Purification, Characterization, and Antiproliferative Activity of a Single-Chain Lectin from Vicia palaestina (Fabaceae) Seeds.

Vicia palaestina Boiss. is an annual herb that grows in dry areas of eastern Mediterranean countries. It belongs to section Cracca subgenus Vicilla, which is characterized by having a high content in the non-protein amino acid canavanine. The seeds from some of these vetches are also rich in lectins. The purification and characterization of a single-chain lectin from the seeds of V. palaestina is described here. This lectin was the most abundant protein in albumin extracts. It has affinity for the glycoconjugate N-acetylgalactosamine and inhibits proliferation of the cancerous Caco-2 and THP-1 cell lines. In addition to their high nutritional value, the seeds from V. palaestina represent a source of lectins with health promoting and pharmacological potential because of their antiproliferative activity.

Physicochemical characteristics and antiproliferative and antioxidant activities of Moroccan Zantaz honey rich in methyl syringate.

Zantaz honey is a monofloral variety produced from the melliferous plant Bupleurum spinosum (Apiaceae), a shrub that grows mainly in the Atlas Moroccan Mountains. Determination of the polyphenol composition revealed that methyl syringate accounts for more than 50% of total polyphenols, which represents a very useful parameter for the characterization of this monofloral honey. Epicatechin, syringic acid and catechin are also abundant. Caco-2 and THP-1 cells were used for determination of antioxidant and antiproliferative activities in Zantaz honey, respectively. All six commercial samples that were used for these studies exhibited antioxidant activity and inhibited cell proliferation. Interestingly, these activities had a positive correlation mainly with the content in methyl syringate and gallic acid. The recognition of health promoting activities in Zantaz honey should increase its commercial value, which would have a positive economic impact on the poor rural communities of Morocco where it is produced.

Characterization of Vicia ervilia (bitter vetch) seed proteins, free amino acids, and polyphenols.

Vicia ervilia is an ancient crop from the Mediterranean Region. It may represent a useful source of proteins for food and animal feed, as well as bioactive components. Seed samples from 39 populations of V. ervilia have been analyzed. Polyphenol contents ranged from 0.09% to 0.19%. Luteolin, kaempferol, apigenin, and quercetin were the major aglycones. The total free amino acid content of the seeds was 0.05% to 0.19% in which canavanine represented 9% to 22%. The protein content was 24.1%. The amino acid composition indicated a high content in acidic amino acids and a deficit in sulphur amino acids. V. ervilia seeds proved to be a good substrate for the preparation of protein isolates. The seed extracts inhibited the proliferation of Caco-2 colon tumor cells, simultaneously, exerting antioxidative effects. Hence, seeds of V. ervilia could represent a source of high-value food and feed components, as well as functional components. PRACTICAL APPLICATIONS: Vicia ervilia (bitter vetch) (Leguminosae) is an ancient crop from the Mediterranean Region. Although it was still grown in many Mediterranean countries at the beginning of the twentieth century, other crops that provide higher and more consistent yield later replaced it. However, V. ervilia seeds may represent a useful source of proteins for human nutrition and animal feeding, and a source of bioactive components with health-promoting properties. Our results show that the seeds of V. ervilia could, indeed, represent a source of high-value food and feed components, as well as functional, health-promoting components. This may result in a revalorization of this neglected crop. The availability of numerous populations in seed banks guarantees the preservation of a genetic diversity in V. ervilia that could be used for the production of new varieties with better nutritional and functional characteristics.

Pectin-rich extracts from olives inhibit proliferation of Caco-2 and THP-1 cells.

Three olive modified pectin extracts have been produced by heat and acid treatment of the major by-product of olive oil production. Their effect on proliferation of the colon carcinoma Caco-2 and the leukemia monocytic THP-1 cell lines has been studied in order to determine possible anti-tumor properties. All extracts inhibited proliferation at some of the concentrations ranging from 1 to 10 mg ml-1. Interestingly none of the extracts inhibited the growth of confluent Caco-2 cells, showing the specificity of the antiproliferative effect for the transformed Caco-2 phenotype. All the extracts inhibited agglutination of red blood cells by galectin-3, a lectin involved in tumor growth, metastasis, and immune cell regulation that has been proposed as a mediator of the anti-tumor effects of modified pectins. In addition, activation of caspase-3 in THP-1 cells indicates that treatment with the pectin-rich extracts triggers apoptosis. These results point to a possible use as health-promoting food ingredients or supplements.

Purification of free arginine from chickpea (Cicer arietinum) seeds.

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.

Influence of peptides-phenolics interaction on the antioxidant profile of protein hydrolysates from Brassica napus.

