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The use of enzymes to recover soluble peptides with functional properties from insoluble proteins could prove to be very expensive, implying high reaction times and low yields. In this study, the insoluble granular protein, previously delipidated, was hydrolyzed using enzymes (trypsin) as a comparison to the proposed alternative method: subcritical water hydrolysis (SWH) using both nitrogen and oxygen streams. The result of the hydrolysis was characterized in terms of the yield and peptide size distribution as well as different functional properties. The SWH of the delipidated granules resulted in a higher recovery yield than that obtained by enzymatic hydrolysis in half of the time. The foaming capacity of the peptides obtained by SWH was higher than that obtained by trypsin hydrolysis, although the foam stability was lower. Slight differences were detected between these peptides in terms of their emulsifying properties.

Potential residues of the potent allergen lysozyme used as a microbial stabilizing agent in wine production might pose a serious health thread to susceptible individuals. Therefore, EU legislation requires the labeling of the allergenic agent, if it is present in the final product. To allow for product testing, an indirect ELISA method to be specifically used in wine analysis was developed and validated. Furthermore, trial wines treated with defined amounts of lysozyme were subjected to an array of different filtration and other enological processing regimes in order to evaluate their potential to deplete the allergen content of the wines. By these means, processing methods ought to be identified that can be integrated in a good manufacturing practice guideline to enable wine producers to utilize lysozyme in their cellars and still provide wines free of allergenic residues. However, among the enological procedures under scrutiny, only bentonite fining proved to be capable of significantly reducing the allergenic residues.

The effect of steady shear and turbulent flow on the formation of amyloid fibrils from hen egg white lysozyme (HEWL) was studied. The conversion and size distribution of fibrils obtained by heating HEWL solutions at pH 2 were determined. The formation of fibrils was quantified using flow-induced birefringence. The size distribution was fitted using decay of birefringence measurements and transmission electron microscopy (TEM). The morphology of HEWL fibrils and the kinetics of their formation varied considerably depending on the flow applied. With increasing shear or stirring rate, more rod-like and shorter fibrils were obtained, and the conversion into fibrils was increased. The size distribution and final fibril concentration were significantly different from those obtained in the same heat treatment at rest. The width of the length distribution of fibrils was influenced by the homogeneity of the flow.

d-Psicose (Psi) might be an ideal sucrose (Suc) substitute for food products due to its sweet taste, easy processing, and functional properties (noncaloric and low glycemic response). In the present study, the effects of Psi on foaming properties of egg white (EW) protein and the quality of butter cookies were analyzed to find a better use of Psi in aerated food systems. The results showed that Psi could improve the foaming properties of EW protein with increasing whipping time in comparison to Suc and d-fructose (Fru). The addition of Psi to butter cookies, as partial replacement of Suc, had no influence on the cook loss while significantly contributing to a color change of the cookie crust through a nonenzymatic browning reaction. Furthermore, Psi-containing cookies possessed the highest antioxidant capacity in all tested cookies using two assays of radical scavenging activity and ferric reducing power. It was found that there was a close correlation between the crust color and the antioxidant activity of the cookie. The results suggest that the addition of Psi enhanced the browning reaction during cookie processing and, consequently, produced a strong antioxidant activity.

Hen's egg allergy represents one of the most common and severe IgE-mediated reactions to food in infants and young children. It persists, however, in many cases also lifelong. Therefore, the aim of this study was the detailed analysis of a technological process used to reduce the allergenic potential of hen's egg. The investigation focused on the pasteurized egg as starting material, intermediate, and final products of a nine-step manufacturing process performed for use of eggs in convenience products appropriate for allergic individuals. The steps consisted of a combination of various heat treatments and enzymatic hydrolyses. The alterations were controlled by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE), immunoblotting, enzyme allergosorbent test (EAST) inhibition, and mass spectrometry. Thereby it could be demonstrated that the allergenic potential of the raw material was reduced from step to step, and despite the known stability against heat and proteolysis of certain egg proteins, the total allergenic potential was finally below 1/100 that of the starting material without a significant change in texture and flavor as evaluated in various products.

Apolipoprotein B (apoB) is a nonexchangeable apolipoprotein that dictates the synthesis of chylomicrons and very low density lipoproteins. ApoB is the major protein in low density lipoprotein, also known as the "bad cholesterol" that is directly implicated in atherosclerosis. It has been suggested that the N-terminal domain of apoB plays a critical role in the formation of apoB-containing lipoproteins through the initial recruitment of phospholipids in the endoplasmic reticulum. However, very little is known about the mechanism of lipoprotein nucleation by apoB. Here we demonstrate that a strong phospholipid remodeling function is associated with the predicted alpha-helical and C-sheet domains in the N-terminal 17% of apoB (B17). Using dimyristoylphosphatidylcholine (DMPC) as a model lipid, these domains can convert multilamellar DMPC vesicles into discoidal-shaped particles. The nascent particles reconstituted from different apoB domains are distinctive and compositionally homogeneous. This phospholipid remodeling activity is also observed with egg phosphatidylcholine (egg PC) and is therefore not DMPC-dependent. Using kinetic analysis of the DMPC clearance assay, we show that the identified phospholipid binding sequences all map to the surface of the lipid binding pocket in the B17 model based on the homologous protein, lipovitellin. Since both B17 and microsomal triglyceride transfer protein (MTP), a critical chaperone during lipoprotein assembly, are homologous with lipovitellin, the identification of these phospholipid remodeling sequences in B17 provides important insights into the potential mechanism that initiates the assembly of apoB-containing lipoproteins.

