Purification of free arginine from chickpea (Cicer arietinum) seeds

© 2015 Elsevier Ltd. All rights reserved. Chickpea is a grain legume widely consumed in the Mediterranean region and other parts of the world. Chickpea seeds are rich in proteins but they also contain a substantial amount of free amino acids, especially arginine. Hence chickpea may represent a useful source of free amino acids for nutritional or pharmaceutical purposes. Arginine is receiving great attention in recent years because it is the substrate for the synthesis of nitric oxide, an important signaling molecule involved in numerous physiological and pathological processes in mammals. In this work we describe a simple procedure for the purification of arginine from chickpea seeds, using nanofiltration technology and an ion-exchange resin, Amberlite IR-120. Arginine was finally purified by precipitation or crystallization, yielding preparations with purities of 91% and 100%, respectively. Chickpea may represent an affordable green source of arginine, and a useful alternative to production by fermentation or protein hydrolysis.This work was carried out with the financial support of Junta de Andalucía (Spain) to the Laboratory of Bioactive and Functional Components of Plant Products (Instituto de la Grasa, C.S.I.C.). Cristina Megias is recipient of a JAE-Doc (C.S.I.C.) contract from the “Junta para la Ampliación de Estudios” program (cofinanced by the European Social Fund). Isabel Cortés-Giraldo is recipient of a JAE-Pre (C.S.I.C) fellowship from the “Junta para la Ampliación de Estudios” program (cofinanced by the European Social Fund). Thanks are due to María Dolores García-Contreras for technical assistance and to Jose Julian Rios for HPLC-MS analyses of purified arginine.Peer Reviewe

Mejora de la extracción proteica de la harina de girasol mediante hidrólisis con alcalasa

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.This work was supported by grant AGL 2001-0526.Peer reviewe

Polyphenol composition and in vitro antiproliferative effect of corm, tepal and leaf from Crocus sativus L. on human colon adenocarcinoma cells (Caco-2)

25 Páginas, 3 Figuras, 3 TablasSaffron is cultivated for production of the saffron spice. Nevertheless, a huge amount of saffron by-products including corms, tepals and leaves with little or no commercial value are generated during the processing of the spice. This biomass contains bioactive compounds whose exploitation can increase the profitability and sustainability of this traditional crop. A significant amount of polyphenols, mainly glycosides of kaempferol, luteolin and quercetin, have been determined in tepals and leaves of saffron. Proliferation of Caco-2 cells was greatly inhibited by the tepal and leaf extracts (ED50 0.42 mg/ml), while the corm extract caused some signs of toxicity and completely abolished proliferation (ED50 0.05 mg/ml). To our knowledge, these are the first data reporting the inhibition of the proliferation of Caco-2 cells by extracts from tepals and leaves of saffron, and polyphenols could be responsible for this effect.This work has been supported by the grant RTA2013-00005-00-00 (INIA, Spain) and is part of a Short Term Scientific Mission (STSM) carried out in the Agricultural University of Athens (AUA) and supported by the grant COST ActionFA1101 (http://www.saffronomics.org). We are grateful to the European Social Fund, Fundación Parque Científico y Tecnológico de Castilla-La Mancha and Junta de Andalucía for additional financing.Peer reviewe

Affinity purification and characterisation of chelating peptides from chickpea protein hydrolysates

A chickpea protein hydrolysate produced with pepsin and pancreatin was used for the affinity purification of chickpea chelating peptides. Three chelating peptide fractions were obtained after affinity chromatography with immobilised copper. These peptide fractions showed a higher chelating activity and histidine contents than the original protein hydrolysate. Chelating activity was positively correlated with the histidine content of the purified fractions. Different subfractions were also obtained after gel filtration chromatography from the affinity purified peptide fractions. Some of these subfractions showed a higher chelating activity and histidine contents than the original fractions. These results suggest that a combination of high His contents, around 20-30%, and small peptide size provide the best chelating activities. Thus sequential purification with affinity and gel filtration chromatography is a useful procedure for the purification of chickpea peptides with high chelating activity. These results show that a range of chelating peptides are generated during digestion of the chickpea proteins that, after metal chelation, may prevent the generation of reactive oxygen species (ROS) and favour metal absorption. © 2011 Elsevier Ltd. All rights reserved.This work was supported by Spanish Ministry of Innovation and Science through Research Project AGL2007-63580.Peer Reviewe