The role of the peptides-phenolic compounds (PC) interaction on the antioxidant capacity profile (ACP) of protein hydrolysates from rapeseed (Brassica napus) was studied in 36 hydrolysates obtained from a PC-rich and PC-reduced protein substrate. The latent profile analysis (LPA), with data of seven in vitro methods and one assay for cellular antioxidant activity (CAA), allowed identifying five distinctive groups of hydrolysates, each one with distinctive ACP. The interaction of peptides with naturally present PC diminished in vitro antioxidant activity in comparison with their PC-reduced counterparts. However, CAA increased when peptides-PC interaction occurred. The profile with the highest average CAA (62.41 ± 1.48%), shown by hydrolysates obtained by using alcalase, shared typical values of Cu(2+)-catalysed ß-carotene oxidation (62.41 ± 0.43%), ß-carotene bleaching inhibition (91.75 ± 0.22%) and Cu(2+)-chelating activity (74.53 ± 0.58%). The possibilities for a sample to exhibit ACP with higher CAA increased with each unit of positively charged amino acids, according to multinomial logistic regression analysis.

Determination of L-canavanine and other free amino acids in Vicia disperma (Fabaceae) seeds by precolumn derivatization using diethyl ethoxymethylenemalonate and reversed-phase high-performance liquid chromatography.

A method for determination of the non-protein amino acid l-α-amino-γ-(guanidinooxy)-n-butyric acid (L-canavanine) and other free amino acids in Vicia disperma 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 were 0.15 and 0.50 µM, respectively. The method has a high intra- (RSD=0.35%) and inter-repeatability (RSD=2.86%), and a 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.

Determination of ß -Cyano-L-alanine, γ -Glutamyl- ß -cyano-L-alanine, and Common Free Amino Acids in Vicia sativa (Fabaceae) Seeds by Reversed-Phase High-Performance Liquid Chromatography.

A method for determination of ß-cyano-L-alanine, γ-glutamyl-ß-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 µM, 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-ß-cyano-L-alanine, ß-cyano-L-alanine, and common free amino acids in eight wild populations of V. sativa from southwestern Spain.

Angiotensin-converting enzyme-inhibitory activity in protein hydrolysates from normal and anthracnose disease-damaged Phaseolus vulgaris seeds.

BACKGROUND: Bean seeds are an inexpensive source of protein. Anthracnose disease caused by the fungus Colletotrichum lindemuthianum results in serious losses in common bean (Phaseolus vulgaris L.) crops worldwide, affecting any above-ground plant part, and protein dysfunction, inducing the synthesis of proteins that allow plants to improve their stress tolerance. The aim of this study was to evaluate the use of beans damaged by anthracnose disease as a source of peptides with angiotensin-converting enzyme (ACE-I)-inhibitory activity. RESULTS: Protein concentrates from beans spoiled by anthracnose disease and from regular beans as controls were prepared by alkaline extraction and precipitation at isolelectric pH and hydrolysed using Alcalase 2.4 L. The hydrolysates from spoiled beans had ACE-I-inhibitory activity (IC(50) 0.0191 mg protein mL(-1)) and were very similar to those from control beans in terms of ACE-I inhibition, peptide electrophoretic profile and kinetics of hydrolysis. Thus preparation of hydrolysates using beans affected by anthracnose disease would allow for revalorisation of this otherwise wasted product. CONCLUSION: The present results suggest the use of spoiled bean seeds, e.g. anthracnose-damaged beans, as an alternative for the isolation of ACE-I-inhibitory peptides to be further introduced as active ingredients in functional foods.

Nutritional quality of protein in the leaves of eleven Asphodeline species (Liliaceae) from Turkey.

The nutritional quality of the protein in the leaves of 11 Asphodeline (Liliaceae) species was investigated by the determination of the amino acid composition and calculation of several nutritional parameters. The average protein content was 4.7% and ranged from 2.5% in Asphodeline damascena ssp. rugosa to 8.2% in A. turcica. The most abundant essential amino acids were Thr (5.7%), Val (6.0%), Ile (4.7%), and Trp (2.1%). The amino acid composition of Asphodeline peshmeniana was well equilibrated according to Food and Agriculture Organisation standards, but Lys and sulphur amino acids were at limiting concentrations in all the other taxa. Determination of the protein efficiency ratio and biological value revealed that the protein in the leaves of Asphodeline species is of high nutritional quality. Hence, the Asphodeline leaves that are typically used in Turkey for the preparation of salads, represent a good source of protein with high levels of several essential amino acids and a good nutritional value. Analysis of the similarity based on the amino acid composition indicated the existence of different clusters that are consistent with the taxonomical classification, area of distribution, and morphological similarities of the Asphodeline species.

Antioxidant and metal chelating activities of peptide fractions from phaseolin and bean protein hydrolysates.