Apolipoprotein B (apoB) is the essential nonexchangeable protein in chylomicrons and very low-density lipoprotein-derived lipoprotein particles, including low-density lipoprotein (LDL). ApoB has been a key target for cardiovascular research because of its essential role in the assembly, secretion, delivery, and receptor binding of LDL. The three-dimensional structure of apoB has not been determined. However, the N-terminal region of apoB is homologous to the lipid storage protein lipovitellin, which allows the modeling of this region based on the X-ray structure of lipovitellin. The model of the N-terminal 17% of apoB (B17) suggests that, like lipovitellin, B17 consists of an N-terminal beta-barrel domain, a helical domain, and a beta-sheet domain (C-sheet). Here we test the validity of this model by limited proteolysis of B17 and the characterization of individual domains expressed in Escherichia coli and insect cell systems that are consistent with the model and proteolysis data. Circular dichroism studies of the individual domains indicate that they are folded and their secondary structures are in agreement with the model. We find that the helical domain and C-sheet of apoB interact with each other in vitro, suggesting a strong interaction between these two domains, even without a covalent peptide bond linkage. Our data suggest that the three lipovitellin-like domains exist in B17. Furthermore, the domains fold independently with secondary structures and stabilities like those of intact B17.

The refined molecular structure of lipovitellin is described using synchrotron cryocrystallographic data to 1.9 A resolution. Lipovitellin is the predominant lipoprotein found in the yolk of egg-laying animals and is involved in lipid and metal storage. It is thought to be related in amino acid sequence to segments of apolipoprotein B and the microsomal transfer protein responsible for the assembly of low-density lipoproteins. Lipovitellin contains a heterogeneous mixture of about 16% (w/w) noncovalently bound lipid, mostly phospholipid. Previous X-ray structural studies at ambient temperature described several different protein domains including a large cavity in each subunit of the dimeric protein. The cavity was free of any visible electron density for lipid molecules at room temperature, suggesting that only dynamic interactions exist with the protein. An important result from this crystallographic study at 100 K is the appearance of some bound ordered lipid along the walls of the binding cavity. The precise identification of the lipid type is difficult because of discontinuities in the electron density. Nonetheless, the conformations of 7 phospholipids and 43 segments of hydrocarbon chains greater than 5 atoms in length have been discovered. The conformations of the bound lipid and the interactions between protein and lipid provide insights into the factors governing lipoprotein formation.

Complementary DNA coding for the channel-forming alpha-subunit of a large conductance Ca(2+)-activated K+ channel (maxi Kca channel) was cloned from bovine aortic smooth muscle cells. This cloned mammalian KCa channel (Bslo) and its homolog from Drosophila (Dslo) were expressed in the HEK293 human embryonic kidney cell line. Both Bslo and Dslo KCa channels were sensitive to inhibition by the internally applied serine proteinase inhibitors: bovine pancreatic trypsin inhibitor (BPTI, KD = 7.0 microM for Bslo and 2.6 microM for Dslo) and chicken ovoinhibitor (OI, KD = 1.5 microM for Bslo and 11.4 microM for Dslo). BPTI and OI are members of the Kunitz and Kazal families of proteinase inhibitors, respectively. The approximately 60-residue inhibitory domains of these proteins have a different tertiary structure except in the region of a loop formed by approximately 6 residues, in which the peptide backbone adopts a similar conformation complementary to the active site cleft of many serine proteinases. At the single-channel level, BPTI and OI were found to inhibit KCa channels by a similar mechanism involving the production of discrete low-conductance events. These two inhibitors also exhibited competitive behavior, suggesting that they bind to an overlapping site. Kinetic characterization revealed that the dissociation rate of BPTI from the bovine KCa channel is fast (k(off) = 0.41 s-1), whereas that from the Drosophila KCa channel is slow (k(off) = 9.0 x 10(-4) s-1) and indicative of a strong molecular interaction. The stable complex of BPTI and trypsin was inactive as a KCa channel inhibitor, further supporting the idea that the trypsin inhibitory loop of BPTI recognizes a specific site on the channel protein. These results lead to the conclusion that the alpha-subunit of maxi KCa channels contains a conserved proteinase inhibitor binding site. We hypothesize that this site corresponds to a C-terminal domain of the channel protein that structurally resembles serine proteinases.