Release of Flavonoids from Lupin Globulin Proteins during Digestion in a Model System

Abstract

Lupin seed globulin proteins form complexes with flavonoids, predominantly apigenin C-glycosides. Enzymes typical for the gastrointestinal tract were used to hydrolyze lupin seed globulins. Release of native flavonoids as a result of the proteolysis reaction was observed. Different analytical methods such as size exclusion chromatography, HPLC-MS, and fluorescence spectroscopy (steady-state fluorescence, fluorescence anisotropy, fluorescence lifetimes) were used for a detailed characterization of this phenomenon. Flavonoids liberated from lupin globulin proteins as a result of pancreatin-catalyzed digestion were bound by γ-conglutin resistant to this enzyme. Two possible mechanisms of this interaction may be suggested: hydrogen bonding between oligosaccharide chains of glycoproteins and the sugar moieties of the flavonoid glycosides or electrostatic attraction between positively charged γ-conglutin and flavonoids partially ionized at pH 7.5.

Digestion susceptibility of seed globulins isolated from different lupin species

“…3b, d, f). Figure 4b (peaks 1 and 2) presents the UV spectra of these compounds which were identical with previously detected apigenin derivatives [23,39]. Domain phenolic compounds present in economically important lupin seeds species (narrow-leaf, yellow and white) are apigenin derivatives [39].…”

“…4b, peaks 1, 2 and 3). Such flavonoids (apigenin derivatives) relisted from narrow-leaf seed globulins during digestion were identified in our previous research [23].…”

“…Extraction of lupin seed globulins was carried out using the method proposed by Czubinski et al [23]. Briefly, albumins were removed from defatted lupin flour using extraction with water pH 8.0 containing 10 mM CaCl 2 , 10 mM MgCl 2 (34 ml per 1 g of lupin flour) for 4 h. Then samples were centrifuged for 1 h at 20,000g, and the pellet devoid of albumin was used for further extraction.…”

“…Digestion of lupin seed globulins was carried out applying the method described previously [23,25]. Firstly, three different combinations of proteolytic enzymes were used in the first stage: pepsin, pancreatin and double digestion model where pepsin followed by pancreatin were used.…”

“…1a, lane 3). Protein bands of these molecular weights correspond to γ-conglutin α (31.4 kDa) and β (16.5 kDa) subunits [23,25,27]. The protein band with a molecular weight of 46 kDa corresponds to not-reduced form of γ-conglutin (coupled α and β subunits) [27].…”