA and MBSA had a concentration of Pnl similar to the Pnl concentration produced in oxidized microsomes. Both DBSA and MBSA significantly (p < 0.05) protected against lipid peroxidation, assessed by the formation of thiobarbituric acid reactive substances (TBARS), and protein damage, evaluated by amino acid analysis, for the three systems assayed, and this protection was always higher than that exhibited by BSA, which was used as control. The order of effectiveness was DBSA > MBSA > BSA and was parallel to the Pnl content in the assayed proteins. These results suggest that antioxidative activity of BSA may also be related to its ability to react with lipid oxidation products and to produce modified BSA with antioxidative activity. This mechanism may also be contributing to the antioxidative activity exhibited by many proteins.
Chemical Research in Toxicology 05/2001; 14(5):582-8. · 3.78 Impact Factor
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ABSTRACT: A method for the determination of proteins in fats and oils is described. Proteins were sequentially precipitated with acetone and hydrolyzed, and the produced amino acids were fractionated and quantificated. This analysis protocol afforded a method of high sensitivity and specificity which was fully evaluated and validated. The data obtained showed good accuracy and linearity with excellent reproducibility and recovery. When the method was applied to 40 olive oils, all of them contained proteins in the range 10-50 microg/100 g of oil, suggesting that proteins are nonpreviously described minor components of these oils. In addition, the proteins precipitated were almost exclusively composed by one polypeptide of apparent 4600 molecular weight, which was isolated from olive drupes and partially characterized by amino acid analysis. Similar polypeptides were also detected in other seeds, suggesting that they may constitute a new class of polypeptides in plants with oleosin-like characteristics. Furthermore, the method was also applied to different fats and oils, and all the samples analyzed contained proteins, suggesting that natural fats and oils always contain polypeptides and/or proteins as minor components. These results also suggest that some peptides are soluble in lipid matrixes, where they might be playing unknown functions. The developed procedure provides a methodology for the determination of these components.
Analytical Chemistry 03/2001; 73(3):698-702. · 5.86 Impact Factor
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ABSTRACT: Recent studies have hypothesized that pyrrole formation and polymerization may be contribute to the nonenzymatic browning produced in both oxidized lipid/protein reactions and the Maillard reaction. To develop a methodology that would allow investigation of the contribution of this browning mechanism, the kinetics of formation of color, fluorescence, and pyrrolization in 4, 5(E)-epoxy-2(E)-heptenal/lysine and linolenic acid/lysine model systems were studied. In both cases similar kinetics for the three measurements were observed at the two temperatures assayed (37 and 60 degrees C), and there was a high correlation among color, fluorescence, and pyrrolization measurements obtained as a function of incubation time. Because the color and fluorescence production in the 4,5(E)-epoxy-2(E)-heptenal/lysine system is a consequence of pyrrole formation and polymerization, the high correlations observed with the unsaturated fatty acid also suggest a contribution of the pyrrole formation and polymerization to the development of color and fluorescence observed in the fatty acid/lysine system. Although the contribution of other mechanisms cannot be discarded, all of these results suggest that when the pyrrole formation and polymerization mechanism contributes to the nonenzymatic browning of foods, a high correlation among color, fluorescence, and pyrrolization measurements should be expected.
Journal of Agricultural and Food Chemistry 09/2000; 48(8):3152-8. · 2.82 Impact Factor
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ABSTRACT: The reactions of 4,5(E)-epoxy-2(E)-heptenal with 4-methylimidazole and N(alpha)-acetyl-L-histidine methyl ester were studied to characterize the adducts produced in the modification of histidine residues by epoxyalkenals and to develop a methodology for the determination of these adducts in protein hydrolysates. The reaction products, which were isolated and characterized, resulted in the Michael adducts produced in the addition of one of the imidazolic nitrogens to the carbon-carbon double bond of the epoxyalkenal. Only some of the theoretical isomers were produced. Thus, in the reaction with 4-methylimidazole, the main product was 4, 5-epoxy-3-(4-methylimidazol-1-yl)heptanol (88%), although the formation of 4,5-epoxy-3-(5-methylimidazol-1-yl)heptanol (12%) was also observed. On the other hand, the reaction with N(alpha)-acetyl-L-histidine methyl ester produced exclusively N(alpha)-acetyl-1-[1'-(1' ',2' '-epoxybutyl)-3'-hydroxypropyl]-L-histidine methyl ester. This last compound was used to develop a procedure for the determination of 4, 5(E)-epoxy-2(E)-heptenal-histidine adducts in protein hydrolysates. When this procedure was applied to the analysis of bovine serum albumin treated with 0.01-10 mM 4,5(E)-epoxy-2(E)-heptenal, the formation of the adduct was observed and its concentration increased with the concentration of the aldehyde and the incubation time, and was parallel to the histidine losses observed in the protein after acid hydrolysis as well as to the formation of protein carbonyls. In addition, the number of histidine residues lost in the protein was very similar to the number of adduct residues produced, suggesting that the addition reaction is the major mechanism for histidine losses suffered by proteins following their reaction with epoxyalkenals.