Bean protein isolate and phaseolin were hydrolysed using pepsin and pancreatin, and the resulting hydrolysates were filtered through a 1kDa cut-off membrane and fractionated by size exclusion chromatography. Three fractions corresponding to MW 0.7-1.0kDa, 0.43-0.7kDa and <0.43kDa (A1, A2, and A3 for protein isolate fractions, and B1, B2, and B3 for phaseolin fractions) were assayed for antioxidant and metal chelating activity and they were also subjected to amino acid and SDS-PAGE analysis. Fractions A1 and B1 had the highest copper chelating activity (78% and 82%, respectively), while iron chelating activity was the highest in fractions A1 and B3 (36% and 16%, respectively). Fractions A2 and B3 had the highest antioxidant activity as determined by inhibition of reducing power and ß-carotene bleaching, while the highest ABTS radical scavenging activity was found in A3 and B3. Thus, fractions coming from the isolate and phaseolin had similar activities except for iron chelation, suggesting that phaseolin is the major contributor to the antioxidant and copper chelating activities of the hydrolysed protein isolate.

Hemagglutinating activity of polyphenols extracts from six grain legumes.

The erythrocyte agglutinating activity of polyphenol extracts from six grain legumes was investigated. Polyphenols are amphipathic molecules that can bind to proteins and lipids through hydrophobic and polar interactions, leading to agglutination of liposomes and bacteria. The extracts from four of the six legumes that were studied caused erythrocyte agglutination at concentrations in the µM range. Soybean extracts had the highest activity, followed by the extracts from lentils, broad bean, and chickpea. As a good representative of these legumes, binding of the polyphenols extracted from lentils to erythrocytes was investigated in more detail, showing that agglutination was mediated by binding of 84% of the polyphenols present in the incubation, which corresponds to 2.42 µg bound polyphenols/mg erythrocytes, and a maximum polyphenol binding of 96% according to Lineweaver-Burk plots. The relatively high concentrations that are required for agglutination justify that polyphenols more probably do not agglutinate erythrocytes in vivo, but the possibility still exists that in vivo binding without agglutination could occur, which could have some effects on the metabolism and health-promoting properties of polyphenols.

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.

Antioxidant and chelating activity of Jatropha curcas L. protein hydrolysates.

BACKGROUND: Antioxidant and chelating activities were determined in protein hydrolysates that were produced by treating a protein isolate of a non-toxic genotype of Jatropha curcas with the protease preparation alcalase. RESULTS: 50 min protein hydrolysate with a degree of hydrolysis of 31.7% showed highest antioxidant and chelating activity. These activities were also determined in six peptidic fractions that were separated by gel filtration chromatography of the 50 min hydrolysate. The lower-molecular-weight peptidic fractions had the highest antioxidant and chelating activities, which correlated with a higher content in antioxidant and chelating amino acids such as tyrosine and histidine. CONCLUSION: Results show that J. curcas represents a good source of bioactive peptides. This may be important for the revalorization of defatted J. curcas flour, a by-product resulting form oil extraction for biodiesel production. This is especially important in Third World and developing countries such as Mexico.

Sunflower protein hydrolysates reduce cholesterol micellar solubility.

Plant protein hydrolysates are a source of bioactive peptides. There are peptides that decrease the micellar cholesterol solubility from bile acids and therefore may reduce in vivo cholesterol absorption. The presence of these peptides in sunflower protein hydrolysates has been studied. Sunflower protein hydrolysates produced with alcalase plus flavourzyme or with pepsin plus pancreatin inhibited in some degree the cholesterol incorporation to micelles. Protein hydrolysates generated after 30 min of hydrolysis with alcalase, and after 30 min of hydrolysis with pepsin, were the inhibitoriest of the cholesterol incorporation to micelles. The average amino acid hydrophobicity of inhibitory peptides in cholesterol micelles was higher than the observed in the corresponding protein hydrolysates. This high hydrophobicity probably favours their inclusion in the lipid micelles. In vivo, this inhibition may translate in a decrease of cholesterol absorption. Reported results show that a combination of different characteristics such as peptide size or hydrophobicity may be responsible of the inhibitory activity of generated peptides.

Chickpea protein hydrolysate as a substitute for serum in cell culture.

The growth of mammalian cells in vitro requires the use of rich culture media that are prepared by combining serum with specific nutrient formulations. Serum, the most expensive component of culture media, provides a complex mixture of growth factors and nutrients. Protein hydrolysates that can support in vitro cell growth and eliminate or reduce the need to use serum have been obtained from different sources. Here we describe the use of two food grade proteases to produce a chickpea protein hydrolysate that has been added to cell culture medium in order to determine whether it can be used as a substitute for serum. Medium containing the hydrolysate has been tested using two human cells lines: the monocytic THP-1 cell line which grows in suspension, and the epithelial Caco-2 cell line which grows as a monolayer. The chickpea protein hydrolysate was a good substitute for serum in the first case, but did not allow growth of Caco-2 cells. Supplementation of culture media with this inexpensive and safe hydrolysate would greatly reduce the cost of cell culture.