Caracterización proteica de las semillas de once especies de amaranto

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 aminoacid 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.Peer reviewe

Chickpea chelating peptides inhibit copper-mediated lipid peroxidation

© 2014 Society of Chemical Industry. BACKGROUND: Transition metals produce radical oxygen species promoting lipid peroxidation processes that favor the development of cardiovascular and neurodegenerative diseases. In addition, the oxidation of lipids present in food may affect the quality of food products. Therefore antioxidants counteracting these metal pro-oxidant effects may have high potential for the pharmacology and food industries. This study investigated the capability of peptide fractions purified from chickpea protein hydrolysate to inhibit copper-mediated lipid peroxidation in three different lipid substrates: β-carotene, unsaturated fatty acid mixture and low-density lipoprotein. RESULTS: Peptide fractions with the highest histidine content were the most antioxidant. This antioxidant effect is mainly due to the capability of histidine to bind copper and act as a hydrogen donor through its imidazole ring. CONCLUSION: The results suggest that chickpea proteins are a potential source of antioxidant peptides that may be included as ingredients in functional foods with beneficial health effects. In addition, these antioxidant peptides may be useful to protect food products from lipid peroxidation processes and thus increase their quality and shelf life.This work was supported by the Spanish Ministry of Innovation and Science through Research Project AGL2007-63580. Cristina Torres-Fuentes is recipient of a JAE-Pre (CSIC) contract from the ‘Junta para la Ampliación de Estudios’ program (cofinanced by the European Social Fund). Thanks are due to María Dolores García-Contreras for technical assistance.Peer Reviewe

Effect of chickpea protein hydrolysates on cell proliferation and in vitro bioavailability

6 páginas, 4 figuras.Bioactive peptides in foods may have health-promoting properties, including effects on cell proliferation. The goal of this study was to determine whether physiological digestion of chickpea protein using pepsin and pancreatin could release this type of peptides. The THP-1 and Caco-2 cell lines were used as in vitro models to determine the effects of the hydrolysates on cell proliferation. Hydrolysates with a high degree of hydrolysis inhibited the growth of Caco-2 cells by up to 45%, suggesting that chickpea-derived peptides might inhibit the growth of tumors in the colon. Proliferation of THP-1 cells was inhibited by up to 78% by direct exposure to the hydrolysates. Nevertheless, bioavailability experiments using differentiated Caco-2 cells as a model of the intestinal barrier, in co-culture with THP-1 cells, showed that proliferation of the monocytic THP-1 cells was actually enhanced by up to 66%, which could represent an immunomodulatory activity.This work was supported by the Ministerio de Ciencia e Innovación of Spain through grants AGL2005-01120 (J.G.C.), and AGL2007-63580 (M.A.), which were partially supported with FEDER funds from the European Union.Peer reviewe

Nutritional and functional properties of Vicia faba protein isolates and related fractions

