Digestion susceptibility of seed globulins isolated from different lupin species

Jarosław Czubiński,Aleksander Siger,Eleonora Lampart-Szczapa

doi:10.1007/s00217-015-2550-7

Jarosław Czubiński, Aleksander Siger + Show 1 more

Open Access

https://doi.org/10.1007/s00217-015-2550-7

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Abstract

The aim of this work was to study the processes taking place during the enzymatic hydrolysis of seed globulins isolated from narrow-leaf (var. Zeus and Bojar) and yellow (var. Lord and Parys) lupin seeds species cultivated in Poland. In lupin seed globulins hydrolysis studies, there were used enzymes typical for the human gastrointestinal tract, such as: pepsin, pancreatin, trypsin and chymotrypsin. The obtained hydrolysates were assessed based on the results of electrophoretic, immunoblotting, as well as chromatographic separations. The evaluation of lupin seed globulins digestion susceptibility was supported by the bioinformatics analyses. Analysis of hydrolysates showed that the proteins present in globulins are completely hydrolysed by: pepsin, double digestion model (pepsin followed by pancreatin) or chymotrypsin. The high specificity of pancreatin and trypsin action results in limited lupin globulins hydrolysis. Protein fraction resistant to the action of these enzymes was γ-conglutin which also retains its antigenic properties. Its insensitivity to the hydrolysis might be associated with the formation of complexes with flavonoids which were released from other protein connections during digestion, as well as with relatively low number of cleavage sites for trypsin. The analysis of the three-dimensional structure of γ-conglutin enabled a very accurate description of the amino acid residues localisation, at which trypsin hydrolysis should occur.

Highlights

Lupin seeds, because of its nutritional and health-promoting properties, have become functional additive used in the manufacture of food products
Pepsin from the porcine gastric mucosa (P7000, activity ≥250 units/mg protein), pancreatin from the porcine pancreas (P1750, activity equivalent to 4× U.S.P. specifications), trypsin from the porcine pancreas, TPCK treated in order to prevent any chymotrypsin activity (T1426, ≥10,000 units/mg protein), chymotrypsin from the porcine pancreas (C4120, ≥40 units/mg protein), Tris(hydroxymethyl) aminomethane, sodium dodecyl sulphate (SDS), N,N,N′,N′-tetramethylethylenediamine (TEMED), trifluoroacetic acid (TFA), glycine, acrylamide, nitroblue tetrazolium (NBT), 5-bromo-4-chloro-3-indolyl phosphate (BCIP), bovine serum albumin (BSA), antirabbit IgG-alkaline phosphatase antibodies and Coomassie Brilliant Blue R-250 were purchased from Sigma-Aldrich
Hydrolysis conducted by pancreatin, which was used in the double digestion model, caused further degradation of the peptides formed in the first step

Summary

Introduction

Because of its nutritional and health-promoting properties, have become functional additive used in the manufacture of food products. It has been confirmed that the health-promoting properties of lupin seeds are due to the presence of γ-conglutin. This protein fraction can be effectively used in the treatment of diabetes [1,2,3]. Based on the exposure of human hepatocyte cell line (HepG2) to γ-conglutin, it was found that the mechanism of this action is similar to that of insulin in the human body. According to Capraro et al [4], this fraction is able to penetrate into HepG2 cells by pinocytosis and undergoes the multiple phosphorylation after uptake

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