Subunit composition affects formation and stabilization of o/w emulsions by 11S seed storage protein cruciferin.

The 11S globulin cruciferin is the major storage protein in Brassicaceae/Cruciferae seeds and exists as a hexamer in its natural configuration. Arabidopsis thaliana cruciferin is composed of CRUA, CRUB and CRUC subunits. Wild type (WT) cruciferin and cruciferins composed only of identical CRUA, CRUB and CRUC subunits were examined for their ability to form and stabilize oil-in-water (o/w) emulsions. All proteins (0.9% at pH 7.4 and 2.0), except CRUC, formed stable canola oil or triolein emulsions with a dispersed phase volume fraction of 22-23%. A fine emulsion was formed by CRUB at pH 7.4 with droplet sizes of 6.8 and 8.6 µm for canola oil and triolein, respectively. The presence of 0.5 M NaCl reduced the level of adsorbed protein and protein load at the interface at pH 7.4, and resulted in emulsions that were less stable. Emulsions of CRUA and CRUB (pH 7.4, zero ionic strength, canola oil or triolein) had higher stability than emulsions with WT cruciferin up to 15 days after formation. CRUC formed a stable emulsion only at pH 2.0. The low solubility, low surface hydrophobicity and compact structure of the CRUC protein may contribute to its inferior emulsifying properties at neutral pH; however, acidic pH-induced dissociation of the hexameric assembly improved these properties. The abundance and exposure of hydrophobic residues in the hypervariable regions, extended loop regions, and solvent exposed surfaces of cruciferin are critical factors affecting o/w interface stabilization.

Structural Properties of Cruciferin and Napin of Brassica napus (Canola) Show Distinct Responses to Changes in pH and Temperature.

The two major storage proteins identified in Brassica napus (canola) were isolated and studied for their molecular composition, structural characteristics and the responses of structural features to the changes in pH and temperature. Cruciferin, a complex of six monomers, has a predominantly ß-sheet-containing secondary structure. This protein showed low pH unstable tertiary structure, and distinctly different solubility behaviour with pH when intact in the seed cellular matrix. Cruciferin structure unfolds at pH 3 even at ambient temperature. Temperature-induced structure unfolding was observed above the maximum denaturation temperature of cruciferin. Napin was soluble in a wider pH range than cruciferin and has α-helices dominating secondary structure. Structural features of napin showed less sensitivity to the changes in medium pH and temperature. The surface hydrophobicity (S0) and intrinsic fluorescence of tryptophan residue appear to be good indicators of cruciferin unfolding, however they were not the best to demonstrate structural changes of napin. These two storage proteins of B. napus have distinct molecular characteristics, therefore properties and functionalities they provide are contrasting rather than complementary.

Structural stability and Sin a 1 anti-epitope antibody binding ability of yellow mustard (Sinapis alba L.) napin during industrial-scale myrosinase inactivation process.

This study investigated the structural stability of yellow mustard (YM, Sinapis alba L.) napin and the changes of its Sin a 1 anti-epitope antibody-binding ability during myrosinase enzyme inactivation process. The food industry uses myrosinase-inactive non-pungent YM for uses beyond spice applications. Napin was isolated from seeds received from an industrial processor before (YM + M) and after (YM - M) myrosinase inactivation. Secondary and tertiary structural features and surface hydrophobicity parameters of napin were analyzed. The Sin a 1 content in YM seeds and the stability of Sin a 1-containing napin during simulated in vitro gastrointestinal (GI) digestion were determined by a non-competitive indirect enzyme-linked immunosorbent assay using the Sin a 1 anti-epitope antibody (AE-Ab) as the primary Ab. YM napin retained the dominant alpha-helical components of secondary and tertiary structure folds during this process. YM - M napin showed changes in hydrophobicity parameters of the molecules and binding ability of AE-Ab: 2.19 ± 0.48 g per 100 g of YM - M seeds vs. 1.49 ± 0.16 g per 100 g YM + M seeds. YM - M proteins were more susceptible for in vitro GI digestion and also showed a 30% reduction in AE-Ab binding ability upon digestion of napins. This suggests that the myrosinase inactivation process has induced the surface modification of napin, exposing Sin a 1 epitope, leading to an increase in AE-Ab binding. However, the epitope region of YM - M napin showed improved susceptibility for hydrolysis during GI digestion resulting in fewer available epitope regions, suggesting a possible reduction in napin immune reactivity.