Donatella Resta

ACE-inhibitory activity of enzymatic protein hydrolysates from lupin and other legumes.

The objective of this investigation was to compare the angiotensin converting enzyme (ACE)-inhibitory activity of the hydrolysates obtained by pepsin digestion of proteins of some legumes, such as chickpea, common bean, lentil, lupin, pea, and soybean, by using the same experimental procedure. The ACE-inhibitory activity was measured by using the tripeptide hippuryl-histidyl-leucine (HHL), as model peptide, and HPLC-DAD, as analytical method. The peptide mixtures of all legumes were active, with soybean and lupin the most efficient, with IC50 values of 224 and 226 μg/ml, respectively. Considering the promising results obtained with lupin, and aiming to identify the protein(s) that release(s) the peptides responsible for the activity, the peptides obtained from the pepsin digestion of some industrial lupin protein isolates and purified protein fractions were tested. The most active mixture, showing an IC50 value of 138 μg/ml, was obtained hydrolysing a mixture of lupin α+β conglutin.

A label‐free internal standard method for the differential analysis of bioactive lupin proteins using nano HPLC‐Chip coupled with Ion Trap mass spectrometry

Quantitative proteomics based on MS is useful for pointing out the differences in some food proteomes relevant to human nutrition. Stable isotope label‐free (SIF) techniques are suitable for comparing an unlimited number of samples by the use of relatively simple experimental workflows. We have developed an internal standard label‐free method based on the intensities of peptide precursor ions from MS/MS spectra, collected in data dependent runs, for the simultaneous qualitative characterization and relative quantification of storage proteins of Lupinus albus seeds in protein extracts of four lupin cultivars (cv Adam, Arés, Lucky, Multitalia). The use of an innovative microfluidic system, the HPLC‐Chip, coupled with a classical IT mass spectrometer, has allowed a complete qualitative characterization of all proteins. In particular, the homology search mode has permitted to identify single amino acid substitutions in the sequences of vicilins (β‐conglutin precursor and vicilin‐like protein). The MS/MS sequencing of substituted peptides confirms the high heterogeneity of vicilins according to the peculiar characteristics of the vicilin‐encoding gene family. Two suitable bioinformatics parameters were optimized for the differential analyses of the main bioactive proteins: the “normalized protein average of common reproducible peptides” (N‐ACRP) for γ‐conglutin, which is a homogeneous protein, and the “normalized protein mean peptide spectral intensity” (N‐MEAN) for the highly heterogenous class of the vicilins.

Evaluation of total quinolizidine alkaloids content in lupin flours, lupin-based ingredients, and foods

Lupin proteins are gaining attention to replace animal proteins and other plants ingredients in several foods such as bakery products, imitation dairy and meat products, and beverages. One of the major safety issues of lupin-based foods is the presence of quinolizidine alkaloids (QAs), bitter compounds produced by lupin plants as a defense mechanism against predators. In mammals, QA intoxication is characterized by trembling, shaking, excitation, and convulsion. Lupanine and sparteine, the most common QAs, show acute oral toxicity due to neurological effects leading to the loss of motor co-ordination and muscular control. In this paper, 27 samples of lupin-based products, i. e., flours, protein isolates, and food (either model or commercially available ones), were analyzed for evaluating the QA content using a method based on GC/MS. All the analyzed samples were safe since they respect the maximum limit of 200 mg/kg fixed by the Health Authorities of Australia, New Zealand, Great Britain, and France, that have regulated this topic. The QA contents were particularly low in protein isolates and in foods containing these ingredients, indicating that their use is a very effective tool for keeping low the daily intake of QAs.

Optimization of the Enzymatic Hydrolysis of Lupin (Lupinus) Proteins for Producing ACE-Inhibitory Peptides.

Recently, the enzymatic hydrolysis of Lupinus albus and Lupinus angustifolius proteins with pepsin was showed to produce peptides able to inhibit the angiotensin-converting enzyme (ACE). The objective of the present work was to test different hydrolytic enzymes and to investigate three lupin species (L. albus, L. angustifolius, Lupinus luteus) with the final goal of selecting the best enzyme/species combination for an efficient production of ACE-inhibitory peptide mixtures. Pepsin gave peptides with the best IC50 values (mean value on three species 186 ± 10 μg/mL), followed by pepsin + trypsin (198 ± 16 μg/mL), chymotrypsin (213 ± 83 μg/mL), trypsin (405 ± 54 μg/mL), corolase PP (497 ± 32 μg/mL), umamizyme (865 ± 230 μg/mL), and flavourzyme (922 ± 91 μg/mL). The three species showed similar activity scales, but after pepsin + trypsin and chymotrypsin treatments, L. luteus peptide mixtures resulted to be significantly the most active. This investigation indicates that lupin proteins may be a valuable source of ACE-inhibitory peptides, which may explain the activity observed in experimental and clinical studies and foresee the application of lupin proteins into functional foods or dietary supplements.

Quinolizidine Alkaloids in Seeds of Lupin Genotypes of Different Origins

The intake of lupin-based foods could imply the exposure of consumers to quinolizidine alkaloids. The objectives of this study were to assess the genetic variation among and within 11 geographic regions of Lupinus albus ecotypes, verify the quinolizidine alkaloids amount of alkaloid-poor L. albus and Lupinus angustifolius varieties, and assess the effect of two climatically contrasting Italian environments on the alkaloid content. The quantitation was performed by GC-MS, and in all samples lupanine was the most abundant quinolizidine alkaloid, followed by albine and 13alpha-hydroxylupanine for L. albus and by 13alpha-hydroxylupanine and angustifoline for L. angustifolius. Some regions tended to have a high (Azores) or low (Egypt, Near East, Maghreb) total alkaloids content, but the variation among ecotypes within regions was larger than that among regions following the estimation of variance components. Alkaloid-poor varieties tended to have higher total alkaloid contents when grown in the subcontinental climate site, exceeding in some cases the limit of 0.200 mg/g.

Changes of isoflavones during the growth cycle of Lupinus albus.

The objective of this study was to monitor the changes in isoflavone content in different plant organs (leaves, stems, roots) during the crop growth stage of three cultivars of Lupinus albus (white lupin) under field conditions, taking into account sowing time effects (autumn and early spring) and cultivar effects. Three sampling dates (from late vegetative to late grain growth stages) were evaluated. Seven isoflavones and four flavonoids were identified by LC-ESI-MS analysis. The isoflavone content was higher in leaves than in stems, and it was highest before flowering, whereas it decreased during maturity. Autumn-sown plants showed higher isoflavone content than early spring-sown plants, especially in late vegetative and early reproductive stages. Genistein 7- O-glucoside was the main isoflavone of leaves and stems in the late vegetative stages of early spring sowing, whereas genistein was the main isoflavone under autumn sowing. Variation among cultivars affected only marginally the total isoflavone content. No isoflavones were detected in seeds.