Julio Girón-Calle

Production of ace inhibitory peptides by digestion of chickpea legumin with alcalase

Abstract Short peptides from different sources have proved to be very efficient inhibitors of the angiotensin I-converting enzyme, an enzyme with a major role in the regulation of blood pressure. These peptides are of therapeutic value, so that the possibility of obtaining such peptides by treatment of chickpea legumin with the protease alcalase has been explored. Legumin is the main storage protein in chickpea seeds. Treatment of legumin with alcalase yielded a hydrolysate that inhibited the angiotensin I-converting enzyme with an IC 50 of 0.18 mg/ml. Fractionation of this hydrolysate by reverse phase chromatography afforded six inhibitory peptides with IC 50 values ranging from 0.011 to 0.021 mg/ml. All these peptides contain the amino acid methionine and are also rich in other hydrophobic amino acids. These results demonstrate that hydrolysates of chickpea legumin obtained by treatment with alcalase are a good source of peptides with angiotensin I-converting enzyme inhibitory activity.

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 β-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.

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.

Stabilization-immobilization of carboxypeptidase A to aldehyde-agarose gels: A practical example in the hydrolysis of casein

Abstract A procedure of covalent multipoint attachment to modified 10 BCL agarose gels has been performed for stabilization–immobilization of carboxypeptidase A (CPA). All the enzyme offered to the support was immobilized, yielding an intrinsic activity of 55% as compared to the soluble enzyme. The multipoint derivative of CPA is more than 1000 times more stable than the soluble enzyme in absence of autolysis phenomena, as shown by thermal inactivation of the soluble and immobilized enzymes. In order to test the activity of this new immobilized derivative, it was used for hydrolysis of casein previously treated with chymotrypsin. Results show specific release of amino acids depending on the time of action of CPA. This immobilization–stabilization might play a very important role to allow the use of expensive enzymes in specific applications where time, temperature or the use of chemical reagents are limiting factors.

Effect of chickpea aqueous extracts, organic extracts, and protein concentrates on cell proliferation.

Pulses should be part of a healthy diet, and it is also becoming clear that they have health-promoting effects. Nevertheless, most studies on the bioactive or health-promoting properties of pulses have been carried out using soybeans. We have studied cell growth-regulating properties, which may be responsible for anti-cancer properties, in chickpea seeds. Chickpea seeds are a staple in the traditional diet of many Mediterranean, Asian, and South and Central American countries. In addition, chickpea seeds have industrial applications since they can be used for the preparation of protein concentrates and isolates. The cell lines Caco-2 (epithelial intestinal) and J774 (macrophages) have been exposed to chickpea seed extracts and protein preparations in order to screen the different chickpea fractions for effects on cell growth. Both cell growth-promoting and cell growth-inhibiting effects were found. Most interestingly, a fraction soluble in ethanol and acetone specifically and almost completely inhibited the growth of Caco-2 cells exhibiting a cancerous phenotype. It is concluded that chickpea seeds are a source of bioactive components and deserve further study for their possible anti-cancer effect.

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 β-carotene oxidation (62.41 ± 0.43%), β-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.

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.

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.