Chemical Research in Toxicology 08/1999; 12(7):654-60. · 3.78 Impact Factor
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ABSTRACT: epsilon-N-Pyrrolylnorleucine was determined in different fresh food products to study its presence as a normal component of food proteins. Twenty-two different products were screened: cod, cuttlefish, salmon, sardine, trout, beef, chicken, pork, broad bean, broccoli, chickpea, garlic, green pea, lentil, mushroom, soybean, spinach, sunflower, almond, hazelnut, peanut, and walnut. Foods were homogenized, their proteins were precipitated with trichloroacetic acid and hydrolyzed with 2 N NaOH for 20 h, and the epsilon-N-pyrrolylnorleucine content was determined by capillary electrophoresis. The epsilon-N-pyrrolylnorleucine, which was identified by HPLC/MS in sardine muscle hydrolysate, ranged in the 22 foods analyzed from 0.24 to 6.36 micromol/g. This concentration was correlated with the protein content of the food (r = 0.687, p = 0.00041). In addition, the epsilon-N-pyrrolylnorleucine/lysine ratio was found to be a function of the lipid, iron, and protein contents of the food (r = 0.881, p < 0.0001) and was directly correlated with lipid and iron contents and inversely correlated with the protein content. These results are in agreement with the oxidative stress origin proposed for epsilon-N-pyrrolylnorleucine and suggest that the epsilon-N-pyrrolylnorleucine/lysine ratio is a characteristic of each food. In addition, epsilon-N-pyrrolylnorleucine seemed to be a normal component of many fresh food products, in which it may be acting as a natural antioxidant.
Journal of Agricultural and Food Chemistry 05/1999; 47(5):1942-7. · 2.82 Impact Factor
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ABSTRACT: Bovine serum albumin (BSA) was incubated for 24 h in the presence of 10 mM ribose (RI), methyl linoleate hydroperoxides, or the secondary products of methyl linoleate oxidation (SP), at five temperatures (25, 37, 50, 80, and 120 degrees C) and different pHs (4, 7, and 10), to study the influence of these variables in the browning, fluorescence, amino acid losses, and pyrrolization of the modified proteins. All treated proteins exhibited similar colors and fluorescence spectra, and the spectra of their Ehrlich adducts were also analogous. However, at 25-50 degrees C the proteins treated with oxidized lipids exhibited higher color changes, amino acid losses, and pyrrolization than the BSA treated with RI, and these effects were much higher in proteins treated with RI at 80-120 degrees C. The effect of pH was similar in proteins treated with RI or SP. These results suggested a similarity for browned proteins obtained from both carbohydrates and oxidized lipids. In addition, both reactions seem to be complementary, because melanoidin formation derived from oxidized lipids can be produced under conditions different from those carbohydrate/protein reactions.
Journal of Agricultural and Food Chemistry 03/1999; 47(2):742-7. · 2.82 Impact Factor
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ABSTRACT: The antioxidative activity of nonenzymatically browned bovine serum albumin (BSA) produced by reaction with ribose (RI), hydroperoxides of methyl linoleate oxidation (HP), and secondary products of methyl linoleate oxidation (SP), at different pHs (4, 7, and 10) and temperatures (25, 37, 50, 80, and 120 degrees C), was studied to compare the antioxidative effects of carbohydrate- and oxidized lipids-modified proteins. The modified proteins (RIBSA, HPBSA, and SPBSA) were tested for antioxidative activity (at 100 ppm) in soybean oil using the thiobarbituric acid-reactive substances (TBARS) assay. All of them decreased significantly (p < 0.05) the TBARS formation in the oil and exhibited different effectiveness as a function of the temperature and the pH of the medium. In addition, there was a good correlation between the antioxidative activity of the protein and the amino acid losses produced during the nonenzymatic browning. These results are in agreement with an analogous and complimentary contribution of both Maillard and oxidized lipid/protein reactions to the antioxidative activity produced in foods during processing and storage.
Journal of Agricultural and Food Chemistry 02/1999; 47(2):748-52. · 2.82 Impact Factor
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F. J. Hidalgo,
R. Zamora, M Alaiz,
A. Garrido Fernández,
L. Rejano Navarro,
A. Heredia Moreno,
J. M. Castellano,
C. Gómez Herrera,
J. Vioque,
A. Guinda,
R. Borja Padilla,
P. García García
Grasas y Aceites. 01/1999;
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Grasas y Aceites. 01/1999;
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M.ª C. Duran Quintana,
E. Graciani,
F. J. Hidalgo,
R. Zamora,
J. L. Ruiz Barba,
A. G. Pérez Rubio,
A. Garrido Fernández,
A. de Castro,
J. Vioque,
M. Brenes Balbuena, M Alaiz,
W. Moreda Martine
Grasas y Aceites. 01/1999;
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ABSTRACT: The Ehrlich reaction was optimized to determine pyrrolized proteins produced as a consequence of lipid peroxidation and oxidative stress. The procedure consisted of the treatment of the modified protein with p-(dimethylamino)benzaldehyde at a controlled acidity and temperature, and the determination of adducts produced against the blank obtained in the absence of the reagent. The extinction coefficient of Ehrlich adducts was calculated by using epsilon-N-pyrrolylnorleucine (Pnl) as standard and was 35,000 M-1 cm-1. The response was linear and reproducible within the range 0. 16-20 microM Pnl. The assay was applied to determination of pyrrole content in bovine serum albumin, bovine alpha-globulins, bovine gamma-globulins, and mixtures of them, incubated overnight with 1 mM of 4,5(E)-epoxy-2(E)-heptenal, obtaining results similar to those from determination of Pnl by capillary electrophoresis after basic hydrolysis of the protein. The method was also applied to pyrrole determination in bovine plasma proteins either incubated with epoxyalkenals, hydroxyalkenals, lipid hydroperoxides, and secondary products of lipid peroxidation, or oxidized with Fe3+/ascorbate. All these treatments produced pyrrolization of plasma proteins and all Ehrlich adducts gave very similar absorbance spectra with the exception of that produced in the treatment with hydroxyalkenals. The above results suggest that protein pyrrolization is a normal consequence of the lipid peroxidation process and of oxidative stress, and that Ehrlich adducts may be valid to determine this pyrrolization.
Analytical Biochemistry 10/1998; 262(2):129-36. · 3.00 Impact Factor
