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Sirtori E.,University of Milan | Isak I.,University of Milan | Resta D.,HPF Nutraceutics Srl | Boschin G.,University of Milan | Arnoldi A.,University of Milan
Food Chemistry | Year: 2012

Pea protein is considered to be an emerging alternative for the formulation of numerous food products, including functional foods, because of its purported hypolipidemic, anti-hypertensive and hypoglycaemic activities. The present investigation was designed to evaluate the effects of thermal and mechanical treatments on an industrial pea protein isolate (Pisane®) from Pisum sativum L. As a preliminary step, the main protein components of P. sativum seed were identified, using the canonical proteomic approach, including 2D-separation and mass spectrometry. Most of the main spots were assigned to the major pea storage proteins: legumin, vicilin and convicilin. Differential scanning calorimetry and proteomic techniques were used to investigate the effects of processing on the protein profile and to assess the availability of stable peptides. After prolonged treatments, no proteins were present in their native forms and the protein solubility was greatly decreased; however, some intense spots were still present on 2D-gels. The spot identity was confirmed by HPLC-Chip-MS/MS, showing that the vicilin 30 kDa fragment and legumin acidic subunits were resistant and some specific peptides remained intact. DSC thermograms showed that strong treatments decreased the denaturation enthalpy. The mechanical treatments, instead, did not modify (in a relevant way) either the protein profile or DSC thermograms. © 2012 Elsevier Ltd. All rights reserved. Source


Lammi C.,University of Milan | Zanoni C.,University of Milan | Scigliuolo G.M.,HPF Nutraceutics Srl | D'Amato A.,Polytechnic of Milan | Arnoldi A.,University of Milan
Journal of Agricultural and Food Chemistry | Year: 2014

Previous experiments in suitable animal models and in mild hypercholesterolemic individuals have shown that the consumption of lupin proteins may be useful for controlling total and low-density lipoprotein (LDL) cholesterol levels. With the objective of providing evidence that peptides deriving from the hydrolysis of lupin proteins may be responsible of the observed activities and for investigating the mechanism of action, HepG2 cells were treated with lupin peptides obtained by either pepsin (P) or trypsin (T) hydrolysis, and molecular and functional investigations were performed on the LDL receptor/SREBP2 pathway. For the first time, this paper provides experimental evidence that lupin peptides are able to interfere with the HMGCoAR activity, up-regulating the LDL receptor (136 and 84% vs the control for P and T peptides, respectively, at 1 mg/mL) and SREBP2 proteins (148 and 73% vs the control for P and T peptides, respectively, at 1 mg/mL) via the activation of PI3K/Akt/GSK3β pathways and increasing the LDL uptake at HepG2 cell line (40 and 50% vs the control for P and T peptides, respectively, at 1 mg/mL). These results may be useful in explaining the activities observed in vivo in animals and humans treated with lupin protein. © 2014 American Chemical Society. Source


Resta D.,HPF Nutraceutics Srl | Brambilla F.,HPF Nutraceutics Srl | Arnoldi A.,University of Milan
Food Chemistry | Year: 2012

In food science there is a growing demand of methods for the absolute quantification of proteins, such as allergens or bioactive proteins, and shotgun proteomics based on mass spectrometry is a promising analytical tool in this area. This paper describes an innovative label-free method for the absolute quantification of γ-conglutin, one of the most relevant lupin seed proteins, which is hypoglycaemic and a major allergen. The main features of the method are: (a) the chromatographic separation was performed on an HPLC-chip system coupled with an ion trap mass spectrometer; (b) five proteotypic peptides of γ-conglutin were selected and analysed with a multiple reaction monitoring (MRM) method; (c) absolute quantification was obtained by the standard addition method after purification of a reference sample of γ-conglutin from lupin seed; (d) the matrix effect was overcome by spiking with an exogenous protein, i.e. BSA, as internal standard. © 2011 Elsevier Ltd. All rights reserved. Source


Sirtori E.,University of Milan | Resta D.,HPF Nutraceutics Srl | Arnoldi A.,University of Milan | Arnoldi A.,HPF Nutraceutics Srl | And 2 more authors.
Food Chemistry | Year: 2011

Peanut-allergic individuals may also react to lupin, which, for this reason, has been included in the EU list of food allergens. Since there is not yet any general consensus on the major allergen/s in lupin, the objective of this investigation was to compare the reactivity of the main lupin proteins towards the IgE of the sera of allergic patients. Both Lupinus albus and Lupinus angustifolius were investigated. ELISA's, Western blotting and mass spectrometry, including also de novo sequencing of the unknown lupin proteins, were used for identifying the IgE-binding proteins. Significant differences in the protein reactivities were observed. In particular, there was a direct relationship between the level of peanut-specific IgE's and the cross-reactivity to lupin proteins; also the reactivity of each serum appeared to be unique. Although numerous lupin proteins bind IgE's, our data suggest a predominant contribution of α-conglutin in the reactivity of both L. albus and L. angustifolius. © 2010 Elsevier Ltd. All rights reserved. Source


Boschin G.,University of Milan | Scigliuolo G.M.,HPF Nutraceutics Srl | Resta D.,HPF Nutraceutics Srl | Arnoldi A.,University of Milan | Arnoldi A.,HPF Nutraceutics Srl
Journal of Agricultural and Food Chemistry | Year: 2014

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 IC 50 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. © 2014 American Chemical Society. Source

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