The goal of this research was the characterisation of Vicia faba (broadbean) protein isolates and related fractions in order to determine whether this grain legume could be used for production of high quality protein products and other fractions rich in functional components. Alkaline extraction of the defatted seed flour, followed by precipitation at the isoelectric pH, yielded a 92% protein isolate with a high oil absorption capacity. The contents of the favism-inducing glycosides, vicine and convicine, in the isolate were reduced by more than 99% as compared to the original flour, although the amino acid composition was similar to that of the flour. Some of the by-products of protein isolate production may also be of interest from a nutritional and functional point of view. Thus, the oil resulting from hexane extraction of the flour is rich in unsaturated fatty acids, and polyphenols (resulting from extraction of the defatted flour with acetone) showed a high ABTS radical-scavenging activity. In addition, the solid residue (resulting from protein solubilisation) was high in fibre and showed good water absorption. These results show good nutritional and functional properties in V. faba protein isolates and related fractions, which may favour the revalorisation of this traditional bean crop. © 2011 Elsevier Ltd. All rights reserved.This work was carried out with the financial support of Junta de Andalucía (Spain) to the Laboratory of Bioactive and Functional Components of Plant Products (Instituto de la Grasa).Peer Reviewe

Isoflavones in chickpea (Cicer arietinum) protein concentrates

28 Páginas; 3 Tablas; 4 FigurasThe isoflavones in chickpea concentrates were analysed and compared to the original flour. Protein concentrates were prepared by alkaline extraction and precipitation of protein at the isoelectric pH. HPLC analysis revealed that the concentrates were enriched in isoflavones. The concentration of total isoflavones was 45 and 10 µg/g in concentrates and flour, respectively. Isoflavones in flour were hydrolyzed to the corresponding aglycones during alkaline extraction. While hydrolyzable derivatives of biochanin A represent 90% (w/w) total isoflavones in flour (9 µg/g), biochanin A aglycone was the major isoflavone in concentrates (31 µg/g). Minor components were formononetin, genistein, and the flavonol kaempferol. Thus, chickpea protein concentrates represent an even better source of health-promoting isoflavones than the original chickpea.This work was carried out with the financial support of Junta de Andalucía to the Laboratory of Bioactive and Functional Components of Plant Products (Ayudas interanuales to AGR 257, Instituto de la Grasa, C.S.I.C.). Cristina Megías is recipient of a JAE-Doc contract from the “Junta para la Ampliación de Estudios” program (C.S.I.C. and European Social Fund). Isabel Cortés-Giraldo is recipient of a JAE-Pre fellowship from the “Junta para la Ampliación de Estudios” program (C.S.I.C. and the European Social Fund). We are thankful to J.J. Rios for HPLC/MS analyses.Peer reviewe

Determination of β -Cyano-L-Alanine, γ -Glutamyl- β -Cyano-L-Alanine, and common free amino acids in vicia sativa (Fabaceae) seeds by reversed-phase high-Performance liquid chromatography

© 2014 Cristina Megías et al. A method for determination of β-cyano-L-alanine, γ-glutamyl-β-cyano-L-alanine and other free amino acids in Vicia sativa is presented. Seed extracts were derivatized by reaction with diethyl ethoxymethylenemalonate and analyzed by reverse-phase high-performance liquid chromatography. Calibration curves showed very good linearity of the response. The limit of detection and quantification was 0.15 and 0.50 M, respectively. The method has high intra- (RSD = 0.28-0.31%) and interrepeatability (RSD = 2.76-3.08%) and remarkable accuracy with a 99% recovery in spiked samples. The method is very easy to carry out and allows for ready analysis of large number of samples using very basic HPLC equipment because the derivatized samples are very stable and have very good chromatographic properties. The method has been applied to the determination of γ-glutamyl-β-cyano-L-alanine, β-cyano-L-alanine, and common free amino acids in eight wild populations of V. sativa from southwestern Spain.This work was carried out with the financial support of Junta de Andalucía (Spain) to the Laboratory of Bioactive and Functional Components of Plant Products (Instituto de la Grasa, C.S.I.C.). Cristina Megías and Isabel Cortés-Giraldo are recipients of a JAE-Doc (C.S.I.C.) contract and a JAEPre (C.S.I.C.) fellowship from the “Junta para la Ampliación de Estudios” program (cofinanced by the European Social Fund), respectively.Thanks are due to Mar´ıa Dolores Garc´ıa Contreras for technical assistance.Peer Reviewe