Partial purification and immobilization/stabilization on highly activated glyoxyl-agarose supports of different proteases from flavourzyme.

The fractioning of some components and their immobilization of Flavourzyme, a commercial protease/aminopeptidase preparation, has been investigated to improve its specificity and stability. Adsorption of Flavourzyme on two ionic exchangers yielded two fractions with endoprotease activity and one fraction containing aminopeptidase activity. The use of an amine agarose gel has made it possible to purify a 43 kDa protein with only endoprotease activity. Immobilization of this endoprotease and the original Flavourzyme preparation onto glyoxyl-agarose provided derivatives that were more thermostable than their soluble counterparts. Tests using immobilized Flavourzyme and immobilized purified endoprotease for the hydrolysis of chickpea proteins showed that both preparations can be used for the production of protein hydrolysates and compare very favorably with the original crude Flavourzyme in terms of reducing the production of free amino acids. This was especially so in the case of immobilized endoprotease, which produced only 0.2% free amino acids. Keeping free amino acids content low is very important in protein hydrolysates for nutritional use to avoid excessive osmotic pressure.

Affinity purification of copper-chelating peptides from sunflower protein hydrolysates.

Copper-chelating peptides were purified from sunflower protein hydrolysates by affinity chromatography using immobilized copper. A variety of protein hydrolysates were obtained by incubation with the proteases Alcalase and Flavourzyme for different periods of time. Chelating activity was indirectly determined by measuring the inhibitory effect of hydrolysates on the oxidation of beta-carotene by copper. Copper-binding peptides purified from the two hydrolysates that inhibited oxidation by copper the most contained 25.4 and 42.0% histidine and inhibited beta-carotene oxidation 8 and 3 times more than the original hydrolysates, which had 2.4 and 2.6% histidine, respectively. Thus, histidine content is not the only factor involved in antioxidant activity, and probably other factors such as peptide size and amino acid sequence are also important. This work shows that affinity chromatography can be used for the purification of copper-chelating peptides and probably other metals of nutritional interest such as calcium, iron, and zinc. In addition to their antioxidant potential, chelating peptides are of nutritional interest because they increase bioavailability of minerals.

Affinity purification of copper chelating peptides from chickpea protein hydrolysates.

Chickpea protein hydrolysates obtained with alcalase and flavourzyme were used for purification of copper chelating peptides by affinity chromatography using copper immobilized on solid supports. The chelating activity of purified peptides was indirectly measured by the inhibition of beta-carotene oxidation in the presence of copper. Two protein hydrolysates, obtained after 10 and 100 min of hydrolysis, were the most inhibitory of beta-carotene oxidation. Purified copper chelating peptides from these protein hydrolysates contained 19.7 and 35.1% histidine, respectively, in comparison to 2.7 and 2.6% in the protein hydrolysates. Chelating peptides from hydrolysate obtained after 10 min of hydrolysis were the most antioxidative being 8.3 times more antioxidative than the hydrolysate, while chelating peptides purified from protein hydrolysate obtained after 100 min were 3.1 times more antioxidative than its hydrolysate. However, the histidine content was higher in peptides derived from the 100 min hydrolysate (19.7 against 35.1% in 10 min hydrolysate), indicating that this amino acid is not the only factor involved in the antioxidative activity, and other factors such as peptide size or amino acid sequence are also determinant. This manuscript shows that affinity chromatography is a useful procedure for purification of copper chelating peptides. This method can be extended to other metals of interest in nutrition, such as calcium, iron, or zinc. Purified chelating peptides, in addition to their antioxidative properties, may also be useful in food mineral fortification for increasing the bioavailability of these metals.

Production of Brassica carinata protein hydrolyzates with a high Fischer's ratio using immobilized proteases.

Brassica carinata protein isolates were hydrolyzed using the digestive enzymes trypsin, chymotrypsin, and carboxypeptidase A in order to obtain hydrolyzates with a high Fischer's ratio. The proteases were immobilized using two glyoxyl-agarose supports of different porosity, 4 and 10% agarose gels, in order to evaluate the effect of substrate diffusion into the support containing the enzyme on the hydrolytic process. Reaction time, substrate concentration, and the enzyme to substrate ratio were optimized in an attempt to increase the Fischer's ratio in the resulting hydrolyzates. Gel filtration chromatography of a hydrolyzate with a degree of hydrolysis of 36% yielded a fraction that represented 31% of the total hydrolyzed proteins and had a Fischer's ratio of 28.3 with a phenylalanine + tyrosine content below 1.5%. This material could be used for preparing special diets when there is a need to increase the supply of branched amino acids and/or reduce the intake of aromatic amino acids.